Annual Report 2012 - Rajiv Gandhi Centre for Biotechnology

Transcription

Annual Report
2012
Rajiv Gandhi Centre for Biotechnology
Thiruvananthapuram, Kerala
Phone: +91 471 2341716, 2347975
Fax: +91 471 2348096, 2346333
E-mail: [email protected]
Website: www.rgcb.res.in
Contents
Director’s Report
05
CANCER BIOLOGY
Cancer Research Program - 1
09
11
13
t
o
N
s
t
n
e
t
n
Co Done
Cancer Research Program – 7
Cancer Research Program – 8
16
18
20
22
24
26
CARDIOVASCULAR & DIABETES BIOLOGY
Cardiovascular Disease Biology
32
Diabetes Disease Biology
36
PATHOGEN BIOLOGY
Mycobacterium Research Group - 1
38
Mycobacterium Research Group – 2
40
Molecular Virology
42
Viral Disease Biology
44
Cholera Biology
50
REPRODUCTIVE BIOLOGY
Molecular Reproduction - 1
52
Molecular Reproduction - 2
54
NEUROBIOLOGY
Molecular Neurobiology
56
Neural Stem Cell Biology
60
Human Molecular Genetics
62
Neurobiophysics
63
CHEMICAL & ENVIRONMENTAL BIOLOGY
annual report - 2011
3
Chemical Biology - 1
65
67
Molecular Ecology
69
Environmental Biology
71
PLANT DISEASE BIOLOGY
Plant Disease Biology & Biotechnology - 1
73
75
76
78
Laboratory Medicine And Molecular Diagnostics
Regional Facility for DNA Fingerprinting
Instrumentation Engineering
Library and Information Services
4
Animal Research Facility
84
Distributed Information Sub –Center 86
Administrative Division
88
Staff List
89
Invited Speakers at RGCB
92
rajiv gandhi centre for biotechnology
DIRECTOR’S REPORT 2012
“Destiny is not a matter of chance; it is a matter of choice.
It is not a thing to be waited for; it is a thing to be achieved.”
William Jennings Bryan
Seven years we decided not to wait for destiny
to happen but go ahead and make it happen.
And we did. However these are now times of
turbulent budget cuts and increasing pressure
to transform our style of research and delivery.
There is increased demand for better performance
and delivery from both the government who funds
the institute in toto as well as the public, whose
tax money goes into this funding. Unless these
expectations are actively met, institutes, such as
ours will find it difficult to move on, let alone excel.
Now is the right time to act. At RGCB we are
fortunate to have at the heart of every scientist,
student and staff member, a deep commitment to
discovery and innovation for the welfare of India
and mankind all over. As we finish one more year
of existence, this passion has become stronger
than ever.
The master plan for the RGCB Bio-Innovation
Center to be located in second campus at
Aakulam is ready. Since construction of the
above facility will take time owing to detailed
government procedures involved, we decided to
set up the core facilities of the innovation center at
our new transit facility, a “ready to use” building
in the KINFRA Video Park adjacent to the Sainik
School at Kazhakootam. High tech equipment
to set up the central core facilities in bio imaging
and genomics has been purchased for the bioinnovation center. This transit facility should be
ready by December 2013.
We did well in the year, both in fundamental
research and innovation for process & product
development. RGCB embarked on a large
multifaceted research program into diabetes with a
large number of investigators within and outside the
institute. The year saw an important finding from
our Molecular Reproduction group that suggested
molecular signaling set off by ERα and CrkL
association to have a central role in pregnancy and
cancer, two events which share many parallels in
growth, adhesion, invasion and immune tolerance.
Our Cancer Research Program elucidated
the biological properties of a novel hydroxyl
porphyrin-THPP, a compound synthesized by
our collaborators at the National Institute of
Interdisciplinary Science and Technology. The
compound is water-soluble, generates appropriate
singlet oxygen and has high absorption spectra
in the near infrared red (NIR) region - all key
requirements for a successful photodynamic
agent. Further our studies demonstrated that
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THPP uniquely exhibited high photo-cytotoxicity,
and localized specifically in the nucleus strongly
supporting its potential application both as efficient
photo-sensitizer for photodynamic therapy (PDT)
and as a NIR imaging agent.
Our Molecular Ecology group provided
important evidence to show presence of the
fungus Batrachochytrium dendrobatidis (bd)
in swabs taken from skins of various frog
species in various reserve forest locations of the
Western Ghats. This was the first such screening
reported from the Indian sub continent, which
until now, was generally believed to be free of
the deadly fungus. The skin infecting fungus
causes lesions and shedding of top-layer which
then cause the amphibians absorb water and
various salts through the skin eventually causing
the amphibian heart to stop and finally death.
The International Union for Conservation of
Nature (IUCN) has called this amphibian
chytridiomycosis the worst infectious disease
ever recorded among vertebrates in terms of the
number of species impacted, and its propensity
to drive them to extinction. It is believed that the
fungus originated in Africa and traveled all over
the world piggyback riding on the African clawed
frog that was exported for various studies and
clinical testing.
Our plant biotechnologists provided more evidence
this year for molecular pathways involved in
fungal disease of ginger, a most important cash
crop of Kerala. Expression signature of key genes
involved in major defense signaling pathways
was examined after inoculation with the fungal
pathogen, Pythium in both cultivated ginger and
its wild relative Z. zerumbet. Results indicated
a key role for the modulation of reactive oxygen
species signaling in governing Pythium resistance
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in Z. zerumbet. The transcript accumulation of
ROS scavengers was found to be significantly
higher in the resistant host. Moreover, transcript
levels of ROS scavengers were significantly higher
even at later time periods, indicating a persistent
ROS scavenging activity in the incompatible host,
whereas in the compatible host, transcripts of
ROS scavengers were comparatively weak and
occurred only at an early time period. RGCB
plant biotechnologists also reported this year
characterization of a novel Type III polyketide
synthase, quinolone synthase (QNS), from Aegle
marmelos that is involved in the biosynthesis
of quinolone alkaloid. Using homology-based
structural modeling, two crucial amino acid
residues (Ser-132 and Ala-133) were identified
at the putative QNS active site. Mutagenic studies
suggest that this protein may have evolved from
an evolutionarily related member of chalcone
synthase super family by mere substitution of
two active site residues. The identification and
characterization of QNS offers a promising
target for gene manipulation studies toward the
production of novel alkaloid scaffolds.
International recognition of RGCB’s capabilities
in elucidating disease biology also came our way
during the year. RGCB together with Mayo Clinic
received a major National Institutes of Health
grant to study vaccine and natural infection
immune responses to the pandemic H1N1 flu. The
European Union has given us a large multi centre
grant to study viral involvement in development
of head & neck cancer.
Currently used drug screening strategy utilizes the
NCI-60 panel for end-point metabolic activity.
However, our present understanding of the cancer
and the signaling pathways involved has led to the
realization that screens for apoptosis are far more
superior to assays of cellular metabolic status.
Miniaturization of apoptosis screens however
remains a formidable challenge. Our cancer
researchers have developed a novel strategy to
achieve such a miniaturization of apoptosis screen
using Fluorescence Resonance Energy transfer
(FRET)-based tools for apoptosis detection.
These FRET-based sensors contain Cyan and
Yellow fluorescent proteins; the energy transfer
between which indicates activation of caspases,
proteases involved in apoptosis, with untreated
cells appearing green and caspase-activated cells
appearing blue due to loss of energy transfer.
The researchers then developed a panel of cancer
cell lines bearing the FRET-based sensors and
used these for high throughput image based,
simultaneous quantitation of caspase activation
and multiple other apoptosis events, thereby
successfully miniaturizing apoptosis detection.
Our Neurobiology group has developed a new/
alternate technique for detection of intracellular
release of free calcium, which is now ready for
interested users to license. Its main application
would be in measuring the activity of calcium
channel proteins since activity of calcium channel
proteins leads to release of intracellular free calcium.
The Molecular Diagnostics Group have developed
a “point of care” diagnostic kit to detect swine fever
meant for use by farmers and veterinarians who
rear and manage pigs, a major large and small-scale
industry in North East India. This kit has completed
field-testing and we hope it will be marketed soon.
RGCB continued as in the previous years to
support societal needs of the country and state.
We performed numerous DNA Fingerprinting
analyses for crime and legal institutions and
DNA barcoding for the Forests Department
mainly to identify illegally hunted game meat. Our
Laboratory Medicine & Molecular Diagnostics
facility continues to be mainstay for molecular
viral diagnosis in the state especially during
influenza and other viral fever pandemics.
The RGCB Governing Council and the Scientific
Advisory Council have always strongly supported
the institute and provided much needed mentoring.
I must here write a few words about our first
Chairman of the RGCB Governing Council
and former Secretary of the Department of
Biotechnology who recently retired. Padma
Bhushan Maharaj Kishen Bhan was always an
enigma for RGCB. Having been initially not in
favor of making RGCB a national institution
under the Department of Biotechnology, once
this happened, he subsequently led the Governing
Council from the front, consistently raising the
performance bar. Governing Council meetings
were never routine administrative meetings but
full-fledged discussions on science, science quality
and science management. The enormous scientific
talent of the Governing Council members further
enhanced the quality of these discussions. No
matter how prepared the Director of the institute
went for these meetings, fully armed with fact
and figures, it would take only a few minutes
for Dr Bhan to rip them apart and point out
how standards must improve. In short, he was
responsible for shaking RGCB out of that comfort
zone in which the institute for long placed itself. I
would therefore use my grandmother’s definition
of the gooseberry to best phenotype Dr Bhan. First
a bitter taste, then a little sour, which then gives
way to a lasting sweetness. Thank you Dr Bhan.
We will continue Discovery and Innovation with
top performance.
I have to place on formal record, RGCB’s and
my personal heartfelt gratitude to Mr. Oommen
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Chandy, Honorable Chief Minister of Kerala
and Mr. Adoor Prakash, Honorable Minister
for Revenue for having taken RGCB’s
land ownership at no cost to the Council of
Ministers for formal approval. RGCB is today
the proud and sole owner of land that houses
the present campus at Poojapura and the land
allotted for its second campus at Aakuluam.
In this matter, I must also place on record
the dedicated efforts of my good friend and
colleague, Mr. Mohanan Nair who patiently
and consistently articulated RGCB’s case
with the state government and finally obtained
this most favorable decision for RGCB.
Last but in no way the least, I would also like
to acknowledge the contributions to RGCB
by three excellent officers, Mr. Rajan Panicker,
IA&AS (Retd), our Controller of Administration,
Ms C. Jayatheeswary, our Finance Officer and
Major K.P Sanjeev (Retd), our Security Officer
all of who retired this year. Mr. Rajan Panicker
came to us from his position as Deputy Accountant
General of Kerala. His administrative experience
and outstanding sincerity stood in great stead for
RGCB, guiding the institute to its development,
particularly in implementation of key staff welfare
policies. Ms Jayatheeswary came on deputation
from the Accountant General’s office and played a
critical role in managing and balancing the increasing
RGCB budget. Major Sanjeev joined RGCB after a
successful career in the Indian Army. A postgraduate
in Telecommunication Engineering, he played a
major role looking after RGCB’s construction work
in addition to his duties of managing security of the
institute. It was a pleasure to have worked with
Mr. Rajan Panicker, Ms C. Jayatheeswary and
Major Sanjeev and we will be always grateful
to them. I also take this opportunity to welcome
into our senior management, Mr. K.M. Nair,
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The Honorable Minster of Revenue, Government of
Kerala, Mr. Adoor Prakash handing over the land title
deeds of the RGCB Campus 1 and Campus 2 to the
RGCB Director, Professor M. Radhakrishna Pillai in the
presence of the Additional Secretary to Government
of Kerala (Revenue), Mr. Vijaya Kumar and the RGCB
Senior General Manager (Institute Development
Division), Mr. Mohanan Nair.
IRS as Chief Controller and Mr. BabuMathai
as Finance Officer. Mr. Nair came from his
position as Chief Controller of Vikram Sarabhai
Space Centre of the Indian Space Research
Organization. MrBabuMathai also joined RGCB
from the Vikram Sarabhai Space Centre, where
he was Senior Head of Internal Audit. Our
administration and accounts are in safe hands.
Jai Hind
Professor M. Radhakrishna Pillai
FRCPath, PhD, FAMS, FASc, FNASc
Director, RGCB
Cancer Research Program: Laboratory - 1
Dr. T.R. Santhoshkumar
Scientist E II
[email protected]
Santhosh Kumar has a Ph.D in Tissue Engineering from Sree Chitra Tirunal Institute for
Medical Sciences and Technology, Trivandrum and joined RGCB in 2000. His current
research interests include understanding molecular signaling involved in cancer drug
resistance and cell based assay development for drug screening.
PhD Students:
Project Personnel
Praveen K. S.
Deepa I.
Krupa Ann Mathew
Asha Lakshmi
Shankara Narayanan V.
Anjana Soman
Prakash R. Pillai
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A High-Throughput Image Based Screen for the Identification of
Bax/Bak Independent Caspase-Activators against
Drug-Resistant Cancer Cells
Mahendra Seervi, Praveen K. Sobhan, Krupa Ann Mathew, Jeena Joseph, Prakash
Rajappan Pillai and T. R. Santhoshkumar
Despite the use of new generation novel target
specific drugs or combination treatments, clinical
findings suggest that drug-resistance caused
by defective apoptosis signaling remains as a
major challenge in cancer treatment. A common
apoptotic defect in drug-resistant tumor is the
failure of cancer cells to undergo Bax/Bak dependent
mitochondrial permeabilization due to impaired
Bcl-2 family proteins signaling. Therefore, Bax and
Bak independent caspase-activating compounds
appear to be effective in killing such tumor cells.
An image-based cellular platform of caspasesensors in Bax and Bak deficient background
allowed us to identify several potential Bax/Bak
independent caspase-activating compounds from
a limited high-throughput compound screening.
FRET based caspase probe targeted at the nucleus
enabled accurate and automated segmentation
without intermediate cell processing yielding
good Z-value. Some of the positive hits identified
Figure 1: [a] Schematic representation of mitochondrial mediated apoptosis and its regulation by Bcl-2 family
proteins- Stress induced Proapoptotic bax and bak translocation to mitochondria triggers cytochrome c release
mediated caspase activation and antiapoptotic Bcl-2 proteins inhibit apoptosis by blocking the action of bax and bak.
[b] (i) Schematic representation of the strategy for high-throughput drug screening for bax/bak independent caspase
activating compounds. The positive hits were further evaluated in antiapoptotic Bcl-2 family protein over expressing
cells to get most potential compounds. (ii) Development of cellular platform by stably expressing caspase sensor
FRET probe (SCAT3) in MEF WT and DKO cells. Knock out status for bax and bak is confirmed by western blot. (iii)
Drug induced caspase mediated FRET loss was read out by using BD pathway Imager. The schematic representation
describes major three steps of the screening strategy i.e. Automated image acquisition, Segmentation and Analysis.
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showed promising activity against drug-resistant
human cancer cells expressing high level of Bcl2 or Bcl-xL. Using this approach, we describe
thiolutin, CD437 and TPEN as the most successful
drug candidates for addressing drug-resistance
caused by aberrant expression of Bcl-2 family
proteins in tumor cells. The screen also enables
quantification of multi-parameter apoptotic events
with caspase activation in HTS manner in live
mode, allowing characterization of non-classical
apoptosis signaling.
Figure 2: Validation of caspase sensor FRET probe (SCAT3 NLS) expressing cellular platform- SCAT3 NLS expressing
MEF cells were treated with anticancer drug etoposide and PAC-1 and Image acquisition was carried out using
BD pathway imager (objective: 20X).
[a] For analysis, segmentation was optimized for demarcating the cell nucleus expressing SCAT3 NLS.
[b] As noticed in merged images, both etoposide and PAC-1 induced massive FRET loss (blue cells) in WT cells.
In DKO cells, only PAC-1 was able to induce FRET loss. The FRET ratio images are shown with color scale bar
for indicating the dynamics of ratio change. The quantification of FRET ratio was demonstrated by scatter plots.
[c] The cleavages of FRET probe and caspase-3 were confirmed by western blot.
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Transition of Cancer Cells into Cancer Stem like Phenotype during
Hypoxia and Its Role in Drug Resistance and Invasion
Krupa Ann Mathew, T.R. Santhoshkumar and M. Radhakrishna Pillai
A rather diminutive subpopulation of cancer
cells called the cancer stem cells (CSCs) or tumor
initiating cells (TICs) plays decisive role in the
response of cancer cells to therapeutic regimens
and subsequent recurrence or metastasis. CSCs
evade anticancer therapy using the characters they
share with normal stem cells such as quiescence,
multi drug resistance-pump activity and low levels
of reactive oxygen species (ROS). Identification
and characterization of CSCs present a prospect
to target them for effective cancer therapy. There
is increasing evidence that microenvironment
factors influence the behavior of these rare cancer
cells. We have recently showed that treatment of
cancer cells with therapeutic agents selected cells
with cancer stem like properties subsequent to
reactivation of antioxidant signaling, indicating
the dynamic nature of such cells and their ability
to switch between multiple states in response to
environmental cues.
In this study, in order to study the influence of
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hypoxic microenvironment on the dynamics of stem
cell compartment, multiple cancer cell lines were
exposed to either hypoxia mimetics (CoCl2 and
DFO) or physiological hypoxia (3% O2). Both short
term and long term hypoxia altered the equilibrium
of stem cell to non-stem cell compartment in cancer
cell lines. The stem cell fraction recognized on
the basis of functional properties such as multi
drug resistance-pump activity (Fig. 1A & 1B), ROS
level (Fig. 1C), ALDH1 expression (Fig. 1D) etc.
were found to be enriched as a result of hypoxia
treatment. Hypoxia treated cell lines also showed
enhanced tumor invasion potential (Fig. 1E)
and resistance to antitumor drugs (Fig. 1F). This
phenotypic transition is rather due to secondary
acquisition of stem like traits than an enrichment
of preexisting cell populations subsequent to
elimination of sensitive populations. The study
provides evidence for transient acquisition of stem
cell-functional traits by cancer cells during hypoxia
aiding in invasion and resistance to therapy.
Figure 1: Cell lines were subjected to different concentrations of CoCl2 and DFO each for various time points. The
fold change in SP (A) and Rhodamine low population (B) for HeLa after hypoxia treatment is presented as graphs
(n=3). Asterisk annotations represent *p < 0.05, **p < 0.005 and ***p < 0.0005 compared between hypoxia and
normoxia using student’s t-test. Cells were treated with CoCl2 and DFO for 72 h and surviving cells were recovered
under normoxia before using them for intracellular ROS level analysis (C) using CellROX® Deep Red reagent and
ALDH1 level assay (D) using ALDEFLUOR™ fluorescent reagent. DEAB, a specific ALDH1 inhibitor was used along
with, in order to confirm ALHD high cells. Representative scatter plots for each case are given. (E) Representative
images of normoxic and hypoxic Hela cells that invaded matrigel matrix after 48 h of incubation are given. (F)
HeLa cells expressing FRET based caspase sensor, DEVD were subjected either normoxia or physiological hypoxia
simultaneous with indicated anticancer drugs. After 48 h of incubation, ECFP/EYFP ratio imaging of the cells was
carried out using BD PathwayTM 435 Bioimager. Representative ratio image of each treatment is given.
13
Role of Cell Cycle in Cell Signaling Response Heterogeneity: Studies
Using Cells Expressing Cell Cycle Indicator
Asha Lekshmi, Shankara Narayanan V., Jeena Joseph and T. R. Santhoshkumar
Tumors are diverse and heterogeneous but
all share the ability to proliferate beyond the
constraints of limiting growth in normal tissues.
This heterogeneity is even evident in single cell
expanded clones suggesting for possible epigenetic
mechanism that can establish differential protein
density within a short period of time in a growing
culture. The heterogeneity is spread over multiple
signaling responses such as cell survival, cell death,
calcium flux and redox regulation. Identification of
the key regulators that confer this heterogeneity in
a cancer population may help to identify critical
compounds that can be potentially employed
for drug intervention. Multiple proteins undergo
rapid post translational modification and ubiquitin
modification while travelling from one cell cycle
phase to another. Since these proteins posses
diverse signaling roles, their abundance and
modification may play a critical role in cell signaling
heterogeneity in a cell cycle dependent manner. It
is proposed to identify such oscillating proteins
that confer cell cycle dependent heterogeneity in
various signaling responses.
A critical hall mark of apoptosis, actin remodeling
was analysed by stably expressing actin mRFP
protein in FUCCI expressing cells (Fig. 1). Studies
using these probes substantiated that cell cycle
phase plays a critical role in providing heterogeneity
in apoptotic response (Fig. 2).
Figure 1: U2OS cells stably
expressing FUCCI probe and
β-actin mRFP. The recombinant
cells were treated with 500 nM
docetaxel and imaged for 48
hours. Actin remodeling is
an indicator of apoptosis and
FUCCI probe indicates the
progression through cell cycle.
Images taken at the beginning
and at the 18th hour after drug
treatment are given.
Figure 2: Graph representing
the average cumulative cell
death in each phase of cell
cycle in U2OS FUCCI cells
expressing actin mRFP after
500 nM of docetaxel treatment
over a period of 30 h.
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Organelle Remodeling during Cell Death: Role of
Bcl-2 Family Proteins and Caspases
Deepa Indira and T. R. Santhoshkumar
Apoptosis accompanies distinct morphological as
well as biochemical changes of membrane bound
structures such as mitochondria ,endoplasmic
reticulum, golgi, lysosomes etc. Activation of
caspases is the prime cause of these changes.
Upstream caspases are activated through various
internal and external stimuli leading to the
activation of downstream caspases such as caspase
3 which cleave many intracellular target proteins to
induce apoptotic cell death. Most of the organelle
changes and its kinetics are not well characterized.
Green fluorescent protein and its variants were
used to track diverse organelles. Caspase sensor
FRET probe was expressed at the nucleus. This
enables us to kinetically study the organelle
remodeling and caspase activation in live cells (Fig.
1A). As shown in figure 1G, cisplatin treatment
leads to lysosomal membrane destabilization and
caspase activation. Time lapse studies show that
lysosomal destabilization initiates before caspase
Figure 1: (A) OVCAR8 cells with fluorescently labeled mitochondria (Mito-DsRed) and endoplasmic reticulum (EREGFP) and SCAT3 NLS
(B & C) SW 48, SW 48 Bax/Bak double knock out cells were treated with cisplatin, camptothecin and thiolutin. After
24 hours cells were lysed and western blot analysis of OPA1, DRP1 was carried out with β- actin as loading control.
(D & E) SW 48, SW 48 Bax/Bak double knock out cells were treated with cisplatin, camptothecin and thiolutin. After
24 hours western blot analysis was carried out for caspase 3. Hsc 70 was used as loading control.
(F) SW 48, DLD and their Bax/Bak double knock out cells were analyzed for PUMA expression level.
(G) OVCAR 8 cells expressing lysosomes and FRET probe with nuclear localization signal (SCAT3-NLS) were treated
with cisplatin. Time lapse imaging was carried out and representative ratio images are given.
15
activation indicating that at least in some forms
of apoptosis, lysosomal destabilization
may
contribute for mitochondrial permeabilization.
Our initial studies identified significant early
mitochondrial remodeling during apoptosis. Further
to understand the importance of pro-apoptotic
Bcl-2 family proteins in mitochondrial remodeling,
key structural proteins of mitochondria such as
OPA1, DRP1 and PUMA were analysed in Bax and
Bak knock out phenotype (Fig 1B-1F). The study
suggests that Bax and Bak regulate apoptosis and
mitochondrial remodeling by modifying PUMA
and OPA1.
PUBLICATIONS
»» Sobhan PK, Seervi M, Deb L, Varghese S, Soman
A, et al. Calpain and Reactive Oxygen Species
Targets Bax for Mitochondrial Permeabilisation
and Caspase Activation in Zerumbone Induced
Apoptosis. PLoS ONE, 2013, 8(4): e59350.
doi:10.1371/journal.pone.0059350
»» Praveen K.Sobhan, Mahendra Seervi, Jeena
Joseph, Saneesh Varghese, Prakash Rajappan
Pillai, Divya Mundackal Sivaraman2, Jackson
James, Roshin Elizabeth George, K.E .Elizabeth,
T.R. Santhosh Kumar1 and M.Radhakrishna
Pillai 1. Immortalized functional endothelial
progenitor cell lines from umbilical cord blood
for vascular tissue engineering. Tissue Eng Part C
Methods. 2012 Nov;18 (11):890-902.
Publications in collaboration
with other RGCB Groups
»» Thomas SA, Vasudevan S, Thamkachy R,
Lekshmi SU, Santhoshkumar TR, Rajasekharan
KN, Sengupta S. Upregulation of DR5 receptor by
the diaminothiazole DAT1 [4-amino-5-benzoyl2-(4-methoxy phenyl amino) thiazole] triggers
an independent extrinsic pathway of apoptosis
in colon cancer cells with compromised pro and
antiapoptotic proteins. Apoptosis. 2013 Feb 24.
[Epub ahead of print]
»» Mohanlal S, Maney SK, Santhoshkumar
TR, Jayalekshmy A. Tricin 4’-O-(erythro-βguaiacylglyceryl) ether and tricin 4’-O-(threoβ-guaiacylglyceryl) ether isolated from Njavara
(Oryza sativa L. var. Njavara), induce apoptosis
in multiple tumor cells by mitochondrial pathway.
J Nat Med. 2012 Oct 9. [Epub ahead of print]
»» Haneef J, M P, Thankayyan RSK, Sithul H,
16
Sreeharshan S. Bax Translocation Mediated
Mitochondrial Apoptosis and Caspase Dependent
Photosensitizing Effect of Ficus religiosa on
Cancer Cells. PLoS ONE, 2012, 7(7): e40055.
doi:10.1371/journal.pone.0040055
»» Divya MS, Roshin GE, Divya TS, Rasheed VA,
Santhoshkumar TR, Elizabeth KE, James J, Pillai
RM. Umbilical cord blood-derived mesenchymal
stem cells consist of a unique population of
progenitors co-expressing mesenchymal stem cell
and neuronal markers capable of instantaneous
neuronal differentiation, Stem Cell Res Ther. 2012
Dec 19 3(6):57. [Epub ahead of print]
»» Thomas SA, Thamkachy R, Ashokan B,
Komalam RJ, Sreerekha KV, Bharathan A,
Santhoshkumar TR, Rajasekharan KN, Sengupta
S. Diaminothiazoles Inhibit Angiogenesis
Efficiently by Suppressing Akt Phosphorylation.
J Pharmacol Exp Ther. 2012 Mar 13. [Epub ahead
of print]
»» Sankaran D, Pakala SB, Nair VS, Sirigiri DN,
Cyanam D, Ha NH, Li DQ, Santhoshkumar
TR, Pillai MR, Kumar R. Mechanism of MTA1
protein overexpression-linked invasion: MTA1
regulation of hyaluronan-mediated motility
receptor (HMMR) expression and function. J
Biol Chem. 2012 Feb 17;287(8):5483-91. Epub
2011 Dec 27
»» Marzook H, Li DQ, Nair VS, Mudvari P, Reddy
SD, Pakala SB, Santhoshkumar TR, Pillai MR,
Kumar R. Metastasis-associated protein 1 drives
tumor cell migration and invasion through
transcriptional repression of RING finger protein
144A. J Biol Chem. 2012 Feb 17;287(8):5615-26.
Epub 2011 Dec 19.
CANCER RESEARCH PROGRAM: LABORATORY - 2
Dr Ruby John Anto
Scientist EII
[email protected]
Ruby John Anto took her Ph.D in Biochemistry from Amala Cancer Research Centre, Thrissur
and did post doctoral training at RGCB and MD Anderson Cancer Centre, Houston, Texas,
before joining RGCB in 2004.
PhD students:
Vinod B.S., SRF
Arun Kumar T.T., SRF
Jayesh Antony, SRF
Haritha H. Nair, JRF
Lekshmi R. Nath, SRF
Minakshi Saikia, JRF
Project Personnel
Sai Shyam Narayanan, SRF
17
MAJOR TOPICS OF RESEARCH:
1. Chemosensitization
2. Bioprospecting for Novel Anticancer Principles
Chemosensitization: In this study, we aim at identifying potential chemosensitizers,
which can enhance the chemotherapeutic efficacy of conventional chemotherapeutic
drugs. We are also investigating better modes of drug delivery so that the efficacy
of drug administration can be improved.
a. Curcumin-Mediated Enhancement of 5-Fu-Induced Apoptosisis
Independent of Akt and Mapk Pathways in Breast Cancer Cells
BS Vinod, Jayesh Antony, Haritha H Nair, Minakshi Saikia, Sai Shyam Narayanan, and
Ruby John Anto
The antimetabolite agent 5-fluorouracil (5-FU)
is widely used in the treatment of many types
of cancers including breast cancer. Our previous
studies have shown that curcumin enhances
5-FU-induced cytotoxicity in a panel of breast
cancer cell lines of diverse receptor status through
thymidylate synthase dependent down-regulation
of NF-κB. In addition to these pathways Akt and
MAPK pathways are also reported to be involved in
5-FU chemoresistance. In this part of the study we
checked for the involvement of these two survival
pathways in regulating the synergism between
5-FU and curcumin. Our results indicate that there
is a time-dependent transient phosphorylation of
Akt upon treatment with 5-FU and when curcumin
18
was given in combination there was a significant
down-regulation of the same. These results indicate
that curcumin could be an effective modulator
in down-regulating 5-FU- induced activation of
Akt. It was also observed that 5-FU transiently
phosphorylate the three MAPKs viz: p42/44, JNK
and p38, which is extensively down-regulated by
curcumin. 5-FU also induce nuclear translocation
of AP-1, the downstream target of MAPKs and
curcumin down-regulate the same. But it was
very interesting to note that these molecules do
not have any role in regulating the synergism as
evidenced by the existence of synergism even after
the inhibition of Akt and MAPK pathways using
the corresponding inhibitors.
Fig 1: The synergism of 5-FU and curcumin is independent of Akt and MAPK pathways.
b) Folic acid conjugated nanocurcumin and nanopaclitaxel induce more
cytotoxicity in HeLa cells compared to their free counterparts
Arun Kumar T, Thulasidasan and Ruby John Anto
In vitro and in vivo studies from our group have
shown that cervical cancer cells can be sensitized
by curcumin to paclitaxel, the most effective
anticancer drug ever been isolated from plants.
However the common disadvantage of in vivo
administration of both these compounds is their
insolubility in aqueous medium. Recent studies have
shown that polymeric nanoparticle-encapsulated
formulations of paclitaxel and curcumin is readily
dispersed in aqueous media and is exhibiting all
biological activities produced by free paclitaxel
and curcumin. In this study, we are exploring
better modes to improve the efficacy of the drug
delivery systems for paclitaxel and curcumin,
which are capable of increasing their therapeutic
index, devoid of adverse effects. We are evaluating
the chemosensitizing efficacy of curcumin in
paclitaxel chemotherapy using the most effective
19
formulations of paclitaxel and curcumin loaded
polymer nanoparticles by both in vitro and in vivo
experiments. Biodegradable polymer nanoparticles
(nanocurcumin and nanopaclitaxel) synthesized
using poly (lactic acid) and poly (lactic/glycolic)
acid (PLGA) coated with Polyethyleneglycol
(PEG), which is conjugated to folic acid are being
studied for their biological activity in comparison
with that of their free counterparts. Confocal
microscopic pictures clearly indicate that cellular
uptake of nanocurcumin suspended in aqueous
medium is equivalent or slightly more than that of
curcumin dissolved in DMSO. Both nanocurcumin
and nanopaclitaxel produced better cytotoxicity
and inhibited the clonogenic potential of HeLa
cells compared to their free counterparts. From
these results we conclude that nanocurcumin and
nanopaclitaxel formulations resolve the problem of
insolubility of these compounds in aqueous media.
They also show better efficacy compared to their
DMSO counterparts, in vitro. Studies are going
on to identify a synergistic combination of these
formulations in HeLa cells.
Fig.2: Nanocurcumin and nanopaclitaxel are more effective in inducing cytotoxicity, and down-regulating the
clonogenic potential of HeLa cells compared to the free drugs.
20
a) Isolation and identification of a potential anticancer compound,
tryptanthrin from the indigenous medicinal plant, Wrightia tinctoria
Jayesh Antony, Minakshi Saikia, Lekshmi R Nath, Sophia Margaret Joseph and
Ruby John Anto
We isolated a potent anticancer compound from the
leaves of the medicinal plant, Wrightia tinctoria,
which belong to the family, Apocyanaceae. The
powdered leaves were extracted using solvents of
varying polarity. The DCM extract [IC50- 20 µg/ml]
was found to induce maximum cytotoxicity in human
cancer cell lines of various origins. Since DCM extract
was the most cytotoxic towards the melanoma cell
line A375 [Fig 3a: cytotoxicity of the extracts in
A375], the activity of the extract was checked in
different skin cancer cell lines (Melanoma & Nonmelanoma) and the result also proved that A375 is
the most sensitive cell line (Fig: 3b). The extract was
further fractionated and purified by three successive
rounds of silica gel column chromatography, which
lead to the isolation of a pure compound, as analyzed
by Thin layer chromatography (TLC) (Fig :3c) as well
as HPLC (Fig: 3d). The compound exhibited much
higher cytotoxicity compared to the extract [IC50-
8.8µg/ml] and characterization using the different
spectroscopic analyzes such as UV-Visible, IR, Mass
and NMR [1H & 13C] identified the compound as
Tryptanthrin (Fig: 3e). The compound does not
induce significant cytotoxicity in the normal human
epidermal melanocytes (HEMA-LP) even at higher
concentrations, indicating its pharmacological
safety does not induce significant cytotoxicity in
the normal human epidermal melanocytes (HEMALP), while being highly toxic to the melanoma cells
(Fig: 3f &g). Cell cycle analysis by flow cytometry
revealed that the compound is inducing significant
cell cycle arrest (< 0. 001) in the G2/M phase, both
in a time and concentration dependant manner (Fig:
3j). The compound also induced apoptotic bodies
[Fig: 3g], membrane flip-flop [Fig: 3h] and cleavage
of caspases and PARP (Fig: 3i) in A375 cells proving
that it is inducing apoptosis in melanoma cells.
Fig. 3: Tryptanthrin from Wrightia tinctoria induces apoptosis and cell cycle arrest in melanoma cells, while being
non-toxic to normal melanocytes.
21
b) F-17 isolated from Chromolaena odorata induces caspase-dependent
apoptosis in cervical cancer cells.
Lekshmi R Nath, Arunkumar TT, Jayesh Antony, Sophia Margaret Joseph and
Ruby John Anto
Chromolaena odorata (Family-Asteraceae) is a
common shrub, traditionally used to treat wounds,
uterus- related problems and early stages of cancer.
The present study was intended to evaluate the
anticancer activity of its organic extracts. The
powdered leaves were successively extracted using
solvents of increasing polarity and the cytotoxicity
of these extracts was checked by MTT assay in
five cancer cell lines of various origins. Among the
four extracts screened, the DCM extract was found
to be cytotoxic to almost all cancer cells, except
HL-60. We selected HeLa for further studies since
it turned out to be the most sensitive cell line in
which, a dose-dependent cytotoxicity (IC50-37.5
µg/ml) was induced by the DCM extract, which
produced around 16 spots as observed in TLC,
when Chloroform:methanol was used as the
solvent system. Among 100 fractions collected by
column chromatography, the 17th fraction eluted by
DCM: Chloroform showed maximum cytotoxicity,
which produced two peaks in the UV spectrum
and two distinct spots in TLC in the solvent
system, Chloroform:methanol. We compared the
morphological effects of F-17 in HeLa cells and
normal fibroblasts. While F-17 induced phenotypic
changes characteristic of apoptosis in HeLa cells
Fig.4: F-17 induces caspase-dependent apoptosis in HeLa cells leading to PARP cleavage
22
from 5 µg/ml onwards, no significant change was
observed in normal fibroblast up to 100 µg/ml .The
percentages of cells in the early and late stages of
apoptosis were also evaluated after treating HeLa
cells with F-17, which induced a dose dependant
increase in the extent of apoptosis. Initiation of
early stage of apoptosis by F-17 was evaluated and
confirmed by Annexin V staining using fluorescence
microscopy and FACS analysis. We also observed
that F-17 induces dose dependent cleavage of the
initiator caspases, effector caspases, and the DNA
repair enzyme, PARP, which clearly indicate that
F-17 induces classical apoptotic program in HeLa
cells. The present study provides, for the first time,
a clear evidence for the pro-apoptotic effect of a
DCM fraction of Chromolaena odorata, which
offers a scientific basis for its use in traditional
medicine. The work is ongoing to characterize the
active principle responsible for anticancer effect.
LIST OF ONGOING EXTRA MURAL GRANTS
Sl.
No.
Title of Project
Funding Agency
1
Isolation and identification
of anticancer principle from
the mistletoe growing on
Chrysophyllum spp
CSIR,
Govt. of India
2
Comparison of the
chemopreventive efficacy of free
curcumin and biodegradable
polymer based nano curcumin
in Benzo[a] pyrene–induced
lung carcinogenesis
DST,
Govt. of India
List of Publications:
2012-2013
Peer Reviewed Journals
Total
Approved
Cost of
the Project
(In Rs.)
Output
Publication/
Patents
2012 - 2015
23,55,400/-
Nil
2013 - 2016
51,56,200/-
Duration
From - To
Nil
Anto, Synthesis of piperazinyl benzothiazole/
benzoxazole derivatives coupled with 1,3,4
oxadiazole-2-thiol: novel hybrid heterocycles
as anticancer agents. Med Chem Res., DOI
10.1007/s00044-013-0510-y, 2013.
»» BS Vinod, J Antony, HH Nair, VT Puliyappadamba,
M Saikia, S Shyam Narayanan, A Bevin
and R John Anto. Mechanistic evaluation
of the signaling events regulating curcuminmediated chemosensitization of breast cancer
cells to 5-fluorouracil. Cell Death Dis., 4, e 505;
doi:10.1038/cddis.2013.26, 2013.
»» Kumar SN, Nambisan Bala, Sundaresan A,
Mohandas C and Anto RJ, Isolation and
identification of antimicrobial secondary
metabolites from Bacillus cereus associated with
a rhabditid entomopathogenic nematode. Ann
Microbiol., DOI: 10.1007/s13213-013-0653-6,
2013.
»» Balachandran S Vinod, Tessy T Maliekal
and Ruby John Anto, Phytochemical As
Chemosensitizers: From Molecular Mechanism
to Clinical Significance. Comprehensive Invited
Review. Antioxid. Redox Sign., 18, 1307–1348,
2013.
»» Deepa G, Arun Kumar T Thulasidasan, Ruby
John Anto, J Jisha Pillai, GS Vinod Kumar. Crosslinked acrylic hydrogel for the controlled delivery
of hydrophobic drugs in cancer therapy. Int J.
Nanomed., 7, 4077 - 4088, 2012.
»» M. S. R. Murty, B. Ramalingeswara Rao, Mohana
Rao Katiki, Lekshmi R. Nath & Ruby John
»» Lekha Nair K, Arun Kumar T Thulasidasan, Deepa
G, Ruby John Anto, GS Vinod Kumar. Purely
aqueous PLGA nanoparticulate formulations of
23
curcumin exhibit enhanced anticancer activity
with dependence on the combination of the
carrier. Int J Pharm., 4:425 (1-2), 44-52, 2012.
»» Haritha H Nair, Balachandran S Vinod, Jayesh
Antony, Minakshi Saikia, Ruby John Anto,
Curcumin sensitizes breast cancer cells to 5-FU
through thymidylate synthase-dependent downregulation of NF-κB (Abstract). J. Carcinogen.,
Vol.11, Supplement 1, 2012, pp-S31.
»» Non-Peer Reviewed Journals
»» Haritha H Nair and Ruby John Anto. Triple
negative breast cancer: The unusual one among
the many. Amala Research Bulletin (2012) 32,
100-108.
List of Conference
Presentations: 2012-2013
»» CN Sreekanth, BS Vinod, Jayesh Antony, Haritha
H Nair and Ruby John Anto, Novel approaches
for cancer chemosensitization., International
Symposium on Recent Advances in Cancer
Research: Therapeutics to Chemoprevention,
Central University of Gujarat (CUG),
Gandhinagar, India, 8-9th Feb-2012 (Invited
Talk).
»» Arun Kumar T Thulasidasan, Lekha Nair K,
Deepa G, G.S.Vinod Kumar and Ruby John
Anto. Curcumin-entrapped PLGA nanoparticles
and curcumin-entrapped poly acrylic acid based
gel nanoparticles improve the efficacy of drug
administration in vitro. International Conference
on Advances In Biological Sciences, 15-17 March
2012, Kannur (Poster presentation).
»» Jayesh Antony, Sophia Margaret Joseph, Lekshmi.
R. Nath, Harsha Chandran and Ruby John
Anto. Isolation, identification and molecular
characterization of a potential anticancer
compound from the indigenous medicinal plant,
Wrightia tinctoria. International Conference
on Advances in Biological Sciences held at
24
Kannur University, during March 2012 (Poster
presentation).
Antony, Minakshi Saikia, Ruby John Anto.
Curcumin sensitizes breast cancer cells to
5-FU through thymidylate synthase-dependent
down-regulation of NF-κB. 3 rd International
Conference [Carcinogenesis-2012] on frontiers
in carcinogenesis and preventive oncology:
molecular mechanisms to therapeutics, November
19-21, 2012, New Delhi, India. (Poster and Oral
presentations)
»» Lekshmi.R.Nath, Sophia Margaret Joseph, Jayesh
Antony and Ruby John Anto. Identification
of novel medicinal plants having anticancer
potential, International seminar on World
Bioheritage concerns over climate change, with
special reference to ethnic vegetables (Botanica 2012)
organized by Department of Botany, Sreenarayana
college, Chempazhanthy, Thiruvanathapuram,
Kerala, India, September7-8, 2012 ,2012. (Oral
presentation).
Awards, Honors etc:
»» Jayesh Antony, Sophia Margaret Joseph, Lekshmi
R Nath, Harsha Chandran and Ruby John Anto,
Isolation and identification of a novel anticancer
principle from the indigenous medicinal plant,
Wrightia tinctoria, International Conference on
Advances In Biological Sciences, 15-17 March
2012, Kannur. [Best Poster Award].
Antony, Minakshi Saikia, Ruby John Anto,
Curcumin sensitizes breast cancer cells to
5-FU through thymidylate synthase-dependent
down-regulation of NF-κB. 3 rd International
Conference [Carcinogenesis 2012] on frontiers
in carcinogenesis and preventive oncology:
molecular mechanisms to therapeutics, November
19-21, 2012, New Delhi, India. [Best Poster
Award, Best presenter award]
INTEGRATED CANCER RESEARCH III: LABORATORY - 3
Dr. Suparna sen gupta
Scientist EII
[email protected]
Suparna Sen Gupta received her Ph.D. in Biochemistry from Bose Institute, Calcutta. She
did her post doctoral training at University of Kansas, USA and as a CSIR Pool-Officer at
National Institute of Immunology, New Delhi, before joining RGCB in 2000.
Program scientist:
Anasuya Ray
(DST-Women Scientist Scheme)
PhD students:
Shashikala S
Smreti Vasudevan
Reshma Thamkachy
Rohith Kumar N.
25
Fodrin Localizes in the centrosome with Gamma-tubulin: Implications
Shashikala S., Rohith Kumar.N and Suparna Sengupta
Nucleation is the intermediate stage of microtubule
assembly from tubulin. Centrosomes are the
nucleation centres for microtubules in eukaryotic
cells. Gamma-tubulin primarily nucleates
microtubules from the centrosome along with some
other proteins. The major amount of gamma tubulin
complex is believed to be located in the centrosome
before the onset of mitotic division. However, a
considerable amount is found in the cytoplasm
in the form of a complex, the function of which
is not known. Cytoplasmic gamma tubulin ring
complex was purified from brain in our laboratory.
Non-erythroid spectrin or fodrin, a 235 kDa protein
was found to be associated with this brain gammatubulin ring complex. Immunohistochemical
analysis in mammalian brain tissue sections
confirmed the association.
Further studies by immunocytochemistry in
neuroblastomal and glioblastomal cells showed that
there was co-localization of spectrin and gammatubulin in both centrosomes and cytoplasm. To
rule out the possibility that the association pattern
is specific to cancer cell lines, we have further
checked the association of fodrin with g-tubulin in a
primary culture of cells extracted from 18 day (E18)
old mouse embryonic brain. Glial and fibroblast
cells showed co-localization in centrosomes but
the association was not found in the centrosomes of
differentiated neurons and astroglial cells (Figure
1). This shows that presence of g-tubulin and fodrin
in the centrosome is not required in non-dividing
cells.
This association was further checked in centrosomes
purified by sucrose density gradient from goat
brain tissue and brain specific cell lines. The 55%
centrosomal fraction showed the presence of nonerythroid spectrin along with gamma-tubulin and
other centrosomal markers in brain specific cell
lines, but not in HeLa which is a cervical cancer
cell line. Immuno pulldown of centrosomal fraction
also confirmed that g-tubulin and fodrin interact to
each other in centrosomes.
During the onset of mitosis there is an evident
increase in the localization of g-tubulin in the
centrosomes along with other proteins. To check
the status of fodrin’s association to g-tubulin
throughout cell cycle, their co-localization pattern
Figure 1: Colocalization of fodrin with gamma-tubulin in centrosomes of primary mouse embryonic brain cells.
26
Figure 2: Mitotic cells in primary cell culture were immunolabelled with a-fodrin (red), g-tubulin (green) and
a-tubulin (cyan). Arrows illustrate absence of co-localization.
in the centrosome was studied in neuroblastoma
cells at interphase and at different stages of mitosis.
During interphase and prophase, fodrin was found
to be co-localized with g-tubulin in centrosomes,
but by metaphase and throughout anaphase,
telophase and cytokinesis, the presence of fodrin
at centrosomes clearly diminished. Figure 2 shows
a cell in anaphase where fodrin is absent in the
centrosome. From these observations, it is implied
that fodrin is not required in the centrosome during
mitosis when enough accumulation of g-tubulin is
achieved.
We also analyzed whether the association of
Fodrin and g-tubulin in centrosome was actin or
tubulin dependant. Neuroblastomal cells were
treated with microtubule depolymerizing agent
nocodazole or microfilament depolymerizing
agent Cytochalasin B . Nocodazole treatment did
not have any effect on the co-localization pattern
of fodrin and g-tubulin both at the centrosomic
and cytoplasmic levels even though under this
condition, microtubule cytoskeleton deploymerized.
However, upon Cytochalasin B treatment, when the
actin cytoskeleton depolymerized, centrosomal
localization of fodrin was lost. The results point out
that fodrin localization in the centrosome is actin
dependent as depolymerisation of microfilaments
stops fodrin localization. Image analysis revealed
that γ-tubulin concentration also decreased
considerably in the centrosome under this
condition. This indicates towards a role of fodrin
as a regulatory transporter of γ-tubulin to the
centrosomes for normal progression of mitosis.
Analysis of Fodrin Association with Gamma Tubulin Complex in
Mammalian Brain
Rohith Kumar, Nisha E Thomas, Shashikala Sasidharan and Suparna Sengupta
Microtubule nucleation normally occurs from the
microtubule organizing centres of cells. Gamma
tubulin has been implicated as the major protein in
the nucleation and orientation of microtubules. Its
nucleation capacity is highly efficient in association
with some other proteins in the form of Gamma
tubulin Ring complex (g-TuRC). Five other proteins
known as gamma tubulin complex proteins (GCPs)
are normally reported in various mammalian cells.
The GCPs binds directly to the gamma tubulin and
helps in the nucleation efficiency of the g-TuRC.
All these proteins were found to possess some
common motifs called GRIP motif. The GRIP2 motif
has been recently acknowledged to be the region
responsible for the direct binding with gamma
tubulin. In our laboratory, nonerythroid spectrin
or Fodrin was identified as a novel component of
the gamma tubulin ring complex isolated from
brain. This association has been further verified in
neuronal and glial cells.
To determine the direct binding partner of fodrin
within the g-TuRC, fodrin was purified from goat
brain homogenate using standard protocol and
used for far western analysis. A 52 kDa band
was observed and identified as gamma tubulin
(Figure 3). Since fodrin is a heterodimer with high
molecular weight, in silico methods were applied
to determine the probable interacting region in
fodrin. Amino acids sequence 1907-1990 of alpha
fodrin showed high homology with GRIP 2 motif.
Fodrin sequence was then modelled and docked
27
with gamma tubulin and interaction was observed
within the identified region (Figure 4). Molecular
dynamic simulations validated the interaction
further.
Spectrins are ribbon like protein with some
conserved domains. The different domains of nonerythroid spectrin or fodrin would be cloned to
confirm the interaction with gamma-tubulin and
to see the effect of this interaction on the cellular
functions of gamma-tubulin.
Figure 3: A. γ-TuRC complex from goat brain was run on 8% PAGE, transferred to PVDF membrane and incubated
with purified fodrin and developed with anti fodrin antibody. B. The same blot was stripped and developed with
anti gamma-tubulin antibody.
Figure 4: The modelled fodrin structure was docked with gamma tubulin followed by molecular dynamic simulation
and the interacting regions were visualized. Hydrogen bonds are shown as white dotted lines.
28
III Anticancer Activities of Diaminothiazoles
Reshma Thamkachy and Suparna Sengupta
Cancer cells grow a resistance towards cells’
natural way of dying, apoptosis. The tumor
suppressor protein p53 may play major role in many
ways in the activation of apoptosis. It is involved
in the activation of apoptosis either through the
extrinsic pathway with protein Bid acting as the
connector protein or through the mitochondrial
pathway leading to caspase activation. Since its
role in apoptosis is important, dysfunction of this
transcription factor leads to tumorigenesis. Most
anticancer drug’s action is through the activation
of p53 which in turn will lead to activation of
apoptosis. But cancers that have mutant forms of
p53 are resistant to these anticancer drugs. Thus
drugs which work independely of p53 are required
to combat these kinds of cancers. Diaminothiazoles
are a group of antimitotic compounds that have
shown cytotoxic and anti angiogenic properties.
Earlier studies in this lab have shown that
apoptosis mediated by DAT1 is mostly contributed
by DR5 activation. We have further found that the
lead diaminothiazole DAT1 is equally effective in
cell lines with disfunctional p53. The apoptosis
caused by DAT1 is triggered by an upregulation of
the death receptor DR5 in a p53 independent way. It
was also found that DAT1 could act synergistically
with vinblastine, a potent drug used in the
treatment of various kinds of cancer, to induce
cytotoxicity in p53 knocked out cells.
Mechanism of Resistance of Cancer Cells against Antimitotic Agents
Smreti Vasudevan and Suparna Sengupta
Antimitotic agents represent a rapidly evolving
cancer chemotherapeutic area, with newer drugs
being added and undergoing different phases of
clinical trials. But acquired drug resistance is the
major challenge that these drugs face in clinics.
Over-expression of ATPase Binding Cassette
(ABC) transporter proteins, differential tubulin
isotype expression, altered apoptotic signaling and
impaired mitotic machinery are prime antimitotic
resistance mechanisms.
We are studying antimitotic drug resistant cell line
models to gain insights of the various resistance
parameters. The efficacy of a lead diaminothiazole
(DAT1) in these drug resistant cell line models
is being explored. Since resistance mechanisms
developed by cells against small molecules (like
diaminothiazoles) and that against large molecules
(like Taxol) differ considerably we have developed
two drug resistant cell lines namely HCT116/TaxR
which is resistant to Taxol and HCT116/DAT1R
which is resistant to the compound DAT1. We
found that DAT1 being small molecular weight
compound is able to bypass most of the drug
resistance mechanisms. It is not a substrate of
P-glycoprotein efflux pump as found by preliminary
docking studies supported by the experimental
finding that DAT1 is efficacious in P-glycoprotein
expressing multidrug resistant cancer cell lines.
Figure 5 shows that taxol resistant HCT116 variant
– HCT116/TaxR over-expresses P-glycoprotein
whereas the DAT1 resistant sub line – HCT116/
DAT1R does not express it. DAT1 is able to induce
mitotic block, which is followed by apoptosis in
HCT116/TaxR cells. Tubulin isotype composition
is an important determinant rendering anti-mitotic
drug resistance. We have determined the tubulin
isotype composition of drug sensitive HCT116 cell
line, its taxol resistant variant HCT116/TaxR and
DAT1 resistant variant HCT116/DAT1R cells. As
shown in Figure 6, in HCT116/TaxR cells there
is over-expression of βII and βIII tubulin isotypes
and down-regulation of βIV tubulin isotype
compared to parental HCT116 cells; whereas in
HCT116/DAT1R cells there is down-regulation
of both βII and IV tubulin isotypes. There are also
increases in the ratio of antiapoptotic protein and
proapoptotic proteins in the case of HCT116/TaxR
which DAT1 can reverse in 0.5 µM concentration,
a concentration where it is effective in wild type
HCT116 cells also. Taxol however, needed much
higher concentration to combat the change in
apoptotic parameters. In future we want to extend
our studies in primary colon cancer cells and in
xenograft tumour models.
29
Figure 5: Expression of P-gp in taxol and DAT1 resistant cell lines
Figure 6: Expression of β-tubulin isotypes in taxol and DAT1 resistant cell lines
V 6 Shogaol Shows efficacy against breast cancer cells and spheroids
Anasuya Ray and Suparna Sengupta
6 shogaol, a dehydrated product of gingerol
is exclusively found in dried ginger. 6 shogaol
displayed cytotoxicity against colorectal carcinoma,
gastric carcinoma, hepatocarcinoma, non small cell
lung carcinoma, ovarian carcinoma etc. and has
been implicated to induce cell death via multiple
mechanisms. Spheroids developed under special
media conditions serve as a model system to study
the properties of stem cells in culture. Earlier we
have reported the efficacy of 6 shogaol to inhibit
MCF-7 monolayer cells and also against spheroids
formed from this cell line. Further we have checked
antiproliferative activity of 6 shogaol against
another breast cancer cell line MDA-MB 231 and
its spheroid (Table 1). Interestingly, MDA-MB 231
30
cells formed comparatively loose and less rounded
spheres than those of MCF 7 cells. Paclitaxel
treatment could not exert any inhibition against
both type of spheroids (Table 1).
Effect of 6 shogaol on MCF-7 cell cycle also has
been investigated. MCF-7 cells after 24 and 48
hrs of growth under 6 shogaol treatment were
stained with PI and analysed for the DNA contents
by flow cytometer. As evident from DNA profiles,
6-shogaol increased cell cycle arrest at G2/M
phase. After 48 hrs treatment, it increased from
23 % (control) to 30 and 51 % at 8 (IC50) and 16
μM (2x IC50) respectively (Figure 7). However, at a
relatively higher concentration, such as at 3x IC50,
the percentage of G2/M accumulated cells were
diminished, which is likely due to increased cell
death under such condition (Figure 7).
There was significant cell death found in MCF7 monolayer cells when we have used different
concentrations of 6 shogaol for different time
points. The cell death mechanism involved in these
cells is being analysed.
Although cancer stem cells are present in a very
low percentage (0.2-1%) in tumor, they have a
crucial role in cancer recurrence. Several reports
suggested that normal stem cells and cancer stem
cells are regulated by similar signaling pathways
including Notch, Wnt/β-catenin and Hedgehog.
Thus, targeting these self-renewal pathways may
be a useful strategy to target CSCs and thereby
inhibit cancer resistance and recurrence. We are
trying to assesss the signaling pathway altered by
6 shogaol in MCF-7 and MDA-MB 231 spheroids
Cell lines
6 shogaol (μM)
Taxol (nM)
MCF-7 cell line
7.94±0.57
4.92±0.71
MDA-MB 231 cell line
5.67±0.73
2.86 ±0.66
MCF-7 spheroid
39.52±0.62
> 50 μM
MDA MB 231 spheroid
11.18±0.83
> 20 μM
Table 1. IC50 values of 6 shogaol and Taxol in breast cancer cells
Figure 7: Effect of 6-Shogaol on the cell cycle of MCF-7 cells
31
Papers published
»» Sannu A. Thomas, Smreti Vasudevan, Reshma
Thamkachy, Swathi U. Lekshmi, Thankayyan
R. Santhoshkumar, Kallikat N. Rajasekharan
& Suparna Sengupta (2013): Upregulation of
DR5 Receptor by the Diaminothiazole DAT1
[4-amino-5-benzoyl-2-(4-methoxy phenyl amino)
thiazole] Triggers an Independent Extrinsic
Pathway of Apoptosis in Colon Cancer Cells with
Compromised Pro and Antiapoptotic Proteins:
Apoptosis, DOI 10.1007/s10495-013-0826-6
Patents, etc (if applicable)
»» DAT1: Suparna Sengupta and K.N.Rajasekharan:
US Patent US 8,158,806 B2: Granted in 2012
Conference Presentations
Oral Presentation:
»» Vasudevan S, Thomas SA, KC Sivakumar,
Rajasekharan KN and Sengupta S:
32
“Diaminothiazoles, a novel class of tubulin
inhibitors showing efficacy in multidrug
resistant cancer cells.” in the Proceedings of
3rd International Conference on Stem Cells
and Cancer (ICSCC-2012): Proliferation,
Differentiation and Apoptosis”, New Delhi,
India, 27-30 October, 2012.
»» Rohith Kumar N, Nisha Elizabeth Thomas,
Shashikala Sasidharan, Sivakumar K.C and
Suparna Sengupta: “Fodrin Binds to Gamma
Tubulin for its association to the Gamma
Tubulin Ring Complex” in the Proceedings of
International Conference on Biomolecular Forms
and Functions in Bangalore, Karnataka, 8-11
January, 2013.
Ph.D. Awarded
»» Sannu Ann Thomas (2012): Study of the
Mechanism of Action of a Diaminothiazole, A
Potential Anticancer Agent: University of Kerala
DR. S. ASHA NAIR, Ph.D
Scientist E II
[email protected]
Asha Nair took her Ph.D. from the Regional Cancer Centre, Trivandrum, Kerala. She trained
as a post doctoral fellow at Harvard Medical School and M.D. Anderson Cancer Centre
Houston, Texas, USA before joining RGCB in 2006.
Ph.D Students
Saneesh Babu P. S.
Diana David
Dhanya K.
Chithra J. S.
Keerthi Sugathan
Project Fellows
Chandraprabha M. G.
Nisha Asok Kumar
Senior Technical Officer
Indu Ramachandran
Technical Assistant
PrameelaKumary T. K.
33
FOXM1: A potential therapeutic target for photodynamic therapy
SaneeshBabu P. S., D. Ramaiah* S. Asha Nair, and M. Radhakrishna Pillai
Collaborators: *Photochemistry and Photonics Division, National Institute for Interdisciplinary
Science and Technology (NIIST), Thiruvananthapuram.
Photodynamic therapy is a promising antitumor
treatment modality approved for the management of
both early and advanced tumors. The mechanisms
of its antitumor action include generation of singlet
oxygen and reactive oxygen species that directly
damage tumor cells and tumor vasculature. A
number of mechanisms seem to be involved in
the protective responses to PDT that include
activation of transcription factors, heat shock
proteins, DNA damage repair genes, antioxidant
enzymes and antiapoptotic pathways. Elucidation
of these mechanisms might result in the design of
more effective combination strategies to improve
the antitumor efficacy of PDT. For investigating
this,we did a real-time PCR for eight ROS resistant
genes influenced by PDT with a sub lethal dose of
squaraine PDT in MDA MB 231 cells. According
to our results, we observed a fold induction of ROS
scavenger enzymes (Catalase, MnSODandPRDX3),
34
DNA repair genes (BRCA 2 and XRCC1) heat shock
proteins (HSP 70 and HSP a1a) which are………
FOXM1 was identified as the transcription factor
for these genes in ROS responses. This ROSregulatory function of FoxM1 protects proliferating
tumor cells from oxidative stress and promotes
survival. Thiostreptone , a known chemical
inhibitor for FOXM1 was employed for assessing
FOXM1 knock down as a therapeutic strategy to
enhance the effect of PDT. To check this strategy
in squaraine PDT, we treated the cells with 1M
and0.5M thisotreptone 24h prior to PDT and we
observed twofold enhanced cytotoxicity using
0.5M and about five times enhancement with 1M
of thiostreptone in breast cancer MDA MB 231 cells.
These preliminary results demonstrate FOXM1 to
be a potential therapeutic target to enhance the
efficacy of PDT.
Smurf2 inhibition downregulates proliferation of MDA- MB-231 cells
in a TGF-β/Smad independent manner.
Diana David and S. Asha Nair
Smad ubiquitin regulatory factors (Smurfs) belong
to the HECT- family of E3 ubiquitin ligases and
comprise mainly of two members, Smurf1 and
Smurf2. Initially, Smurfs have been implicated in
determining the competence of cells to respond
to TGF-β/BMP signaling pathway. Nevertheless,
the intrinsic catalytic activity has extended the
repertoire of Smurf substrates beyond the TGF-β/
BMP super family expanding its realm further to
epigenetic modifications of histones governing the
chromatin landscape. The present study was done
to analyse the role of Smurf2 in regulating breast
cancer cell proliferation and cell cycle progression
after silencing the expression of Smurf2 using
Smurf2 specific siRNAs and its association with
TGF-β/Smad pathway.
35
In our study we silenced the expression of Smurf2
in MDA-MB-231 cells using Smurf2 specific siRNAs
and analysed cell proliferation at 24, 48 and 72 hrs
post-transfection using BrdU assay, MTT assay
and clonogenic assay (Figure 1). We observed that,
Smurf2 silencing inhibits cell proliferation in a time
dependent manner with maximum inhibition at
48 hrs post-siRNA transfection and also induces
accumulation of cells in the G0/G1 phase of the cell
cycle with significant alteration in the expression
levels of specific cell cycle regulatory proteins
(Figure 2). However, there is no alteration in the
expression levels of the effectors of TGF- β pathway
after Smurf2 inhibition (Figure 3). Our studies thus
indicate that, Smurf2 downregulation inhibits
proliferation of MDA-MB-231 breast cancer cells
in a cell cycle dependent manner by accumulating
the cells in the G0/G1 phase of the cell cycle which
may be independent of TGF-β/Smad pathway.
Forkhead box Transcription factor, FoXM1 binds to cdk1 promoter
Dhanya K., S. Asha Nair
Forkhead box (Fox) proteins are family of
evolutionarily conserved transcriptional regulators,
which control a wide spectrum of biological
processes. A loss or gain of Fox function can
alter cell fate and promote tumorigenesis as well
as cancer progression. FoxM1 is a proliferationassociated Forkhead transcription factor which
regulates genes important for G1/S-transition,
S-phase progression, G2/M transition and
progression through M-phase. Here we show that
FoxM1 directly binds to the cdk1 promoter and
regulate its expression. Expression of CDK1 is very
essential for cell cycle progression. Abnormal up
regulation of Fox M1 has been reported in majority
of solid human cancers including liver, breast,
lung, prostate, cervix, colon, pancreas and brain.
Cell cycle dependent regulation of FoxM1 is very
essential for the controlled expression of cell cycle
regulators
Previous studies from our lab reported that increased
expression of CDK1 occurred when skp2 was
overexpressed. This led us to hypothesize that there
may be an involvement of Forkhead box protein,
FoxM1 in regulating the cdk1 gene expression. This
may be because skp2 targets TIS21, an inhibitor of
36
FoxM1.Inorder to rule out this possibility, Foxm1
gene silencing was performed by FoxM1 siRNA.
Interestingly, a significant deduction at both
mRNA and protein levels of CDK1 were observed.
These results indicate a co-ordinate expression
of FOXM1 and cdk1.Insilico analysis was done to
predict whether Foxm1 has any binding site on
the cdk1 promoter. Three FoxM1 consensus sites
(TAAACA) were observed upstream of the cdk1
transcription start site.ChlPassay was performed
using FoxM1 antibody and the pulled out chromatin
fragments were amplified using PCR primers to
DNA sequences in the vicinity of the potential
FoxM1 binding sites on cdk1 promoter region. ChIP
results indicated that FoxM1 protein directly bound
to the human cdk1 promoter region.
Insilico analysis prediction of foxm1 consensus
sequence on cdk1 promoter region followed by
pull down of foxm1 binding site of cdk1 promoter
employing chromatin immunoprecipitation (ChIP)
analysis clearly indicates the possibility of cdk1
being controlled by foxm1 at the transcriptional
level. Further experimental validations need to be
conducted to confirm the transcriptional activity
of foxm1 on cdk1 gene transcription.
Endometrial cancer stem cells express Metastasis Tumor Antigen -1
Chitra J.S., S. Asha Nair and M. RadhakrishnaPillai
Endometrial cancer (EC) is the most frequent
among infiltrating gynecologic malignancy of the
female genital tract and is most common in postmenopausal women in the developed countries.
Although the incidence of EC is lower in East Asian
than in Western countries, it tends to increase
markedly in recent years due to increased incidence
of obesity and altered life style. EC is generally
classified as type I endometrioid EC or type II non
endometrioid EC based on etiology and clinical
variables. The majority of EC are of type I, which
are associated with good prognosis. However,
myometrial invasion and distant metastasis
decreases the survival rates of patients after
surgical treatment. In contrast, type II EC is often
related to poor prognostic factors, such as high
grade or deep myometrial penetration. Several
studies have shown that the cells which undergo
invasion possess stem cell characteristics like selfrenewal and the ability to differentiate into the
tissue of origin. However the mechanism of invasion
by these cells needs to be explored in detail. There
could be altered signaling pathways or proteins in
these cells conferring invasiveness to them which
in turn can be associated with myometrial invasion,
histologic grade and metastasis.
In our study 48 samples from 16 patients were
analyzed using immunohistochemistry to study
the expression of MTA1 which are well known
mediators of invasion Grade 1endometrial cancer
showed 50- 60 % nuclear expression and mild
cytoplasmic expression, Grade 2 showed 70-75%
nuclear expression while Grade 3 is shown to have
90-95% of nuclear expression. All the corresponding
nor mal endometrium showed mild nuclear
expression of MTA1 (30-35%). The endometrial
cancer stem cell like cells were isolated from
endometrial cancer cell lines Ishikawa and HEC1A
based on the functional marker Hoechst 33342
dye exclusion by these cells and we analyzedthe
expression pattern of MTA1 using western blot
and cellular localization was studied using
immunocytochemistry. The side population (SP)
cells were found to overexpress MTA1 while
compared to main population (MP). MTA1
expression was confined to the nucleus in both
SP and NSP. In SP cells high expression of MTA1
was found to localize at the nuclear membrane.
Understanding the molecular mechanism of MTA
mediated invasion of CSCs will help to develop
new therapeutic approaches to prevent tumor
progression and relapse in endometrial cancer.
37
Indirubins sensitizing the frontline therapies in
Chronicmyelogenousleukemia: A key solution for resistance
Chandraprabha M.G. and S. Asha Nair
It is well established at this point that the major
regulators of the eukaryotic cell cycle are the
cyclin dependent kinases. And the basic idea is
that a series of cdks and cyclins complexes are
activated in a specific sequence during the cell
cycle to trigger the events of the cell cycle in the
appropriate order.
The scientific interest on Indirubin originated
from its role as an anti-leukemic agent among
an 11 herbal constituents containing traditional
chines medicine. Recent studies show that the
mechanism of action Indirubin and its derivatives
is complex. Apart from cdk inhibition, other
targets include glycogen synthase kinase-3, c-src,
STATS, aryl hydrocarbon receptor etc. interaction
with these multiple targets may contribute to the
antiproliferative and cell death including effects of
Indirubins and avoid the appearance of Indirubinresistant cells.
Induction of cell cycle blockade and cdk inhibition
is among the anticancer action of Indirubin with
unclear mechanisms. In an attempt to reveal the
mechanism of action of Indirubin-3’-monoxime-5’sulphonate (IRD), we steered our studies to analyse
38
the effect of the same on major cell cycle events.
This approach was put forth with an intention to
identify relevant targets pertaining to cell cycle
regulations. The first phase of the study
• The antiproliferative and apoptosis inducing
effect of Indirubin derivative on K562 cells was
evident from the results.
• The cell cycle of tumor cells treated with
Indirubin derivative is arrested in the G0/
G1 phase, resulting in inhibition of cell
proliferation, and ultimately, induction of
apoptosis
• Combination of Imatinib and IRD induced
apoptosis at lower doses.
After the initial phase of the study, we checked
the effect of IRD on key effector molecules that
comes downstream to the Bcr-Abl contributing
to tumorigenesis. One prominent result was the
accumulation of p27 and inversely correlative
downregulation of the E3 ligase, SKP2. Further
to when explored the role of Skp2-p27 axis in
IRD mediated cytotoxic effect on CML cells, the
following results were obtained
• The Indirubin derivative transcriptionally down
regulates SKP2
• P27kip1upregulation and SKP2 downregulation
contribute to the antiproliferative effect of IRD.
• IRD antagonizes Skp2/p27 interaction via
FOXM1 mediated transcriptional inhibition.
• The cdk inhibitory activity of IRD can be
explained with its FOXM1 inhibitory effect.
As FOXM1 is suppressed, the phosphatase
cdc25B downstream to it, is inhibited, which
in turn prevents the removal of two inhibitory
phosphate groups at the Thr 14 and Tyr15
residues of CDK.
Conclusively, the much celebrated imatinib which
was a milestone to the targeted therapy had its
own place in the history of cancer therapy. But the
relapse and mutations generated by the disease
has outsmarted the drug. It has again brought
us back to the old wine method of disarming
the enemy before attacking. And Proteasome
machinery is a classical target for cancer treatment.
Eventhough ubiquitin mediated proteolysis and cell
cycle, progress in different dimensions; cell cycle
checkpoints are controlled by ubiquitin mediated
timely degradation of key regulatory proteins.
Combinatorial therapies with Imatinib are worth a
shot to address the resistance in frontline therapies
of CML treatment.
In vitro evaluation of Peptide based nanoparticle for
the delivery of 5-Fluorouracil
Nisha Asok Kumar, Ashwanikumar, G.S. Vinod Kumar, S. Asha Nair &
M. RadhakrishnaPillai
Colon cancer is the third most commonly diagnosed
cancer and the second cause of cancer-related death
in men and women combined in the US. According
to American cancer society, it is expected to cause
about 50,830 deaths during 2013.5-Fluorouracil (5FU) remains one of the most frequently prescribed
chemotherapeutic drugs for the treatment of colon
cancer. But the major problem in this conventional
chemotherapy is their adverse side effects, lack
of site-specific delivery and the development of
chemoresistance and disease relapse. In order
to circumvent these limitations, numerous other
delivery systems are being developed. However,
the main obstacle to their application remains
their poor cellular uptake and the lack of selective
delivery into malignant cell/tissues. Therefore
there is a growing need to develop a potent
delivery system to ensure slow release of the
drug at its target site. The self-assembling of biomolecules is a common phenomenon in biology.
This property of the biomolecules is being taken
advantage of in the field of tissue engineering,
drug delivery and microfluidics. Short peptides
(8–16 residues) are chemically synthesized and
they form β-sheet structures in water. Depending
on the pH and the ionic strength of the medium,
these peptides self-assemble into nanofibers, which
in turn form a hydrogel. The peptide CH3CONH-
RADARADARADARADA- CONH2 was modified
at the 6th position to form Phenylalanine instead of
Alanine and was named as RADAF6 [CH3-CONHRADARFDARADARADA-CONH2].The aromatic
ring of the phenylalanine helps better π-π stacking
of the polymer and the drug and hence contributes
to higher drug loading efficiency. In this study
we have investigated the potency of 5-FU loaded
RADAF6 as drug delivery systemfor the slow
release of 5-FU in vitro.
Cell cytotoxicity assayrevealed that 5-Fu loaded
RADAF-6 exhibited higher cytotoxicity than
that of free 5-FU over a range of experimental
concentrations in the colon cancer cell line
HCT-116. The Rhodamine entrapped RADAF-6
was completely internalized as was found to be
localized in the cytoplasm of the cell after 4 hours
of incubation. The effect of the peptide nanoparticle
on cell cycle progression in HCT-116 cells was also
assessed and it was found that there is a timedependent arrest in various phases of cell cycle.
At 24hrs, a delayed S phase was seen whereas it
progressed to G2/M arrest at 48hours of treatment
in cells treated with free 5-Fu as well as with
5-Fu loaded RADAF-6. The ability of the peptide
nanoparticle to induce apoptosis was checked
by Acridine Orange/Ethidium Bromide staining
as well as by cleavage of the PARP enzyme. The
39
cells treated for 48hours with 5-Fu loaded RADAF-6
showed more number of early apoptotic cells
whereas cells treated with 5-FU showed greater
number of late apoptotic cells . The control cells
stained bright green in color which indicated
viable cells. Cleavage of the PARP enzyme was
found after 48 hours of treatment with free-5FU as
40
well as 5-FU loaded RADAf-6. The results clearly
demonstrate the efficiency of 5FU loaded peptide
nanoparticle RADAF-6 to inhibit cell proliferation,
arrest cell cycle and to induce apoptosis mediated
cell death. RADAF-6 can be used as an effective
biocompatible pH sensitive drug delivery system.
EXTRA MURAL GRANTS
Principal Investigator
No
Project Title
Period
Funding agency
1.
Tumor primordial cells in colorectal cancer:
implications for surgical margins and minimal
residual disease(multicentric project)
2008 - 2012
Department of Biotechnology,
Govt. of India
2.
Indirubin, a CDK inhibitor, to enhance cytotoxicity
and apoptosis in CML-blast crisis cells- a preclinical
evaluation
2010-2013
Indian Council of Medical
Research, Govt. of India
Co-P. Investigator
No
Project Title
Period
Funding agency
1
Design, development, in vitro and in vivo studies of
novel sensitizers for photodynamic therapy
2007-2014
Department of Science and
Technology, Govt. of India
A novel site specifically targeting nanoparticle
based oral-drug and siRNA releasing polymer
systems for colon cancer
2010-2013
Govt. of India
2.
Publications
»» Thomas, A. P.; SaneeshBabu, P.; Asha Nair, S.;
Ramakrishnan, S.; Ramaiah, D.; Chandrashekar,
T. K.; Srinivasan, A.; RadhakrishnaPillai, M.
meso-Tetrakis
»» ( p - s u l f o n a t o p h e n y l ) N - C o n f u s e d
PorphyrinTetrasodium Salt: A Potential Sensitizer
for Photodynamic Therapy. Journal of Medicinal
Chemistry 2012, 55, 5110-5120.
»» Simon A M, Jagadeeshan S, Abraham E,
Ashalatha. A,.Pillai, J,. Kumar N A, Nair. A
S, Kumar G.S.V (2012) Poly (D,L-lactic-coglycolide) nanoparticles for the improved
therapeutic efficacy of all-trans-retinoic acid:
A study of acute myeloid leukemia (AML) cell
differentiation in vitro. Medicinal Chemistry
8,805-810
»» Karunakaran, S. C.; Babu, P. S. S.; Madhuri, B.;
Marydasan, B.; Paul, A. K.; Nair, A. S.; Rao, K.
S.; Srinivasan, A.; Chandrashekar, T. K.; Rao, C.
M. RadhakrishnaPillai, M. In vitro Demonstration
of Apoptosis Mediated Photodynamic Activity
and NIR Nucleus Imaging through a Novel
Porphyrin. ACS Chemical Biology 2012.
Oral presentation
»» Diana David, JemPrabhakar, S. Asha Nair,
M. Radhakrishna Pillai. Smurf2 inhibition
modulates proliferation of breast cancer cells in
a TGF-β/Smad independent manner. 32nd Annual
Convention of Indian Association for Cancer
Research & International Symposiun: Infection &
Cancer, Dr. B.R. AmbedkarCenter for Biomedical
Research (ACBR), University of Delhi, February
13th-16th, 2013.
Poster Presentations
»» SaneeshBabu P.S, D Ramaiah, S. Asha Nair, and
M.RadhakrishnaPillai
»» “Implications of a squaraine dye as photosensitizer
for photodynamic therapy in cancer”, on 32nd
Annual Convention of Indian Association for Cancer
Research (IACR) & International Symposium
atInfection & Cancer at Dr. B.R. AmbedkarCenter
for Biomedical Research (ACBR) University of
Delhi, North Campus on Feb 13-16, 2013.
41
»» DhanyaK.andS. Asha Nair “Silencing of forkhead
box protein, foxm1 b leads to the down regulation
of cdk1-a gatekeeper of M phase entry”, on3rd
International Conference on Stem Cells and Cancer
(ICSCC 2012),October 27-30, 2012: New Delhi,
India.
»» Chitra J.S.,S. Asha Nair and Prof. M. Radhakrishna
Pillai. “Metastasis tumor antigen - a master co
regulator in invasion of endometrial cancer stem
like cells” on3rd International Conference on Stem
Cells and Cancer (ICSCC 2012) October 27-30,
2012: New Delhi, India
42
»» Chandraprabha M.G. and S. Asha Nair “Pawns
equally threatening as Queen: Targets other than
BCR-ABL in CML therapy” on 3rd International
Conference on Stem Cells and Cancer (ICSCC
2012) October 27-30, 2012: New Delhi, India.
»» NishaAsok Kumar and S. Asha Nair “Off targets
of Imatinib mesylate: A gut instinct” on 3rd
International Conference on Stem Cells and Cancer
(ICSCC 2012) October 27-30, 2012: New Delhi,
India.
Cancer Research Program: Laboratory – 5
Dr.Priya Srinivas
Scientist E II
[email protected]
Priya Srinivas is a Ph.D in Biochemistry from Regional Cancer Centre, Thiruvananthapuram,
Kerala. She joined RGCB in 2000. She had worked as a Visiting Scientist for a year from
2009 at the Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester,
Minnesota, USA.
PhD students:
Rakesh S
Veena Somasundaram
Sreelatha KH
Reshma RS
Revathy
Satheesh Kumar S
Technical Staff:
Arya Nagendran
Technical Personnel:
Usha V
43
The influence of BRCA1 on the characteristics and therapeutic
interventions of Breast Cancer Stem cells.
Veena Somasundaram and Priya Srinivas
The cancer stem cell hypothesis has been in the
foray since a century or so but gained momentum
when Bonnet D and Dick J E isolated cells that
displayed the stem like properties of self renewal
and multilineage differentiation from leukemia
samples (Bonnet and Dick 1997), followed by Blaire
et al., 1997, Pardal et al., 2003 and Reya et al., 2001.
These putative stem-like cells were later called the
‘cancer stem cells’ (CSCs). Although the concept of
CSCs is not entirely new, advances made over the
past two decades in our understanding of normal
stem cell biology in conjunction with the recent
application of these concepts to experimentally
define CSCs has resulted in the identification of
CSCs in several human malignancies (Tan et al.,
2006).
tumor. BRCA1 plays a cardinal role in maintaining
genetic stability within a cell and has also been
speculated to be involved in the differentiation of
mammary cells. The characteristics of CSCs from
BRCA1-defective breast cancers (BCSCs) have not
been analyzed well.
BRCA1 expression is involved in normal embryonic
development, mammary stem cell differentiation,
and cancer development. Inactivation of BRCA1
plays an important role in cancer stem cells and also
leads to the development of high-grade, basal-like
The mammospheres of HCC 1937/WT BRCA1
showed a higher expression of the EMT markers
while the HCC 1937 showed a higher expression
of the stem cell markers. Thus, the presence of
a functional BRCA1 could indicate increased
invasiveness (Figure 1).
A comparison and contrast of the HCC 1937
(BRCA1-defective) and the HCC 1937/wt BRCA1
(full length BRCA1 reconstituted) breast cancer
cell lines by microarray, immunofluorescence and
western blotting brought to light the possibility of
a unique relationship between BRCA1, β-catenin
and the predominantly intestinal stem cell marker,
CDX2 which could add a new dimension to the
close association between BCSCs and BRCA1.
Figure 1: Immunoflurescence analysis shows better expression of EMT markers in HCC1937/WT BRCA1 BCSCs
and stem cell markers in HCC 1937 BCSCs (Left panel). A similar trend in expression of EMT and stem cell markers
was obtained after microarray analysis (Right panel).
44
We had earlier reported estrogen dependent
signaling events leading to increased sensitivity
for apoptosis in BRCA1 knockout, BG-1, ovarian
cancer cells to plumbagin (PB) in comparison to
other chemotherapeutic drugs (Thasni et al., 2008).
Plumbagin is a naphthaquinone with hydroxyl
and methyl groups at the 5th and 2nd carbons
respectively.
With the objective of extending this study to see
if plumbagin could act specifically on BRCA1
defective Breast cancer Stem Cells (BCSCs), we
had initially studied the effects of plumbagin on the
ability of MCF 7 (BRCA1 wild type) and HCC 1937
(BRCA1 defective) cells to form mammospheres
(composed predominantly of BCSCs) in nonadherent culture conditions as well as on a direct
reduction of the ALDH1+ stem cell population
after a 48hr treatment protocol. Plumbagin shows
a heartening ability to target breast cancer stem
cells. The study was further shifted to a more
reproducible and uniform scenario with the
comparison and contrast between HCC 1937
and HCC 1937/WT BRCA1 cell lines that differed
exclusively with regard to the expression and
functioning of BRCA1.
In vitro experiments to study the effects of
plumbagin on BCSCs gave promising activity
when compared to the standard drug carboplatin
which caused an increase in the ALDH1+ stem
cell numbers post treatment possibly due to the
rapid killing of the bulk cells thus triggering
the CSCs to proliferate and increase in number
dramatically. A similar increase in ALDH1+
cells was observed in HCC 1937/WT BRCA1 cells
treated with plumbagin while HCC 1937 cells
with a mutated BRCA1 showed a reduction in
the ALDH1+ stem cell population. This could not
be correlated with difference in the expression of
ABCG2 multidrug resistance protein as it could
be for other cell lines previously analyzed in the
lab (Figure 2). Knockdown of the BRCA1 protein
showed a possibly direct effect of the C-terminus
of BRCA1 (absent in HCC 1937 cells) in the action
of plumbagin.
Figure 2: Plumbagin significantly reduces the ALDH1+ stem cell population in HCC 1937 cell line while HCC
1937/WT BRCA1(HCCR) shows an enrichment of the ALDH1+ stem cells under identical experimental conditions.
45
Effects of Cancer Associated Fibroblasts (CAFs) on BRCA1+/- breast
cancer cells: Relation to aggressiveness
Sreelatha KH and Priya Srinivas
(In collaboration with Dr. Jem Prabhakar, Additional Professor, Department of Surgical
Oncology, and Dr. Thara Somanathan, Assistant Professor, Department of Pathology, Regional
Cancer Centre, Thiruvananthapuram, Kerala)
The development of an altered stromal
microenvironment in response to carcinoma is
an early event during carcinogenesis and is a
prerequisite for tumor invasion and metastasis.
Earlier it was thought that cancer development
is an independent event which occurs due to
mutations in cancer cells. But later it was proved
that the stroma that are seen in immediate
vicinity of the cancer cells contribute a lot for
tumor progression. Among the cells in the tumor
stroma, Cancer Associated Fibroblasts (CAFs) are
considered to be the master regulators of paracrine
signaling driving the progression of various
cancers through release of various soluble factors
and extracellular remodeling. These activated
fibroblasts are involved in creating a niche for
cancer cells, promoting their motility. Indeed, CAF
show some degree of plasticity controlled by tumor
cells themselves, undergoing a differentiation
process called mesenchymal-mesenchymal
transition. Although the notion that CAF acquire a
phenotype similar to myofibroblasts (MF) is widely
accepted, the agents driving this transition in vivo
are not yet fully elucidated.
CAF secrete proteins that may stimulate adhesion
and motility, and escape from the localised growth
control, as well as induce de novo angiogenesis. CAF
have also been reported to exert a prometastatic
effect on pancreatic tumor cells. In parallel to the
ability of activated fibroblasts to stimulate cancer
progression toward an aggressive phenotype,
tumor cells themselves stimulate activated stromal
cells to release biological agents for their growth
and dissemination. Studies had found that the
co-culture of the cancer cells along with the CAF
helped in cancer progression. Factors such as TGF
β and IL6 could be involved in activation of the
CAFs and once activated; these cells could induce
increased stemness in the cancer cells through
Epithelial-Mesenchymal Transition.
Influence of tumor microenvironment on BRCA1
defective cancer progression has not been analyzed
till date. The cancer cells become more aggressive
when more mutations are generated due to
46
genomic instability. It has also not been studied
whether there is any difference in the gene/ protein
expression (eg. growth factors, chemokines) by
the fibroblasts in the presence of BRCA1 mutated
cancer cells versus BRCA1 wild type cancer cells.
A study of the gene/protein expression of BRCA1
associated CAF will help to target the CAF for the
treatment of BRCA1 defective cancers and will be the
first study of this kind. Also stromal cells particularly
CAF are found to be genetically stable, so therapies
that target these cells are less likely to become
ineffective because of acquired drug resistance.
CAF being an important component in tumor
progression, we aim to know the reciprocal effect
of CAF on BRCA1 mutated breast cancer cells. For
this we isolated CAF from human breast cancer
tissues by enzyme dissociation method. The
characterization of these cells was done using the
specific markers by Immunofluorescence, Western
blotting and Flow cytometry. The reciprocal effects
of CAF and breast cancer cells on proliferation were
analyzed by cell proliferation assay (Figure 3). We
also analyzed the effect of CAF on the invasive
and migratory potential of breast cancer cells by
invasion assay and migration assay respectively
using the 3D culture method (Figure 4). There have
been reports that the altered fibroblasts were able to
induce the breast cancer metastasis, so we suspect
the presence of a more aggressive CAF which
induces tumor invasion, which may be generated
by the influence of mutated cancer cells. So we are
on the track to standardize methods to generate
aggressive CAFs by co-culturing BRCA1 mutated
breast cancer cells and the altered fibroblast cells.
Our observations indicate that the proliferation rate
of CAF was highly dependent upon the genetic
alteration in the cancer cells. It has also been
observed that the invasive and migratory potential
of these cancer cells are also influenced by CAF.
From our study it is clear that CAF can influence
breast cancer cells’ proliferation, migration and
invasion which depend on the mutational status
of the cancer cell eventually leading to tumor
progression and metastasis.
Figure 3: Cell proliferation assay of breast cancer cells after 48 hours of treatment with the conditioned medium
obtained from CAFs
Figure 4: Invasion and Migration potential of breast cancer cells in the presence of CAF. (HCCR- HCC 1937/wt BRCA1)
Studies on Regulation of Cell Growth by BRCA1/2 in Prostate Cancer
Cells: Influence of Certain Selected Quinones
Reshma RS and Priya Srinivas
Prostate cancer is the second most frequently
diagnosed cancer of men and the fifth most
common cancer overall. Nearly three-quarters of
the registered cases occur in developed countries.
Incidence rates of prostate cancer vary by more
than 25-fold. With growing life expectancy, the
overall prevalence of cancer has increased.
Environmental and geographical factors also
play some role. About 10-15% of patients have
positive family history. Majority of families with a
clearly dominant predisposition to breast and/or
ovarian cancer have a greater chance of BRCA1/2
mutation. The prostate is the most consistently
reported site for cancer susceptibility in male
carriers of BRCA1/2 mutation. Prostate gland is
controlled by steroid hormones and thus BRCA1/2
proteins could be of functional importance since
BRCA1 and 2 are reported to influence Estrogen
receptor (ER)/Androgen receptor (AR) expression.
Even though prostatic cancer is increasing the
etiology of BRCA1/2 related prostatic cancer and
their risk remains poorly understood, also their
47
optimal clinical management is not yet defined.
Recently Plumbagin is shown to be active against
prostate cancer cell lines. However, none of those
studies have looked into action of Plumbagin on
the functional consequence of BRCA1/2 absence
in prostate cancer cells. Therefore, this study was
planned to analyze the cellular effects of Plumbagin
and some closely related quinones in BRCA1/2
blocked prostate cancer cells.
The main objective of the study is to analyze the
effect of Plumbagin and related quinones like
1,4-Naphthaquinone, Menadione, and Lawzone
in comparison to Tamoxifen and Finasteride in
prostate cancer cells with respect to BRCA1/2
absence/ presence. Two prostate cancer cell lines,
PC-3 (ERα+, ERβ+, AR-) and DU-145 (ERα-, ERβ+,
AR-) were used for the study.
Molecular docking studies of various compounds
Plumbagin, 1,4 - Naphthaquinone, Menadione,
Lawzone and Tamoxifen were done on the following
three receptors Estrogen Receptor α, Estrogen
Receptor β and Androgen Receptor using glide
docking program. The native ligand binding
pocket of respective receptor molecules received
from Protein Data Bank (PDB ids 1X7J, 2PIP,
3DT3) was considered as docking site, grid were
generated around this region and docking studies
were performed. The Glide score was used to rank
the order of the ligand molecules in each receptor.
It was observed that the native ligand molecules
exhibits better binding score when compared to
that obtained for other molecules.
Cell proliferation studies were carried out using
MTT assay. Both PC 3 and DU 145 cells were
Immunofluorescence (PC 3 cells)
Figure 5: Immunofluorescence studies confirm the silencing of BRCA1 and BRCA2 proteins in PC3 cells.
Figure 6: Annexin V-FITC staining of PC 3 cells. The control cells show no fluorescence due to the lack of Annexin’s
ability to penetrate the intact cell membrane. Treatment with Plumbagin (4μM), 1,4 NQ (15μM), Menadione (15μM)
and Lawsone (80μM) shows flipping of plasma membrane as shown by binding of Annexin-FITC to phosphatidyl
serine.
48
more sensitive to Plumbagin when compared to
other compounds used for the study. The anticancer activities were in the order Plumbagin>1,
4-Naphthaquinone, Menadione>Lawsone. The
IC50 values of different compounds on both
cell lines proved that the quinones used for the
study are more effective in PC 3 cell lines than
DU 145 cell lines. We confirmed BRCA1/ BRCA2
gene silencing by Immunofluorescence (Figure
5). Comparitive proliferation studies of BRCA1/2
wild type and silenced cell lines after treatment
by the compounds were analyzed by MTT assay.
Plumbagin, Menadione and finasteride showed
increased sensitivity in BRCA1/2 blocked castrate
resistant cells.
To confir m whether the naphthaquinone
derivatives induce cell death via apoptosis,
loss of mitochondrial membrane potential was
checked using JC1 dye. The number of cells which
emitted green fluorescence was less in the case of
Menadione when compared with cells treated with
Plumbagin and 1,4 naphthaquinone. The extent
of loss of mitochondrial membrane potential was
more in Plumbagin-treated PC 3 cells than DU 145
cells. Flip-flop of phosphatidyl serine to the outer
surface of the membrane represents early stage
of apoptosis and was detected as bright green
fluorescence when stained with Annexin V-FITC.
Plumbagin, 1, 4-Naphthaquinone and Lawsone
shows comparative flipping of plasma membrane
(Figure 6).
βhCG and BRCA1 in Gestational Trophoblastic Diseases
Revathy and Priya Srinivas
(In collaboration with Dr Nirmala C, Professor and Head of the Department, Dept of O&G,
SAT Hospital, Medical College, Thiruvananthapuram and Dr. Jayshree V Vaman Additional
Professor, Dept of O&G, T D Medical College, Alappuzha)
Gestational Trophoblastic Diseases comprises
of a range of pregnancy related tumors from the
premalignant disorders of complete and partial
hydatidiform moles to the malignant disorders
of invasive mole, choriocarcinoma, placental
site trophoblastic tumor and the epithelioid
trophoblastic tumors. The reported incidences of
GTD vary widely with the highest incidence being
in South-East Asia, especially India with rates of
2 to 12 per 1000 pregnancies. The incidence of
GTD is very high especially in Kerala with 5.1 per
1000 deliveries as reported in a survey between
1990 to 2007 (Lybol et al., 2011). The disease
biology being vague, methods to determine the
malignant potential being limited and the chances
of recurrence of GTDs being high, there is a need
to unravel the molecular mechanisms behind GTD
to find a treatment for this disease.
hCG is a glycoprotein hormone synthesized by the
trophoblast cells in the placenta. βhCG level peaks
up during pregnancy, hence used as a pregnancy
marker, but it shoots up much beyond this higher
level during GTD cases. The levels of glycosylation
vary depending upon the stage of pregnancy and
also the type of tumor (in case of GTD). hCG is
involved in both pregnancy induced breast cancer
protection and pregnancy maintenance. BRCA1
Figure 7: The levels of βhCG in first trimester placenta (S1 & S2), second trimester placenta (S3 & S4) and GTD
Vesicular mole samples (VM1 & VM2).
49
plays the role as the growth and morphogenetic
regulator of mammary epithelial cells but the loss of
its function may lead to increased tumor aggression
and metastasis. Though BRCA1 plays a significant
role in DNA repair pathways, its particular role in
cancer development especially in breast/ovarian
cancers is still not known completely. Various
positive and negative modifiers of BRCA1 have
been identified, the best characterized stimulant
being estrogen, thus controlling proliferation and
malignancies of hormone regulated tissues like
breast, ovary and prostate. Tumor progression
in BRCA1 defective conditions couldn’t be
controlled by inhibiting estrogen/estrogen receptors
(Hosey et al., 2007, Fan et al., 2002). Apart from
estrogen, progesterone and βhCG are important in
differentiation of the breast tissue, also βhCG and
estrogen can induce progesterone synthesis. Thus,
we hypothesis that the other possible reason for
the selective tumorigenesis by BRCA1 might be
the influence of βhCG which has not been analysed
till date.
Analyzing GTD is an ideal system to study the effect
of βhCG on BRCA1as βhCG peaks to the maximum
in this condition. We performed the initial studies
using age matched normal placental controls and
GTD samples. The tissues were analysed for protein
levels of βhCG and BRCA1 which showed the
concomitant decrease in BRCA1 level with increase
in βhCG level. The GTD samples which have higher
amount of βhCG showed to have lower levels of
BRCA1 in them as compared with the normal age
matched placental controls by western blotting.
Further studies to reveal the relation between βhCG
and BRCA1 in GTD samples will be dealt with in
the future study.
Reciprocal regulation of BRCA1 and β-hCG: An in vitro study
Satheesh Kumar S and Priya Srinivas
BRCA1 germ line mutations have been identified
in nearly 50% of hereditary breast cancers and
approximately 80% of cases with both hereditary
breast and ovarian cancers. Also decreased BRCA1
expression due to hyper methylation of the BRCA1
promoter or loss of BRCA1 allele has been observed
in 30-40% of sporadic breast cancers. Thus,
BRCA1 may generally play a role in breast cancer
development. Hormonal therapy occupies a critical
position in breast cancer treatment but tumor
progression in BRCA1 defective condition could not
be controlled by inhibiting estrogen/progesterone
receptors as majority of BRCA1 defective cases are
triple negative. Though estrogen and progesterone
mediated abnormal signaling and proliferation is
absent in BRCA1 defective triple negative cases,
the cancer is critically aggressive and the treatment
is highly complicated. Apart from estrogen and
progesterone, hCG is thought to be critical for
development and differentiation of breast tissue
and is secreted mainly during pregnancy. Though
the involvement of intact hCG in cancer is not
urged, a subunit of hCG called β-hCG is abundantly
secreted during some cancerous conditions and the
recent studies show that it could be a tumorigenic
factor though the exact involvement is not clear
50
yet. Also some reports show that it could be a
biomarker for detection of cancer. Therefore, we
hypothesize that the other possible reason for the
severe tumorigenesis of BRCA1 defective triple
negative cases might be the influence of b-hCG.
Though the influence of estrogen/progesterone on
BRCA1 has been analyzed in detail, influence of
β-hCG on BRCA1 had not been analyzed yet which
is the rationale of our study.
We have used the triple negative cell lines
with varied BRCA1 status where, in HCC1937,
BRCA1 is deficient and HCC1937/wt BRCA1,
BRCA1 is over expressed. We have checked
the beta-hCG expression in these cell lines by
immunofluorescence and we found that both the
cell lines express b-hCG (Figure 8). By ELISA, we
made it clear that the b-hCG is secreted and the
level of secretion is higher in HCC1937 cells where
BRCA1 is deficient than in HCC1937/wt BRCA1
where BRCA1 is proficient (Figure 9). From our
preliminary study, it is partially clear that there
exists a regulation between BRCA1 and b-hCG
and further revealing the complete story between
BRCA1 and b-hCG will be our future interest.
Figure 11: Expression of β-hCG in HCC1937 and HCCR cells.HCCR:HCC1937/wt BRCA1.
Figure 12: β-hCG level in HCC1937 and HCCR cell supernatant. HCCR: HCC1937/wt BRCA1
Molecular evidence for the anticancer activity of Plumbagin in
BRCA1 defective breast cancer
Rakesh S and Priya Srinivas
Collaborators: Dr Chuxia Deng, NIDDK, NIH, USA, Dr Mahesh Kumar J, Centre for Cellular
and Molecular Biology, Hyderabad, India and Dr. T.V. Anilkumar, Experimental Pathology
Laboratory, Sree Chitra Tirunal Institute of Medical Science and Technology, BMT Wing,
Trivandrum, India
Standard chemotherapeutics have been developed
that are used to target the repair deficient cancers,
especially BRCA1 mutated breast and ovarian
cancers. The recent emergence of PARP inhibitors
against Homologous recombination deficient
tumors is an example for the event in the race for
new drugs. Clinical and preclinical trials indicated
that the resistance caused by a subset of BRCA
less tumors to this novel therapeutic is due to the
emergence of varied allelic alterations and reversal
mutations. Thus PARP inhibitors stumble in
breast cancer chemotherapy. The present study is
centered up on deriving novel molecules that target
BRCA defective tumors. It had been proved from
our laboratory that a naphthaquinone derivative,
Plumbagin had been effective against BRCA less
51
tumors of ovarian origin. The invitro evaluation of
Plumbagin resulted in the induction of reactive
oxygen species in BRCA1 defective breast cancer
cells (Figure 10A). The preclinical evaluations of
Plumbagin were performed in conditional knock
out mouse models for Brca1. The Brca1 knock out
models was generated from two different strains of
Cre driven promoters (WAP ad MMTV) (Figure 10B).
Plumbagin induced apoptosis in mammary tumor
in both the knock out mouse strains. The toxicity
profile showed by Plumbagin in various tissues of
transgenic mice studied, provided evidence for its
safer disposition as a therapeutic lead. The present
work from our laboratory provides the foundation
for the future use of plumbagin in a properly
scheduled phase trials in human.
Figure 10: A. Reactive oxygen species induced by Plumbagin in BRCA- mutated breast cancer cells. Plumbagin
(Pb) treated in both MX1 and HCC-1937 cells and compared to untreated control (C). B. Cartoon representing the
generation of Brca1 knockout mice
Extra-mural funding (Current Projects)
No.
Investigator(s)
Title
Funding Agency
Duration
1
Priya Srinivas (I)
HCG and BRCA1 in Gestational
Trophoblastic Disease
KSCSTE
2013-2016
2
Priya Srinivas (I)
Studies on regulation of cell growth by
brca1/2 in prostate cancer cells: influence
of certain selected quinines.
BRNS
2009-2012
Papers published:
»» Thasni KA, Ratheeshkumar T, Rojini G,
Sivakumar KC, Rakesh Sathish Nair, Srinivas
G, Asoke Banerji, Veena Somasundaram, Priya
Srinivas. Structure activity relationship of Plumbagin
52
in BRCA1 related Cancer Cells. Molecular
Carcinogenesis: 52, 392-403 (2013).
»» Sutapa Sinha, Krishnendu Pal, Ahmed Elkhanany,
Shamit Dutta, Ying Cao, Gourish Mondal,
Seethalakshmi Iyer, Veena Somasundaram, Fergus
J Couch, Viji Shridhar, Resham Bhattacharya,
Debabrata Mukhopadhyay and Priya Srinivas.
Plumbagin inhibits tumorigenesis and angiogenesis of
Ovarian Cancer cells in vivo. Int. J. Cancer: 132,
1201-1212 (2013).
»» Somasundaram, V. and Srinivas, P. (2012),
Insights into the targeted elimination of BRCA1defective cancer stem cells. Med. Res. Rev.,
32: 948–967.
»» Krishnakumar G, Rameshkumar KB, Priya
Srinivas, Satheeshkumar K, Krishnan PN.
Estimation of camptothecin and pharmacological
evaluation of Ophiorrhiza prostrata D. Don and
Ophiorrhiza mungos L. Asian Pacific Journal of
Tropical Biomedicine S727-S731, http://dx.doi.
org/10.1016/S2221-1691(12)60304-9, 2012. Conference Presentations:
»» Veena Somasundaram, Sreelatha K H, Reshma
R S, Rakesh S and Priya Srinivas. ‘Defects in
BRCA1-Novel clues to targeting Breast Cancer
Stem Cells.’ Paper Presented at the 32nd Annual
Convention of the Indian Asociation for Cancer
Research, University of Delhi (North Campus)
from February 13-16, 2013.
»» Sreelatha KH, Reshma RS, Veena S, Rakesh SN,
Thara Somanathan, Jem Prabhakar, and Priya
Srinivas. Mammary Cancer Associated Fibroblast
(CAF) mediated breast tumor progression, an in
vitro study. Poster Presented at the 32nd Annual
Convention of the Indian Asociation for Cancer
Research, University of Delhi (North Campus)
from February 13-16, 2013.
»» Reshma RS, Sreelatha KH, Veena Somasundaram,
Rakesh SN, Priya Srinivas. Castrate Resistant
Prostate Cancer and Naphthaquinones. Poster
Presented at the 32nd Annual Convention of
the Indian Asociation for Cancer Research,
University of Delhi (North Campus) from
February 13-16, 2013.
»» S Veena, KH Sreelatha, RS Reshma, SN Rakesh, S
Priya. ‘Carboplatin and ALDH1+ Breast Cancer
Stem Cells: Unseen Facts. Poster Presented at
the 11th International Congress on Targeted
Anticancer Therapies, Paris, France, March 4-6,
2013. Annals of Oncology 24 (Supplement 1):
i30–i32, 2013.
»» S Priya, RS Reshma, SN Rakesh, KH Sreelatha,
S Veena. ‘Novel Phytochemical to target BRCA2
Related Prostate Cancer. Poster Presented at
the 11th International Congress on Targeted
Anticancer Therapies, Paris, France, March 4-6,
2013. Annals of Oncology 24 (Supplement 1):
i23–i26, 2013
»» K. H. Sreelatha, R. S. Reshma, S. Veena, S. N.
Rakesh, S. Thara, P. Jem, S. Priya. Influence of
Cancer Associated Fibroblast (CAF) on human
breast cancer cells in an in vitro co-culture
model. Poster Presented at the 11th International
Congress on Targeted Anticancer Therapies,
Paris, France, March 4-6, 2013. Annals of
Oncology 24 (Supplement 1): i30–i32, 2013
53
CANCER RESEARCH PROGRAM: LABORATORY- 6
S Sreeja, Ph.D
Scientist EI
[email protected]
Dr. Sreeja received a Ph.D in Biotechnology from University of Kerala for work on Molecular
mechanisms of Estrogen action. She joined RGCB as a scientist in 2001.
PhD students :
Jazir Haneef
Parvathy M
Project Fellows :
Hima Sithul
Juberiya .M.Azeez
54
Technical Personnel :
Savitha
An in vitro investigation on the role of Estrogen and Reactive Oxygen
Species in the invasion of Thyroid Cancer Cells with emphasis on
TGF-β signaling
Hima Sithul, Juberiya .M.Azeez, S.Sreeja
Thyroid disorders are the most common endocrine
malady in India and are very frequent in Kerala, the
southern-most region of India. According to various
reports, thyroid cancer occurs three times more
frequently in females than in males, and in females,
the incidence decreases after menopause. However,
the molecular basis for this gender difference
is still poorly understood. The marked variation
between male and female disease incidence has
led to speculation on whether gender is a possible
epidemiologic risk factor. This gender difference
suggests that the growth and progression of thyroid
cancer may be influenced by female sex hormones,
particularly estrogens. Experimental data have
clearly demonstrated that estrogens can influence
cancer cell growth. In this study, we hypothesize
that E2 and ROS will modulate thyroid cancer cell
growth, adhesion, migration, and invasion and
these phenotypic changes are associated with
functional ER interspersing with coregulators and
its cross talk with other growth-regulating signal
transduction pathways particularly through Smad
mediated TGF β.
Intracellular levels of ROS
Figure No: 1: Intracellular levels of ROS measured by DCFDA for Normal thyroid cells (NThy) and papillary thyroid
carcinoma cells (PTC) under 20 nM 17-beta estradiol, SFM and NAC for 48 hrs.
Figure No 2: Western blot analysis of proteins from Normal thyroid (NThy ) and Papillary thyroidcarcinoma (PTC)
cells treated with 20nM 17 beta Estradiol (E2) for 48 hrs
55
An in vitro study on differentially expressed genes in the luteal phase of
the Menstrual Cycle:
Indhu Hariharan**, Hima Sithul, Juberiya. M. Azeez, Jem Prabhakar*
S Sreeja and M Radhakrishna Pillai**
Collaborators: * Division of Surgical Oncology, Regional Cancer Centre, Thiruvananthapuram,
** CRP - 7, Rajiv Gandhi Centre for Biotechnology.
There are concerns that the timing of surgery
in the menstrual cycle could affect the chance
of tumor cells spreading to other parts of the
body. If genes involved in various aspects of
carcinogenesis are hormonally regulated, their upregulation at specific times in the menstrual cycle
might pre-dispose seeding cancer cells towards
better survival, faster growth rate or increased
metastatic potential. The main objective of this
study was to give a scientific explanation through
a molecular biology approach to understand the
important events that occur at the time of surgery
to determine long term prognosis. If the timing
of surgical treatment during a particular phase
of the menstrual cycle plays a significant role in
survival from pre menopausal breast cancer, this
could possibly extend and/or save a great number
of women’s lives. As a part of this study, we had
assessed variations in estrogen and progesterone
receptor expression rates in breast cancer patients,
56
who were operated in different phases of the
menstrual cycle. By Microarray, we found out that a
number of genes are differentially expressed in both
phases of menstrual cycle. Out of those genes CDH:
(a gene involved in cell adhesion, was up regulated
by 4 fold), ING1: (a tumor suppressor that induces
cell growth arrest and apoptosis was up regulated
by 6 fold), KRT8: (a gene known to maintain cell
integrity and cell differentiation was up regulated
by 2 fold), CST6: (with an anti-metastatic function
was found to be up regulated by 3.4 fold). We also
observed that a few genes which were expressed
exclusively in the luteal phase such as CDH1 and
TOB1 had significant anti invasive properties.
We have done a real time PCR for validating
the expression of these genes. Currently we are
examining the functional studies of TOB 1 which
is expressed exclusively in the luteal phase by an
in vitro system having ± Progesterone. Studies
are in progress.
Role of Mitochondrial uncoupling protein 2 , Ezrin and Oxidative
stress in theanti-estrogenic effects of methanolic extract of Punica
granatum (PME) in estrogen receptor positive breast cancer cells
Juberiya .M.Azeez, Hima Sithul, S.Sreeja
Several mechanistic studies in cell culture and
mouse models suggest possible estrogen receptormediated and non-estrogen receptor-mediated
benefits of pomegranate juice with respect to
breast cancer risk. We had reported that PME binds
to ER and induced a dose dependent decrease
in cell proliferation , was an anti-estrogenic in
the mammary gland, without compromising the
beneficial effect of estrogen in the cardiovascular
and skeletal system and had no estrogenicity in
the uterus. Pomegranate peel extracts have been
shown to possess significant antioxidant activity
in various in vitro models. Antioxidants are capable
of stabilizing, or deactivating, free radicals before
they attack cells. The phytochemical tests indicated
the presence of alkaloids, glycosides, tannins, and
flavonoids are in the crude methanolic extract.
These compounds are known to possess potent
antioxidant activity. Mitochondria are the main
site of intracellular oxygen consumption and the
main source of ROS formation. Uncoupling proteins
(UCPs) are a family of proteins that can prevent
ROS formation by modulating mitochondrial
transmembrane proton gradient.The main objective
of this study is to find out the role of plasma
membrane localized Ezrin and how mitochondrial
uncoupling protein 2 and ROS regulates the anti
estrogenic effects of methanolic extract of Punica
granatum (PME) in estrogen receptor positive
breast cancer cells in a mechanistic way. Studies
are in progress.
B
Figure : Panel A: - Matrigel invasion assay of MCF -7 cells treated with 10 nM 17- beta estradiol , 160 μg PME,
Estradiol + PME for 48 hrs, cells were stained with Hoechst dye.and nuclei were counted using fluorescence
microscope. Panel B: - Percentage invasion of MCF-7 cells treated with PME. Panel C - PME increases expression
of ER beta and reduces the expression of metastatic protein Ezrin at varying concentration of PME and time. 1control ,2- 10 nM 17- beta estradiol for 24 hr, 3 - 160 μg PME for 24 hr, 4 - 10nM 17 - beta estradiol for 48 hr and
5- 160 μg PME for 48 hr .
57
Extra Mural Funding
Sl No
1
2
Investigators
Title
Duration
Dr. S. Sreeja
Role of CNP and PAI - I in vascular
remodeling in estradiol deficiency
and atherosclerosis: Experiments in
ovariectomized Rabbit
Indian Council
Of Medical
Reasearch
2010-2013
Dr. S. Sreeja
Survey and evaluation of Ethno
Botanical Resources of Western Ghats,
Special emphasis to breast cancer and
phytoestrogenic activity
Planning &
Economic Affairs
Department
(WGDP)
2010-2013
Publications
»» Jazir Haneef, Parvathy M, Santhoshkumar
R Thankayyan, Hima Sithul, Sreeja S. Bax
Translocation Mediated Mitochondrial Apoptosis
and Caspase Dependent Photosensitizing Effect
of Ficus religiosa on Cancer Cells. PLoS One,
doi:10.1371/ journal.pone.0040055
List of conference
presentations:
»» “Differential gene expression in breast tumor
tissue at different stages of menstrual cycle: Special
emphasis on timing of breast cancer surgery and
58
Funding Agency
prognosis”- Indu. H, Hima. S, Jem. P, Sreeja. S,
Pillai. M.R at the International Conference
on Repromics-Omics in Reproduction & 23rd
Annual Meeting of the Indian Society for the
Study of Reproduction and Fertility, 7-9 February
2013, Rajiv Gandhi Centre for Biotechnology,
Kerala, India.
»» “Can Lymphocytic thyroiditis be a predisposing
factor for Papillary Thyroid Carcinoma?”Prathibha R, Sreejith S, Hima ,, John K, Sreeja
S, M.R. Pillai at the 3rd International Conference
on Stem Cells and Cancer (ICSCC-2012):
Proliferation, Differentiation and Apoptosis”,
27-30 October 2012, New Delhi, India.
Dr.K.B.Harikumar
Scientist C
[email protected]
Harikumar did his Bachelors and Masters degree in Biochemistry from Nagpur University.
He took the PhD from the MahatmaGandhi university while working at Amala Cancer
Research centre.Harikumar trained as a Post Doctoral fellow at M D Anderson Cancer
Centre.Houston, USA and Virgenia Common Wealth University,Richmond,USA.Harikumar
is a recipient of the Department of Biotechnology’s Ramalingaswamy Re-entry Fellowship.
Research Fellow:
Sabira Mohammed
Technical Assistant:
Shirly James
59
Role of Sphingosine kinase 1 in TLR dependent
innate immune response
Sabira Mohammed, Shirly John, K.B.Harikumar
The long term focus of this proposal is to gain
insight into the role a potent bioactive lipid S1P in
innate immune response with the hope that this
knowledge eventually will lead to the discovery
of improved therapeutic approaches for viral
infection. Sphingolipids are a class of bioactive
lipid mediators characterized by the presence of a
serine head group with one or two fatty acid tails.
Sphingosine, one of the major sphingolipids, and
its phosphorylated product S1P, have emerged as
the modulators of multiple cellular processes, such
as cell growth, survival, differentiation, and T cell
egress, and have therapeutic potential. Sphingosine
is phosphorylated by two enzymes Sphingosine
kinase 1 (SphK1) and 2 (SphK2). The role of the
SphK/S1P system in viral pathogenesis is not well
understood. The available literature indicated
that there is a differential role of Sphingolipid
metabolizing enzymes in viral pathogenesis. Most
of the studies conducted to date have focused
primarily on the role of the sphingolipid system on
viral replication and apoptosis and little is known
about the role this system plays in host response.
Our preliminary investigation suggested that
SphK1 might be playing a role in TLR7/9 dependent
interferon production. Inhibition of SphK1 by
pharmacological approach decreased the type I
interferon production after treating with TLR7/9
agonist in vitro. Further studies are underway to
elucidate role of SphK1 in innate immune response.
A computational based approach to analysis MicroRNA and
transcription factor (TF) co-regulatory networks in pancreatic cancer
K.B.Harikumar, Sona Charles*, G.Reshmi*
*Collaborators, Viral Disease Biology, RGCB
Pancreatic cancer (PaCa) is the fourth leading
cause of cancer-related death with average 5-year
survival rate is only 6%. The mortality-incidence
ratio of PaCa is about 0.99. There is always a
need to identify better diagnostic and prognostic
markers of PaCa for early detection and better
clinical management. MicroRNAs are regulators of
gene expression and often deregulated in human
cancers. In the present study we are taking the
advantage of a computational platform to construct
miRNA-TF based co-regulatory network to identify
potential targets that can be used as early detection
or prognostic markers of human PaCa. Starting
with known genes that have been verified for
aberrant expression and functions in PaCa and
those implicated in literature, we combined gene
expression profiles with functional microarray
gene expression data sets (GEO) to generate a
network containing a number of genes linked by
specific functional associations. Using a random
60
permutation algorithm and training sets of known
PaCa specific genes, we assigned weighted scores
to each screen and total score is calculated for
each gene, providing an indication of the gene’s
involvement in self renewal.
From the microarray data we identified 198 genes
which are differentially expressed in patients
with good and bad prognosis. We also identified
18 microRNAs and 7 transcription factors which
regulate the expression of above mentioned genes
(Table 1).Further analysis revealed the presence of a
positive feedback loop mediated by miR-200c, and
the transcription factor ZEB1 through E-cadherin
(figure 1).miR-200c inhibits ZEB1 Transcription
Factors through E-cadherin thereby inhibiting
Invasion in patients with good prognosis.We are
now validating these targets in human samples of
pancreatic cancer
Table 1 :miRNAS AND THEIR TARGETS ASSOCIATED WITH PDAC
microRNAs
Targets
microRNAs
Targets
miR-22
ESR1
miR-22
SP1
miR-486
CD40
miR-34
BCL2
let-7
RAS
miR-34
NOTCH1
miR-200
RAS
miR-221
CDKN1B
miR-10a
HOXB1
miR-107
CDK6
miR-10a
HOXB3
miR-155
TP53
miR-21
PTEN
miR-194
EP300
miR-21
RECK
miR-200b
EP300
miR-200c
ZEB1
miR-224
CD40
Double positive feedback loop in good prognosis samples
miR-200
miRNA
ZEB1
TF
Gene
E-cadherin
Invasion
Figure 1: Double positive feedback loop in good prognosis samples of pancreatic cancer samples identified using
system biology apparoch
Spice derived phytochemicals-Sesamin and Cardamonin- for colorectal
cancer chemoprevention
Sabira Mohammed, Shirly John, K.B.Harikumar
Colorectal cancer (CRC) is the third most
common cancer in men and second in women
worldwide. Several factors have been attributed
for the causation of CRC in humans. Several
epidemiological evidences support the roles of diet,
lifestyle, and medication in reducing the risk of
colorectal cancer.There is a considerable increase
in the pharmacological effects of nutraceuticals
for cancer treatment and prevention. Many of
the nutraceuticals are potent anti-inflammatory
agents. Therefore these molecules have a potential
in prevention and treatment of colon cancer. In this
project we are mainly focusing on nutraceuticals
derived from spices. We are mainly focusing
61
on Sesamin which derived from Sesame and
cardamonin from Cardamom. Our initial studies
showed that Sesamin inhibited the growth human
colon cancer cells in vitro. A combination of
Sesamin and capecitabine was found to be more
effective in inducing cell death in colon cancer
cells in culture as compared to either agent alone.
At present we are studying the effect of Sesamin in
preclinical models of colitis and colitis associated
colorectal cancer.
Research Grants Extra-Mural Funding
K.B. Harikumar - Principal Investigator
No
Title
Duration
1
Spice derived phytochemicals Sesamin
and Cardamonin- for colorectal cancer
chemoprevention
Department of Biotechnology (DBT)Ramalingaswami fellowship
2012-2017
2
Novel aspects of sphingosine
1-phosphate (S1P) in innate immune
responses and host defense mechanism
Dept. of Science and Technology
(DST), Government of India
2013-2016
Publications (2012-2013 May)
»» 1: Liang J, Nagahashi M, Kim EY, Harikumar
KB, Yamada A, Huang WC, Hait NC,Allegood
JC, Price MM, Avni D, Takabe K, Kordula T,
Milstien S, Spiegel S.Sphingosine-1-phosphate
links persistent STAT3 activation, chronic
intestinalinflammation, and development of
colitis-associated cancer. Cancer Cell. 2013,
23(1):107-20.
»» 2: Price MM, Oskeritzian CA, Falanga YT,
Harikumar KB, Allegood JC, Alvarez SE,
»» Conrad D, Ryan JJ, Milstien S, Spiegel S. A
specific sphingosine kinase 1inhibitor attenuates
airway hyperresponsiveness and inflammation
in a mastcell-dependent murine model of
allergic asthma. J Allergy Clin Immunol. 2013,
131(2):501-11
62
Funding Agency
»» 3: Van DN, Roberts CF, Marion JD, Lépine S,
Harikumar KB, Schreiter J, Dumur CI,
»» Fang X, Spiegel S, Bell JK. Innate immune
agonist, dsRNA, induces apoptosis in ovarian
cancer cells and enhances the potency of
cytotoxic chemotherapeutics.
»» FASEB J. 2012; 26(8):3188-98.
Conference/Seminar
presentations
»» Harikumar KB, Charles S, Reshmi G. A
computational based approach to analysis
MicroRNA and transcription factor (TF) coregulatory networks in pancreatic cancer. Poster
presented at 32nd Annual convention of Indian
Association for Cancer Research, New Delhi,
February 2013.
Dr. Debasree Dutta
Scientist C
[email protected]
Debasree has a Masters degree in Biochemistry from the University of Calcutta Followed
by a MTech and PhD from Jadavpur University.She trained as a Post Doctoral fellow from
2007 to 2012 at the University of Kansas Medical Center in US
Research Fellows:
Rahul Sanawar
63
Role of Histone chaperones in inducing pluripotency
Rahul Sanawar and Debasree Dutta
The generation of induced pluripotent stem cells
(iPSCs) from somatic cells demonstrated that
adult mammalian cells can be reprogrammed to a
pluripotent state with the enforced expression of
different transcription factors. This new technology
has enabled researchers to derive disease-specific
stem cells for the study and possible treatment of
degenerative disorders with autologous cells. iPSC
derivation is ethically and legally less problematic
and technically more feasible. In general, iPSCs
are almost of similar nature to Embryonic Stem
Figure. MEFs showing the efficiency of transfection by
lentiviral particles (GFP expression).
Mouse embryonic fibroblasts were transfected with viral
particles having transcription factors responsible for
maintaining pluripotency. The figure mentioned below
depicts the efficiency of transduction for the expression
of Oct4 and Nanog in MEFs.
64
Cells (ESCs) although with numerous subtle but
substantial molecular differences. Till now, iPSCs
have been derived from different species and from
other somatic cell populations with varied protocols
with each one having its own advantages and
disadvantages. In effect, iPSC technology has
several unsolved issues; low efficiency of iPSC
generation, genetic alterations, the possibility
of tumor formation in vivo, and uncontrolled
growth of the remaining cells that are partially
reprogrammed and refractory to differentiation.
To address these problems,
we should understand the
molecular mechanisms involved
in generation of iPSCs. Along
with a transcriptional regulation,
reprogramming entails global
epigenetic remodeling. Recent
studies have implicated
histone modifying enzymes in
iPSC generation. So on these
contexts, the proposed study
will focus on the role of histone
modifier, histone chaperones, in
iPSCs generation. Chaperones
associate with histones upon
their synthesis, escort them into
the nucleus, and aid in their specific association
with DNA during different processes such as
DNA replication, repair, or transcription. They
also interact with histone modification enzymes
and modulate different PTMs involved in various
cellular processes. We will analyze how they can
affect the efficiency of generation of iPSCs, and how
do they regulate the histone modification status
both globally and within the genes implicated in
inducing pluripotency.
Our initial data revealed that downregulation of
Histone chaperone, Asf1, in mouse ES cells induced
the expression of genes related to pluripotency.
So, we tried to knockdown Histones chaperones
in mouse embryonic fibroblasts to generate iPSCs
from them. The standardization of lentiviral
mediated knockdown in fibroblasts is presently
being worked out in the laboratory.
The standardization of using the particle
concentration is still being worked out.
Nano
g
Oct
4
Hoesch
t
Phas
e
Figure.
Transduction
efficiency
viral
particles for
generating
iPSC from
Figure. Transduction
efficiency
of viral
particlesoffor
generating
iPSC
from MEFs.
MEFs.
Hemogenic endothelium- regulation and reprograming
Debasree Dutta
Hematopoietic stem cells (HSCs) are multipotent
stem cells that give rise to all the blood cell types
from myeloid and lymphoid lineages. Under different
physiological stress and disease related condition,
HSCs are transplanted, which are usually derived
from bone marrow, peripheral blood, or umbilical
cord blood. Patients suffering from certain cancers
of the blood or bone marrow, such as multiple
myeloma or leukemia or even haemolytic anaemia
usually undergo this transplantation. So, in these
cases, the recipient’s immune system is generally
destroyed with radiation or chemotherapy before
the transplantation. Graft-versus-host disease
is a major complication of transplantation
related to HSCs. This complication can be
avoided if we can derive in vitro developed
patient specific HSCs. But, even in this way,
when the target is lysis of blood cells, a direct
transplantation might not help. Hence, in this
proposal we will try to address this problem
while deriving an option in overcoming this
hurdle. Existence of Hemangioblast, the
putative bipotential progenitor of endothelial
and HSCs, is controversial. Rather, nowadays,
it is considered that HSCs originate from
Hemogenic endothelium, composed of
endothelial cells. Hemogenic endothelium
exists in yolk sac, dorsal-aorta in the
Figure.
Mouse
differentiated
for the of HE (48h).
Figure.
Mouse
ESCsESCs
differentiated
for the Formation
Formation of HE (48h).
65
aortagonad-mesonephros (AGM) region in vivo.
At the molecular level, transcription factor Runx1 is
required for the transition towards HSCs generation
while in the differentiated endothelial cells. HoxA3
is needed to maintain the endothelial phenotype
by downregulating Runx1. But, the targets of these
factors as well as the nucleoprotein structure
that forms at the locus of cell surface markers
expressed on cells from hemogenic endothelium,
is entirely unknown. The nucleoprotein structure
involves histone modulators, histone modificatons
and the transcription factors at the particular loci.
Hence, understanding the mechanistic regulation
of genes implicated in hemogenic endothelium
will ensure a better formulation of generation of
Hemogenic endothelium from fibroblasts. So, I tried
to differentiate mouse ESCs towards hemogenic
endothelium using a cocktail of growth factors/
inhibitors. The trial of generating the perfect HE in
culture condition is still being worked out.
Research Grants Extra-Mural Funding
Debasree Dutta- Principal Investigator
Sl. No
Title
1
Role of Histone chaperones in inducing
pluripotency
Dept. of Biotechnology,
Government of India.
2012-2015
2
Transcriptional regulation of vascular
endothelial growth factor receptor 3
Council of Scientific & Industrial
Research, Government of India.
2013-2016
3
Hemogenic endothelium- regulation
and reprograming
Dept. of Science and Technology,
2013-2016
Publications
(2012 April- 2013 May)
»» Dutta D. (2013). Signaling pathways dictating
pluripotency in Embryonic Stem cells. Int J Dev
Biol. (Accepted).
»» Dutta D, Hong J, Rajendran G, Ray S, Home
P, Saha B, Paul A, Weiss ML, Paul S. (2013).
Inhibition of protein kinase C signaling maintains
rat embryonic stem cell pluripotency. J Biol
Chem. (Under revision).
»» Hong J, He H, Bui P, Ryba-White B, Rumi MA,
Soares MJ, Dutta D, Paul S, Kawamata M, Ochiya
T, Ying QL, Rajanahalli P, Weiss ML. (2013). A
focused microarray for screening rat embryonic
stem cell lines. Stem Cells Dev. 22:431-43.
»»Home P, Saha B, Ray S Dutta D, Gunewardena
S, Yoo B, Larson M, Wolfe MW, Petrof
M, Gallaghar PG, Scholz V, White KL,
Golos TG, Behr B, Paul S. (2012) Altered
subcellular localization of transcription factor
Tead4: an evolutionary conserved mechanism
66
Funding Agency
Duration
to regulate first mammalian cell lineage
commitment. Proc Natl Acad Science
109:7362-7367.
»» “Instem Mouse Embryology Workshop: Stem cells
to organogenesis” 2013 at NCBS, Bangalore,
India (10th March- 23rd March). Abstract was
selected for a 2-week workshop on mouse
embryology. Presented poster on “Histone
modifiers in developmental biology”.
»» Annual meeting of “International Society for
Stem Cell Research” (ISSCR) 2013 at Boston,
USA (12th June- 15th June). Abstract selected for
poster presentation.
Awards
»» International travel grant from Department
of Biotechnology, Government of India for
attending ISSCR conference at Boston, USA.
RGCB – GEORGE WASHINGTON UNIVERSITY
JOINT PROGRAM IN CANCER RESEARCH
Dr Rakesh Kumar
Visiting Distinguished Professor
[email protected]
Upon the recommendation of the RGCB Governing Body, Professor Rakesh Kumar was
invited to join the Rajiv Gandhi Center for Biotechnology as a Visiting Distinguished
Professor of Biotechnology to establish and co-direct a joint cancer research program with
Professor Radhakrishna Pillai. RGCB and the George Washington University have signed a
Memorandum of Understanding for a variety of mutually beneficial academic and scholarly
activities.
PhD students:
Parvathy M
Hezlin Marzook
Deivendran S
67
Role of p21 Activated Kinases in Oral Carcinogenesis
Parvathy M, S. Sreeja, Rakesh Kumar and M Radhakrishna Pillai P21 Activated Kinases (PAKs) are a family of
serine/threonine kinases involved in numerous
physiological processes. A large body of work
has firmly established mechanistic roles of PAKs
in cell survival, motility, angiogenesis and gene
regulation, all of which are highly important in
the context of cancer. This study aims to elucidate
the contribution of PAK1 and PAK4 in oral cancer
progression. The effect of serum growth factors on
the localization of PAK1 and PAK4 was studied in
the Oral Squamous Cell Carcinoma (OSCC) cell
line, HSC4. It was observed that in serum-free
condition, PAK1 was seen mostly in the nucleus;
however upon serum stimulation it translocates
from the nucleus to cytoplasm as a function of time
(Fig. 1). In contrast, PAK4 predominantly resides in
the cytoplasm with albeit nuclear localization in
HSC4. These observations suggest that localization
of PAK1 but not PAK4 may be regulated by serum
growth factors and this in-turn, might affect
cytoplasmic functions of PAK1, especially its
cytoskeletal remodelling activity. Next, status of
cytoskeleton remodelling was used as a functional
endpoint of PAK1 signalling and assessed by
Phalloidin staining of actin. Results suggested a
possible role for growth factor signalling in actin
remodelling in OSCC, presumably due to PAK1
signaling (Fig. 2). To implicate a mechanistic role
for PAK1 in the observed cytoskeletal remodelling
of OSCC cells, we examined the effect of selection
knockdown of PAK1 on serum-induced cytoskeleton
remodelling. It was observed that PAK1 knockdown promotes notable changes in the actin
structure as compared to the control cells (Fig. 3).
Even under light microscopy, it was evident that
PAK1-knock down cells exhibited an aberrant
morphology and higher rate of apoptosis, when
compared to the control siRNA transfected cells.
This indicates that the changes in cytoskeletal
structure leading to morphological changes may
be responsible for increased rate of apoptosis in
these cells. Further studies continue to elucidate
the biological role of PAKs in oral carcinogenesis.
Figure 1: Differential localization pattern of PAK1 and PAK4 under serum stimuli
(Green: PAK1, Blue: Nuclei, and Merged: PAK1 and Nuclei)
Figure 2: Phalloidin staining of OSCC cells that were subjected to serum stimuli
(Green: F-Actin filaments and Blue: Nuclei)
68
Figure 3: Phalloidin staining showing difference in actin structure in the control siRNA transfected and PAK1
knock-down OSCC cells (Green: F-Actin filaments and Blue: Nuclei)
Epigenetic Regulation of Cancer by MTA1
Deivendran S, T. R. Santhoshkumar, M. Radhakrishna Pillai and Rakesh Kumar
The Metastatic Tumor Antigen 1 (MTA1) is a
major overexpressed oncogene in human cancer.
Mechanistically, MTA1 is a chromatin modifier
and modifier of transcription by virtue of its dual
functional activity both as a corepressor and
coactivator. Recent studies suggest that MTA1’s
corepressive activity depends on its interaction
with the Nucleosome Remodeling complex (NuRD),
while coactivator activity is independent of the
NuRD complex and profoundly affected by the
NuRF complex. Since MTA1 has a BAH domain
and because this BAH domain is associated with
the methylation of DNA, we hypothesize a role of
MTA1 or MTA1-containing complexes in target
gene methylation.
MTA1 promotes invasion through influencing the
transcription of its target genes via modifying
the status of chromatin as well as interacting
with transcription factors. To explore the role of
MTA1 in tumor promotion, we hypothesize that
MTA1 may inhibit the expression of metastasis
suppressor genes (MSG). These genes act in such
a way that it can inhibit metastasis at one or more
steps of a process during metastatic progression.
To experimentally test this notion, we selected
44 MSG genes from previous published literature
and examined whether MTA1 is recruited to the
regulatory elements of such genes using a MTA1
based ChIP-on-ChIP genome wide studies in MCF7 cells.The ChIP-on- ChIP data revealed that MTA1
is recruited onto the promoter of 21 MSG genes
among the 44 shortlisted candidate genes. Further,
we performed in silico analysis for all these 21 genes
for presence of CpG islands. Eight genes were seen
to possess CpG Islands. Subsequent, data mining
was performed to define the correlation between
those MSG and MTA1 in the available datasets.
Interestingly, we noticed that the eight genes had
negative correlation with the expression level of
MTA1. The selected candidate genes were then
validated by semi-quantitative PCR in breast
cancer cells with or without MTA1 knockdown
or MTA1 overexpression. Results indicated that
regulation of three MSGs (IGFBP-3, Claudin-1 and
N-Wasp) were upregulated by MTA1. In addition,
we also found that Claudin-1 and IGFBP3 proteins
were regulated by MTA1 when analyzed by
Western blot analysis. Further analysis of regulation
of target genes by MTA1 is in progress.
69
Upstream Regulators and Downstream Effectors of MTA1
during Tumor Progression
Hezlin Marzook, T. R. Santhoshkumar, M. Radhakrishna Pillai and Rakesh Kumar
Cancer involves delicate transcriptional control of
numerous genes that drive cell growth, survival,
invasion, and metastasis via dynamic interaction
of transcriptional factors with chromatin. The
highly condensed structure of chromatin provides
a formidable obstacle for sequence-specific
transcription factors and RNA polymerase
gaining access to nucleosomal DNA in eukaryotic
transcription. Therefore, chromatin modifying
factors are needed to create a dynamic chromatin
environment that modulates DNA accessibility to
transcriptional machinery for appropriate gene
transcription.
One newly added group of ubiquitously expressed
chromatin modifiers is the metastasis-associated
protein (MTA) family, members of which play an
integral role in the nucleosome remodeling and
histone deacetylation (NuRD) complexes. MTA1,
the founding member of the MTA family, was
originally isolated by differential cDNA library
screening using a rat mammary adenocarcinoma
metastatic model as an up-regulated gene in highly
metastatic cells..
The goal of the present project is to examine the
role of MTA1 in tumor progression under stressed
conditions such as serum-starvation or hypoxia.
Preliminary experiments in breast cancer cell lines
laid a proof-of-concept about the alterations in the
sub-cellular distribution of MTA1 during serum
starvation and/or hypoxia. A previous data has
shown that MTA1 interacts with HIF1α, promotes
its degradation via de-acetylation, and stimulates
its transcriptional activity, leading to increased
expression of VEGF-A, with implication for cancer
progression and metastasis. Our observations of
nuclear-to-cytoplasmic redistribution of MTA1
under stress-induced signaling highlight the
significance of functional cross-talk between two
distinct pathways. It is possible that MTA1 may
have a protective role against stress, presumably,
due to its ability to regulate a sub-set of relevant
genes in the nuclear compartment. In contrast,
MTA1 is expected to exert distinct functions in the
cytoplasm involving putative interacting partners,
yet to be deciphered.
»» Parvathy M, Sreeja S, MR Pillai and Rakesh
Kumar. Functional Role of p21 Activated Kinases
in Oral Cancer. 3rd International Conference of
70
Stem Cells and Cancer (ICSCC), New Delhi,
October 2012.
M Radhakrishna Pillai
Professor
[email protected]
Radhakrishna Pillai joined RGCB in 2005 moving from the Regional Cancer Centre at
Trivandrum where he was Professor of Molecular Medicine. Dr. Pillai is a Fellow of the
Royal College of Pathologists, London, the National Academy of Medical Sciences, India,
the National Academy of Sciences, India and the Indian Academy of Sciences.
Visiting Scientist:
Tessy Thomas Maleikal, PhD
Program Scientist:
Reshmi G, PhD
Research Associate:
Srinivas K P, PhD
Research Students and Fellows:
Sumithra Shankar
Janki Mohan Babu V
Hezlin Marzook
Deivendran S
Sajitha I S
Prathibha R
Data Manager & Systems Analyst:
Lekshmi R
Project Assistants:
Asha V S
Preetha V Rajan
Roshini Vasudevan
Jinu Austin
71
Biological manifestations of e6 knockout in HPV16 infected cells using
engineered nucleases
Sumitra Shankar and M. Radhakrishna Pillai
Gene targeting using synthetic nucleases is a
new technology, which brings about a knockout
at genomic level by inducing double stranded
breaks at specific region in the genome. Though
these nucleases have to be administered only
once, they make a permanent targeted damage
in the genome. Since the E6 based siRNA
approach has been shown to induce apoptosis,
and senescence in HPV 16 positive cells, an
effective knockout of the viral E6 gene would be
of therapeutic significance. Zinc-finger nucleases
(ZFNs) are artificial restriction enzymes generated
by fusing a zinc finger DNA-binding domain to a
DNA-cleavage domain. They can target unique
sequences within complex genomes. This part of
the study focused on extensive standardization of
protocols to achieve optimum ZFN activity. The
Zinc Finger Nucleases (ZFNs) against the E6 gene
were administered as pairs. ZFNs were incubated
with the plasmid containing E6 gene and higher
activity was achieved at 30˚ C in NEB buffer 2 than
at 37˚C as seen in Lane 6.
was done to detect Non- Homologous End Joining
(NHEJ) events. T7E1 assay detects mismatches
generated by the NHEJ as 2 cut products as
against the control, which has the PCR product
alone. About 0.1% indels were obtained in SiHa
cell line and around 3.8% indels were found in
Caski (Indels were calculated by Image J software).
ZFNs activity at 37˚C resulted in a low percentage
of Indels ~1-4%. To improve the activity of ZFNs
several strategies are currently being employed.
We then cloned ZFNS into a strong promoter. ZFN
expression in HEK cell line showed that the ZFN
expression under CAG promoter was stronger than
CMV promoter.
Following this, ZFNs have been cloned into a
vector containing improved FokI variant containing
sharkey mutations. Currently ZFN treated cells are
being analyzed for editing efficiency. ZFNs have
been transfected into cell lines and incubated
at 30˚C for 3 days. Mismatch endonuclease and
restriction digestion assays are currently being
performed.
After 3 days treatment, genomic DNA was isolated
followed by PCR and finally an endonuclease assay
Fig 1 In vitro cleavage assay of ZFNS in different buffers
72
Lane 1: plasmid with E6 gene
Lane 2: plasmid treated with ZFNs
in NEB buffer 1 @ 37 ˚C
in NEB buffer 1 @ 30˚C
Lane 8: 100bp ladder
Fig 2. Transfection efficiency in SiHa and Caski cell line
Fig3 a) Diagram of T7E1 assay, b) ZFN editing activity in HPV cell lines through T7E1 assay
Fig4. ZFN cloning into pCAGIG vector for strong expression
73
Cell death Mechanisms during in vitro photosensitization
with a novel metalloporphyrin
Saneesh Babu P.S, Betsy Marydasan* D Ramaiah* S. Asha Nair
and M. Radhakrishna Pillai
Collaborators: *Photochemistry and Photonics Division, National Institute for Interdisciplinary
Science and Technology (NIIST), Thiruvananthapuram.
Photodynamic therapy (PDT) is an emerging
treatment modality for various kinds of tumors and
it involves the inactivation of living cells by the
combined action of light and a photo sensitizer. A
variety of photo sensitizers have been examined
for PDT, including porphyrins, phthalocyanines
and metallophthalocyanines. However, there are
several disadvantages with these first generation
photo sensitizers. Hematoporphyrin derivative
is a mixture of at least nine components and its
preparation is highly sensitive to the experimental
conditions. It also causes cutaneous photosensitivity,
immunosuppression and more importantly has only
a weak absorption in the red region of the spectrum.
To circumvent these drawbacks, biocompatible
metal complexes with their versatile coordination
geometry, as well as varied spectral and redox
properties, could be suitably designed as potent
PDT agents. Our collaborators at NIIST Trivandrum
synthesized a novel Metalloporphyrin BMR 202
with high absorption spectra in NIR region, suitable
water solubility and appropriate singlet oxygen
74
generation capability. The cytotoxicity ofBMR
202 in breast cancer cells (MDA-MB-231) were
investigated both in the presence and absence
of light. Cytotoxic studies revealed that BMR
202 is essentially noncytotoxic in the absence of
light but interestingly on the other hand exhibits
high Photocytotoxicity and showed that the
IC50 value of 7mM. Further we investigated cell
death mechanism behind PDT using Annexin V
apoptotic assay, TMRM, Hoechst and combined
staining of acridine orange and ethidium bromide.
Chromatin condensation and permiabilization of
plasma membrane was evident from Hoechst and
combined staining of acridine orange and ethidium
bromide. Loss of mitochondrial membrane
potential from TMRM assay indicates cell death
induced by BMR 202 via mitochondria mediated
apoptotic pathway. PDT with BMR 202 showed
44.6 % and 51.2% Annexin positivity in 5 and 10mM
concentrations. This demonstrates that BMR 202
induced apoptosis in a concentration dependent
manner.
Research Grants
M Radhakrishna Pillai – Principal Investigator
Sl No
Title of the project
Funding Agency
Duration
1
Development of novel sensitizers based on
NIR dyes- Metal based drug including leads
from traditional medicine.
Technology, Government of
India.
2007-2013
2
Computational biology for design of optimum
Vaccine candidates and development of
quantum dot based diagnostics for classical
swine fever virus.
3
4
5
9
Role of human papillomavirus infection and
other co-factors in the aetiology of head and
neck cancer in Europe and India (HPV –
AHEAD
Experimental studies on therapy of cancers
expressing hCG / hCGB with a recombinant
highly immunogenic vaccine against hCG.
Whole genome survey of microRNA target
site accessibility based on conserved local
RNA secondary structure and protein binding
site overlaps: Creating a Freely Accessible
Web Resource
Effects of topical Midodrine Hydrochloride
(“Midodrine”) on psoriasis.
PUBLICATIONS
»» Primary Publications from Laboratory
»» Hariharan R, Simon R, Pillai MR, Taylor TD.
Comparative analysis of DNA word abundances
in four yeast genomes using a novel statistical
background model. PLoS One 8(3): e58038. Doi:
10.1371/journal.pone.0058038, 2013.
»» Santhi WS, Prathibha R, Charles S, Anurup
KG, Reshmi G, Ramachandran S, Jissa VT,
Sebastian P, Pillai MR. Oncogenic micro RNAs
as biomarkers of oral tumorigenesis and minimal
residual disease. Oral Oncology 2013 , vol 49(6),
p-567-575
»» James P, Reshmi G, Charles S, Pillai MR.
Methfinder – A software package for prediction
of human tissue - specific methylation status of
CpG islands. Bioinformation 9 (1): 61 – 64, 2013.
»» Suneesh C. Karunakaran, P. S. Saneesh Babu,
2008-2013
2011-2013
European Union
2012–2014
2012- 2015
Government of India
Mayo Foundation for Medical
Education and Research
2011- 2012
Bollapalli Madhuri, Betsy Marydasan, Albish K.
Paul, Nair AS, K. Sridhar Rao, A. Srinivasan,
Tavarekere K. Chandrashekar, Ch. Mohan Rao,
Pillai MR and Danaboyina Ramaiah. Water
Soluble Neutral Porphyrin Derivative: An
Efficient Sensitizer for Photodynamic Therapy
and NIR Nuclear Imaging Applications. ACS
Chemical Biology 8: 127 – 132, 2013.
»» Thomas AP, Saneesh Babu PS, Nair AS,
Ramakrishnan S, Ramaiah D, Chandrashekar
TK, Srinivasan A, Pillai MR. Meso-Tetrakis(psulfonatophenyl)N- Confused Porphyrin
Tetrasodium Salt: A Potential Sensitizer for
Photodynamic Therapy. Journal of Medicinal
Chemistry. 55 (11): 5110-20, 2012.
»» Reshmi G, Charles S, James P, Jijith VS, Prathibha
R, Ramachandran S, Divya R, Ramadas K,
Pillai MR. OrCa-dB: A complete catalogue
of molecular and clinical information in oral
carcinogenesis. Oral Oncology 48 (2012) e19.
75
»» Preethi N R, Thulaseedharan JV, Wesley R,
Jayaprakash PG, Lalitha P, Pillai MR. A casecontrol nutrigenomic study on the synergistic
activity of folate and vitamin B12 in cervical
cancer progression. Nutrition and Cancer 64 (4):
550 – 558, 2012
»» Prabhu PR, Jayalekshmi D, Pillai MR. Lung
Cancer and Human Papilloma Viruses (HPVs):
Examining the Molecular Evidence. Journal of
Oncology. 2012:750270, 2012.
»» Reshmi G and Pillai MR. Interplay Between
HPV Oncoproteins and MicroRNAs in Cervical
Cancer, Human Papillomavirus and Related
Diseases - From Bench to Bedside - Research
aspects, Davy Vanden Broeck (Ed.), ISBN: 978953-307-855-7, InTech, 2012.
Publications in Collaboration
with Other RGCB Laboratories
»» Nair SA, Jagadeeshan S, Indu R, Sudhakaran PR,
Pillai MR. How intact is the basement membrane?
Role of MMPs. Advances in Experimental Medical
Biology 749: 215 – 232, 2012.
»» Sobhan PK, Seervi M, Joseph J, Varghese S,
Pillai PR, Sivaraman DM, James J, George RE,
Elizabeth KE, Santhoskumar TR, Pillai MR.
Immortalized functional endothelial progenitor
cell lines from umbilical cord blood for vascular
tissue engineering. Tissue Enggineering Part C
Methods 18 (11): 890 – 902, 2012.
»» Ramachandran S, Venugopal A, Sathisha K,
Reshmi G, Charles S, Divya G, Chandran NS,
Mullassari A, Pillai MR, Kartha CC. Proteomic
profiling of high glucose primed monocytes
identifies cyclophilin A as a potential secretory
marker of inflammation in type 2 diabetes.
Proteomics 12 (18): 2808 – 2821, 2012.
»» Ashwani N, Kumar N A, Nair A S, Kumar G.S.V,
Pillai MR. Methacrylic based nanogel for the pH
sensitive delivery of 5-Flourouracil. International
Journal of Nanomedicine 7: 5769 – 5779, 2012.
»» Krishnan A, Johnson A, Gopinath VR, Nair AS,
76
Pillai MR. Cell cycle analysis and MN induction
frequency reveals G0/G1 blockers to be weak MN
inducers. Drug and Chemical Toxicology 39 (2):
249 – 254, 2013.
»» David D, Nair SA, Pillai MR. Smurf E3 ubiquitin
ligases at the cross roads of oncogenesis and
tumor suppression. Biochim Biophys Acta 1835
(1): 119 – 128, 2013.
»» Rakesh Kumar, Anelia Horvath, Raja Mazumder,
Masakazu Toi, Fumiaki Sato, Pillai MR, Luis
Costa, Maria Carmo-Fonseca, Stefan Knapp,
Amit Dutt, Sudeep Gupta, Rajendra Badwe.
The Global Cancer Genomics Consortium’s
Second Annual Symposium: Genomics Medicine
in Cancer Research. Genes & Cancer doi:
0.1177/1947601913484582, 2013
CONFERENCES / WORKSHOPS
»» Saneesh Babu P.S, D Ramaiah*, S. Asha Nair,
and M. Radhakrishna Pillai. “Implications of a
squaraine dye as photosensitizer for photodynamic
therapy in cancer “ 32nd Annual Convention
of Indian Association for Cancer Research
(IACR) & International Symposium at “Infection
& Cancer at Dr. B.R. Ambedkar Center for
Biomedical Research (ACBR) University of
Delhi, North Campus on Feb 13-16, 2013
»» Sona Charles, Reshmi G, M Radhakrishna
Pillai. “The Revelation of Transcription FactormicroRNA Regulating Networks in Human Stem
Cells” in 3rd International Conference on Stem
Cells and Cancer (ICSCC-2012): Proliferation,
Differentiation and Apoptosis, New Delhi during
27th to 30th October 2012 at New Delhi, Delhi,
India.
»» Janki Mohan Babu, Paul Sebastian, Ramkumar
Hariharan and M.Radhakrishna Pillai. Tumors
of the oral tongue in patients giving no history
of tobacco and alcohol habits have well defined
biological characteristics. 32nd Annual Convention
of Indian Association for Cancer Research at Dr.
B.R. Ambedkar Center for Biomedical Research
(ACBR)
HUMAN PAPILLOMAVIRUSRESEARCH PROGRAM
Chief Investigators
Dr R. Sankaranarayanan
Head, Early Detection & Prevention
Section (EDP) and Head, Screening
Group (SCR) International Agency for
Research on Cancer (WHO-IARC
Dr Michael Pawlita
Dr Masimmo Tomassimo
Division of Genome Modifications Infections and Cancer Biology Group,
International Agency for Research
and Carcinogenesis,
on Cancer (WHO-IARC)
Research Program Infection and
Cancer, German Cancer Research
Center (DKFZ), Heidelberg, Germany.
Dr Tarik Gheit
Dr. M. Radhakrishna Pillai
Infections and Cancer Biology
Group, International Agency for
Research on Cancer (WHO-IARC)
Professor of Disease Biology &
Camcer Research, RGCB
Research Fellows:
Janki Mohan Babu
Priya Prabhu
D. Jayalakshmi
Roshini Vasudevan
Jinu Austin
77
Evaluation of the effectiveness and safety of 2 versus 3 doses of HPV
vaccine in preventing cervical cancer: an Indian multi-centre study
Cancer of the uterine cervix is the second most
common cancer among women globally and
most common cancer among Indian women.
India accounts for one-fourth (130,000 new
cases and 70,000 deaths) of the annual global
burden of cervical cancer (530,000 cases and
275,000 deaths), yet lacks organized cervical
screening and vaccination programs to control
this cancer. Cervical cancer is caused by persistent
infection with one of the 15 high-risk monogenic
Human Papillomaviruses (HPV) and, therefore,
interventions to prevent HPV infection will prevent
cervical cancer. Currently effective prophylactic
vaccines are available to prevent HPV 16 and 18
infections, which cause more than 70% of cervical
cancers. HPV vaccination is a major strategy
for controlling cervical cancer by preventing
persistent HPV infection and it is currently being
given through national immunization programs
in over 50 countries, including low- and middleincome countries (LMICs) such as Bhutan,
Malaysia, Fiji, Eastern Timor, Rwanda, Panama,
Mexico, Argentina, Peru and Colombia, with high
participation rates and excellent safety profile.
Currently HPV vaccination is administered in
3-doses spread over 6 months and no proof is
available as to whether fewer than 3-doses will
be as effective. In order to catalyse and advance
the introduction of HPV vaccination in LMICs
at a faster pace, vaccination costs need to be
significantly reduced both by lowering the costs
of the vaccine itself, as well as the costs of its
delivery. Vaccine delivery costs are a substantial
78
component of any vaccination program and the
use of fewer than 3- doses, such as 2-doses, can
substantially reduce vaccination costs. However,
the efficacy and safety of a two-dose regimen needs
to be established before such a recommendation
can be made.
A multi-centre cluster randomised trial to evaluate
the comparative efficacy of 2- versus 3- doses of
HPV vaccination in preventing persistent HPV
infection and cervical neoplasia was initiated in
2009 as a collaborative project of 11 institutions
in India (Tata Memorial Hospital, Mumbai;
Nargis Dutt Memorial Cancer Hospital, Barshi;
Jehangir Clinical Development Centre, Pune;
Christian Fellowship Community Health Centre,
Ambillikai; Gujarat Cancer Research Institute,
Ahmedabad; All India Institute of Medical
Sciences, New Delhi; MNJ Institute of Oncology
and Regional Cancer Centre, Hyderabad, Cancer
Foundation of India, Kolkata, Civil Hospital,
Aizwal; STNM Hospital, Gangtok; Rajiv Gandhi
Centre for Biotechnology, Thiruvananthapuram)
and the International Agency for Research on
Cancer (IARC) of the World Health Organization,
Lyon, France. The study aimed to recruit 20,000
unmarried girls aged 10-18 years from different
regions of India and randomly allocate them to
groups of 10 000 girls each to receive either two
doses on days 1 and 180 or three doses standard
schedules on days 1, 60 and 180 of quadrivalent HPV
vaccine. The study was planned to be monitored
and evaluated by way of outcomes related to long-
term immunogenicity, HPV infection frequency
and persistence of both vaccine included HPV
types and all oncogenic types, incidence rate of
high-grade cervical intraepithelial neoplasia (CIN
2 and 3) caused by vaccine included HPV types and
non-included types and possibly invasive cancer
over several years of follow-up by linking with
population-based cancer registries. However, the
suspension of HPV vaccination studies in India in
April 2010 (due to events unrelated to our study)
led to a number of girls receiving only one dose (in
both 2- and 3-dose arms) or receiving 2 doses on
days 1 and 60 (in the 3-dose arm) by default, which
led to the loss of randomization, thus rendering
the study, in effect, as an observational study of
4955 girls with 1 dose, 3963 with 2 doses on day 1
and 60, 4920 with 2 doses over 180 days and 4337
with 3-doses over 180 days.Despite the suspension
of vaccination and loss of randomization, all
vaccinated girls are visited annually and their
health enquired into. Details regarding marriage,
pregnancy, antenatal events, delivery and perinatal
events are collected and appointments are fixed for
plasma sample collection to study immunogenicity
and cervical cell collection to establish HPV
infection frequency and persistence as per protocol.
The plasma samples and cervical cell samples are
analysed for HPV immunogenicity by competitive
luminex immunoassay (CLIA) and HPV genotyping
by PCR bead-based multiplex genotyping(TS-MPG)
at the RGCB’s dedicated state-of-art laboratories.
Technical assistance and external quality assurance
has been provided from the German Cancer
Research Centre (DKFZ), Heidelberg (Dr M. Pawlita)
and Infections and Cell Biology Group (ICB) of IARC
(Dr M. Tommasino and Dr T. Gheit). Staff from
RGCBT has been trained in the above institutions
through technology transfer programs supported by
IARC. Analysis of plasma samples from girls with
different vaccine doses collected at baseline, 7, 12
and 18 months suggest that the immunogenicity
of 2-dose schedules over 180 days was non-inferior
to the 3-dose schedules at 7 months or 1 month
after the last dose and at 18 months from the first
dose, however immunogenicity following default
1-dose and 2-doses over 60 days were inferior to
that of 3-doses over 180 days. Analysis of cervical
cell samples and plasma samples collected at 24
and 36 months is progressing which will provide
valuable information immunogenicity over 3 years
and interim HPV infection frequencies following
different dose schedules
Biological significance of microRNAs in HPV associated cancers.
Janki Mohan, Smita Joshi*, Tarik Gheit**, Massimo Tomassimo** R.Sankaranarayanan**
and M.Radhakrishna Pillai
Collaborators: *Hirabai Cowasji Jahangir Medical Research Institute, Pune, India and
**International Agency for Research on Cancer, Lyon, France.
Human papillomavirus (HPV) is the most common
sexually transmitted infection (STI) worldwide, and
the principal etiological agent of cervical cancer,
the second most common cancer among women.
Over the last two decades or so, the associations
between high risk HPV with other forms of cancer
(oral and ano-genital malignancies), have also
been confirmed. Over 100 HPV types have been
identified, about 20 of which are termed highrisk types, because of their propensity to disrupt
control of normal cell-cycles and thereby accelerate
development of cervical malignancy. Human
Immunodeficiency virus (HIV), another STI with
extensive public health impact, shares many
common behavioral risk factors with HPV and the
infections interact in important ways. Female sex
workers (FSW) have a particularly high risk for both
HIV and HPV infection, and interactions between
these infections are especially pronounced in
this population group. Women with HIV infection
commonly have a broader range of HPV infection
spectrum, often with multiple concurrent HPV
infections. HIV disease also influences the natural
history of HPV by increasing the likelihood of
persistent infection and virulence, and hastening
the time-course of HPV disease. Thus, compared
with HIV-negative women, HIV-positive women
are more likely to progress to cervical cancer, have
a worse prognosis and a higher risk of recurrence.
MicroRNAs (miRNAs) are small, non-coding RNA
molecules, able to regulate expression of target
genes post-transcriptionally, are associated with
regulation of diverse physiological processes
including cell differentiation and cell division.
About one thousand seven hundred human
miRNA molecules have been identified so far. Each
79
miRNA is believed to regulate multiple genes, with
predictions that greater than one third of all human
genes may be regulated by miRNA molecules.
A number of miRNAs have been shown to be
intrinsically involved in cancer pathogenesis and
progression. There is sufficient evidence to show
that some of these “Oncomirs” possess a tumor
suppressive/anti-apoptotic role while others exhibit
potent pro-apoptotic/proliferation promoting roles
in the cell. Taking the above facts into consideration
and knowing the importance of miRNAs as key
regulators of gene expression, studying the miRNA
deregulations in these patient subsets is likely
to yield molecular insights into carcinogenesis.
This study will also address identifying the most
common HPV subtype prevalent in HIV positive
patients. Genotyping results from the cervical
samples of 600 HIV-patients revealed 44% of HPV
80
Fig 2: The above expression image shows compact
view of heirarchial cluster tree. Tree further classified
on the basis of miRNAs expression. Red color shows
over expressed miRNAs (>0) & Blue color shows underexpressed miRNAs (<0).
positivity. The most predominant sub-types found
were HPV-16, 31 and 18. The miRNA array analysis
identified 22 differentially expressed miRNAs that
most accurately discriminates between patients
infected with only HIV (Group-I) and infected with
both HIV and HPV (Group-II). Our results also
suggest that patients infected with both HIV and
HPV have deregulation of miRNAs involved in cell
cycle, angiogenesis, apoptosis, immune response,
HIV latency and various other important biological
processes.
RGCB Visiting Scientist
Tessy Thomas Maliekal
[email protected]
Tessy Thomas Maliekal has a Ph.D in Biotechnology from University of Kerala in 2010 and
did her post-doctoral training at Regional Cancer Centre, Thiruvanananthapuram and
then at National Centre for Biological Sciences, Bangalore. She joined RGCB as a Visiting
Scientist in 2009.
Project Fellows:
Madhumathy Nair G.,
Kochurani K.J.
81
TGF-b mediated regulation of self-renewal and chemoresistance in oral
carcinoma
In collaboration with Division of Surgical Oncology, Regional Cancer Centre,
Thiruvananthapuram
A major hurdle in the therapeutic outcome of
cancer is recurrence thought to be mediated by
cancer stem cells (CSCs) possessing self-renewal
ability and chemoresistance. Recent evidence
shows that the tumor niche formed by stromal cells,
tumor cells and CSCs play a role in the induction
of CSC properties. We evaluatedthe role of TGF-b
signaling in modulating self-renewal ability and
chemoresistance. Even though the classical TGF-b
mediators are Smad proteins, other mediators
like TIF1g are also implicated in TGF-b mediated
responses. We evaluated the role of Smad 4 and
TIF1g in oral carcinoma. The self-renewal ability
of CSCs can be marked by ALDHHi cells or the
expression of ALDH1A1. The cells sorted from HSC4 oral carcinoma cell line as ALDHHi and ALDHLo
showed that while ALDHHi cells have the ability to
self-renew and differentiate, the ALDHLo cells die
off gradually (Fig 1 & 2).
Figure 1. a) Morphological characteristics of sorted cells after 6 days as shown by DIC images. b) the trypan blue
exclusion results after 9 days of serial sorting
Our initial analysis showed that ABCG2 expression
can be used as a marker for chemoresistance.
Majority of oral cancer samples analyzed showed
expression of ALDH1A1 as well as ABCG2 in
subsets of tumor cells as shown in Fig 3.
82
The samples were also analyzed for the expression
of Smad4 and TIF1g. Majority of the samples
showed Smad4 loss, and if present was limited to
cytoplasm, where as TIF1³ expression was more
or less uniform in most samples and a nuclear
component was evident (Fig 4).
Figure 2. ALDH Hi cells have the ability to self-renew and differentiate to keep the percentage of ALDH Hi cells
constant, while ALDH Lo cells loose the ALDH Hi cells gradually.
Figure 3. OSCC section was probed for
ALDH1A1 and ABCG2. Hoechst was added to
stain the nuclei.
83
Fig 4. Immunohistochemical analysis of Smad4 or TIF1³ on OSCC sections.
PUBLICATIONS
»» Vinod, B. S, Maliekal, T. T, and Anto R. J.
(2013). Phytochemicals as chemosensitizers: from
molecular mechanism to clinical significance.
Antioxid Redox Signal 18, 1307-1348.
84
CARDIOVASCULAR DISEASE BIOLOGY
Professor C Chandrasekharan Kartha, MD
Professor of Eminence
[email protected]
Chandrashekaran Kartha is a MD in Pathology from All India Institute of Medical Sciences,
New Delhi. He worked as Senior Grade Professor & Head, Division of Cellular & Molecular
Cardiology, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Trivandrum
before joining RGCB in January 2009.
Program Scientists:
Sumi S, PhD
Surya Ramachandran, PhD
PhD students:
Ajith Kumar G S, MVSc
Ann Mary Johnson, MSc
Binil Raj SS, MPharm
Shammy S, MSc
Project Fellows:
Kshemada Kappagantu
MPharm (SRF)
Vinitha A, MSc (SRF)
Project Assistant:
Athira G, MSc
Animal Handler:
Aswathy
SriHari
85
Microvascular endothelial cell remodeling in pressure overload
cardiac hypertrophy
GS Ajithkumar, SS Binilraj, Santhosh kumar.S, Sanjay.G* and CC Kartha
*Dept. of Cardiology, SCTIMST, Thiruvananthapuram
During the evolution of heart failure, remodeling
of the heart occurs, which consists of adaptive
mechanisms to normalize the pump function.
But continuous remodeling will lead to severe
cardiac hypertrophy and contractile dysfunction.
Paracrine and autocrine interactions involving
cardiac endothelial cells and cardiac myocytes
could significantly modulate remodeling events in
a diseased heart. We aim to analyze the changes
that occur in the cardiac endothelial cells during
evolution of pressure overload cardiac hypertrophy
and cardiac failure.
Animal experiments were done after obtaining
clearance from institutional animal ethics
committee. Experiments were conducted in
pressure overload heart failure model in male
Wistar rats. Surgical procedure for creating pressure
overload heart failure model by ascending aortic
constriction was standardized. Cardiac hypertrophy
was confirmed grossly and at the molecular level.
A total of 90 Wistar rats underwent surgical
procedures in two categories- ascending aortic
banding and sham operation. These experimental
animals were divided in to six groups depending
upon the sacrifice period post surgery (1 month to
6 months). Out of the six groups, aortic constricted
and sham operated rats of first and second month
groups post surgery were sacrificed. Heart weight/
body weight ratio and ventricular weight/ body
weight ratio were significantly higher in rats
with aortic constriction (Fig.1). Before sacrifice,
echocardiographic and blood pressure parameters
were recorded. A significant increase in inter
ventricular septal thickness and left ventricular
posterior wall thickness were observed in rats
which had aortic constriction. There was also a
decrease in left ventricular lumen size in rats with
aortic constriction compared to the left ventricle of
control rats in echocardiography (Fig.2& Table.1).
Figure 1: Heart weight: Body weight ratio and LV weight: body weight ratios in rats with aortic constriction and
control rats sacrificed after 1 month and 2 months post surgery.
Figure 2: Representative pictures of trans-thoracic M- mode echocardiographic parameters of functional status of
heart at the time of sacrifice, after 1 month and 2 months post surgery. Fig.2A & Fig.2B are ECHO of the heart in
rats with aortic constriction and sham operated heart of rats of 1st month group. Fig.2C & Fig.2D are ECHO of the
heart in rats, 2 months post surgery.
86
Blood and tissue samples have been collected from
sacrificed animals and microvascular endothelial
cells from the left ventricle have been isolated. An
increase in the intensity of cardiac hypertrophy
from first month to second month group has been
observed in rats which underwent constriction
of aorta. Quantitative real time PCR analysis
in microvascular endothelial cells revealed a
significant increase in the level of expressions of
eNOS and endothelin (Fig.3).
Figure 3: Graphical representation of mRNA level of eNOS & ET by quantitative real time PCR in endothelial cells
isolated from rats sacrificed 2 months after constriction of aorta and sham operation.
Table.1: Trans-thoracic M- mode echocardiographic parameters of functional status of heart of experimental rats at
the time of sacrifice, 1 month and 2 months post surgery. EF= ejection fraction; FS= fractional shortening; PG=
pressure gradient; IVST= inter ventricular septal thickness; LVPWT= left ventricular posterior wall thickness;
LVID= left ventricular internal diameter at diastolic state; AAC= ascending aortic constriction.
Expression of ELAV-like protein, HuR is increased in lungs of rats with
left ventricular hypertrophy and associated
pulmonary vascular remodeling
Binil Raj SS, Ajithkumar GS, Santhoshkumar S, CC Kartha.
Pulmonary hypertension associated with left heart
disease (PH-LHD) represents the most common
form of PH and is characterized by lung endothelial
dysfunction and vascular remodeling. 68 to 76 %
of patients with LHD have PH which serves as an
independent predictor of morbidity and mortality
in heart failure. Left ventricular or valvular disease
results in passive backward transmission of
elevated left atrial pressure and partial obstruction
to pulmonary venous drainage. This hemodynamic
disturbance in circulation causes increased shear
stress and turbulent flow in pulmonary circulation.
Vascular endothelium senses this hemodynamic
stress acting on luminal surface by various
87
mechanosensors. It initiates signals that adapts
endothelium to its new environment by functional
and structural changes in pulmonary vasculature.
In LHD induced pulmonary vascular remodeling
there appears to be hemodynamic stress induced
endothelial dysfunction leading to dysregulation
of vasoactive mediators and growth factors. The
mechanisms that lead to endothelial dysfunction
during pulmonary vascular remodeling and
associated PH are however unclear. Strong rationale
exists for the study of mechanosensitive genes in
pulmonary endothelial cells and how these factors
affect endothelial function. We analyzed the level of
expression of a shear sensitive factor HuR, an ELAVlike protein and its regulator molecules such as
Kruppel like factor 2 (Klf2), endothelial nitric oxide
synthase (eNOS) and bone morphogenic protein 4
(BMP4) in lung tissues of rats with left ventricular
hypertrophy. Left ventricular hypertrophy was
induced in male Wistar rats by banding ascending
aorta. Five months after aortic banding, significant
increase in heart weight to body weight ratio
and left ventricle + septum weight to heart
weight ratios were observed in rats indicating left
ventricular hypertrophy. In ventricular tissues of
aorta banded rats, five months after surgery there
was a shift from MYH6 (Myosin heavy chain 6) to
MYH7. Aortic banding also produced an increase
in right ventricular weight to body weight ratio
demonstrating right ventricular hypertrophy.
Compared to sham operated controls, increase in
pulmonary arterial wall thickness was observed in
rats which underwent aortic banding, representing
pulmonary vascular remodeling (Fig.1). The aortic
banding led to significant increase in HuR mRNA
levels in the lung tissues. We also analyzed the HuR
regulating anti-inflammatory and antiproliferative
genes Klf2 and eNOS as well as proliferative
gene, BMP4 in lung tissue. We observed that Klf2
was down regulated with no significant change
in the expression of eNOS. BMP4 mRNA levels
were found to be increased. Our study reveals
that LHD associated hemodynamic stress in
pulmonary circulation induces the activation of
HuR and results in downstream dysregulation of
various endothelial mediators. Down regulation of
Klf2 and upregulation of BMP4 could change the
endothelium to an inflammatory and proliferatory
phenotype during pulmonary vascular remodeling.
Our results indicate a critical role for HuR in
pulmonary vascular remodeling, secondary to left
ventricular hypertrophy.
Figure 1. Photomicrograph of lung of rats with left ventricular hypertrophy associated with pulmonary vascular
remodeling (Verhoeff-van Gieson staining). A- Sham operated controls B- Rats with aortic banding. An increase in
pulmonary arterial wall thickness is seen in rats which underwent aortic banding.
88
IGF-1 promotes cardiac stem cell proliferation through AKT-1/
FOXO3A signaling pathway
Ann Mary Johnson and C C Kartha
IGF-1 is a small peptide that belongs to Insulin like
growth factors which are structurally homologous to
proinsulin. IGF-1 is expressed in many tissues and
has autocrine, paracrine and endocrine functions.
Anversa etal., identified a cycling population of c
kit+ resident cardiac stem cells (CSCs) expressing
IGF receptor-1 in human hearts implying the
necessity of growth factor mediated signaling in
the cardiac stem cell niche. The proliferation rate
of cardiac stem cells in vivo and in culture depends
on the availability of growth stimulants such as
growth factors, cytokines, etc. Growth factors
such as EGF, bFGF, HGF and IGF-1 are known
to accelerate the proliferation of cardiac stem
cells in culture. Among these factors, IGF-1 has
proliferative effects in CSCs. But the mechanism
through which IGF-1 promotes proliferation in
CSCs remains to be elucidated. Binding of IGF-1 to
its receptor IGFR-1 leads to the activation of PI3/
Akt-1 pathway and regulates cell proliferation,
differentiation, and apoptosis. A few related studies
indicate Akt-1 phosphorylates and inhibits the
winged-helix family of transcription factors, such
as FOXO3a, which is a key negative regulator of cell
cycle progression. FoxO proteins are important in
cardiac biology. Among them FoxO1, FoxO3a and
FoxO4 are vital factors in mediating stress response
Figure: 1Population doubling time: Control- CSCs
maintained in basal medium without IGF-1 and CSCs
treated with IGF-1 (100ng/ml) in basal medium. *p <0.05
vs without IGF-1 (n=3).
and maintains cardiac function during pathological
condition. One way in which Akt-1 promotes cell
survival and proliferation is by phosphorylating
FOXO3a, a major substrate of Akt-1 resulting in
cytoplasmic sequestration of FoxO3a. We speculate
that the survival and growth of cardiac stem cells
in heart greatly depends on the intrinsic level of
active FoxO3a and its target proteins.
In our previous study, we observed nuclear
localization of active FoxO3a in quiescent cardiac
resident c kitPOS stem cells from murine heart. On
treatment with IGF-1, an increased proliferation
of cardiac stem cells and sequestration of FoxO3a
in the cytoplasm of these cells was observed
indicating their inactivation and nuclear export
of FoxO3a (Fig 1). Further, we found FoxO3a as
one of the targets of IGF-1 signaling. IGF-1 posttranslationally modifies FoxO3a by phosphorylation
at Ser-253 through Akt1 and inhibits FoxO3a
nuclear translocation. Thus, it is possible that
IGF-1 mediates cardiac stem cell proliferation by
inhibiting FoxO3a and their target proteins which
are involved in quiescence maintenance (Fig 2).
Understanding this mechanism may give an insight
into an essential role of FOXO3a in the regulation
of CSC proliferation.
Figure: 2Western blots of pFoxO3aS-253 and pAktTh308 in CSCs: Increased phosphorylation of FOXO3a
(inactivation) and Akt-1 (activation) upon IGF-1
stimulation for 6h. For blocking pAkt-1 function the CSCs
were pretreated with LY294002 (LY) for 30 min prior to
IGF-1 stimulation. NC- Cardiac stem cells cultured in
CSC medium containing 10%serum.
89
Expression analysis of cardiac developmental genes in the
myocardium of patients with tetralogy of Fallot
S Shammy, R Suresh Kumar* and C C Kartha
*Collaborator Dr.K.M.Cherian Heart Foundation, Kerala.
Tetralogy of Fallot (ToF) is a complex cyanotic
congenital heart defect characterized by a
constellation of four major anatomical defects:
pulmonary stenosis, perimembranous ventricular
septal defect, biventricular connection (over ridding)
of aorta and right ventricular hypertrophy. These
malformations occur presumably due to unequal
division of the conotruncus or erroneous alignment
of the ascending aorta during embryogenesis. The
exact cause of the disease still remains unknown.
The advances in developmental biology and
molecular genetics have led to studies which
suggest that complex interplay of numerous
genetic and environmental factors (multifactorial),
during intrauterine development contribute to ToF
The mature heart consists of different cell types,
including myocardial cells, endothelial cells,
fibroblasts, smooth-muscle cells and specialized
conducting cells. These cell lineages have their
origin from the different layers of mesoderm,
which give rise to four types of cardiac progenitor
cells such as primary or first heart field (FHF),
secondary heart field (SHF),neural crest cells (NC)
and proepicardium with distinct characteristics
and fate. These precursor cells attain their target
fate with the aid of various signaling molecules
and their pathways, which are regulated by
defined transcription factors. These signaling
pathways are expected to operate in a highly
orchestrated manner to control various processes
such as migration, proliferation and differentiation
of cardiac precursors. Since in tetralogy of Fallot
the defects are localized to the outflow tract of
the right ventricle and its septation, secondary
heart field’s cells and their regulation are most
likely to play a critical role in the development of the
defect. We analyzed the spatial expression pattern of
transcription factors such as ISLET1, HAND2,GATA4,
MEF2C and ligand molecule JAG1which operates in
the regulatory network of SHF.
For gene expression analysis, 50-100mg tissue
samples were collected from right ventricular
outflow tract (RVOT) of 120 patients with ToF
(age 2-15 years), who underwent surgical repair
at Madras Medical Mission hospital, Chennai.
Institute ethical committee clearance was obtained
for the study and informed consent was collected
from all the patients. RNA was isolated from the
tissue resected from RVOT of 10 patients and 3
control samples. The expression of candidate genes
was detected by semiquantitative RT-PCR both
in tissues from outflow region of right ventricle of
normal and in cardiac biopsies of patients with ToF.
Gene expression values were normalized against
the tissue levels of the housekeeping gene GAPDH
We observed a 4 fold increase in the expression
of ISL1 in tissue samples of patients with ToF
when compared to control samples. There was a
3-6 fold decreased expression of HAND2, GATA4,
MEF2C and JAG1in the disease samples. Our study
reveals over expression of progenitor marker ISL1
in RVOT tissues from patients with ToF , which
indicates the presence of progenitor cells in RVOT.
Down regulation of transcription factors such as
HAND2, GATA4, MEF2C , which are required for
the maintanence of homeostasis and functioning of
myocardium may also be responsible for the right
ventricular dysfunction in ToF.
Figure 1: Expression of cardiac developmental genes in right ventricular outflow myocardium patients with Tetralogy
of Fallot
90
Can Amalaki Rasayana Attenuate Cardiac Dysfunction Associated
With Cardiac Failure And Aging?
Kshemada and C C Kartha
Amalaki Rasayana is an ayurvedic preparation
which belongs to the rejuvenator class of drugs.
This study aims to evaluate the effect of this
Rasayana on aging associated cardiac dysfunction
and reversal of cardiac remodeling during cardiac
failure in male Wistar rats. The effect of Amalaki
Rasayana on various molecular mechanisms and
pathways involved in cardiac failure will also be
studied.
Approval was granted from the animal ethics
committee of the institute to perform animal
experiments. Accordingly, 40 animals were
collected at the age of one month and assigned
cages. These animals were then classified into
groups each having 10 animals. Groups A, B, C
are the test groups and group D is the control
group. The animals were fed with diet and water
and regularly monitored. Amalaki Rasayana
prepared by Vaidyaratnam P. S. Varier’s Arya
Vaidya Sala, Kottakkal, Kerala was collected
and stored. At the age of 3 months the animals
were labelled with picric acid. Body weights,
baseline echocardiographic parameters and
ECG were measured. After measuring all the
above parameters the animals are being fed with
calculated doses of Amalaki Rasayana. Group A
receives 250 mg/kg BW; group B receives 500 mg/
kg BW; group C receives 750 mg/kg BW and group
D receives vehicle only.
Molecular Pathogenesis of Varicose Veins
S. Sumi, G. Athira, N. Radhakrishnan*, B.L. RaviKumar**, S.R. Kalpana***, G
Kamalapur***, C.C. Kartha
Collaborators:* St.Thomas Hospital, Changanacherry, Kerala
**Kempegowda Institute of Medical Sciences, Bangalore
***Sri Jayadeva Institute for Cardiovascular Sciences & Research, Bangalore
Structural failures of vein such as valve weakness,
vein wall dilatation, or deep vein damage result in
venous retrograde flow in limb, leading to sustained
periods of distal high venous pressure causing
varicose veins. The genetic basis and molecular
mechanisms underlying the pathogenesis and
progression of the disease remain uncertain.
Results from studies in varicose-associated
diseases and knock-out animal models suggest
that mutations in FoxC2 gene have an important
role in the disease pathogenesis.
We initially sequenced the 1.5 kb single coding
exon of FoxC2 gene from DNA isolated from whole
blood samples of 382 patients with varicose veins
and 372 control subjects. Sequencing revealed the
presence of only two variations (C354T and G426A
with low frequency of 0.02 of 0.04 respectively) and
hence these variations were not followed up for
further studies.
We extended the analysis to approximately 3Kb
region of 5’ untranslated region (UTR) of FoxC2
gene as well as the 3’UTR region of gene too.
Seven FoxC2 polymorphisms were observed in
patients with varicose veins and normal subjects.
Two known variants [C-512T (rs34221221) and
A-1538G (rs4843162)] and two novel variants
A-2647T and G1632A were found to be significantly
associated with patients. C-512T polymorphism
is possible pathogenically important since it is
present in the immediate early promoter of the
FOXC2 gene which is a highly conserved region
of the gene. C-512T polymorphism may alter
transcription factor binding and subsequent gene
expression. Real time PCR analysis confirmed the
91
elevated expression of FoxC2 in vein specimens
of patients compared to control vein specimens.
Immunoblot analysis confirmed that the overexpression of FoxC2 transcript was associated with
increased FoxC2 protein level in varicose veins. We
investigated whether there was any correlation
between C-512T and FOXC2 expression at both
mRNA and protein levels in venous tissues from 22
patients with varicose veins and normal saphenous
vein tissues from 20 normal subjects. Patients with
TT genotype had significantly higher expression
of FoxC2 mRNA compared to heterozygous CT
genotype (p<0.05), while the difference was not
significant at the protein level. Our study indicates
that transcriptional modulation of FoxC2 expression
by regulatory polymorphisms has an impact on the
development of varicose veins.
Varicose veins histology indicated smooth muscle
cells (SMC) proliferation with loss of intimal
medial distinction. Immunohistochemical staining
revealed an increased expression of FoxC2 protein
in varicose vein tissues (Figure 1). FoxC2 is a key
inducer of epithelial mesenchymal transition
(EMT). It has been reported that common molecular
pathways are seen in EMT and endothelial
mesenchymal transition (EndMT). FoxC2 may
well be implicated in EndMT. Wee propose that
over-expression of FoxC2 in vein wall may leads
to EndMT and may contribute to venous wall
remodeling by affecting SMC proliferation and
vein wall architectural pattern. Mechanism of
FoxC2 based EndMT induction is to be further
investigated.
Figure 1: Immunohistochemical staining with FoxC2 antibody in venous tissue sections from patients with varicose
veins and controls (Anti FoxC2 Ab 1:100): (A) Histological analysis of normal saphenous vein (B) varicose vein tissue
histology indicated a loss of intimal medial distinction and increased SMC proliferation. (C) Isotype control in normal
saphenous veins and (D) varicose vein tissues. (E) FoxC2 expression is localized to tunica media in normal venous
tissues. (F) FoxC2 is uniformly expressed in intima and media of varicose vein tissues.
92
Plasma Cyclophilin A as a Marker of Inflammation in Type 2 Diabetes
Surya Ramachandran, Vinitha A, Anila Venugopal, Divya G, V Chitrasree*, N S Pratap
Chandran**, Ajit Mullassari*, K R Santosh***, Ramankutty V****, M Radhakrishna Pillai
and C C Kartha
Collaborators *Madras Medical Mission, Chennai, India ,**Indian Institute of Diabetes,
Thiruvananthapuram, India, ***PRS Hospital, Thiruvananthapuram, India,****Achutha
Menon Centre, SCTIMST, Thiruvananthapuram, India
Hyperglycemia is involved in the development
of cardiovascular complications associated with
diabetes. Key pathways, factors, and mechanisms
implicated in this process include oxidative
stress, advanced glycation end products, aldose
reductase, reductive stress, carbonyl stress and
protein kinase C. Adhesion of monocytes to the
endothelium followed by transmigration into the
subendothelial space is one of the key early events
in the pathogenesis of atherosclerosis. Various
studies have demonstrated increased leukocyte–
endothelial interactions in animal models of
diabetes and with monocytes from diabetic
individuals. High glucose (HG) treatment of THP1 monocytes induced changes in the expression
levels of multiple cytokines, chemokines, and
related molecules. In our earlier study, we reported
for the first time the proteome changes in human
monocytes primed with high glucose. Using
2-dimensional gel electrophoresis experiments
such as, MALDI TOF MS analysis together with
bioinformatics tools we screened for secreted
monocyte proteins under hyperglycemic conditions.
We identified Cyclophilin A, an immunophilin as
a secretory protein from high glucose activated
monocytes.
In order to assess the potential of Cyclophilin as
a screening marker of inflammation in diabetes
mellitus, we conducted a study in 556 study
subjects consisting of 101 patients with diabetes
mellitus (DM), 122 patients with coronary artery
disease (CAD) and 121 normal healthy volunteers.
We also included 103 patients with diabetes
diagnosed with CAD (DM+CAD5y) within 5 years
and 109 patients diagnosed with CAD after 10
years (DM+CAD10y). Diabetes was assessed
by recording HbA1c levels and coronary artery
disease was diagnosed by a positive treadmill test.
Plasma samples were collected using EDTA and
centrifuged for 10 min at 2,500 g within 30 min
of collection, and Cyclophilin A was measured
immediately by using a Cyclophilin A ELISA Kit.
The study subjects comprised of 68% males and
32% females. Median age of the study group was 57
years. Median HbA1c values of the study subjects
was 6.8 (range=4.2-15.5). Triglycerides levels were
higher in patients with CAD (median value=126.5
mg/dL) and DM+CAD (median value= 137 mg/
dL) in comparison to DM (median value= 91 mg/
dL) and normal controls (median value=99 mg/
dL). HbA1c levels were higher in patients with
93
DM (median value=8.5) than in normal controls
(median value=5.3). We observed increased
Cyclophilin A levels in patients with diabetes
(median values=16.5 ng/mL, range= 10.0-32.9)
and patients with DM+CAD (median= 19.39 ng/
mL, range=10.0-56.4) when compared to normal
controls (median= 13.3 ng/mL, range= 5.1-19.9).
Cyclophilin levels were observed to be highest in
patients with CAD (median=20.0, range= 9.0-
57.2) than in other study subjects (Figure 1). Our
preliminary analyses indicate increased levels of
secretion of cyclophilin A in the plasma of patients
with both diabetes and coronary artery disease as
compared to patients with CAD alone and normal
controls. Detailed multivariate logistic regression
analysis is being carried out to estimate the
association between plasma cyclophilin levels and
vascular disease in patients with diabetes.
Research Grants Extra –Mural Funding
Sl.No.
Name of Grant
Duration
1
A proteomic analysis of circulating cells for
the discovery of biomarkers of increased risk
of atherosclerosis in patients with type 2
diabetes mellitus
Indian Council for Medical
Research
2010-2013
2
Remodeling of cardiac endothelium in
progressive heart failure
Department of
Biotechnology
2011-2014
3
Molecular mechanisms of pulmonary
microvascular endothelial dysfunction under
fluid shear stress
Glaxo Smithkline
Foundation, UK
2011-2014
4
Can Amalaki rasayana attenuate cardiac
dysfunction associated with cardiac failure
and aging
Technology
AWARDS AND HONOURS
»» Surya Ramachandran was awarded a three month
Visiting Scientist fellowship from Oklahoma
Medical Research foundation, Oklahoma, USA
(May 2012-August 2012)
PUBLICATIONS
»» Original Publications in Journals
»» Padmaja Parameswaran Nampi, Cheranellore
Chandrasekharan Kartha, Gin Jose, Anil Kumar
Pallikkaveedu Rajan, Anilkumar Thapasimuthu,
Harikrishna Varma. Sol-Gel Nanoporous Silica
As Substrate For Immobilization Of Conjugated
Biomolecules For Application As Fluorescence
Resonance Energy Transfer (FRET) Based
Biosensor. Sensors & Actuators: B. Chemical
(In Press)
»» Ramachandran S, Venugopal A, K S, G R,
Charles S, G D, Chandran NS, Mullassari A,
Pillai MR, Kartha CC. Proteomic profiling
94
Funding Agency
2012-2015
of high glucose primed monocytes identifies
cyclophilin A as apotential secretory marker of
inflammation in type 2 diabetes. Proteomics.
2012; 12(18):2808-21
»» Ramachandran S, Kartha CC. Cyclophilin-A: a
potential screening marker for vascular disease in
type-2 diabetes. Can J Physiol Pharmacol. 2012
Aug;90(8):1005-15
CONFERENCE PRESENTATIONS
»» Raj SSB, Ajithkumar GS, Santhoshkumar S,
Kartha CC. Expression of ELAV-like protein,
HuR is increased in lungs of rats with left
ventricular hypertrophy associated pulmonary
vascular remodelling. 6th PVRI Workshops
& Debates, Istanbul, Turkey; 21-25th January,
2013.
»» Kartha CC. Aesthetics of Biology and Biology
of Aesthetics. Ramalingaswamy Conclave 2013,
Raviz Kollam, 19-22 January, 2013
DIABETES RESEARCH PROGRAM
Rajiv Gandhi Centre for Biotechnology
Diabetes, a chronic inflammatory disease is not
only among the most common and costly health
problems, they are also among the most silent.
It is fast becoming a global epidemic causing
steadily escalating mortality rate, thus demanding
immediate attention. There is a two to four fold
increase in risk of stroke and atherosclerosis in
patients with diabetes. Diabetes is the leading
cause of renal failure in many populations in
both developed and developing countries. It is
also one of the main causes of visual impairment
and blindness. Early detection of diabetes and its
associated complications could reduce mortality
rate by timely intervention. A focus shift from the
currently available diagnostic clinical markers
towards the development of novel screening
markers is imperative for the early identification
of such diseases. Recent advancements in high
throughput technologies such as proteomics,
metabolomics and genome-wide association
study (GWAS) can help to identify novel markers
of disease delineate the mechanism of disease
progression and understand the underlying
pathophysiology of its vascular complications.
Understanding the magnitude of this health
problem, a Diabetes Research Program was initiated
in July 2012 by Professor M Radhakrishna Pillai,
Director, Rajiv Gandhi Centre for Biotechnology
under the leadership of Professor C C Kartha
as a flagship program of the Cardiovascular
Disease Biology Laboratory. The diabetes research
team brings together the expertise and skills of
scientists, clinicians and epidemiologists into
a comprehensive and complementary diabetes
research effort.
In the past one year, we have identified key
research questions and assigned personnel to
navigate various research themes. We have also
established multi site research collaborations with
physicians, molecular epidemiologists, national
and international research institutes to explore
our areas of interests. Outlined below is a brief
description of plans and programs for the Diabetes
Research Program.
Metabolic changes associated with the risk of
developing type2 diabetes.
Abdul Jaleel K. A PhD
Scientist E II
Type 2 diabetes mellitus is not only a disorder
of metabolic dysregulation but also is the most
commonly diagnosed metabolic disease. Metabolic
diseases are often present for years before
becoming clinically apparent. Given that effective
interventions are nowadays available for delaying
or preventing the onset of type 2 diabetes, and
the escalating burden of the condition in India,
earlier identification of individuals at risk has
became an increasing necessity. Emerging
technologies have augmented the feasibility of
acquiring high-throughput profiles of a whole
organism’s metabolic status (metabolite profiling,
or metabolomics). These advancements allowed
assessment of large numbers of metabolites
that are substrates, products and by-products
of biochemical pathways of body metabolism
and are particularly appropriate for studying
metabolic diseases such as diabetes. Moreover,
in addition to serving as potential biomarkers of
disease, metabolites may have unexpected roles
as regulatory signals with hormone-like functions
or effectors of the pathogenesis of disease itself.
Some cross-sectional studies, which used
metabolomics tools, recently have documented
95
differences in blood metabolite profiles of various
small molecule metabolites before and after
glucose loading and in obese compared with lean
individuals. These studies bring up the possibility
that alterations in plasma metabolite levels could
predict the onset of diabetes and therefore aid in
the identification of ‘at risk’ individuals by adding
information over standard risk factors. We launched
a project “Metabolic Profiling of Normal Healthy
people in Kerala”by recruiting normal healthy
people from a village of Alleppey district in Kerala
with the aim of profiling the blood metabolites
using mass spectrometry based metabolomics
tools. The ultimate objective of the project is to
delineate the distinct metabolic pathways specific
for the specific “at risk” group of individuals such
as those having higher body mass index, people
who have family history of diabetes, and lifestyle
changes such as unhealthy diet, lack of physical
activity etc. The recruitment of participants, human
studies and various laboratory tests are underway.
The preliminary data on the clinical and various
biochemical and hormonal measurements so far
obtained are discussed in a separate section of this
annual report.
Metabolic characterization of Maturity Onset Diabetes of
Young (MODY) in India
Early onset type 2 diabetes is an aggressive
phenotype and is a prevalent form of type 2
diabetes in India. Both MODY and classical type 2
diabetes are included in the early onset categories
of type 2 diabetes. MODY is an autosomal dominant
diabetes mellitus comprises a heterogeneous group
of monogenic disorders characterised by β cell
dysfunction. Mutations in at least nine different
genes that result in the MODY phenotype have
been reported. However, the most frequent causes
of MODY are mutations in genes encoding the
transcription factors hepatocyte nuclear factor 1 a
(HNF1A, MODY3) and hepatocyte nuclear factor
4 a (HNF4A, MODY1) and the glucokinase (GCK,
MODY2) enzyme. Although various MODY gene
mutations have distinct phenotypes , differentiating
these from each other and from common forms of
diabetes can be challenging in clinical practice. It
is important to distinguish not only various MODY
types from each other but also MODY from type 2
diabetes in the young because it allows optimal
treatments and prediction of future course of the
illness.
Advances in genetic knowledge alone may not
provide enough information to direct diabetes
management until there is a complete understanding
of disease mechanism. Though gene mapping
identify genetic quantitative trait loci influencing
disease-related phenotypes, characterization
of phenotype is essential to uncover molecular
pathways leading from genotype to clinical trait.
Body metabolite levels are particularly relevant for
type 2 diabetes, characterized by altered energy
96
metabolism and often preceded by a metabolic
syndrome. Metabolites therefore serve as important
predictive intermediates that link gene expression
and clinical measurements. We, therefore,
believe that these metabolic alterations could be
identified by performing mass spectrometry based
metabolomics analysis in the blood of patients of
common MODY types and type 2 diabetes. Given
the different genetic aetiologies and distinct
metabolic pathways affected, we hypothesize that
subjects with MODY 1, 2, & 3 could have a different
pattern of plasma metabolic profile between them
and also when compared to subjects with normal
healthy people and type 2 diabetes. To this effect, a
concept proposal titled “Metabolic characterization
of Maturity Onset Diabetes of Young (MODY) in
India” has been submitted to the Indian Council
for Medical Research with the following specific
aims; (1) to estimate the prevalence of MODY using
clinical criteria from a registry of 5000 diabetic
patients who were enrolled during the last 3 years
in the diabetes clinic of our clinical collaborator,
(2) to measure the prevalence of MODY 1, 2 & 3
mutant types among the MODY subjects by genetic
analysis, (3) to determine whether the routine
biochemical, endocrine, inflammatory markers
and plasma metabolites distinguish the different
genetic subtypes of MODY and (4) to determine
the basal and postprandial metabolic landscape
in people of different MODY types, early onset type
2 diabetes and normal healthy controls by mass
spectrometry based metabolomics.
Metagenomic analysis of gut microbiome from
Type 2 Diabetic subjects in Kerala
Harikrishnan K PhD
Scientist C
The human gastrointestinal tract harbors a complex
microbial ecosystem. The ability to aid the host
in extracting energy and nutrients from food is
considered an important benefit that has resulted
in a long history of co evolution between humans
and gut microbes. Recent evidence shows that the
gut microbiota plays a crucial role in modifying the
metabolic phenotype and predisposition to diseases
in the host. It’s been known for some time that
type 2 diabetes can be caused by a combination
of genetic and lifestyle factors. Gut microbiota
was recently proposed as an environmental factor
responsible for the control of body weight and
energy metabolism, which is closely linked to
obesity and metabolic disorders such as type 2
diabetes. But the nature of this interaction and the
microbiome’s role in determining susceptibility to
diabetes hasn’t been properly explored.
Assessment and characterization of gut microbiota
has become a major research area in human disease,
including type 2 diabetes, the most prevalent
endocrine disease worldwide. Studies based on
large-scale 16S rRNA gene sequencing and other
molecular biology techniques have shown a strong
relationship between composition of the intestinal
microbiota and metabolic diseases like obesity and
diabetes. However, the molecular mechanisms of
interaction between microbiome variation and
human physiology have not been adequately
described, nor have the key microbiota contributors
been identified yet. Characterization of the gut
microbiota has been hindered by the refractory
cultivation of most species. Metagenomic analysis
of microorganisms by direct extraction and cloning
of DNA from an assemblage of microorganisms and
16S rRNA sequencing, provides insight into the
genetic potential and diversity of complex microbial
communities, including uncultured species.
Studies on gut microbiota of Indian population
in general and particularly that from type 2
diabetic population are inadequate. The incidence
of diabetes in Indians, particularly in Kerala
population is exhibiting an increasing trend.
A comprehensive insight on the human gut
microbiota, in terms of phylogenetic composition
as well as genetic and metabolic potential, is
essential to understand the dynamics and possible
mechanisms involved between gut microbiota and
pathogenesis of disease. Studies in this direction is
totally lacking in Kerala. Therefore, it is worthwhile
to look into the role of gut microbiota as an etiology
in high incidence of diabetes apart from the lifestyle
and genetic predisposition. The present pioneering
attempt to analyse the gut microbiome of type
2 diabetic subjects in Kerala through cultureindependent metagenomic approach may help in
generating insights into the gut microbial diversity
and the baseline data generated may also help in
monitoring individuals at risk of such disorders.
Monocyte proteins as markers of vascular disease in type 2 diabetes
Surya Ramachandran PhD
Program Scientist
Chronic inflammatory diseases such as diabetes
and atherosclerosis can be considered as long
term effects of innate immunity in the context
of individual disease susceptibility. Turning
down innate surveillance may delay or prevent
inflammatory diseases. There is however, a need
to study cellular and molecular mechanisms of this
surveillance in detail in order to understand the
importance of immune response and inflammation
in the development and progression of chronic
diseases. The research strategy is to contribute to
the understanding of early vascular disease and
associated co-morbidities of diabetes by focusing
on two keys of equal importance namely, early
detection of vascular disease in diabetes mellitus
through development of screening markers and
97
development of diagnostic markers to predict
progression of vascular lesions in diabetes mellitus
in order to build up effective interventions.
In an earlier study, we examined for the first
time the proteome changes in human monocytes
primed with high glucose. Using 2-dimensional
gel electrophoresis experiments such as, MALDI
TOF MS analysis together with bioinformatics
tools we screened for secreted monocyte proteins
under hyperglycemic conditions. We detected
five proteins that have secretory function either
by classical/nonclassical or exosomal pathway.
Of these five proteins, cyclophilin A was found
in plasma of patients with type 2 diabetes and
coronary artery disease in higher levels than in
healthy volunteers. Our demonstration that high
glucose activates monocytes to secrete cyclophilin
A suggests its role as a potential marker of early
inflammatory disease in type 2 diabetes. In order to
use this secreted protein as a clinically viable and
specific marker of early vascular disease in type 2
diabetes, we need to understand factors affecting
its secretion levels and its mechanism of secretion.
Our preliminary observations (during a three
month study with Dr Michael Kinter, Free Radical
Biology & Aging Program at Oklahoma Medical
Research foundation, Oklahoma, USA) using
Selected Reaction Monitoring (SRM) carried
out in a LC coupled tandem Mass spectrometer
indicated that cyclophilin is an oxidative stress
induced secretory factor in hyperglycemia. The
SRM assay uses a highly multiplexed method for
quantitatively estimating expression of prominent
proteins participating in the oxidative stress related
pathway. A peptide library integrating 28 key
proteins in the oxidative stress pathway has already
been created and established. Peptide sequences
of key players in oxidative stress pathways such as
catalases, peroxidases, peroxiredoxins, thioredoxin
and heat shock proteins have been identified and
described. We have also studied the expression
pattern of cyclophilin A protein in the presence of
Nrf2, the master regulator of oxidative stress. Our
results reveal an association between cyclophilin
expression and Nrf2. When the transcription factor,
Nrf2 is knocked down it leads to increased catalase
production, indicating increased ROS production
under high glucose conditions. Comparative
studies on the quantitative expression patterns
of Cyclophilin and other oxidative stress proteins
under hyperglycemic conditions is required to
reveal whether Cyclophilin is a key player of
the oxidative stress pathway. To this effect, a
project proposal titled “How does Cyclophilin A,
an Oxidative Stress Induced Secretory Protein
Modulate Vascular Disease Progression in Type
2 Diabetes”? has been submitted to the Indian
Council for Medical Research. The primary aim
of this project is to explore at the molecular and
genetic level, the mechanism of glucose induced
secretion of cyclophilin A from monocytes, its
effects on proteins in the oxidative stress pathway
and the role of genetic variations in the cyclophilin
gene in secretion of cyclophilin from monocytes.
The secondary aim will be to elucidate the role
of secreted cyclophilin A in the development of
macro vascular disease in an animal model of type
2 diabetes.
Gut microbiota induced epigenetic alterations causal for
type 2 diabetes mellitus
Sumi S PhD
Program Scientist
Genetic susceptibility to diabetes is generally
studied on the basis of gene variations. Recent
studies focus on the involvement of epigenetic
mechanisms as an interface between the effects of
genetic predisposition and environmental factors
in diabetes mellitus. Gut microbiota has been
implicated as an epigenetic factor contributing
to chronic diseases such as obesity and diabetes.
These indigenous microbiota produce multiple
98
low-molecular weight (LMW) substances that can
interact with different targets in the cells, tissue,
organs and organism on the whole. The ability of
the gut microbiota to produce folate, cobalamin and
methylamines could affect host DNA methylation
patterns, while acetate and butyrate produced from
microbial fermentation could modify chromatin
structure and gene transcription via histone
acetylation. Gut microbiota imbalance may produce
different epigenetic abnormalities which may play
a role in the pathogenesis of diabetes mellitus. We
hypothesize that gut microbiota in patients with
diabetes mellitus produce an altered LMW profile.
These LMW molecules may cause abnormal DNA
methylations and histone modifications in genetic
pathways of type 2 diabetes mellitus.
Research Collaborations
Dr K Sreekumaran Nair MD, PhD. Professor of Medicine, Mayo Clinic, Minnesota, USA
Dr Michael T Kinter PhD. Associate Member, Oklahoma Medical Research Foundation,
Oklahoma City, USA
Dr Ramankutty V MD. Professor, Achutha Menon Centre for Health Science Studies,
Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram,
India
Dr G Vijayakumar MD, Director, Medical Trust Hospital and Diabetes Centre, Kulanada,
Pathanamthitta, India
Dr K R Santosh MD, Interventional cardiologist, PRS Hospital, Thiruvananthapuram,
India
Indian Institute of Diabetes, Pulayanarkota, Thiruvananthapuram, India
99
Dr. Abdul Jaleel K.A
Scientist E-II
[email protected]
JRF:
Lekshmi Padmakumar
Metabolic Profiling of Normal Healthy people in Kerala
Lekshmi Padmakumar, G. Vijayakumar*, V Raman Kutty**, Abdul Jaleel
Collaborators * Medical Trust Hospital & Diabetes Centre, Kulanada, Pathanamthitta
** Achutha Menon Centre for Health Science Studies, SCTIMST, Trivandrum
We have a strong interest in understanding
the genetic and environmental interaction
in the mechanism of pathogenesis in type 2
diabetes (T2D). We believe that, investigating
the environmental impact (mainly nutrition and
physical activity) on body metabolic pathways
is a starting point towards understanding the
metabolic transition process associated with the
onset of T2D. The metabolic alterations, which may
be the foundation for metabolic diseases such as
diabetes, could be identified by performing mass
spectrometry based metabolomics analysis in the
blood of normal healthy study participants who are
at the risk of developing T2D (e.g., people having
family history of diabetes, Obesity, etc). Tools of
metabolomics measures chemical phenotypes that
are the net result of genomic, transcriptomic, and
proteomic variability, therefore, provide the most
integrated profile of biological status. Such studies
are likely to offer substantial data and rationale for
developing hypothesis based mechanistic studies.
The recruitment of study participants for this
project is currently underway. So far 81 normal
healthy people were recruited and human studies
were performed on them. Though all study
participants are normal healthy people, we have
been selecting and recruiting participants in two
100
groups of Low and High socio-economic strata
(SES). Epidemiological data on the prevalence
of T2D in Kerala as well as in other parts of India
indicate that the prevalence of T2D is significantly
high in people of High-SES when compared to LowSES, irrespective of the setting of the study whether
it is rural or urban or combined. Life style changes
such as lack of physical activity and consumption
of more energy rich foods due to rapid prosperity
are reported and proposed as factors contributing
to the high prevalence of diabetes in high-SES. It
can be assumed that those factors create a distinct
metabolic transition state leading to a pre-diabetes
state well in advance in those populations and
could be identified with our study design.
The study participants were recruited for both the
SES groups and the participants were matched for
their age, sex and body mass index (BMI). Family
assets and possession of various household items
were taken as indicators of their socio-economic
position. Only healthy people without any known
illness, including diabetes (screened by fasting
blood sugar and HbA1c, Table-1) were recruited
for the study. The anthropometric and other routine
blood biochemical measurements were shown in
the Table -1. As expected the lipid profile of women
were found better when compared to men.
Table – 1. Clinical and biochemical profile of study participants. Values are presented as mean ±
standard deviation. * indicates significant difference between male & female
Parameters
All
Female
Male
Total number
81
41
40
28.13 ± 6.37
(range 18 - 40)
29.41 ± 6.37
26.90 ± 6.20
0.07
BMI (Kg/m2)
22.56 ± 2.87
22.04 ± 2.65
23.09 ± 3.03
0.07
Waist circ. (cm)
76.72 ± 8.70
73.95 ± 7.0
79.55 ± 9.35
0.003 *
FBS (mg/dl )
87.73 ± 8.76
89.41 ± 8.87
86.00 ± 8.40
0.07
HbA1c (%)
5.39 ± 0.31
5.45 ± 0.34
5.32 ± 0. 2.69
0.05 *
176.95 ± 32.60
169.7 ± 30.66
177 ± 32.6
0.30
TGL (mg/dl )
101.8 ± 87
67.1 ± 31.81
137.4 ± 109.5
0.0003 *
HDL (mg/dl )
39.59 ± 10.79
44.12 ± 10.72
34.95 ± 8.79
0.00006 *
LDL (mg/dl )
114.2 ± 27.4
114.23 ± 23.41
114.27 ± 31.26
0.99
VLDL (mg/dl )
20.39 ± 17.42
13 ± 6.4
28.0 ± 22
0.0002 *
Age (years)
T. CHOL (mg/dl )
p value (M vs. F)
Postprandial metabolism.
Much of one’s life is spent in the postprandial state,
that is, the period that comprises and follows a
meal. The physiology of postprandial state involves
numerous finely regulated motor, secretary,
hormonal and metabolic events, where insulin
plays a major role. Thus to really understand why
certain groups or population having particular life
style and diets are relatively protected from diabetes
or prone to diabetes, studies of the postprandial
state are needed. Towards that objective each study
participant were given a mixed meal (25 kcal/kg
ideal body weight; 55% carbohydrate, 30% fat,
15% protein) in the morning of the study day after
an overnight fast. Blood samples were collected
from hand vein before the meal and then every
30 min after meal ingestion till 120 minutes. One
of our objectives is to perform mass spectrometry
based metabolomics on plasma samples collected
at different time intervals post meal. Though we
did not see any difference in the plasma glucose
response to meal challenge between the SES
groups, we could find a significant difference in the
plasma glucose values when compared between
male and female participants, showing a better
insulin sensitivity for females (Figure-1A). We also
observed a similar trend for those who have a BMI
of <23 kg/m2 (20.98 ± 1.64) when compared to
people whose BMI is >23 kg/m2 (25.72 ± 2.11) as
shown in Figure-1B.
101
Diabetes & Inflammatory Markers
Since we have observed an insulin sensitivity
difference between men and women as measured
by plasma glucose response to a meal challenge,
we thought of analyzing the values of selected
inflammatory and diabetes markers between
men and women. Though both the male and
female participants are normal healthy people, we
found that markers such as Adiponectin, Gastric
inhibitory polypeptide (GIP), Leptin, Plasminogen
Activator Inhibitor type 1 (PAI-1),), and Resistin
were significantly different between men and
women (Figure-2). However, plasma levels of IL6, TNF α, C-peptide, Insulin, Glucagon, Ghrelin,
Glucagon-like peptide-1 (GLP-1), and Visfatin were
not different (data not shown).
The above data between male and female
particpants prompted us to analyze those paramters
between participants who has lower BMI and
higher BMI as well. We catogorized participants
into two BMI groups, whose BMI are less than 23
kg/m2 and whose BMI are more than 23 kg/m2.
Though the people in the group of higher BMI
are not obese, their lipid profile were significantly
different from the normal or lower BMI group
(table-2). higher BMI group (Figure-3), whereas
adiponectin level is significantly lower. The levels
of diabetes markers such as C-peptide, glucagon
and visfatin are significantly high in the higher BMI
group. These measurements of markers indicate
that even though people are not obese, BMI higher
than normal can lead to a low grade inflammation
and insulinPlasma levels of inflammatory markers
such as IL-6 and TNF-α are significantly higher in
resistance (Figure-3).
Figure -2. Levels of plasma diabetes markers measured in men and women. Assay for all the markers were performed
by a magnetic bead assisted immuno-fluorescent multiplex assay. Only markers having values of significant
differences are shown. All values are in pg/mL and are represented as mean ± SEM.
Table -2. The lipid profile parameters of study participants based on their BMI (between BMI < 23
kg/m2 and BMI >23 kg/m2). All differences are significant. Values are represented as mean ± SEM.
BMI < 23 kg/m2
BMI >23 kg/m2
p Value
BMI (kg/m2)
20.92 ± 0.26
25.72 ± 0.41
0.001
T.CHOL (mg/dL)
170.64 ± 4.16
191.07 ± 5.58
0.005
TGL (mg/dL)
89.92 ± 1.03
141.70 ± 23.01
0.049
HDL (mg/dL)
42.17 ± 1.86
35.85 ± 1.95
0.023
LDL (mg/dL)
110.33 ± 3.75
126.51 ± 6.30
0.033
VLDL (mg/dL)
18.03 ± 2.20
28.35 ± 4.60
0.050
Parameter
102
Figure-3. Levels of plasma inflammatory and diabetes markers between participants of normal BMI (< 23 kg/m2)
and higher BMI (>23 kg/m2) groups. All values are in pg/mL. Parameters showing only significant differences are
shown.
Family history of Diabetes
Among the 81 study participants we recruited
and studied, there were 19 participants, who have
family history of diabetes (FHD+) as their first
degree relative(s) were dignosed having T2D. We
catogorized those particiants as a group having
FHD and matched them with a control group with
age, sex and BMI but having no FHD. We did not
see any difference in the values of fasting blood
Figure -4. Levels of plasma diabetes markers measured between participants having positive family history of
diabetes (FHD+) and participants of no-FHD. Subjects were matched for the age, sex and BMI and assay for all the
markers were performed by a magnetic bead assisted immuno-fluorescent multiplex assay. Only markers having
values of significant differences are shown. All values are in pg/mL and are represented as mean ± SEM.
103
sugar or lipid profile between the participants
who has FHD and those who have no FHD. No
differences in the levels of inflammatory markers
such as IL-6, TNF α, and adiponectin were observed.
However, levels of hormones such as insulin,
glucagon, C-peptide, Ghrelin, GLP-1, and visfatin
were higher in those who have positive FHD when
compared to people who have NO-FHD (Figure-4).
These measurements observed with respect to FHD
104
are in contrast to what is observed for higher BMI.
Together, these preliminary data indicate that
impact of genetic and environmental factors
trigger different routes of mechanism towards the
pathogenesis of type 2 diabetes. We believe that
we will able to deliniate the altered biochemical
pathways and various mechanisms by performing
mass spectrometry based metabolomics analsysis.
Mycobacteria Research Group I
Sathish Mundayoor, Ph.D.
Scientist G
[email protected]
Sathish Mundayoor obtained his Ph.D from All India Institute of Medical Sciences and did
Post- Doctoral training at Forshungsinstitut Borstel, Germany & Washington University
in St Louis, Missouri. He was a Visiting Scientist at Centres for Disease Control, Atlanta,
Georgia and joined RGCB in 1995.
PhD Students: Madhavilatha G K
Biljo V Joseph
Sunil Kumar V J
Dhansooraj D
Mahesh P P
Annapoorna K
Technical Officer:
Laiza K Paul
105
Tuberculosis is a major problem in the country and the efforts of the laboratory are
focussed on three main areas : Mycobacteria-macrophage interactions, Development
of novel Vaccine systems and Molecular epidemiology of the disease.
Theme 1: Mycobacteria-macrophage interactions
Identification of genes of Mycobacterium tuberculosis involved in the
downregulation of macrophage Scavenger Receptors:
Sunil Kumar V J, Satheesh Mundayoor
Mycobacterium tuberculosis – macrophage (MΦ)
interaction is central to TB infection, latency,
disease reactivation and transmission. The entry
of Mycobacterium tuberculosis (Mtb) into the host
macrophage and its survival in the intracellular
milieu are key components in pathogenesis of
tuberculosis. Signals from microbial products,
phagocytosis and cytokines result in changes to the
surface and secretory properties of macrophages.
It has already been reported that the ingestion
of live Mtb downregulates the expression of
macrophage receptors such as CR1, CR3 and
MR known to mediate phagocytosis of opsonized
and non-opsonized Mtb respectively. But the
SR intensity on THP 1 cells
106
expressions of macrophage Scavenger Receptor
(SR) have not yet been studied. We analyzed the
differential expression of macrophage SR in live
Mtb and live Mycobacterium smegmatis (Msmeg)
infected macrophages at different time points
post-infection using confocal microscopy, western
blotting and Real-Time PCR and showed that live
Msmeg showed up regulation of SR.
We made a library of M. tuberculosis genomic
DNA in an E coli-mycobacteria shuttle vector and
electroporated into M smegmatis. We used this
library to infect THP 1 cells and using Fluorescence
Activated Cell Sorting, isolated low fluorescing cells
that had decreased intensity
of SR. The live M smegmatis
was recovered from the lowfluorescing cells, enriched and
used for repeated rounds of
infection and FACS analysis. We
were able to obtain 4 colonies
that showed downregulation
of SR. Infection of these clones
into THP1 cells and confocal
microscopy confir med this
observation. The genes were
amplified, sequenced and
compared. Interestingly all the
four clones isolated came from
the same genomic area and the
largest clone showed maximal
decrease in fluorescence as
shown in the figure. Studies
to identify the mycobacterial
genes and further characterize
them are in progress.
Theme II: Development of novel Vaccine systems
Development of a vaccine delivery system using hepatitis B core
antigen based VLPs to deliver mycobacterial antigens
Dhansooraj D, Sathish Mundayoor
Growing prevalence of TB and the emergence
of XDRTB have stimulated substantial efforts to
develop better vaccines for TB. Recent researches
have shown that some of the antigenic proteins
and fusion of different proteins produced by
Mycobacterium tuberculosis can give protection in
animal models when administrated with specific
adjuvants. In the present study, we explored the use
HBcag-VLPs for delivery of tuberculosis antigens.
Nano-sized hepatitis B virus core virus-like particles
(HBc-VLP) are suitable for uptake by antigenpresenting cells. Mycobacterium tuberculosis
antigen culture filtrate protein 10 (CFP-10) is an
important vaccine candidate against tuberculosis.
The purified antigen shows low immune response
without adjuvant and tends to have low protective
efficacy. The present study is based on the
assumption that expression of these proteins on
HBc nanoparticles would provide higher protection
when compared to the native antigen alone. The
cfp-10 gene was expressed as a fusion on the major
immunodominant region of HBc-VLP, and the
immune response in Balb/c mice was studied and
compared to pure proteins, a mixture of antigens,
and fusion protein-VLP, all without using any
adjuvant. The humoral, cytokine, and splenocyte
cell proliferation responses suggested that the
HBc-VLP bearing CFP-10 generated an antigenspecific immune response in a Th1-dependent
manner. By virtue of its self-adjuvant nature and
ability to form nano-sized particles, HBc-VLPs are
an excellent vaccine delivery system for use with
subunit protein antigens identified in the course of
recent vaccine research.
CFP-10 specific IFN-γ response to VLP immunization.
Splenocyte from different groups of mice were
stimulated with CFP-10 (5 µg/ml). Con A (5 µg/
ml) was used as control. (A) IFN-γ-secreting cells
detected by ELISPOT assay (B) Images represent
splenic ELISPOT responses. G1- buffer immunized
group, G2- CFP-10 immunized group, G3- HBc-
VLP immunized group, G4- group in which HBc
+ CFP-10 immunized and G5- fusion protein
immunized group. SFC=Spot forming units. Con
A= Concanavalin A. The data are expressed as
the mean ±SD. Statistical significance between
groups is indicated. *** P < 0.001.
107
Theme III: Molecular Epidemiology of TB
Molecular epidemiology of Mycobacterium tuberculosis isolates from
Kerala, India using IS6110-RFLP, spoligotyping and MIRU-VNTRs.
Biljo V Joseph, Sathish Mundayoor
Tuberculosis (TB) continues to be a major health
problem in India, and there is very little information
about the prevalent genotypes of tubercle bacilli
that cause TB in India, especially in Kerala. Our
aim was to study the different circulating strains
of Mycobacterium tuberculosis (MTB) that are
prevalent in Kerala, India. We analyzed 168 MTB
isolates from as many pulmonary TB patients
using IS6110-RFLP, spoligotyping and MIRU-
VNTRs. The results of IS6110-RFLP revealed that
majority of isolates had null copy (10.89%) or
single copy (44.87%) of IS6110 insertion. Low copy
(<6) isolates accounted for 71.5% in the isolates
studied. Genotypic clade designations were done
by comparing with the SITVIT2 database which
showed 68 patterns; of which 51 corresponded to
different shared types whereas 17 patterns were
orphans. Among the 51 SITs recorded, 42 SITs
matched a preexisting SIT
in the SITVIT2 database,
whereas 9 SITs were
newly-created. Majority
of the isolates (64.28%)
belonged to the ancestral
East-African Indian (EAI)
lineage. MIRU-40 and
31 (HGDI = 0.6555 and
0.6524) showed highest
discrimination, while
MIRU-2 and 20 (HGDI =
0.0354 and 0.0696) had
the least discriminatory
power. ETR-A and B
(HGDI 0.7382 and 0.6743)
discriminated better
as compared to other
MIRU loci. The overall
HGDI for MIRU-VNTRs
at 0.9735 (calculated for
166 isolates) showed a
better discriminator y
power than spoligotyping
used alone. This study
of MTB genotypic
diversity provides a first
snapshot of circulating
MTB genotypic clones
in Kerala.
Interrelationship and Clade Designation of strains from Kerala: A minimum spanning tree (MST) illustrating
phylogenetical relationships between the Indian spoligotypes and MIRU data: (A) spoligotyping based tree (B) 12loci MIRU-based tree (C) Combined spoligotyping + MIRU-based tree
108
Publications
»» G. K. Madhavilatha, Biljo V. Joseph, Laiza K. Paul,
R. Ajay Kumar, Ramkumar Hariharan and Sathish
Mundayoor. Whole genome sequence of two
clinical Isolates of Mycobacterium tuberculosis
from Kerala, South India. J. Bacteriology. 194
(2012):4430
»» Sunil V. Joseph, G. K. Madhavilatha, R. Ajay
Kumar, and Sathish Mundayoor. Comparative
Analysis of Mycobacterial Truncated Hemoglobin
Promoters and the groEL2 Promoter in FreeLiving and Intracellular Mycobacteria Appl
Environmental Microbiology 2012.78(18):64996506
»» Biljo V Joseph, Smitha Soman, Indulakshmi
Radhakrishnan, Véronique Hill, D
.
Dhanasooraj, R. Ajay Kumar, Nalin Rastogi,
Sathish Mundayoor. Molecular epidemiology
of Mycobacterium tuberculosis isolates from
Kerala, India using IS6110-RFLP, spoligotyping
and MIRU-VNTRs. Infection, Genetics and
Evolution 16 (2013) 157–164
»» D Dhanasooraj, R Ajay Kumar, Sathish
Mundayoor. Vaccine delivery system for
tuberculosis based on nano-sized hepatitis B
virus core protein particles. Int J Nanomedicine,
2013. 8: p. 835-43.
From Collaborations:
»» Sharrel Rebello, A K Asok, S V. Joseph, B. V.
Joseph, L Jose, S Mundayoor & Jisha M.S. 2012
Bioconversion of Sodium Dodecyl Sulphate to
Rhamnolipid by Pseudomonas aeruginosa: A
Novel and Cost-Effective Production Strategy.
Appl Biochem Biotechnol. DOI 10.1007/s12010012-9988
Posters published
»» D Dhanasooraj, R Ajay Kumar, Sathish
Mundayoor: Development of a vaccine delivery
system using hepatitis B core antigen based
VLPs to deliver mycobacterial antigens. BMC
Infectious Diseases 2012 12(Suppl 1):P9.
Conferences/Meetings
»» David Abraham, Cork, S.C., Venugopal, K.P.,
Shah, A., Madhavilatha, G.K., Anusree, B.S.,
and Mundayoor, S. Strain identification of Mycobacterium tuberculosis complex isolates
from rescuedsloth bears (melursus ursinus)
in Southern India. International Wildlife
Conference, Kruger National Park, South Africa.
9-12 September 2012
109
MYCOBACTERIUM RESEARCH GROUP – 2
R. Ajay Kumar, Ph.D.
Scientist E II
[email protected]
Ajay Kumar received his PhD in Microbiology at Madurai Kamaraj University, Madurai. Postdoctoral training at Indian Institute of Science, Bangalore, Sree Chitra Tirunal Institute for
Medical Sciences and Technology, Trivandrum, and University of Massachusetts Medical
School, Worcester, USA.
PhD Students:
Leny Jose
Roshna Lawrence
Gomez
Aneesh C
Ranjit Ramachandaran
110
T.R. Kannan
Mycobacterium tuberculosis is the causative agent of tuberculosis (TB) and is responsible
for approximately 1.4 million deaths a year across the world. It is an intracellular pathogenic
bacterium and resides in professional phagocytes. The success of M. tuberculosis as a
recalcitrant pathogen of humans is attributed to its ability to invade macrophages, the very
cells that are meant to eliminate pathogens from the host system. This process is achieved
by hijacking the host machinery involved in the immune response and ultimately leading to
the prevention of maturation of phagosome. In eukaryotes gene expression and repression
are the result of modification or remodelling of the chromatin. Chromatin structure and
accessibility to DNA sequences are regulated by modifications of both DNA and histones
which are collectively termed ‘epigenetic modifications’. The mechanisms of epigenetic
modifications include histone acetylation, histone methylation, DNA methylation etc., and
these modifications provide a way to control the expression of genes by regulating access of
transcriptional regulators to regulatory DNA sequences such as promoters and enhancers.
Histone acetylation is a key component in epigenetic regulation and is maintained by two
kinds of enzymes, histone acetyl transferases (HAT) and histone deacetylase (HDAC). Most
studies thus far have focused on diseases such as cancer, but our understanding of the
role of epigenetics in host-pathogen interactions is still at its infancy. The major interest of
our lab is to understand how M. tuberculosis, upon infection, regulates gene expression in
macrophages through chromatin modifications.
1. Isolation, identification and functional characterization of a
Mycobacterium tuberculosis protein with histone acetyl
transferase activity.
Leny Jose and R. Ajay Kumar
Macrophages derived from human monocyte cell
line THP1 were infected with virulent laboratory
strain M. tuberculosis H37Rv. Chromatin of infected
and non-infected cells was immunoprecipitated
(ChIP) and the proteins of the precipitate were
subjected to 2-D electrophoresis. A unique protein
present in the infected sample was identified as
a mycobacterial hypothetical protein (hereafter
referred to as MTX). The gene for MTX was
amplified from M. tuberculosis H37Rv, cloned in
E. coli, expressed and the protein was purified to
homogeneity. Employing immunoprecipitation,
western blot and confocal microscopy analyses we
showed that the protein is indeed transported into
the nucleus of the macrophages and binds to the
chromatin (Fig 1).
Bioinformatics predictions using ab initio modelling
revealed that this hypothetical protein could
possess acyl transferase activity as its most
probable function. Since it was found bound to
the chromatin, we presumed MTX might possess
histone acetyl transferase activity (HAT) and
therefore subjected it to HAT assays. The results
revealed that MTX indeed possesses HAT activity.
The target of activity was found to be histone H3
and acetylation occurs at amino acid residues K9/
K14. Anacardic acid, an inhibitor of HAT activity,
prevented histone acetylation activity of MTX. ChIP
sequencing analysis revealed that MTX is recruited
to nine promoters of THP1-derived macrophages.
The role of MTX in the regulation of the expression
of these genes is the current focus of our study.
Fig 1. MTX protein binds to chromatin of macrophage:
Representative image from the Z-stacked confocal
images shows MTX protein (red) bound to the chromatin
(green).
111
2. Survival strategies of M. tuberculosis by inducing epigenetic
modifications in macrophages.
Aneesh. C and R. Ajay Kumar
Our goal in this study is to understand the
correlation between intracellular M. tuberculosis,
host chromatin modifications, and transcription of
host defense genes.
Our preliminary studies have revealed that
infection of THP1-derived macrophage cells
with M.tuberculosis led to an increase in total
histone H3 acetylation (fig 2 A) and a decrease
in the expression of histone deacetylase HDAC1
at 12 hours post infection (PI) (fig 3 A), as in
the case of other bacterial infections. However,
quite interestingly, at 24 hours PI, we observed a
significant decrease in total histone H3 acetylation
(fig 2 B) and a concomitant increase in HDAC1
expression (fig 3 B) in the macrophages.
Combination of these two activities – upregulation
of total histone H3 acetylation and downregulation
of HDAC1 expression at 12 hours PI - may represent
the first response of macrophages leading to the
expression of various immune response molecules
in order to eliminate bacterial pathogens from
the system. Therefore we propose that the
significant upregulation of HDAC1 expression
and the corresponding decrease in the histone H3
acetylation that we observed at 24 hours PI could be
responsible for the downregulation of the expression
of a number of immunity-related host genes during
M. tuberculosis infection. Enhancement of HDAC1
expression may be a strategy employed by the
successful intracellular pathogen to silence the
host defense gene expression by deacetylating
histones at the regulatory sequences such as
promoters and enhancers of defense genes to
facilitate its survival inside the host.
Interestingly, our observations indicate that
these modifications are brought about only by
metabolically active, and not by heat-killed, M.
tuberculosis. This suggests that thermolabile
mycobacterial factors might play a direct or indirect
role in chromatin modifications (acetylation and
deacetylation of histones) in the host macrophages
thereby modulating the host gene expression to help
the bacteria survive in such a hostile milieu and
perhaps promote the disease. We are interested in
delineating the mechanism of bacterially provoked
epigenetic modifications and identify the bacterial
factors, if any, involved in these modifications.
Fig: 2. Status of histone H3 acetylation in macrophages upon M. tuberculosis infection. (A) Increase in histone
H3 acetylation in macrophages infected with M. tuberculosis at 12 hrs PI as visualized by confocal microscopy.
(B) Decrease in histone H3 acetylation in macrophages infected with M. tuberculosis at 24 hrs PI. (C) Histone H3
acetylation status upon M. tuberculosis infection. Change of levels of acetylated histone H3 at 12 hrs and 24 hrs PI
by western blot analysis. Note the increase in H3 acetylation at 12 hrs PI and the decrease at 24 hrs PI.
112
Fig 3: Differential expression of HDAC1 upon M. tuberculosis infection.. (A) & (B) Immuno-cytochemical imaging
shows expression of HDAC1 at 12 hrs & 24 hrs PI in macrophages infected with dead or live M. tuberculosis. (C) At
12 hrs PI, HDAC1 expression is downregulated in macrophages infected with live M. tuberculosis. (D) At 24 hrs PI
HDAC1 expression is significantly more in macrophages infected with live M. tuberculosis.
Their identification and functional characterization
would be of importance because such factors
could be novel targets for drug intervention for the
treatment of tuberculosis.
Another interest of our lab is dormancy in M.
tuberculosis. In a healthy person the innate
immune system normally eliminates the bacilli
in the lungs. It is believed that one third of the
world population harbours latent M. tuberculosis
and they have a 2-23% lifetime risk of developing
reactivation TB. Fortunately, majority of people
infected with M. tuberculosis never develops
TB although they are asymptomatic carriers of
the bacilli. In such individuals the bacilli are
contained by the immune system of the body
in special structures called ‘granuloma’ which
segregate the infection to prevent the spread of the
bacilli to the rest of the lungs and to other organs.
Granuloma also helps the body to concentrate the
immune response directly at the site of infection.
In granuloma they remain in a non-replicative
(dormant or persistent) form and show a phenotypic
resistance to anti-mycobacterial drugs as these
drugs target growth-related functions. The latently
infected population is the major hurdle to TB control
efforts. Once favourable conditions return, these
dormant bacteria can get reactivated to cause
‘reactivation TB’. The current anti-TB drugs and
those in the pipeline are relatively ineffective in
treating latent TB. For long-term success of TBcontrol programs, consideration of latent infection
is essential. This has rekindled the interest in drug
discovery against dormant M. tuberculosis.
As M. tuberculosis is exposed to diverse stress
conditions in granuloma, its adaptation to such
stress through regulation of genetic programs is an
essential feature of dormancy. A number of genes
are known to be specifically expressed during
dormancy in M. tuberculosis. The mycobacterial
factors involved in the regulation of expression
of these specific genes can be ideal targets for
therapeutic intervention to treat latent TB.
113
3. Expression, purification and characterization of a stress-related
transcriptional regulator of Mycobacterium tuberculosis
Roshna Lawrence Gomez and R. Ajay Kumar
Many genes in M. tuberculosis are reported
to be up-regulated when exposed to different
stress conditions like high temperature, hypoxia,
nutrient starvation, and in vitro infection models.
We selected a putative mycobacterial regulatory
gene, Rv3334 which gets up-regulated during
stress conditions similar to those found inside a
granuloma. In order to understand its functional
role during persistence and virulence, the gene was
cloned from M. tuberculosis H37Rv and expressed
in E. coli and purified to homogeneity.
The gene product, as expected of a transcriptional
regulator, undergoes dimerization under nonreducing conditions in vitro (Fig 4). MALDI-TOF
analysis confirmed both these forms represent
Rv3334.
Since many transcriptional regulators control their
own expression, we tested if this protein can bind
to its own promoter. Employing electrophoretic
mobility shift assay (EMSA) we observed that
Rv3334 protein can bind to its own promoter
(fig 5) suggesting a possible autoregulatory role.
Performing EMSA with DNA fragments of varying
lengths, we found that the binding site is situated
within the 60 bases upstream of the ORF. We are
currently involved in the identification of the
consensus binding site of Rv3334 protein.
Promoter activity was assayed by cloning the
putative promoter region into the promoterless
shuttle vector pFPV, and electroporating into
nonpathogenic M.smegmatis (the gene is absent
in this species). Significant expression of GFP
was observed when it was cloned downstream of
Rv3334 promoter (Fig 6).
Fig 4: Rv3334 protein undergoes dimerization
in vitro. Lane 1. Rv334 protein under reducing
condition. Lane 2. Mol wt markers (KDa). Lane 3.
Rv3334 under non-reducing condition.
Fig 5: EMSA shows Rv3334 protein binds to its putative promoter region. Lane 1: Putative promoter region (213 bp),
without protein; Lane 2-6: Putative promoter + increasing concentrations of Rv3334 protein (2, 4, 6, 8 and 10μM)
114
Fig 6: Rv3334 Promoter is functional in M. smegmatis. M. smegmatis was transformed with pFPV
vector containing GFP without (I) or with the putative promoter (II). A) Qualitative analysis by confocal
microscopy; B) quantitative analysis by measuring GFP fluorescence. a. GFP; b. DIC; c. merged.
Sl.No
Investigators
Title of Project
1
R. Ajay Kumar (PI)
Abdul Jaleel
Satheesh Mundayoor
Identification of transcriptional
regulators expressed in
Mycobacterium tuberculosis during
reactivation from dormancy in vitro,
and identification of their target
sequences
2
R. Ajay Kumar (PI)
Sabu Thomas
Isolation and characterization of
antimycobacterial molecules from
Actinomycetes
FundingAgency
Duration
DBT
2012-2015
OSDD (CSIR)
2012-2015
Publications
Conferences, Presentations:
»» Organic agricultural products may be a source
of vitamin B12. T.R. Kannan, E.V. Soniya, M.
Radhakrishna Pillai and R. Ajay Kumar. National
symposium on Innovative Approaches and
Modern Techniques for Crop Productivity, Food
safety and Environmental Sustainability. 19-20
November 2012. Organized by Society of Applied
Biotechnology, Thrissur.
115
Viral Disease Biology Program
E. Sreekumar, MVSc., PhD
Scientist EI
[email protected]
E.Sreekumar is a post-graduate in Veterinary Immunology, and a Ph.D in Biotechnology
from University of Kerala. He joined RGCB in 2004.
PhD Students:
Anoop M
Rachy Abraham
Aiswaria Padmanabhan
Prashant Mudaliar
Anupriya M.G
Sneha Singh
116
Technical Staff:
Unnikrishnan V.R.
We study two mosquito-borne viral infections: Chikungunya and Dengue. The current
activities are summarized in Fig.1
Fig.1 THEME I. VIRUS EVOLUTIONARY DYNAMICS
1. Evolutionary dynamics of Dengue virus in Kerala
Dengue illnesses are caused by any of the four
Dengue virus serotypes (DENV1, -2, -3, and
-4) of the positive stranded RNA virus (genus
Flavivirus, family Flaviviridae). The disease
spectrum can vary from mild dengue fever (DF) to
life-threatening dengue haemorrhagic fever (DHF)
or dengue shock syndrome (DSS). We carried out
the molecular characterization of dengue virus
strains circulating in Kerala for the last 6 years
(2007-2012). A total of 773 serum samples from
patients suspected of dengue fever were obtained
from Hospitals, Primary Health Centers and by
various field-based surveillances and were used
in the study. Both specific gene-based as well as
complete genome based analysis were done for
the molecular characterization of Dengue virus.
Out of 186 (24%) samples which came positive for
Reverse Transcriptase -PCR, 33% of the samples
were of DENV1, 33% were DENV2, 19% were
DENV2, 19% were DENV3 and 1.6% was DENV4.
Whole genome analysis of DENV1 showed that
it belongs to a separate clade from the Indian
isolates and had closer associations with strains
from Singapore and Brunei. Since this was the first
whole genome analysis of DENV1 from India, this
data would also serve as a reference sequence for
future studies on this serotype in the sub-continent.
Phylogenetic analysis using the envelope coding
region sequences of DENV2 and DENV4 found the
co-circulation of strains belonging to two separate
clades having distinct amino-acid differences as
signatures. Our study also found the emergence of
lineage IV of DENV3 in India for the first time, along
with the circulation of lineage III strains (Fig.2). The
overall results indicate that a genetically distinct
set of Dengue viral strains circulates in Kerala along
with DENV strains with significant similarity to
the strains circulating in the rest of the country.
A sustained local evolution and also frequent
exchange of viral strains with countries of the
Indo-pacific rim could be the reasons for the unique
genetic picture of viral strains circulating in this
region were dengue has become hyperendemic.
117
Fig.2. Phylogenetic analysis of the envelope protein coding region sequences of DENV3 strains indicating the
lineage-shift
118
2. Evolutionary Dynamics of Chikungunya virus in Kerala
Chikungunya virus (CHIKV), an enveloped positivestrand RNA virus belonging to genus Alphavirus
of the Togaviridae family, occasionally causes
epidemics of acute febrile illness with severe and
prolonged arthralgia. It is a major public health
problem especially in the tropical countries where
the vector mosquitoes of the species Aedes are
abundant. Re-emerging strains of Chikungunya
virus present interesting prospects to study
adaptive evolution of alphaviruses. In our study,
we characterized the genetic changes in CHIKV
strains isolated from Kerala, South India during
epidemic (2006-2008) and post-epidemic (20092013) period spanning seven consecutive years.
Nucleotide sequence analysis of the entire stretch
of the envelope proteins E2-E1 coding region
(2769bp) was carried out in representative isolates
for different years. Comparison of these sequences
with that of earlier isolates from Kerala and rest of
India confirmed the circulation of the ECSA strains
with the E1A226V mutation during these years.
Presence of several signature mutations in the
envelope protein coding regions, identified to be
functionally important in reference strains such as
SFV/SINV, implicates their adaptive role in infection
and spread of these strains in mosquito and human
hosts. The study reiterates the prevailing evolution
of the virus in geographic regions with ample
mosquito vectors and susceptible population
favoring re-emergence of newer strains.
THEME II. HOST-VIRUS INTERACTION
1. Human glioblastoma U87-MG cells as an in vitro model for CNS
cell infectivity of Chikungunya virus
Chikungunya virus (CHIKV) is primarily an
arthritogenic alphavirus. But the recent outbreaks
witnessed the incidence of atypical clinical
manifestations including central nervous system
(CNS) infections, especially in very young and very
old patients. The molecular mechanisms of CNS
inflammation induced by CHIKV are not clear. Our
laboratory focused on the susceptibility of astrocyte
cells, one of the major immune cells of the brain
parenchyma that mediates inflammation, to CHIKV
infection. In our studies, CHIKV was found to infect
and activate U87-MG cells, a glioblastoma cell line
of human astrocyte origin and produce the viral
proteins and progeny virions as detected in 12h
post-infection (p.i.). The infection resulted in
apoptosis detectable from 48h p.i. evidenced
by PARP cleavage, loss of mitochondrial
membrane potential, nuclear condensation;
and visible cytopathic effects in a dose and
time dependent manner (Fig.3). Endoplasmic
reticulum stress, as indicated by XBP1 splicing,
and increased autophagy response was
also detected upon CHIKV infection. CHIKV
infection led to the enhanced expression of
the mRNA transcripts of pro-inflammatory
cytokines such as IL-1β, TNF-α, IL-6 and CXCL 9
within 24h p.i. and up-regulation of the proteins
of RLR pathway, indicating the activation of the
cytoplasmic-cellular innate immune response.
This cell line of CNS origin could be a potential
in vitro model to study molecular pathways of
inflammation induced by CHIKV.
Fig.3.Cytopathogenicity of CHIKV in U-87 MG cells
119
2. Elucidation of the cellular mechanisms of
Vascular Permeability in Dengue
Dengue virus, member of Flaviviridae family,
causes mild dengue fever and severe dengue
hemorrhagic fever and shock syndrome. Vascular
leakage leading to shock is a major complication
in Dengue that results in mortality. In order to
understand the molecular mechanisms behind this
phenomenon we carry out studies to understand
the endothelial cell dysfunction. As a first step
to identify a virus strain that efficiently infects
the endothelial cells, we screened isolates from
120 dengue virus positive (of serotype1, 2, and
3) clinical samples collected over a period of five
years (2007-2012), and identified a few isolates
that indicate infection in endothelial cells. Virus
infection in these cells was identified by dengue
virus specific RT-PCR and further confirmation
by immuofluorescence studies are being carried
120
out. For this purpose we have generated antiNS3 protease and anti-envelope protein domain
III antibodies in rabbits. Also, in collaboration
with the pediatric hospital of the Medical College,
Thiruvananthapuram we have collected 215 serum
samples from dengue patients having varying
degrees of disease severity for our further studies
in vascular permeability. We have also carried out
preliminary PCR based analysis for the presence of
PAR-1 and S1P transcripts in EA.hy926 endothelial
cells which detected the basal level expression
of these transcripts. The modulation of these
transcripts upon virus infection will be looked
into. Also, trans-well assays for endothelial cell
permeability are being standardized at present.
THEME III. ANTIVIRALS SCREENING
1. Evaluation of natural products for anti-chikungunya virus activity
using Plaque reduction assay based screens
There are no antiviral drugs currently available
to treat chikungunya. The disease is treated
symptomatically with paracetamol, NSAID and
chloroquine, prolonged use of which may cause
side effects. The work focuses on screening CHIKV
inhibitors with low cytotoxicity from natural
products. We have investigated the inhibitory
effects of several compounds on the replication of
the virus in vitro. Antiviral activity was estimated
by the reduction of the cytopathic effect on infected
Vero cells and by virus titre reduction in plaque
forming assay. We were successful in screening and
isolating a crude plant extract with low cytotoxicity
which could reduce the replication of the virus to
more than two log dilutions in the standard plaque
reduction assay (Fig.4). Further evaluations of this
extract are in progress.
Fig.4.Plaque reduction assay showing the effectiveness of the plant extract
Research Grants
No.
1.
Title
Characterization of Neurovirulence
of Chikungunya virus in cellular and
animal models
Investigator(s)
Dr.E.Sreekumar &
Dr.Jackson James
Funding Agency
Department of
Biotechnology,
New Delhi
Duration
2012-2015
121
PUBLICATIONS
From the laboratory:
»»M. Anoop, Ashish J. Mathew, B. Jayakumar,
Aneesh Issac, Sajith Nair, Rachy
»»Abraham, M.G. Anupriya, E. Sreekumar
(2012). Complete genome sequencing
and evolutionary analysis of Dengue virus
serotype 1 isolates from an outbreak in
Kerala, South India. Virus Genes 45:1-13
From Collaborations:
»»Zinia T Nujum, Vijayakumar K, Pradeep
Kumar AS, Anoop M, Sreekumar E, Ramani
Bai JT, Dalus D, Lalitha Kailas, Saritha N,
Anitha Abraham, Anto Varghese, Raji RT ,
Sudheesh Kumar TK(2012). Performance
122
of WHO probable case definition of dengue
in Kerala, India, and its implications for
surveillance and referral. Dengue Bulletin
36: 94-104
CONFERENCES/ SEMINARS
»» Sreekumar E., Changing Molecular Profiles
of Re-emerging Arboviruses in India. Third
Annual Conference of International Association
of Medical and Pharmaceutical Virologists
(IAMPV) on “Challenges and Strategies in the
Prevention and Management of Viral Infections”
on 9th and 10th November 2012, Chennai, India
»» Anoop M.,Sreekumar E., Genetic Characterization
of the Dengue Virus serotype 2 isolates from
Kerala (2008-11) International Conference on
Dengue and Emerging Infections, on 21-23rd
Nov.,2012, Singapore.
PATHOGEN BIOLOGY :
Viral Disease Biology Program
Joshy Jacob
M. Radhakrishna Pillai
Suja M.S.
R. Radhakrishnan Nair
Vijesh Sreedhar M.D. (Pediatrics) Iype Joseph, MBBS, MPH
Sara Jones, PhD.
Faculty Advisers:
Joshy Jacob, PhD.,
Senior Adviser to RGCB in Virology &
Immunology (Emory University, Atlanta, USA)
M. Radhakrishna Pillai, FRCPath, PhD.
Professor of Disease Biology
R. Radhakrishnan Nair, PhD.,
Reshmi G., PhD.
Program and Research Scientists:
Suja M.S., Ph.D.
Vijesh Sreedhar Ph.D.
Iype Joseph, MBBS, MPH
Sara Jones, PhD.
Reshmi G., PhD.
Project Officer:
Pradip Fulmali MSc.
PhD Student:
Sona Charles
Scientist E1
123
Manipulation of the cell death macinery by West Nile Virus
and its variants
Pradip Fulamli, R. Radhakrishnan and M. Radhakrishna Pillai
This study investigates the nature of cell typespecific restriction of West Nile virus (WNV)
infection by analyzing replication of WNV (804994)
strain in neuroblastoma (IMR), macrophage (THP)
and Human Kidney cell lines. All these cell lines
supported growth of the virus equally and attained
its peak titer by 4th post infection day ( PID). The
virus titer in IMR and THP cells was >1 log less
than the HEK after 1st PID. After 2nd PID, IMR
and HEK grown virus attend the similar titer.
However, in THP cell showed significantly low
titer of the virus until 3rd PI. We also demonstrated
differential cell viability pattern in these cell lines
and showed correlation between the increased in
viral genomic copies with decrease in the percent
viable cell. The WNV 804994 strain took <60hr post
infection (p.i.) to induced 50% cell death in the HEK
(epithelial) cell line and in IMR (neuronal) cell line
>72 hrs p.i. However, in THP (macrophage) cell
line, it did not show 50% cell mortality even after
96 hrs p.i. For mock-infected cells, number of viable
cells remained relatively constant throughout the
experiment. These studies indicated that difference
in cell type affects replication potential of the west
Nile virus and thus the cell death induced by it.
a) CPE based titration WNV 804994
b) plaque based titration WNV
Virus titers of the tissue culture pools of WNV
Virus
Titre(TCID50)
Titre(PFU/l)
804994
1 X 10 7
6 X 10 6
Network Modeling of Dengue Viral Infection Using Time Course
Microarray Data
Sona Charles, Reshmi G and M Radhakrishna Pillai
Dengue virus causes ~50–100 million infections
per year and thus is considered as a major
international public health concern. During
its replication, dengue virus induces dramatic
alterations in the interaction between various
genes/ proteins that are essential to generate
cellular responses, the study of which could
enable the alteration of a cellular behaviour
without any side effects. Modelling of dynamic
regulatory pathways using Dynamic Bayesian
124
Networks (DBNs) on microarray time-series can
provide invaluable insights about the dynamic
interactions among genes. This study proposes to
demonstrate the effectiveness of DBNs through
the analysis of dengue virus infection time series
data. Bayesian networks are a type of graphical
models for capturing complex relationships among
a large amount of random variables by the directed
acyclic graph. The time series microarray data
(GEO: GSE34628) which includes 6 time series
matrix files was used as the sample data for our
analysis. The time points of the initial changes
in the expression of genes based on their time
series expression data was calculated as >1.2-fold
(up-regulation) and <0.70-fold (down-regulation)
compared to baseline gene expression as the cutoffs (Zou et al, 2005). The potential gene regulators
were identified by using the transcriptional time
lag expression values. The potential regulators and
their target genes were converted into a matrix
of regulators and target genes and estimated
transcriptional time lag. Conditional probabilities
of each target gene in relation to its regulator genes
were calculated based on the data matrices which
in turn were used to calculate marginal likelihood
scores. The potential regulators of the target genes
that gave the highest log marginal likelihood were
selected as the interacting genes.
Sero-epidemiological and molecular characterization of Seasonal and
Pandemic Influenza A (H1N1) 2009 virus outbreaks in Kerala
Sara Jones, , Joshy Jacob and M. Radhakrishna Pillai
Influenza viruses are among the most virulent
pathogens and responsible for 3–5 million clinical
infections and 500,000 fatal cases annually.
Currently influenza A viruses of the H1N1 and
H3N2 subtypes are responsible for the seasonal
outbreaks of influenza. The emergence of a novel
H1N1 strain challenged public health officials and
scientists to make prompt decisions about how best
to prevent transmission in the face of an imminent
pandemic. The viral antigens hemagglutinin (HA)
and neuraminidase (NA) are the immune protective
targets of the virus and changes (antigenic shift
and antigenic drift) in these HA and NA molecules
results in evasion of the immune system. Our
main objective is to understand the molecular
biology and epidemiology of influenza viruses,
interactions between influenza virus proteins and
gene segments as well as virus and host immunity.
In our study, 30.17% cases were found to be positive
for pandemic H1N1 2009 and 10.49% positivity for
seasonal influenza A virus, which is similar to the
reports from other parts of India. It was observed
that severe disease and mortality in the pandemic
influenza A (H1N1) 2009 infection predominantly
affected relatively healthy adolescents and adults
between the age of 10 and 50years. To fully
characterize the molecular epidemiology of the
H1N1 strains circulating in Kerala, we isolated virus
by infecting embryonated eggs or Madin-Darby
canine kidney (MDCK) cells in culture with samples
taken from the nasal or throat swabs from humans
showing influenza like illness. We established a
RT-PCR method that
amplifies the HA and
NA segments of the
vRNA of the Influenza
A vir us using the
standard WHO primers.
The preliminary
phylogenetic studies
using the maximum
likelihood model reveals
that H1N1 isolates
were clustered with
Singapore and Beijing
strains suggesting
that strains from both
thelineages were in
circulation in Kerala.
Fig: Amplification and phylogenetic analysis of Influenza A (H1N1)2009 virus. A - PCR
reaction using WHO primer pair. Six sets of primers were used to amplify the full HA
segment. B- PCR reaction using primer pair as reported by Hoffmann et al., to amplify
full NA segment. C- Phylogenetic analysis of completed sequence of HA gene from
2009-2012.
125
(Research Scientist)
Suja M. S. [email protected]
Suja took her PhD from National Institute of Mental Health & Neurosciences (NIMHANS),
Bangalore in Neurovirology and did post doctoral training at Department of Molecular
& Integrative Physiology, Kansas University of Medical Sciences, Kansas,USA and also
at Human Brain Tissue Repository (HBTR), Dept of Neuropathology, NIMHANS. Suja is
supported by the ICMR - National Viral Disease Network.
Neuronal dysfunction in Japanese encephalitis - in vivo& in vitro
studies to understand cytoskeltal integrity in Neuronal process.
Death in cases of Japanese encephalitis (JE)
is presumed to result from, dysfunction and
destruction of neurons. Despite dramatic and
severe clinical course, post-mortem examination
of the patients reveals only a few pathological
lesions. Based on pathophysiological examination,
increasing evidence suggests that both neuronal
destruction and dysfunction might explain the
manifestation of JE. The exact mechanism of JE
induced cytopathic effect in neuronal process
(axons& dendrites) remains to be clarified.
Structural integrity of neuronal process (axons
& dendrites) and synaptic buttons are important
for neurotransmission, thus maintaining optimal
neuronal function. In view of this lacunae we intend
to investigate the structural basis of neuronal
dysfunction in JE and to study the morphological
alteration of neuronal process after JE Viral (JEV)
infection on autopsied brain tissues from JEV
cases, animal models and primary neuronal cell
lines inoculated with prototypic strain of JE virus.
PUBLICATIONS
»» Quantitative proteomics for identifying
biomarkers for rabies. Venugopal A K, Ghantasala
SS, Selvan L D, Mahadevan A, Renuse S, Kumar
P, Pawar H, Sahasrabhuddhe N A, Suja M S,
Ramachandra Y L, Prasad T S, Madhusudhana S
126
Our preliminary results from JEV infected human
brain tissues show presence of characteristic
necrolytic lesions localising to grey-white junction
in frontal cortex and hippocampus (sites of learning
and memory). Predominant inflammatory response
– microglia forming nodules surrounding neurons
causing neuronophagia and neuronal dysfunction.
Neurons in frontal and Hippocampal areas of brain
show dystrophic changes reflecting degeneration.
In chronic cases there appears to be neuronal loss
in substantia nigra and thalamus which also are
sites of predilection for necrolytic lesions. Initial
testing for apoptotic markers and autophagy
from JEV infected human tissues revealed
expression of apoptotic markers on endothelial
cells and microglia. Absence of expression of
LC3 (autophagy marker) proteins in neurons,
but presence in microglia suggested a neuronal
protective mechanism. Further studies are in
progress in mice brain infected with the prototypic
strain of JE virus.
N, Hc H, Chaerkady R, Satishchandra P, Pandey
A and Shankar S K. Clinical Proteomics. 2013
Mar 22;10(1):3.
»» Pictorial commentary: Cerebral Rabies. S K
Shankar, Anita Mahadevan and M S Suja. Kerala
Health Sciences Journal, 2012; Vol.1.
Research Scientist (Medical)
Vijesh Sreedhar K.
[email protected]
Vijesh received his MD in Pediatrics from All India Institute of Medical Sciences, New
Delhi and did postdoctoral research at Postgraduate Institute of Medical Education and
Research, Chandigarh before joining RGCB in 2011. Vijesh is supported by the ICMR
National Viral Disease Network.
Molecular characterization of measles viruses circulating in Kerala
India alone accounts for a major portion of global
measles mortality. Though Kerala has better health
parameter indicators and immunization coverage
compared to many other regions of the country,
the state is endemic for measles. Molecular
epidemiological investigations and genetic
characterization of the circulating viral genotypes
is important for measles control and prevention.
We initiated a study to identify the circulating
measles genotypes in Kerala. Using an RT-PCR
assay for detection of measles RNA, we have tested
clinical specimens from 95 children with suspicion
of measles in Thiruvananthapuram. Measles RNA
was detected in 60 specimens. We optimized
protocols for isolation of wild type measles virus
from clinical specimens using Vero-SLAM cell lines
(transgenic cell lines expressing human SLAM, the
receptor for measles virus). Measles virus infection
of the cell lines is identified by the observation of
cytopathic effect of syncytium formation.
We have optimized RT-PCR assay for amplification
of partial N gene recommended for genotyping
the measles virus as per WHO recommendations.
Isolation of virus from clinical specimens in VeroSLAM cell lines and genetic characterization of the
virus is progressing.
Fig : We were able to obtain three viral isolates from throat swab samples of measles suspected children.
127
BIOLOGY OF LEPTOSPIROSIS :
Research Scientist (Medical)
Iype Joseph [email protected]
Iype Joseph obtained his MBBS degree from University of Kerala and then did an MPH
degree from Achutha Menon Centre for Health Science Studies at the Sree Chitra Tirunal
Institute for Medical Sciences and Technology (SCTIMST), Thiruvananthapuram, Kerala,
India,before joining RGCB in 2011. Iype is supported by the ICMR - National Viral Disease
Network.
Epidemiology of Leptospirosis in Kerala, India
Leptospirosis is a complex bacterial disease with
multiple modes of transmission, numerous hosts,
multitude of pathogenic serovars (>200), various
clinical manifestations, and need complex testing
to provide laboratory confirmation. Among the
reported causes of mortality in Kerala due to
communicable diseases, Leptospirosis contributes
the maximum share for the past many years. Casefatality rates for the disease range from <5% to
30%. Humans and animals may become infected
through direct contact with contaminated urine
or indirectly through exposure to contaminated
water or soil.
In the current year, the gold-standard test for
Leptospira diagnosis in man and animals, the
Micro-Agglutination Test (MAT) was established
in this laboratory. Training and reference sera were
received from Microbiology Department, College of
Veterinary & Animal Sciences, Mannuthy. Cultures
of the references strains were established in
Ellinghausen-McCullough-Johnson-Harris (EMJH)
medium. Temporary arrangements for Dark Field
examination of samples have also been established.
The epidemiology of Leptospirosis is being studied
at three levels.
PUBLICATION
»» Anoop Manakkadan, Iype Joseph, Raji Rajendran
Prasanna, Riaz Ismail kunju, Lalitha Kailas and
Easwaran Sreekumar Lineage shift in Indian
128
1. Clinical epidemiology of Human Leptoispirosis:
This aspect is being studied along with
clinical collaborators. The initial study is a
retrospective analysis of the clinical pattern
of the leptospirosis cases admitted to the
hospitals where the collaborators are attached.
All study plans involving human subjects have
been submitted to Institutional Human Ethics
Committee.
2. Carrier state in rodents: Rodents are considered
to contribute significantly to Leptospira
transmission. Wild rodents (rats) are being
trapped, kidneys harvested and cultured.
Necessary clearance from Institutional Animal
Ethics Committee has been received.
3. Exposure to Leptospira among the high risk
population of Animal Handlers: Leptospirosis
is an occupational hazard for Animal Handlers.
In collaboration with the Animal Husbandry
Department, Government of Kerala, work has
been initiated to estimate the exposure among
Veterinarians to the various serogroups. 103
blood samples of Veterinarians currently in
government employment have been collected
for MAT testing.
strains of Dengue virus serotype-3 (Genotype
III), evidenced by detection of lineage IV strains
in clinical cases from Kerala. Virology Journal
2013 10:37.
Research Scientist
(Bioinformatics & Computational Biology)
Reshmi G
[email protected]
Reshmi took her M.Phil and PhD in Bioinformatics from the University of Kerala and then
trained at the Oklahoma Medical Research Foundation in USA. Reshmi is supported by
the ICMR - National Viral Disease Network.
Host-Pathogen Protein Interaction Predictor: A sequence based
computational approach
Several experimental data sets prove that
unknown protein–protein interactions involved
in host-pathogen relations coordinates complex
signaling formation and protein networking
by reversibly interacting with multiple binding
partners. Understanding the selectivity of
such domains helps to elucidate how defects
in proteins and their binding partners are involved
in human diseases. Since experimental methods to
determine interaction specificity of the domains
are expensive and labor intensive, an accurate
computational method is a prime requirement.
We developed a support vector machinebased predictor using dipeptide composition that
was shown to qualitatively predict protein-protein
interaction with a high accuracy rate. We chose
the SVM classifier with a radial basis function
to perform the classification, since it provides
high classification results and is fairly resistant to
feature selection. The software LIBSVM version
3.0 was employed in this work. The regularization
parameter C and kernel parameter ³ in the SVM
were selected by using a grid search approach.
We trained the classifier using H1N1 and DEN-V
proteins interacting with experimentally verified
host proteins. We trained 18 common viral pathogen
interactions with the corresponding host protein
sequences.To verify the reliability of our prediction
model, we tested it with independent validation
datasets from experimentally verified sources.
Our method correctly classifies binding and nonbinding interactions. We anticipate this approach
to provide several diverse classes of potentially
good binders that can serve as a resource for
possible identification of active antiviral targets.
129
Figure: Internal Architecture of proposed tool
Research Grants
Investigators
Title
Computational biology for
design of optimum Vaccine
candidates and development of
diagnostics for classical swine
fever virus.
National Virology Network
Sara Jones
Antibody response to seasonal
and pandemic(H1N1) influenza
virus outbreaks in Kerala
130
Funding Agency
Duration
2008-2013
Government of India
Indian Council of Medical
Research,
Government of India
2011-2016
2011-2014
Kerala Biotechnology
Commission
Cholera and Environmental Microbiology Laboratory
Sabu Thomas, PhD
Scientist E1
[email protected]
Sabu Thomas received his Ph.D in aquatic biology from the University of Kerala and joined
RGCB in 2001.
PhD students:
Wilson Peter Abraham
Divya M.P
Akhilandeswarre D
Project Fellows:
Nimmy Augustine
Karthika .S
Deepa Mathew P
131
Molecular Characterisation of Environmental Vibrio parahaemolyticus
and its Biofilm Formation
Divya M.P. and Sabu Thomas
Vibrio parahaemolyticus, a Gram-negative
bacterium is commonly found in coastal and
estuarine waters. The organism can accumulate
in high numbers in shellfish and frequently
associated with gastroenteritis in humans following
shellfish consumption. The pathogenicity of the
bacterium classically has been correlated with
the production of Thermostable Direct Hemolysin
(TDH) and TDH Related Hemolysin (TRH). Genome
sequencing of the V. parahaemolyticus clinical
strain RIMD2210633 revealed that it possesses
two sets of type 3 secretion system (T3SS) genes:
T3SS1, located on chromosome 1, and T3SS2,
located on chromosome 2 (Makino et al, 2003).
T3SS1 is responsible for the cytotoxicity of V.
parahaemolyticus for eukaryotic cells while T3SS2
is involved in cytotoxicity and enterotoxicity. Till
now, four major serotypes – O3:K6, O4:K68, O1:K25
and O1: KUT (untypeable) have caused a pandemic
of V. parahaemolyticus infection. O3:K6 was first
identified in India in 1996. Pandemic serogroups
possess the group-specific (GS) gene sequence
in the toxRS operon and ORF8, of the 10 known
open reading frames (ORFs) of the O3:K6-specific
filamentous phage f237. We collected samples
(water, plankton and sea-food) from different
locations of the coastal areas of Kerala. We have
identified 556 isolates of V.parahaemolyticus
by biochemical and molecular tools. Virulence
profiling revealed that18 isolates were found to
possess tdh (tdh+trh-) while 2 have trh (tdh-trh+).
The 2 trh+ strains and 17 tdh+ strains gave
positive amplification with GS-PCR, indicating
their pandemic potential (Table 1). Further work
will characterise the T3SS in these isolates and
understand their pathogenic effect of the strains
on Caco-2 intestinal epithelial cells.
132
Table 1: Summary of potential pathogenic strains
Strain
toxR
tdh
trh
GS
E1
+
+
-
+
E2
+
-
-
+
E3
+
+
-
+
E4
+
+
-
+
E5
+
+
-
+
E6
+
+
-
+
E7
+
+
-
-
E8
+
+
-
+
E9
+
+
-
+
E10
+
+
-
+
E11
+
+
-
+
E12
+
+
-
+
E13
+
+
-
+
E14
+
+
-
+
E15
+
+
-
+
E16
+
+
-
+
E17
+
+
-
+
E18
+
+
-
+
E19
+
+
-
+
CCB12
+
-
+
+
CCB13
+
-
+
+
Quantification of Pathogenic Gram Negative Bacteria
using Infrared Thermography
Divya M. P. and Sabu Thomas
Collaborator: Dr. John Philip, Indira Gandhi Centre for Atomic Research (IGCAR), Kalpakkam,
Tamil Nadu
Detection of bacteria is of fundamental importance
to all microbiological investigations. Conventional
method (standard plate count) is time consuming
whereas newer rapid methods (fluorescent in situ
hybridisation, flow cytometry, biosensors) require
altering the bacteria by staining. The need of the
hour is a simple, fast and sensitive technique that
eliminates modification of the samples. All living
organisms, including bacteria, produce heat due
to basic metabolic processes. This emanated
heat energy can be detected by thermal sensing
technique, Infrared Thermography (IRT). The
present study uses IRT video, thermodynamics
laws and heat transfer mechanisms to directly
measure, in real-time, the amount of energy
lost as heat from the surface of a liquid sample
containing bacteria when the specimen cools to a
lower temperature over 2min. Four Gram negative
bacteria Vibrio cholerae O1, V. mimicus ATCC
33653, Pseudomonas aeruginosa ATCC 27853
and Proteus mirabilis ATCC 7002 were used for
the experiments. 10-fold dilutions of the bacteria
ranging from 101-1011 were prepared and aliquots
inoculated in triplicates into appropriate wells of
the microtiter plates. The microtiter plates were
Fig.1: Infrared images showing
temperature of the 96-well
microtiter plates containing V.
cholerae after (a) 60 and (b) 120 s
incubated at 370C for 20 minutes. After thermal
equilibrium was established, the plates were
placed inside a chiller chamber (2.2⁰C) and the
surface temperature of each well was measured
by the IR camera over 2min. Immediately after
the IRT experiment, the bacterial samples were
processed by conventional plate count method for
determination of the viable counts. It was found that
the temperature of the wells decreased with time
due to energy loss (Figs 1& 2). The heat generated
was directly proportional to a dimensionless
quantity called Energy Content (EC), which is the
ratio of the heat generated by bacterial metabolic
activities to the heat lost from the liquid medium
to the surrounding. As bacterial concentration
increases, the amount of heat generated will also
increase. This linear correlation between EC and
viable count can be seen in Figs 3 & 4. The observed
variation in the EC with species could be attributed
to their metabolism and morphology. Our results
showed that IRT could be employed as a real-time,
non-contact and non-labour intensive alternative
for quantification of bacteria and has the potential
for automation and high throughput
Fig.2: Temperature fall as a function of time for the sample
133
Fig.3: The energy content (EC) values at three time intervals (0, 60 and 120s) for different bacterial concentration
for (a) V. cholerae, (b) V. mimicus, (c) P. mirabilis and (d) P. Aeruginosa wells of V. mimicus
Fig.4: Plot of EC vs. log10 (viable count) of the bacteria
Identification of potential targets for inhibiting biofilm formation in the
pathogenic members of Vibrionaceae family
Akhilandeswarre D and Sabu Thomas
Collaborators: Dr.R. Sowdhamini, National Centre for Biological Sciences, Bangalore ,Dr.
Abdul Jaleel K.A, RGCB
Treating the infection caused by Vibrio pathogens
are difficult due to the biofilm mode of life which
allow them to be highly resistant to antimicrobial
agents and host immune responses. Vibrionaceae
family includes four human pathogens viz., V.
cholerae, V. parahaemolyticus, V. vulnificus and V.
alginolyticus, which have different modalities of
infection and clinical manifestations. The present
134
study focus on identifying and understanding
the conserved genes involved in the formation,
maintenance and dispersal of biofilms in the
pathogenic members of Vibrionaceae family to
come up with potential targets for inhibiting
biofilm formation. These targets could be used
in Virtual high throughput screening technique
for developing drug candidates with antibiofilm
activity. For identifying the conserved potential
targets, we sought to determine the conserved
genes involved in the various stages of biofilm
formation in the Vibrionaceae family members by
using computational approaches and proteomic
techniques.
The microar ray data at various stages of
planktonic and biofilm stages of representative
Gammaproteobacteria (from Microarray databases)
were analyzed to find the differentially regulated
genes. Gene Set Enrichment Analysis (GSEA) was
done for the differentially regulated genes, thereby
identifying the pathways and genes specific to the
biofilm condition in Gammaproteobacteria (Table 2).
The GSEA results generated from the transcriptomic
data are then combined with the proteomic data
(Wetlab) for identifying the conserved targets
against biofilm formation in Vibrionaceae family.
In connection to this proteomic work, the biofilm
formation assay at various time points were done
for V. parahaemolyticus SC192 (Figure 5 & 6) and the
complete proteome profile of V. parahaemolyticus
during the 12hr, 24hr and 48hr planktonic stage
and 24hr and 48hr biofilm stage was generated
employing Nano-Liquid Chromatography coupled
Tandem Mass spectrometry. From the initial
analysis, it was found that 245 proteins are specific
to the biofilm condition of the V. parahaemolyticus
SC192. Further in silico analysis are in progress to
find the lead proteins that could act as potential
targets for inhibiting biofilm condition.
Table 2: Genes and pathways specific to biofilm formation in Gammaproteobacteria
Biofilm stage
Induced pathways
Monolayer biofilm stage
Hypothetical proteins, Flagella biosynthesis,
Chemotaxis proteins, Type IV Pilin biosynthesis, Two component response
regulators & GGDEF/EAL family proteins
Multilayer biofilm stage
Transporter proteins, Binding proteins and Carbohydrate metabolism
Fig.5: V. parahaemolyticus SC192 biofilm stained
with 1% crystal violet. a – 12hr old biofilm; b –
24hr old biofilm; c – 48hr old biofilm.
Fig.6 : V. parahaemolyticus SC192 biofilm stained with LIVE/
DEAD® BacLight™ Bacterial Viability Kit (Invitrogen) and viewed
using CLSM (Nikon, Melville, NY). a, b – 12hr old biofilm; c, d –
24hr old biofilm; e, f – 48hr old biofilm; a, c, e – Image showing a
single stack (XY plane); b, d & f – 3D image (XYZ plane).
135
Novel Biofilm inhibitors against Vibrio cholerae from selected plants:
Isolation and Characterization
Nimmy Augustine and Sabu Thomas
Collaborators: Dr. A.K.Goel, Defense Research & Development Establishment (DRDO),
Gwalior, Dr. R.Ajaykumar, RGCB
Vibrio cholerae is the causative agent of the
water borne disease cholera that still threatens a
large proportion of world’s population. Cholera is
characterized by severe diarrhea and sometimes
even death. Biofilm development plays an important
role for the survival as well as sustenance of V.
cholerae during and after epidemic period. Biofilms
protect the bacteria against antimicrobial agents.
As multidrug resistance in V. cholerae is increasing,
there is an urgent need for novel compounds
that arrest its pathogenic traits such as biofilm
formation virulence factors rather than killing
the bacteria. Usage of antimicrobial compounds
derived from medicinal plants can be dated back to
many centuries and it is well accepted worldwide.
Though extensive studies have been done on the
antimicrobial properties of medicinal plants, very
limited studies have been done to explore the antipathogenic or anti-biofilm activity of the same. In
this study, we have investigated the antibiofilm
activity of main antidiarrhoeal plants.
Based on the literature available, list of selected
plants used against diarrhoea was prepared for
initial screening. Selected plant materials were
collected from Ayurveda Research Institute (ARI),
Poojappura, Trivandrum and different geographic
locations. The plant materials were thoroughly
washed and shade dried and made into fine
powder using a grinder. The powder of each
sample was later extracted with methanol for 24
hr and extract was concentrated using a rotary
evaporator and filtered through a 0.45µm filter. The
filtrates were later dried in dry bath at 40°C. The
extracts were dissolved in DMSO so as to prepare
a stock of 100mg/ml. Different concentrations of
these extracts were checked for biofilm inhibition
against V. cholerae O1 using crystal violet assay.
The inhibitory activity was again confirmed with
cover slip assay using confocal laser scanning
microscopy (CLSM).
Biofilm assay revealed that sub inhibitor y
concentrations of Elephantopus scaber, Centella
asiatica and H. antidysenterica could inhibit biofilm
formation in V. cholerae O1 significantly. Extract
of E. scaber at 2mg/ml could inhibit upto ~76%
biofilm formation and ~56% at 1mg/ml. Above
2mg/ml, the extract showed bactericidal activity. C.
asiatica extracts at 3mg/ml could reduce upto ~75%
biofilm and ~52% at 2mg/ml. H. antidysenterica
extract at 600µg/ml could reduce the biofilm
formation upto ~55% without inhibiting the
growth of bacteria. All the plants analysed
showed antibacterial activity at higher
concentrations. Confocal laser scanning
microscopy (CLSM) analysis confirmed
the inhibitory activity which showed the
reduction in adhesion of the cells onto
the cover slip surface (Fig.7). The biofilm
inhibitory activity of extracts could be due
to the reactivity of the bioactive compound
present in the extracts with secreted
molecules such as exopolysaccharides
or other adhesins or proteins involved
in biofilm formation. Purification and
characterization of the bioactive compound
would be proceded with the plant showing
most significant biofilm inhibitory activity.
Fig.7: CLSM analysis of V. cholerae biofilm. a) V. cholerae
(untreated-positive control), b-d). Biofilm treated with E. scaber,
C. asiatica and H. antidysenterica extracts.
136
Characterization of Polymicrobial Communities and its Biofilm Matrix
Associated Components in Chronic Wound Infections
Karthika S and Sabu Thomas
Clinical Collaborator: Dr. N. Harrison, Sr. Surgeon, S.K. Hospital, Trivandrum
Biofilms are structured community of heterogeneous
bacterial cells enclosed in a self-produced
polymeric matrix and their inherent resistance
to antimicrobial agents is the root cause of many
persistent and chronic bacterial infections in
humans. Among these pertinacious infections,
chronic wound infection is a major worldwide
health problem causing significant morbidity
and mortality. Research conducted so far have
revealed the implication of polymicrobial biofilms
in preventing the wound healing process which
eventually develop into chronic wounds. The
extracellular matrix associated with the biofilm
makes the polymicrobial community a stable
microconsortium and renders antimicrobial
resistance, hence making the infection difficult to
eradicate by conventional antibiotic therapy. In
the present scenario, it is significant to understand
the diversity, spatial distribution and organization
of chronic wound microbiota for proper diagnosis
and effective treatment of such chronic infections.
Analyzing the composition and sharing of the
biofilm associated matrix components among the
polymicrobial communities enables the treatment
by focusing the potential targets for biofilm
dispersal, thereby inducing the healing process in
an orderly and timely manner.
The chronic wound samples were collected from
clinical settings by swabbing and debridement
method and cultured onto selective and nonselective differential enrichment media. The isolates
were identified by 16S rRNA sequencing and the
preliminary results showed that Staphylococcus
sp. and Pseudomonas sp. were found to be the
predominant genera associated with the wound
infections analyzed so far. The other species that
are consistently identified are shown in Table 3. The
metagenomic analysis to interpret the uncultured
microbial community associated with the wound
biopsy specimens are in progress. This study
centering the architecture of bacterial biofilms
and the role of differentially expressed proteins in
biofilm mode of life will enable the development
of next generation therapeutics which will focus
on dispersal of mature bacterial biofilms in nonhealing chronic infections.
Table 3. Microbial community composition of chronic wound samples
Sl.
No.
Sample
name
Disease condition
Organisms identified
1
CW1
Diabetic ulcer & Pressure ulcer
Escherichia coli, Staphylococcus epidermidis,
Staphylococcus sp.
2
CW2
Diabetic ulcer ischemic
Escherichia coli, Pseudomonas aeruginosa, Proteus sp.
3
CW3
Diabetic ulcer
Serratia marcescens, Pseudomonas sp., Serratia sp.
4
CW4
Diabetic ulcer
Staphylococcus hominis, Bacillus sp
5
CW5
Diabetic ulcer
Enterococcus faecalis, Staphylococcus hemolyticus,
Streptococcus pyogens
6
CW6
Diabetic ulcer
Morganella morganii, Proteus mirabilis
7
CW7
Diabetic ulcer & Pressure ulcer
Acenitobacter baumannii
8
CW8
Accident wound
Enterobacter sp.
9
CW9
Injury
Pseudomonas sp. Klebsiella pneumoniae
10
CW10
Injury
Pseudomonas sp. Staphylococcus sp., Klebsiella sp.
11
CW11
Pressure ulcer
Stenotrophomonas maltophilia/ Pseudomonas sp
137
12
CW12
Diabetic ulcer, Carbuncle back
Staphylococcus aureus, Staphylococcus sp.
13
CW13
Abscess
Staphylococcus sp.
14
CW14
Accident wound
Bacillus sp.
15
CW15
Diabetic, severe cellulitis
Staphylococcus sp,
Screening of novel genes involved in cold adaptation from
Pseudomonas psychrophilla isolated from the Arctic fjord
Wilson Peter Abraham and Sabu Thomas
The psychrophilic and psychrotrophic bacteria
that could grow at 0°C or below, populate the
cold environments of our planet including Arctic
and Antarctica regions. How these bacteria
cope with low temperatures induced stress is
poorly understood. Also, fundamental questions,
such as what roles the cold-induced proteins in
psychrophilic bacteria play at low temperature,
are waiting to be elucidated. The ability of
these organisms to sur vive and proliferate
at low temperatures implies that they have
overcome key barriers inherent to permanently cold
environments. These challenges include reduced
enzyme activity, decreased membrane fluidity,
altered transport of nutrients and waste products,
decreased rates of transcription, translation and
cell division, inappropriate protein folding and
intracellular ice formation.
The psychrotrophic, gram negative, rod shaped,
aerobic strain 166 isolated from Arctic fjord has
been chosen to study the mechanism towards
extreme low temperature. Isolate was found to grow
at a relatively broad salinity and temperature range.
To screen the differentially expressed genes at low
temperature bacteria was grown at 40C and 370C.
RNA was isolated in two different temperature
conditions and cDNA is prepared and is subtracted
keeping cDNA at 40C as tester and 370C as driver
cDNA. This technique called the suppressive
subtractive hybridization enables selective
amplification of differentially expressed sequences.
Upon sequencing some of the subtracted cDNA
clones we are able to identify five genes and seven
hypothetical proteins as shown in Table 4. Real
time expression analysis of DNA repair protein
(Clone 2SSH) shows that it is a cold inducible
gene in which 16SrRNA is used as the reference
gene (Fig.8). Nearest phylogenetic relation of the
test organism was found out to be Pseudomonas
psychrophila by sequencing 16S rRNA. Future
work include real time analysis of other selected
genes at 4°C and to construct a deletion mutant to
study the functional analysis of the selected gene
towards low temperature. Our study will help the
understanding of the mechanisms by which the
Arctic bacteria respond to cold shock and adapt
to low temperature.
Table 4. Differentially expressed genes and their functions
Gene
Functions
DNA mismatch repair protein
Repairs insertion, deletion & misincorporation of bases
(Ray et al.,2005)
Transport related membrane protein
Reduction of freezing point (Russel et al., 1998)
Periplasmic lipoprotein
Reduction of freezing point
Methyl accepting chemotaxis transducer
Increases methyl branching
(Jagannatham et al., 2000)
RNA polymerase sigma factor
Bacterial transcription initiation factor
Hypotheical proteins (7nos)
Function unidentified
138
Fig.8: Expression profile of the SSH2 (DNA repair protein) at 37°C and 4°C.
Screening of antibiotic resistant genes in bacteria isolated from the Ar
Deepa Mathew P and Sabu Thomas
The emergence of multi-drug resistance in
clinical and environmental strains of pathogens is
becoming a major problem faced by the clinicians
and the scientific community. However, the
emergence of multi-drug resistant bacteria in the
polar ice caps has only been recognized recently.
Both poles of the Earth have been geographically
isolated for millions of years and have experienced
very less anthropogenic influences. In this study,
sediment and water samples were collected from
Ny-Alesund, in the Svalbard Archipelago (79° 55’
N, 11° 56’ E) situated on the shore of Kongsfjorden,
during the Indian Arctic Expedition 2009,
organized by the Ministry of Earth Sciences. The
sediment and water samples were serially diluted
and plated onto nutrient and marine agar plates
and incubated at 10°C for 3-4 days. All isolates
were tested against the common antibiotics
using disc diffusion method according to the
criteria recommended by the EUCAST (2010).
Based on the antibiogram profile, highly resistant
bacteria were selected for plasmid profiling and
screening of antibiotic resistance genes (Table 5).
We selected isolate A73 for further mechanism
based studies. The resistance of A73 to cefoxitin
is suggestive of AmpC beta-lactamase production.
AmpC beta-lactamases are clinically important
cephalosporinases encoded on the chromosomes of
many Enterobacteriaceae and few other organisms,
where they mediate resistance to cephalothin,
cefazolin, cefoxitin and most penicillins and are
not affected by available beta-lactamase inhibitors.
A73 was subjected to shot gun cloning for screening
of AmpC beta lactamase. The total genomic DNA
of A73 and pUC vector was digested with Sau 3AI
and BamHI and ligated and cloned in to MACH1
and plated onto LB plates containing respective
antibiotics. The positive colonies were subjected to
plasmid isolation and sequencing. Results showed
that majority of the isolates showed MDR pattern.
All the bacterial isolates analysed were resistant
to most of the antibiotics tested except a few like
chloramphenicol, ciprofloxacin and polymyxin B.
All the test strains were found to be sensitive to
ciprofloxacin. Plasmids of varying size were present
in all isolates analysed (Table 5). Shot gun cloning
results revealed that AmpC beta lactamase gene
was found in isolate A73 (Fig.9). Further studies are
needed to elucidate the mechanism of antibiotic
resistant gene transfer and its possible origin in
psychrophilic bacteria from this virgin environment.
139
Table 5.Plasmid and Antibiotic resistance profile of bacterial isolates collected from Ny- Alesund in the
Arctic
Sl.No
Bacterial
isolates
1
A228
Water
Nil
A, Cp, Fr, Tr
2
A140
Water
13-14 kb
A, Cp, Fr, Tr ,Cn
3
A511
Water
~9.5 kb
A, C, Cp, E, Fr, Tr ,Cn
4
A508
Water
~9.5 kb
A, Cp, E, Fr, Tr ,Cn
5
A30
Water
~9.5 kb
A, Ak, Cp, E, Fr, N, Tr, T
6
A15
Sediment
~9.5 kb
A, Ak, Ce, Cp, Fr, N, Tr, T
7
A98
Sediment
~9.5 kb
A, Ak, Ce, Cp, E, Fr, N, Tr
8
A73
Snow Ny- Alesund
~12 kb
A, Cp,Cn, E, Fr, Pb, Tr
9
A74
Snow Ny- Alesund
~5.2-5.3 kb, ~9.5 kb
A, Cp, Fr, Tr ,Cn
10
A8
Lake water
~5.2-5.3 kb, ~9 kb
E, Fr, Tr, Cp ,Cn
Source
Plasmid size
Antibiotic resistance profile†
†Ampicillin-A, Amikacin- Ak, Neomycin- N, Cephalexin- Cp, Cephotaxime- Ce, Erythromycin- E, Furazolidone- Fr,
Polymyxin B- Pb, Ciprofloxacin- Cf, Trimethoprim-Tr, Tetracycline- T, Chloramphenicol- C, Cefoxitin-Cn
Fig.9: PCR amplification of ampC gene in isolate A73. Lane M- 1Kb ladder; Lane 1-11&13 - recombinant clones of
A73 without ampC gene; Lane 12- recombinant clones of A73 with ampC gene
Extramural Grants
Title
Investigators
Funding Agency
Duration
Genetic manipulation of
Coirret-for application on coir
for quality improvement
Dr. U. S. Sarma (PI)
Dr. Anita Das (Co-PI)
Dr. Sabu Thomas (Co-PI)
Dr. Hari Krishnan.K (Co-PI)
Coir Board, Ministry of
MSME, Govt. of India
2011-13
Novel Biofilm inhibitors
against Vibrio cholerae from
selected plants: isolation and
characterisation
Dr. Sabu Thomas(PI)
Dr. R. Ajay Kumar (Co-I)
Dr. A.K. Goel (Co-I)
Defense Research
& Development
Establishment, DRDO,
Govt. of India
2012-14
140
Publications
Primary publications from Laboratory
»» 1.Nimmy Augustine, Wilson Peter, Savita Kerkar
and Sabu Thomas, 2012. Arctic actinomycetes
as potential inhibitors of Vibrio cholerae biofilm.
Curr Microbiol,64(4):338-42.
Publications with Collaborators
»» 2. Diana E Waturangi, Ignasius Joanito, Yogiara
Yogi and Sabu Thomas, 2012. Use of REP- and
ERIC-PCR to reveal genetic heterogeneity
of Vibrio cholerae from edible ice in Jakarta,
Indonesia, Gut Pathogens, 4:2
»» 3.Vinothkannan Ravichandiran, Karthi S,
Anupama K, Sabu Thomas, Adline Princy,
2012.Structure-based Structure-based virtual
screening for plant-derived SdiA-selective
ligands as potential antivirulent agents against
uropathogenic Escherichia coli, European Journal
of Medicinal Chemistry,48:200-205
»» 4.B.B. Lahiri, M.P. Divya, S. Bagavathiappan,
Sabu Thomas and John Philip, 2012.Detection
of Pathogenic Gram Negative Bacteria using
Infrared Thermography, Infrared Physics &
Technology,55:485-490.
»» 5. B.Sadia Khan, Anil Kumar, Divya MP, Sabu
Thomas, Deepa Harichandran and Shamsul
Karim,2013. Fatal non-O1/ non-O139 V.cholerae
Septicaemia in A Patient with Chronic Liver
Disease: A Case Report and Review of Literature.
Journal of Medical Microbiology, February 28, 2013
as doi:10.1099/jmm.0.049296-0
Book Chapter
»» S. Thomas, S Jagadeeshan, P Kumar, S. Mundayoor
and Yung Tse, 2012. Drinking Water Associated
Pathology. Hand Book of Environmental
and Waste Management,Volume 1-Air and
Water Pollution Control. (Eds. Yung-Tse Hung,
Lawrence K. Wang, Nazih K. Shammas, USA).
World Scientific Publishing Co. Pte. Ltd,
Singapore.pp175-236.
»» Sudha A, Wilson P.A and Sabu Thomas, 2012.
Psychrophiles – A plethora of perspectives.
Bacterial Biochemistry and Biotechnology.
Lambert Academic Publishing. New Delhi. pp.
158-167.
International Conference Presentation
»» Nimmy Augustine, Akhilandeswarre D and Sabu
Thomas, 2012. Arctic Actinomycetes as potential
source of biofilm inhibitors against Vibrio cholerae.
International Conference on Regulatory Network
Architecture in Bacteria, SASTRA University,
Mar.9-11, Tamil Nadu, India.
»» Praveen Kumar and Sabu Thomas, 2012.
Molecular characterization of Vibrio cholerae
isolated from the aquatic sources in Southern
Kerala. 15th International Congress on Infectious
Diseases (ICID), June 13-16, Bangkok, Thailand
»» Akhilandeswarre D, 2012.Indo-global Education
Summit & Expo 2012 organized by The Indus
Foundation, September 7- 9, Taj Krishna & Taj
Deccan, Hyderabad.
Invited Lecture
»» Karthika S,Wilson PA,Harikrishnan K and
Sabu Thomas,2012. Biotechnology: A Panacea
for Industrial Renaissance, National Seminar
on Sustainable Development through new
Technologies,14th August, Kochi, Organised by
Coir Board, Govt. of India
Seminars organized
»» Sabu Thomas: Organizing Convenor, International
Conference on Regulatory Network Architecture
in Bacteria (RNAB), March 9-11,2012, Organized
by SASTRA University, Thanjavur and Rajiv
Gandhi Center for Biotechnology
Training courses attended
»» Akhiladeswarre D : Workshop on “Microarray
Data Analysis using R/Bioconductor ”,
jointly organized by Rajiv Gandhi Centre for
Biotechnology and Oklahoma Medical Research
Foundation, USA at Rajiv Gandhi Centre for
Biotechnology, Thiruvananthapuram, Kerala,
India during January 16 – 18, 2013.
Genbank Submissions
»» Karthika S and Thomas S. Partial sequence of
16S ribosomal RNA gene of Streptomyces sp.
A742 isolated from Arctic sediment. Accession
no. KC153537,2012
»» Divya M.P., Anil Kumar V and Sabu T. Partial
sequence of 16S ribosomal RNA gene of Vibrio
cholerae non-O1/non-O139 strain isolated from
blood of a patient with chronic liver disease.
Accession no. JX987303,2012.
141
Environmental Biology Laboratory
Dr. Hari Krishnan. K, Ph. D
Scientist C
[email protected]
Hari Krishnan is a Ph.D in Aquatic Biology from the University of Kerala and joined RGCB
in 2001.
PHOTO OF LAB MEMBERS
Ph.D Student:
Arjun. J.K (UGC JRF)
CSIR JRF:
Aneesh. B
Project Fellow:
Kavitha . T
142
Project Assistant:
Geetha. S. L
1. Purification and characterization of L-Asparaginase from
soil metagenomic Library
Arjun. J.K and Hari Krishnan. K
L-asparaginase (L-asparagine amidohydrolase,
E.C.3.5.1.1) is an anti-neoplastic agent used
in the chemotherapy of Acute Lymphoblastic
Leukemia (ALL) and Lymphosarcoma in humans
for the last 25 years. The potential utility of the
enzyme in treating ALL was first noticed when
the Guinea pig serum had an inhibitory effect on
the lymphoma cell proliferation in mice and later
it was found that the L-asparaginase activity of
the serum was responsible for the inhibition on
tumor cell proliferation. Its antileukemic activity
is attributed to the inability of neoplastic blast
cells to synthesize asparagine from aspartic acid
as they are deficient in L- asparagine synthatase.
However lymphatic tumor cells require large
amounts of L – asparagine in order to achieve rapid
malignant growth. L-Asparaginase reduces the
activity of L-asparagine in the circulatory system
by the hydrolysis of L-asparagine to aspartic acid
and ammonia, leading to selective starvation
of tumor cells, inhibiting protein synthesis and
cytotoxicity of lymphoblastic cells. Later it was
found that the action of the enzyme is coupled
with some side effects and moreover the yield of
the enzyme was not enough to fulfil the demands
of the drug. This created the need to discover new
sources and techniques to enhance the yield and
decrease the side effects of the enzyme. Apart from
its therapeutic application the enzyme has been
used in food industries and for diagnostic purposes.
L-Asparaginase is a relatively wide spread enzyme
found in many plants and microorganisms.
Bacteria are the proven potential sources for
L-Asparaginase, since they can be easily cultured
and the extraction and purification of the enzyme
from them are convenient, facilitating the Industrial
scale production. Asparaginases from Escherichia
coli and Erwinia chrysanthemi are currently under
clinical use as effective drugs in the treatment of
ALL. However, all the reported L- Asparaginase so
far is from less than 1% of culturable microorganisms
present in the environment. The vast majority of
the uncultivable fraction is still unexplored. The
harmful side effects reported from the available
drugs from E. coli and Erwinia also demands
the need to isolate serologically compatible and
therapeutically more effective L-Asparaginase from
novel sources.
Metagenomics is the modern approach to explore
the vast majority of uncultivable microorganisms
present in various environments. In this study,
searching for novel and potential L-Asparaginase,
we have generated a metagenomic library from
the soil collected from forest in the biodiversity
rich Western Ghat region. Since the generation
of large insert metagenomic library is ideal to
access the gene clusters involved in the synthesis
of many of the novel molecules produced by
uncultured fraction of microorganisms present
in the environment, the library was constructed
by ligating 35 kb metagenomic DNA in a BAC
(Bacterial Artificial Chromosome) vector. The
library consists of 1368 clones covering an average
of 4.8× 104 kb of metagenomic DNA. The clones
in the library were screened for L- Asparaginase
production with modified M9 minimal media
supplemented with L-asparagine as the sole
nitrogen source and phenol red as indicator. The
asparaginase positive clones produced pink color
after incubation at 37 0C for 48 hours due to
pH change by the formation of ammonia as the
byproduct (Fig 1). Among the clones, five clones
(ASP1 – ASP5) showed considerable enzyme activity
and were subjected to further studies. The clones
were grown in asparaginase production medium for
36 hrs at 370C. Crude enzyme was prepared by cell
disruption using ultrasonication
and the cell free supernatant
obtained after centrifugation
was subjected to enzyme assay.
L-Asparaginase activity was
determined by measuring the
amount of ammonia liberated
by the enzyme. Among the five
clones, Clone ASP 1 showed
maximum specific activity 0.361
143
16S rRNA gene was amplified
using eubacterial primers. The
amplified gene was sequenced
and the sequence compared
with the public database (NCBI
BLAST). Homology search of 16S
rRNA sequence of clone ASP-1
showed 100% similarity with an
uncultured Serratia sp.
U/mg (Fig 2) suggesting that the clone may be a
promising source for a novel L-Asparaginase.
The phylogenetic relationship of the clone ASP
1 was assessed by isolating BAC DNA from the
clone using Nucleobond BAC-100 DNA isolation
kit (Macherey and Nagel, Germany) and the
The present study suggests the
possibility of the clone ASP1 as a promising source of
L-Asparaginase and needs further
characterization. Studies are
progressing to characterise the
cytoplasmic and periplasmic fractions of the
enzyme from the clone, to assess the effect of
various metal ions on enzyme activity and to check
the cytotoxicity of the enzyme on various cell lines.
2. Production and characterization of Penicillin G Acylase from a
Bacillus sp. isolated from forest soil
Arjun. J.K and Hari Krishnan. K
The development of resistance by microorganisms
to penicillin G and other β lactam antibiotics has
prompted the search for new β lactam antibiotics.
Microorganisms are an important source of
Penicillin G Acylase (PGA) (EC 3.5.1.11), which
hydrolyzes Penicillins to 6-aminopenicillanic acid
(6- APA) and Cephalosporin to 7-amino desacetoxy
cephalosporanic acid (7-ADCA), the β lactam
antibiotic intermediates which are widely used
by the pharmaceutical industries as the starting
materials for the manufacture of several semi
synthetic antibiotics. β- Lactam antibiotics, in
particular penicillins and cephalosporins, represent
one of the world’s major biotechnology markets.
β- Lactam antibiotics alone constitute most of
the world’s antibiotic sales: 3×107 kg/year out of
a total 5×107 kg/year produced worldwide and
hence the annual consumption of PGA is estimated
to be in the range of 10–30 million tons. Due to
the rising interest in sustainable development
and environmentally friendly practices, microbial
enzyme transformation processes are generally
preferred over the conventional chemical conversion
144
process. PGA-mediated conversion of β-lactam
antibiotics provides a novel direction for antibiotics
industries and promotes a safer and cleaner
environment. Apart from β-lactam hydrolysis,
recent developments have resulted in multiple
applications of PGA, including peptide synthesis,
resolution of racemic mixture, enantioselective
acylation. The characteristics of PGA isolated from
different biological and environmental sources were
found to be varied in different aspects including,
substrate specificity, optimum pH, temperature
tolerance etc. Therefore, microorganisms have
been extensively screened for isolation of novel
penicillin acylases with higher compatibility with
industrial deacylation requirements. Due to the
high industrial importance of PGA, searching for
microorganisms overproducing this enzyme is
highly warranted.
The study was an attempt to isolate PGA producing
bacterial strains from the forest soil in the Western
Ghats. The penicillin G acylase producing bacterial
isolates were screened and isolated from the soil
steady increasing trend during 2 - 10 hrs of
incubation and reached maximum activity
at 10 hrs (2.22 U/ml). An incubation time
beyond 10 hrs showed a steady decline
in the enzyme activity during which it
reached a value of 1.3 U/ml at 14 hr (Fig 4).
The presence of the gene responsible
for the synthesis of PGA in RG_PGA
269 was further confirmed by PCR using
gene specific primers which yielded an
amplicon of 2.35kb. In order to assess the
phylogenetic affiliation of the isolate, 16S
rRNA gene was amplified, sequenced
and and compared with the sequences
in the public database for homology
by NCBI BLAST. A phylogenetic tree
was constructed using the sequence of
the isolate studied along with selected
sequences from database using Mega V.4
software and the analysis revealed that
the isolate RG_PGA 269 formed cluster
with Bacillus megaterium
(Fig 5). The Bacillus sps.
are known to be a potent
source of PGA and there are
several reports suggesting that
the extracellular production
of the enzyme by Bacillus
megaterium makes the
downstream process much
easier and cost effective.
The results observed in the
present study suggests that
the strain isolated from the
forest environment can be
used as a potent source for the
production of PGA.
following Serratia marcescens overlay technique.
The enzyme producing strains exhibited a clear
zone, since penicillin G supplemented in the
medium was hydrolysed to 6-APA which inhibited
the growth of S. marcescens. While screening
around 300 environmental isolates, six of them
showed PGA activity. Among the six isolates,
RG_PGA 269 was found to be the more active
producer as depicted by a larger clearing zone (Fig
3) and was selected for further characterization.
Penicillin acylase activity was determined by
measuring the 6-APA liberated from Penicillin-G by
spectophotometric method at 415 nm. The enzyme
assay of RG_PGA 269 at 2 h interval showed a
Biological screening methods
have been used for the
isolation and identification of PGA producing
bacteria from different environments. From our
studies it was found that conventional screening
methods in conjunction with PCR amplification of
gene responsible for production may aid in more
accurate and precise screening of PGA producing
environmental isolates. The forests of Kerala are
known to be biodiversity hotspots and the microbial
resources in these environments are yet to be
exploited. As the microbial antibiotic resistance
continues to increase, it is essential to explore the
new sources of PGA residing in the environmental
pools, which are still unexplored.
145
3. Cloning and characterization of PHA synthase gene (phaC)
of Bacillus sp.
Aneesh. B, Kavitha. T and Hari Krishnan. K
Polyhydroxyalkanoates (PHAs) are biodegradable
and biocompatible polymers accumulated by a
wide range of microbes to overcome stresses from
their environment. The properties of PHAs range
from thermoplastics to elastomers which make
them a potential material with many applications
in various fields. Currently the commercial
production of the polymer is limited due to high
cost of production when compared to conventional
plastics. Hence, the biopolymer research world over
is mainly focusing on developing economically
feasible methods for the large scale production of
good quality biopolymer from microbes utilising
cheap and renewable substrates. In bacteria
polyhydroxyalkanoate biosynthetic genes are
clustered together as PHA operon
with phaA, phaB and phaC
genes which code for b Keto acyl
CoA thiolase, NADPH dependent
acetoacetyl CoA reductase and PHA
synthase respectively. Among the
gene products PHA synthase plays
the central catalytic role in PHA
synthesis and granule formation
by catalyzing the polymerization of
hydroxyacyl-CoA monomers to yield
PHA granules. Polyhydroxybutyrate
(PHB) is the most commonly
accumulated PHA in bacteria.
In this study we have screened out
58 PHB accumulating strains by
Sudan Black B staining from 210
bacterial isolates collected from the
diverse environments in Kerala. The
PHB accumulated by the strains
at 370C after 48 hr incubation was
recovered by solvent extraction and
estimated the polymer yield. Among
the isolates 6 comparatively high
yielding strains were subjected to
yield studies at different temperature
conditions. (Fig. 6). The potential
yielding strain (B3) which yielded
55% polymer from dried cell mass
at 37 0C was selected for further
studies. The polymer was confirmed
as PHB by Fourier –Transfor m
Infrared Spectroscopy (FT-IR). The
146
C=O stretching at 1723 cm-1 and C-H bending
1280 cm-1 in the FT-IR spectrum are typical to PHB
proving the accumulated polymer by the isolate B3
is PHB (fig.7).
As a part of the study on the role of the three
genes in the PHA operon for the accumulation of
PHB in bacteria, the PHA synthase gene (phaC)
which plays the pivotal role in PHB synthesis
was amplified from the bacterial strain B3, cloned
and sequenced. The sequence was subjected to
homology search in the public database, Protein
Data Bank (PDB) and protein modelling was
carried out with the sequence showing maximum
homology, using the software Modeller v9.11 (fig.
8). The predicted, partial, 3D structure of PHA
147
Synthase consists of 9 alpha helices and 5 betapleated sheets.
In order to check the phylogenetic affiliation of B3,
the 16S rRNA gene was amplified and sequenced.
Homology search of the sequence in the public
database (NCBI) revealed that the strain showed
maximum identity to a Bacillus sp.
Our long-term goal is to develop rational strategies
for the controlled synthesis of PHA biopolymers.
To achieve this, sequencing, cloning and gene
expression studies are the pre-requisites to
understand the characteristics of the genes
involved in bacterial PHA metabolism. The
availability of sequence information and the cloned
phaC genes from different organisms may help in
engineering new or improved PHA synthases for the
synthesis of novel biodegradable polymers. Further
studies are progressing to clone the PHA synthase
gene from B3 for the expression, purification and
characterization.
Extramural Grants
Title
A novel site specifically
targeting nanoparticle
based oral - drug
and siRNA releasing
polymer systems for
colon cancer
Investigators
Prof. M.Radhakrishna Pillai
(Project Coordinator)
G.S. Vinod Kumar (PI)
Hari Krishnan. K (Co-PI)
Asha Nair. S (Co-PI)
Bramanandam Manavathi (Co-PI)
Publications
»» Arjun. J.K, Aneesh. B and Hari Krishnan. K
(2013). Production and characterization of
penicillin G acylase from a Bacillus sp. isolated
from forest soil. Biotechnology, Bioinformatics and
Bioengineering, 3 (1) (In press)
»» Arjun, J.K., Aneesh, B. and Harikrishnan K
(2012). Production and characterization of
Penicillin G Acylase from a Bacillus sp. isolated
from forest soil. 2 nd National symposium on
Innovative and Modern Technologies for
Agricultural Productivity, Food Security and
Environment Management,19-20November,
2012, Thrissur, India
»» Aneesh, B., Arjun, J.K. and Harikrishnan
K (2012).Cloning and Characterization of
PHA Synthase Gene (phaC) of Bacillus sp.
isolated from the environment. 53 rd Annual
conference of Association of Microbiology of
India, International Conference on Microbial
World: Recent innovations and future trends,
22-25 November, 2012, Bhubaneswar, India
»» Arjun, J.K., Aneesh, B. and Harikrishnan K
(2013). Partial purification and characterization
148
Funding Agency
Department of
Biotechnology,
Govt. of India
Duration
2010-13
of L-Asparaginase from soil metagenomic
library. International Conference on Advances
in Biotechnology and Patenting, 18-21February,
2013, Trichy, India.
Genbank Submissions
»» Arjun,J.K., Aneesh,B., Bijoynandan,S. and
Harikrishnan,K. Prevalance of beta-lactam
resistance genes in Arctic soil. Uncultured soil
bacterium clones beta-lactamase (bla) gene,
partial cds. (Acc Nos. KC428420 to KC428432
– 13 sequences) (2012)
Training Courses Attended
»» Aneesh, B (2012). Mutagenesis: A powerful
Tool in Bacterial system. XXIV UGC NRCBS
Winter School, 4-18 July, 2012, Madurai Kamaraj
University, Madurai, India.
Awards and Honours
»» Hari Krishnan. K has been nominated as a
member of the State Level Expert Appraisal
Committee (SEAC), Kerala by the Ministry of
Environment and Forest, Govt of India
MOLECULAR REPRODUCTION LABORATORY - 1
Pradeep Kumar G
Scientist F
[email protected]
Pradeep Kumar was awarded PhD in Life Sciences from Devi Ahilya University, Indore in
the year 1988 for his Biophysical Studies of Sperm Membranes. He joined the Faculty of
Life Sciences of the same university in the year 1989. He worked as a Fellow-in-residence
at the Centre for Biomedical Research at the Rockefeller University, New York, NY; visiting
faculty in University of Virginia, Charlottesville, VA and as visiting faculty at University of
Florida, Gainesville, FL. His laboratory relocated to Rajiv Gandhi Centre for Biotechnology
in the year 2004.
PhD Students:
Indu S
Bhagya KP
Sreesha Sree
Divya Saro Varghese
Nomesh Yadu
Karthika Radhakrishnan
Soumya A
Devi AN
Technical Staff: Shyni
Jonhy G
Project Personnel:
Anil Kumar TR
Nithya Sekhar
Chinnumol VS
149
Germline stem cell maintenance, division and differentiation in the testis
(1) Isolation, characterization and differentiation of Germline Sem
Cells (GSCs) from neonatal and adult testis
Indu S, Bhagya KP, Devi AN, Nomesh Yadu and Pradeep G Kumar
was maintained in most tubules upto 4 months of
Busulphan treatment, with few tubules showing
regeneration. Effect of busulfan on germ cell
line GC-1 was evaluated. Results indicated the
occurrence of cell cycle arrest at G-2 phase and
subsequent apoptosis in busulfan treated GC-1
cells. Germ cell depletion for 4 months appears to be
good enough to perform a germ cell transplantaion
assay to establish the bona fide stem cell qualities
of GSCs purified. In order to make the outcome of
the GSC transplantation assay
unambiguous, we have entered
into an MOU with Prof. Polani
B. Seshagiri for the use of his
FVB/N-EGFP mouse as donor
and/or recipient of GSCs from a
wild type mouse. We are currently
breeding these FVB/N-EGFP mice
to establish a colony sufficient
enough to carry out the proposed
GSC purification and subsequent
ES-like colonies formed from GSCs
transplantation.
Neonatal and adult testis had a population of
cells expressing pouf51, fut4, plzf, tnap, ret, gfrα1,
integrin α6, integrin β1, ddx4, c-kit, zfp-42, dppa3,
ifitm3, tex19, gbx1, fgf5 and akp2 (stem cell
markers) along with vasa, dazl and sry (germ cell
markers). These cells have been purified using
MACS, and have been cultured under suitable
conditions to produce ES-like colonies that could
be coerced to express markers of all the three germ
layers. In order to establish that these cells can
establish spermartogenesis in
a germ-cell depleted testis, we
have successfully established a
germ cell depleted (GSD) animal
model by treating the animal
with a single intraperitoneal
injection of busulphan (45mg/ kg
body weight). Within 30 days of
Busulphan treatment complete
depletion of germ cells was
achieved. Germ cell depletion
Germ cell depletion in adult testis after treatment with busulfan
(2) miRNA-mRNA networks during the initiation of 1st wave of
spermatogenesis in mouse testis
Sreesha Sree and Pradeep G Kumar
We previously reported the identification of
germline stem cells (GSCs) in neonatal (P8)
mouse testis, and their continued existence in
adult testis (P24 and above) to support continuous
spermatogenesis. We have completed the profiling
150
the miRNA-mRNA networks during the progression
of 1st wave of spermatogenesis in mouse testis.
We identified 67 microRNAs and 3623 mRNAs
differentially expressed in mouse testis along the
1st wave of spermatogenesis. These two datasets
were integrated using Cytoscape (version 2.8.3)
to generate miRNA-mRNA network map. One
complete network consisting of the entire set of
differentially expressed miRNA- gene interactions
and two subnetworks per complete network could
be obtained for relative expression levels between
any two given stages (P16 vs P8 and P24 vs P16). Five
miRNAs (miR-21, miR-290-3p, miR-711, miR-714
and miR-762) were strongly downregulated, with
correlative upregulation of 98 targets. Twenty four
miRNAs (miR-34c, miR-34b, miR-449a, miR-743b3p, miR-742, miR-880 and miR-741, being the top
seven based on fold change) were upregulated that
correlated with the downregulation of 122 targets.
Expression pattern of few of these microRNAs
were validated by Real-time PCR. Also, a few
targets of these microRNAs were verified for their
expression pattern in the three age groups using
RT-PCR, qRT-PCR and western blotting. These
miRNA-mRNA networks during different stages
of sper matogenesis
were further classified
based on the enriched
functional groups
formed by the target
genes after assigning
functional annotations
u s i n g DAV I D v 6 . 7 .
This approach has
generated global
post-transcriptional
regulator y networks
operating in the mouse
Networks created by integrating differentially displayed miRNAs and nRNAs during
testis during the 1st wave
the 1st wave of spermatogenesis
of spermatogenesis.
(3) Functional characterization of critical molecules
regulating male factor fertility
Our past screening of germ cell proteome from
fertile and infertile human males identified >200
differentially displayed proteins which could
be categorized into transmembrane proteins,
receptors, cytoskeletal proteins, ion channels,
transcription factors, cell cycle regulators,
cell adhesion molecules, etc. Introducing a
bioinformatic filter of testis-specificity, we have
shortlisted a set of 19 differentially displayed
molecules for further functional characterization.
At present, we are undertaking functional studies
on 6 such molecules.
(a) Autoimmune Regulator (AIRE)
Bhagya KP, Karthika Radhakrishnan and Pradeep G Kumar
AutoImmune REgulator (AIRE) is one of the testisexpressed proteins identified in our laboratory, the
absence of which associated with spermatogenic
insufficiency. We cloned and expressed Aire from
human and mouse testis. Mouse testis expresses
Aire1a, and both transcripts and protein could be
detected from neonates to adults. Spermatogonia,
haploid round/elongated spermatids, primary
spermatocyte and secondary spermatocytes were
positive for AIRE on RT-PCR and western blots.
Quantitative real time analysis of P5 to P28 testis
showed that the level of Aire expression decreased
with age. Thy1+ cells from the testis (GSCs) were
positive for AIRE, suggesting that Aire could be
a player in pluripotency network in pre meiotic
germ cells.
Aire is a multidomain protein with CARD, SAND,
two PHDs and four LRRs and a bipartite NLS.
Mouse spermatogonial cells (GC1-spg) were
transiently transfected with Aire and its different
functional domains. Aire showed cytoplasmic
filamentous and nuclear speckle-like pattern in
full length and CARD domain constructs. Typical
filamentous pattern was lacking in deletion
constructs lacking 100 amino acids of N-terminus.
151
Annexin labeling and cell cycle analysis showed
that Aire-overexpressing cells were more prone to
cell death than controls. Western blot and qRT PCR
showed an elevated levels of Caspase-3 in GC1
cells over expressing CARD or full length Aire. But
no Caspases were found to be activated in germ
cells overexpessing Aire, suggesting the operation
of alternate death pathways. Though the CARD
domain of Aire was sufficient in sensitizing a cell
towards death by up-regulation of Caspase-3, Aire
expression alone is not sufficient to induce caspase3-mediated cell death.
We detected E3 ubiquitin ligase activity associated
with PHD domain of Aire. In presence of E1 and
E2 ubiquitin ligase, AIRE brought about the
degradation of Cyclin B2, which was inhibited
Over-experssion of AIRE causes apoptosis in GC-1 cells
by MG132, signifying that cyclin B2 is one of its
targets.
Different fractions separated were evaluated for Aire
expression using PCR and western blot analysis.
Aire protein appeared as a closely migrating
doublet in the isolated haploid cells. Upon exposure
to phosphatase, the higher molecular weight band
of the doublet disappeared with a gain in intensity
of the lower molecular component, suggesting a
potential phosphorylation of Aire in germ cells.
Real time PCR with RNA isolated from mouse
testis of different age groups showed an increased
expression of transcript in the early stages when
compared to mature stages. Treatment of adult
mouse with antiandrogen upregulated the
expression of Aire expression in testis, suggesting
that androgen-dependent germ cell differentiation
correlates with suppression of Aire levels.
Co-IP studies using AIRE-overexpressing GC1
cells and subsequent mass spectroscopy identified
hnRNP C1 - a molecule involved in RNA processing,
DNA strand break repair and apoptosis - physically
interacting with AIRE. Significance of this
observation and the functional relevance will
be addressed. We have initiated experiments
to evaluate the epigenetic regulation of gene
expression through the interaction of AIRE with
histones.
(b) TAR DNA Binding Protein 43 (TDP-43)
Divya Saro Varghese and Pradeep G Kumar
TAR DNA-binding Protein (43 kDa) is a ubiquitously
expressed nuclear protein known to regulate
multiple cellular events by transcriptional activation
Effect of silencing of TDP-43 on the expression of CLP-1
152
or repression of its targets.However, the functional
aspects of TDP-43 in spermatogenesis was not
clearly understood but for its transcriptional
repressor role of SP10, a round spermatid
specific acrosomal
protein. The objective
of our study is to
elucidate the function
of TDP43 expression in
testis and male germ
cell development using
murine in vitro cell line
model and semen from
fertile and infertile
human. Extrapolating
our studies on the
functional role of
TDP-43 in infertility in
mouse spermatogonial GC-1 cell lines, expression
profile of genes involved in infertility in TDP-43
over expressed/silenced system was evaluated.
Our findings show that silencing TDP-43 employing
siRNA to TDP-43up regulated the expression of
testicular variant of Cyclin like protein (Clp-1), a
meiotic cell cycle regulator.In the present study,
we have tried to unveil the cross- talk between
TDP-43 and Clp-1 in relation to cell cycle and
spermatogenesis.
(c) Protocadherin 11Y (PCDH11Y)
Anil Kumar TR and Pradeep G Kumar
The work aims at the evaluation of the expression
of Protocadherin X/Y (PCDHX/Y) in the fertile
and various infertile groups by RT- PCR, western
blot analysis and immunolocalization. We have
developed a PCR-based assay to discriminate
between X and Y homologues of Protocadherin11 and evaluated the expression of both of these
genes in the germ cells from fertile and infertile
human males. The study conducted, indicated
a quantitative reduction in the expression of
PCDH11Y at the transcript level in the infertile
samples when compared to their fertile counterparts
while the variant PCDH11X did not show much
variation among the various study groups. When
the normal spermatozoa showed good localization
of the protein, defective localization was noted in
the infertile sperm. To study the functional aspect
of the protein in the animal model N- terminal
extracellular domain of mouse Pcdh11X has been
generated and studies are in progress.
Localization of PCDH11Y on spermatozoa from fertile and subfertile human males
(d) Nephrocystin 1 (NPHP1)
Nephrocystin, coded by the gene NPHP1, is one
of the differentially displayed proteins associated
with male infertility that was identified in our
laboratory. In order to investigate the role of
nephrocystin in relation
to human male factor
infertility, characterization
of human testis expressed
Nephrocystin in
spermatozoa of fertile and
infertile males were carried
out by RT-PCR analysis. Of
the six Normal fertile and
twenty seven infertile cases,
RT-PCR analysis revealed
the absence of NPHP1gene
Expression of NPHP1 in spermatogenic cells from fertile and infertile human males
153
expression at the transcript level in majority of the
infertile males screened. Nephrocystin, having
a possible role at the maintenance of SertoliSpermatid junction, might be related to the
anomalies in the gene expression at the transcript
level in the male infertile cases, associated with
spermatogenic dysfunction and ultimately involved
in the pathogenesis of human male infertility.
(e) T-Complex Testis Expressed (TCTEX or DYNLT1)
We have optimized protocols for removal of somatic
cells and enrichment of germ cells in testicular cell
suspensions. Germ cell population was separated
into various fractions using FACS-based sorting
based on DNA content and HOECHST exclusion.
These fractions were analyzed for the expression
of TCTEX-1 and various markers of stemness and
differentiation using western blot analysis and RTPCR. This indicated strong indication that TCTEX1-positive cells expressed stem cell markers, and
the lack of TCTEX-1 was associated with the
expression of differentiation-related gene(s). We
have generated antisense construct of TCTEX-1.
Overexpression of TCTEX-1 in GC-1 cells has been
achieved, and the silencing experiments are in
progress. We are also optimizing culture conditions
and electroporation protocols to achieve acceptable
levels of changes in expression levels of TCTEX-1
in spermatogonial cell cultures without affecting
their viability.
Expression of sense- and antisense- constructs of TCTEX-1 in GC-1 cells
Biomembrane structure and cell fusion during fertilization
Soumya A, Chinnumol VS and Pradeep G Kumar
We have recently completed a study involving sperm
membrane RAFTS and RAFT-associated proteins.
An extension of this program has been initiated
to delineate the interactions between isolated
membrane rafts and zona pellucida. The lipid rafts
were prepared as detergent resistant membrane
fractions from goat cauda spermatozoa, floated
on sucrose density gradient during an overnight
ultracentrifugation. We have collected intact
oocytes from goat ovaries with clear zona pellucida.
We have cloned all the three zona pellucida
154
genes (ZP1, ZP2 and ZP3)
into eukaryotic expression
vectors. Binding studies
would be undertaken with
both intact zona pellucida
and purified recombinant
ZP protein(s).
Purified rafts
Extra-mural Funding
Sl.No.
Investigator(s)
Title
Funding Agency
Duration
1
Pradeep Kumar G
Molecular evaluation of interactions
between sperm membrane rafts and
zona pellucida proteins
DBT
2011-2014
2
Pradeep Kumar G
Association between stemness and
TCTEX1 expression in testicular
germ cells from adult mouse testis
BRNS
2011-2014
3
Pradeep Kumar G
Role of CLP-1 in cell cycle regulation
in spermatogenic cells
DST
2013-2016
PhD awarded
»» Chandran U (2012) Identification of testisexpressed cell cycle regulating proteins with
special reference to meiosis, University of Kerala,
Trivandrum.
»» Indu S (2012) Identification and functional
evaluation of factors regulating testicular
germline stem cell division and differentiation
(thesis submitted)
Invited Lectures
»» Kumar PG (2012) Germline stem cells - Prospects
in therapeutics. BV Patel PERD Centre,
Ahmedabad, May 26, 2012.
»» Kumar PG (2012) Germlien stem cells - Prospects
in therapeutics. 12th International Conference
on What is new in managing subfertility,
obstetrics and gynaecology, 3-5 August, 2012.
Madras Medical Mission, Chennai.
»» Kumar PG (2013) towards identifying molecular
markers for human male factor subfertility.
ANKUR-SASSM Oration at the Inaugural
conference of the South Asian Society for Sexual
Medicine (SASSM), Indian Institute of Science,
Bangalore, March 29-31, 2013.
Poster Presentations
»» Chandran U and Kumar PG (2012) Molecular
Cloning and Characterization of a Transcript
Variant of cyclin M gene, mtcnnm1, expressed
in mouse Testis. 45th SSR Annual Meeting and
the 18th Ovarian Workshop, August 12-15,
2012, State College, Pennsylvania (won regional
abstract award for papers from India).
»» Sreesha Sree and Kumar PG (2012) The miRNAmRNA map of mouse testis in relation to
Spermatogenesis. 16th ADNAT convention and
Conference on Animal Genetics and Genomics,
16th to 19th December, 2012, National Institute
of Animal Biotechnology, Hyderabad (won 3rd
prize in the poster session)
»» Sreesha Sree and Kumar PG (2013) MicroRNAs
in mammalian spermatogenesis. International
Conference on Repromics and 23rd Annual
Meeting of the Indian Society for the Study
of Reproduction and Fertility, 7-9 February
Trivandrum, Kerala.
»» Devi AN, Kumar ATR, Varghese DS, Thottacherry
JJ and Kumar PG (2013) Expression profiling
and functional characterization of nephrocystin
in relation to spermatogenesis. International
Conference on Repromics and 23rd Annual
Meeting of the Indian Society for the Study
of Reproduction and Fertility, 7-9 February
Trivandrum, Kerala.
»» Radhakrishnan K and Kumar PG (2013) Histone
modifiers and their role in spermatogenesis.
International Conference on Repromics and 23rd
Trivandrum, Kerala.
»» Kumar ATR, Oommen OV and Kumar PG
(2013) Expression profiling, localization and
155
characterization of protocadherin in fertile and
infertile males. International Conference on
Repromics and 23rd Annual Meeting of the
Indian Society for the Study of Reproduction and
Fertility, 7-9 February 2013, Rajiv Gandhi Centre
for Biotechnology, Trivandrum, Kerala.
»» Indu S and Kumar PG (2013) Establishment
of pluripotent nature of adult mouse testicular
germline stem cells. International Conference
on Repromics and 23rd Annual Meeting of the
Fertility, 7-9 February 2013, Rajiv Gandhi Centre
for Biotechnology, Trivandrum, Kerala.
»» Varghese DS and Kumar PG (2013) TDP-43
mediated regulation of spermatogenesis. In
Trivandrum, Kerala (won the best poster award)
»» Bhagya KP and Kumar PG (2013) Functional
evaluation of AIRE in germ cell development. In
156
Trivandrum, Kerala.
Honors and Awards
»» Chandran U and Kumar PG (2012) Molecular
Cloning and Characterization of a Transcript
Variant of cyclin M gene, mtcnnm1, expressed
in mouse Testis. 45th SSR Annual Meeting and
the 18th Ovarian Workshop, August 12-15,
2012, State College, Pennsylvania (won regional
abstract award for papers from India).
»» Sreesha Sree and Kumar PG (2012) The miRNAmRNA map of mouse testis in relation to
Spermatogenesis. 16th ADNAT convention and
Conference on Animal Genetics and Genomics,
16th to 19th December, 2012, National Institute
of Animal Biotechnology, Hyderabad (won 3rd
prize in the poster session)
»» Varghese DS and Kumar PG (2013) TDP-43
mediated regulation of spermatogenesis. In
Trivandrum, Kerala (won the best poster award)
DEPARTMENT OF MOLECULAR REPRODUCTION:
Utero-Embryo Repromics Laboratory
Malini Laloraya, Ph. D.
Scientist F
[email protected]
Malini Laloraya received her Ph.D from Devi Ahilya Vishwavidyalya Indore and was also a
faculty there previously. She was a Fellow-in-Residence at Centre for Biomedical Research,
Population Council, Rockefeller University, New York and was a Visiting Faculty at University
of Virginia, VA and University of Florida, Gainesville, FL, USA.
PhD students:
Jasna J. Mohan
Renjini AP (SRF-ICMR)
Philip Litto Thoma
Meera Krishna B.
Prashanth Narayan
Annu Joseph
Soumya V
Technical Staff:
Sudha B. Nair (Feb 1, 2012-31 Jan 2013)
Usha B (Nov. 14 2011-31 Jan 2013)
G. Sheela (1 Feb 2013 - continuing)
Project Personnel: Anand G (Project fellow, BRNS)
157
One of the most vital events for the continued existence of man-kind, the wide variety
of flora and fauna is the ability to reproduce. The circle of life completes its full cycle
when it creates a new progeny. Any impairment in reproduction can lead to infertility
or production of defective progeny. Pregnancy is central to this process in mammals.
Despite giant leaps in technologies in assisted reproduction, our success rate is
pathetically low. This is because in women being treated for infertility, even when
healthy embryos are transplanted, the failure rate is high: “up to 70 % fail to dock
successfully in the uterine lining”. This is largely due to our inability to understand
embryo implantation. My lab endeavors to understand this complex phenomenon
by focusing our attention on understanding nuclear receptor coregulators, tissue
remodeling, immune tolerance, adhesion, invasion, angiogenesis and embryonic
pluripotency and thereby identify factors critical for successful pregnancy. We also
envisage to understand infertility in polycystic ovarian syndrome - a metabolic
syndrome characterized by oligo/anovulation, hyperandrogenemia, insulin resistance
and accompanying hyperinsulinemia. The endocrine and metabolic defects contribute
to infertility, endometrial disorders leading to implantation failure. We aim to analyze
this complex disorder using a combinatorial miRNA/mRNA profiling coupled with
bioinformatic analysis.
Interacting partners of Estrogen receptor determine the strength of
ERα signaling during Embryo Implantation.
Renjini A.P. and Malini Laloraya
Embryo implantation is effected by a myriad of
signaling cascades acting between embryo and
the endometrium. Steroid hormones play in the
backdrop regulating each module of this event.
A nidatory’ estrogen surge on a progesterone
background determines the ‘window of uterine
receptivity’. Estrogen mediates cellular signaling
via nuclear receptor ERα (NR3A1) in the uterus.
To gain a deep understanding of ERα Partners and
how they modulate estrogen receptor mediated
signaling, we decided to dwelve into the interacting
partners of ERα in the nucleus during window
of implantation. One among them was Signal
transducer and activator of transcription 3 (STAT3),
a key transcription factor during implantation
acting as mediator of various cytokines. Our study
undoubtedly established ERα-STAT3 interaction by
co-immunoprecipitation, co-localization in uterine
sections and human uterine epithelial cell line
RL 95-2; far-western blotting and co-transfection
studies. Disruption of LXXLL motif in STAT3 helped
in identifying the interacting residues of STAT3
in ERα-STAT3 interaction. Far-western analysis
and co-transfection studies proved that the 221st
158
and 224th leucines of 221 LXXLL 225 is important
for its interaction with ERα. The 225th residue and
the LL224AA mutant (combination of the last two
Ls 224 and 225) behaved similar to that of wild
STAT3. Co-transfection studies of wild and mutant
STAT3s along with ERα showed that intact LXXLL
motif is essential for efficient nuclear translocation
of STAT3. Our ERE reporter assay clearly specify
the fact that STAT3 is a functional co-activator of
ERα and the first and fourth L of LXXLL motif is
important for transactivation function (Figure 1).
Mutation in the 225th L and the double mutation
LL224AA enhanced the ERE activity by several
folds suggesting the steric hindrance caused
by 225th leucine during interaction- a possible
mechanism of modulation of ERα function.
This interaction would propose a new signaling
event where ERα activity is controlled by STAT3
during window of implantation to promote cell
differentiation thereby effecting decidualization.
The understanding of implantation regulation
would be a guide to new diagnostic and therapeutic
approaches, in pregnancy and infertility, as well as
in malignant diseases.
Integrin mediated signaling during embryo implantation:
a). Dock180 has the property to bind DNA
Jasna J Mohan, Pradeep Kumar G and Malini Laloraya.
Integrin mediated events are central to the process
of embryo implantation. Integrins present on the
endometrium and blastocyst mediate the embryo
maternal interaction. Dedicator of cytokinesis
(Dock180) involved in integrin receptor activation,
a cytosolic molecule was found to have a nuclear
presence in the uterus. The expression and nuclear
translocation of Dock180 was reported earlier to
be mediated by estrogen and progesterone. The
nuclear function of Dock180 was attributed to its
159
ability to import autoimmune regulator (Aire) into
the nucleus, like an Importinb. Silencing Dock180
caused blockage of nuclear shuttling of Aire
thereby affecting decidualization via its impact on
downstream targets. Our results last year portrayed
an indispensable role for Dock180 in pregnancy
establishment. We report that homology modeling
suggest DOCK180 to have DNA binding ability.
This DNA binding ability of DOCK180 was proved
experimentally by oligolibrary screening followed
by EMSA. The genomic consensus to which
DOCK180 binds was identified. Promoter analysis
by BIOBASE software identified key genes bearing
the DOCK180 consensus in their promoter and this
was proved experimentally by qRT-PCR analysis in
the Dock1 silenced uteri.
b). Structural insights into the Mechanism of SOS1 nuclear entry
via its NLS.
Anand G and Malini Laloraya.
Receptor tyrosine kinase and integrin pathways are
the two key signaling pathways that coordinate the
cellular activities during the process of blastocyst
implantation. DDR1 (a class of tyrosine kinase) null
mice and integrin null mice are unable to reproduce
because of improper blastocyst implantation. In
both these pathways, the protein, Son of sevenless
(SOS), acts as the Guanine nucleotide exchange
factor (GEF) of Ras. SOS is involved in coupling
tyrosine kinases to Ras activation. Disruption
of both the alleles of Sos1 is known to confer
embryonic lethality in mice. Interestingly, we have
earlier found SOS1, a cytosolic protein, entering
the nucleus under estrogen surge during embryo
implantation. No information exists about a nuclear
presence of SOS1, which is classically a cytosolic
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molecule capable of membrane targeting. Our
computational analysis had identified the presence
of simple bipartite karyophilic clusters of arginines
and lysines similar to ones in nucleus-targeted
protein signaling. These peptides have been
termed nuclear localization signals (NLS) and this
imparts theoretical basis for its translocation into
the nucleus under hormonal treatment. PSORT
II analysis predicted two bipartite NLS in the
molecule at positions 744 and 966 and gave an
NLS score of 3.42. Thus, to prove the importance
of this NLS, we proceeded further to mutate all
the residues individually and in combinations and
observed the translocation under hormonal control
in HEK cells. Wild mSOS1- EGFP was observed in
the nucleus after 24 h but NLS (both NL1 & NL2)
mutated mSOS1-EGFP was found in the cytosol
even after estrogen treatment. Results show that
the SOS1 nuclear translocation was hampered in
NLS mutant (4 residues mutated) mSOS1- EGFP
after hormonal treatment (Figure 3). This provides
a proof that NLS mutated SOS1 was unable to
enter the nucleus and thereby validates that the
identified NLS is a functional NLS critical for
SOS1 nuclear translocation. Efforts are ongoing to
decipher the nuclear function of SOS1.
Autoimmune regulator, AIRE - a key molecule in Implantation
Soumya V. and Malini Laloraya
Immunological adaptation of the uterus to tolerate
the genetically foreign foetus is crucial in leading
to a successful pregnancy. Autoimmune regulator
(AIRE) is the master regulator of autoimmunity.
It was first identified as a mutated protein in
autoimmune polyendocrine syndromes (APECED),
a serious autoimmune disorder. The Autoimmune
regulator (AIRE) is a transcription factor which is
expressed mainly in thymus, spleen, lymph nodes
and bone marrow implicating a role in central
and peripheral tolerance. Even though some other
roles are suggesting, it is mainly a transcription
factor which is able to transcribe nearly fifty tissue
specific antigens in thymus. AIRE has a crucial role
in negative selection of immune cells. Extrathymic
expression of Aire
is also reported, including in
the reproductive organs such as ovary and testis
has led to the suggestion that AIRE might also have
a function(s) outside the immune system.
Interestingly though AIRE-/- mice do not differ
significantly in weight, size or maturation from
their AIRE+/+ and AIRE+/- littermates, about 85%
of them are infertile. Since, Aire Knockouts are
infertile and no explanation as to the reason why
AIRE knockouts are infertile is known, we planned
to understand the importance of Aire in female
infertility by studying the impact of silencing of
AIRE during pregnancy. Earlier work in our lab
has revealed the presence of AIRE in the uterus.
No understanding of the functional significance of
role of Aire in pregnancy exists. In vivo silencing
161
experiments showed lack of
implantation sites suggesting
that Aire is crucial for successful
implantation. The microarray
studies disclose the possible
genes affected in the uterus
during implantation, of which
more than 500 fall in significant
range (Figure 4). Microarray
data signaling pathway
analysis showed that many of
them are important in immune
regulation, adhesion pathways,
Tgfβ signaling, Wnt signaling
pathways, RNA splicing and
transcriptional regulation. The
interaction network created in
Genemania reveals that some of
them are significant candidates
which for m the part of the
interaction network since they have
proven roles in either pregnancy or
Aire activity (Figure 5).
Immune tolerance mechanisms during embryo implantation.
Prashanth Narayan and Malini Laloraya
Successful embryo implantation for establishment
of pregnancy involves increased maternal immune
tolerance towards the semi-allogenic embryo
(with its paternal set of genes). Maternal immune
response has to adapt itself to accommodate the
growing semi-allogenic embryo within the gravid
uterus during pregnancy as failure to do so results
in infertility and reproductive pathologies. A
sub-class of CD4+ T-Lymphocytes are known to
tightly modulate maternal immune
response towards the growing embryo
at the maternal-fetal interface during
pregnancy. These cells are the
CD4+CD25+ T-regulatory cells (Treg).
T-regulatory cells are known to
express FoxP3 (a nuclear transcription
regulator) which then endows these
T-regulatory with ability to render
the CD4+T-effector cells anergic by
regulating the transcription of certain
genes crucial for immune response.
FoxP3 is known to be the master switch
within these CD4+CD25+ T-regulatory
cells.
162
It is known that during pregnancy ‘nidatory’
estrogen surge is pivotal for embryo implantation.
Additionally, this hormone estrogen is known to
expand the population of T-regulatory cells in an
organ-specific manner. The goal of this project is
to understand the mechanisms regulating FOXP3
expression and the importance of estrogen in
regulating Treg expansion. In a bid to understand
this we analyzed the FoxP3 sequence and have
found a NR-box (LXXLL) motif within
the N-terminal of Foxp3 amino acid
sequence (Figure 6). This motif is
known to be present in co-regulators
of nuclear receptors and is required
for interaction with ligand binding
domain. Hence we, theorize that
FOXP3 can directly interact with ER-α
and regulate its transcriptional ability.
Estrogen is seen to increase expression
of FOXP3 as well as its nuclear transit
(Figure 7). Attempts are ongoing to
study the interplay between FoxP3
and ER-α under the ‘nidatory’ estrogen
surge and how this interaction affects
immune tolerance.
Understanding Molecular defects in Polycystic Ovarian Syndrome
(PCOS)
Meera Krishna B , Sathy M. Pillai*, KG Madhavan Pillai* & Malini Laloraya
*Clinical Collaborator - SAMAD IVF Hospitals
Polycystic ovarian syndrome is a metabolic
syndrome which can negatively affect female
fertility. The metabolic disorder is characterised
by hyperandrogenemia, oligo/anovulation and
polycystic ovaries. Familial history, altered
steroidogenesis and insulin signaling pathway
are speculated in the aetiology. Higher rate of
implantation failure or higher risk of spontaneous
miscarriage after pregnancy is associated with
PCOS. The reduced reproductive outcome is
not only due to anovulation but could also be
because of aberrant endometrial receptivity in
patients with PCOS. The manifestation of the
disorder involves complex interaction between
environment and genetic component.
But the real players in the chaos are
still evading, hampering the diagnosis.
MicroRNAs are non-coding RNAs
regulating gene expression by targeting
mRNAs to degradation or translational
repression. miRNA regulation of
molecular networking has been
correlated to many human diseases.
The aberrant miRNA expression can
generate a unique transcriptome as
well as proteome fingerprint which
can explicitly reveal the pathogenesis
of PCOS. Hence the identification of
miRNAs and its interplay with gene
expression associated with PCOS will be critical
for understanding the etiology and for developing
diagnostic/therapeutic strategies.
In our study, we have performed miRNA real
time expression profiling on TLDA platform and
transcript microarray on Illumina Human HT12 V4 platform. The results were validated on
a large population with individual miRNA and
mRNA real time assays. The study revealed that
under PCOS condition 30 microRNAs and 1092
genes were aberrantly expressed. Furthermore,
the differentially regulated miRNAs and mRNAs
were shown to be associated with pathways
163
involved in angiogenesis, oxidative stress, lipid
metabolism, androgen signaling, insulin signaling
,TGF β signaling, BMP signaling, B cell receptor
signaling, TCR signaling and Toll like receptor
signaling. Altogether the data indicate towards
immune and metabolic malfunctions of PCOS.
Our results showing differential expression of
164
genes in peripheral blood under PCOS condition
mirror results reported by others in cumulus cells,
granulose cells, oocytes and ovary. Since changes
in ovarian tissues are reflected in blood, examining
gene/protein expression changes in peripheral
blood can serve as a biomarker for PCOS diagnosis
and greater understanding of its etiology.
Significance of Stat5 and Crk alliance in Pancreatic Development and
Diabetes Mellitus
Annu Joseph and Malini Laloraya
High rate of postimplantation fetal loss is
associated in nonobese diabetic (NOD) mice due
to abnormal decidual production of interferon
(IFN) gamma. It is thought that diabetes alters
the natural events associated with successful
implantation. Diabetic NOD (dNOD) mice exhibit
retarded maturation of uterine pinopodes and
overexpressed MUC1 mucin at implantation sites
as compared to normoglycemic NOD (cNOD) mice.
Aberrant expression of endometrial IFNgamma
in dNOD mice is associated with a nonreceptive
endometrial milieu (reduced leukemia inhibitory
factor (LIF) and decreased phosphorylation of
uterine NFkappaBp65 and STAT3 during Day 4.5
postcoitum), contributing to peri-implantation
embryo loss in type 1 diabetes. Insulin is also a key
factor during acquisition of endometrial receptivity.
Diabetes mellitus is caused by a decrease in the
number (Type 1) or inadequate function (Type 2) of
beta cells in the Islets of Langerhans in pancreas. It
has already been reported that Type 1
diabetes is characterized by a mutant
Stat5, and that Stat5 activity in beta
cells influences the susceptibility
to experimentally-induced Type 1
diabetes. To understand the function
of Stat5b we studied the effect of
Stat5b knockdown on pancreatic
cells. Pancreatic beta cell line MIN6
was treated with Stat5b siRNA.
After transfection with control/Stat5b
siRNA, cDNA from the treated MIN6
cells were used to analyze the relative
transcript levels of Stat5b, Stat5a,
Ins1 and Wdr25, with 18SrRNA as the
endogenous control. Stat5b showed
almost 80% knock down indicating
the efficacy of the treatment. Wdr25,
a Stat5b target gene as predicted
by cisRED database, also showed
an 80% down regulation. Wdr13,
another WD repeat gene, has been
reported to be a negative regulator of
beta cell proliferation, where Wdr13
knockout mice showed higher serum
insulin levels. In our study, Ins1 levels
have increased by 41%. More studies
have to be done to identify whether
Wrd25 also shows a similar functional
pattern, and the role of Stat5b in its
regulation.
165
Extra-mural funding
Funding
Agency
Sl.No
Investigator(s)
Title
1.
Dr. Malini Laloraya (PI)
Exploring whether ROS
modulates pluripotency and
differentiation of stem cells using
the mouse ESC line.
Department of
Bio- Technology,
Govt .of India
20102013
2.
Dr. Malini Laloraya (PI)
Deciphering the SOSGRB signaling at embryo
Implantation.
Board of
Research in
Nuclear Sciences
20102013
(2012-2013):
»» Dr. Malini Laloraya. “ERα proteomics identifies
candidate which regulates the strength of
tumorigenesis”. Lecture at International
Conference on “Reproductive Health with
Emphasis on Strategies for Family Planning” held
at New Delhi, 19th – 21st February 2012 under
the auspices of Indian Society for the Study of
Reproduction and Fertility (ISSRF)
»» Renjini A. P. and Malini Laloraya* “ERα- a
modulator of STAT3 function in the uterus
during embryo implantation” Presented at
International Conference on “Repromics – Omics
in Reproduction” held at Thiruvananthapuram,
7th – 9th February 2013 under the auspices of 23rd
Annual meeting of Indian Society for the Study
of Reproduction and Fertility (ISSRF).
»» Meera Krishna B, Sathy M. Pillai and Malini
Laloraya* Differential gene expression profiling
in peripheral blood under polycystic ovarian
syndrome. Presented at International Conference
on “Repromics – Omics in Reproduction” held
at Thiruvananthapuram, 7th – 9th February 2013
under the auspices of 23rd Annual meeting of
Fertility (ISSRF).
»» Philip Litto Thomas and Malini Laloraya* Reactive
Oxygen Species levels exhibit distinctive profiles
in Pluripotent v/s Differentiating Embryonic Stem
cells. Presented at International Conference on
“Repromics – Omics in Reproduction” held at
Thiruvananthapuram, 7th – 9th February 2013
Fertility (ISSRF).
166
Duration
»» Prashanth Narayan and Malini Laloraya*
Estrogen modulates expression and spatiotemporal distribution of Foxp3. Presented at
»» Annu Joseph, Philip Litto Thomas and Malini
Laloraya* Efforts to understand mechanism
of H2O2-mediated Pluripotency In Mouse
Embryonic Stem cells. Presented at International
Conference on “Repromics – Omics in
Reproduction” held at Thiruvananthapuram,
»» Soumya V., Renjini A.P. and Malini Laloraya*
Uterine AIRE depletion impinges upon
decidualization during early pregnancy.
Presented at International Conference on
“Repromics – Omics in Reproduction” held at
Thiruvananthapuram, 7th – 9th February 2013
Fertility (ISSRF).
»» Anand GS, Zyju DP, Malini Laloraya* SOS1, a
guanine nucleotide exchanger enters nucleus
under estrogen trigger and could be part
of the nucleosome complex. Presented at
Molecular Neurobiology Division
R.V. Omkumar
Mayadevi M.
Scientist F
Scientist C
[email protected]
Omkumar received his PhD in Biochemistry
from the Indian Institute of Science,
Bangalore. He did postdoctoral research
at Purdue University and at California
Institute of Technology, USA before joining
RGCB in 1996.
Visiting Scientist:
Dr. Ani. V. Das
Ph.D. students:
Mathew Steephan
Soumya Paul
[email protected]
Mayadevi received her M.Sc. in Chemistry
from the University of Kerala and worked
at Case Western Reserve University and
Baylor Research Institute, USA, before
joining RGCB.
Ramya.R.Prabhu
Archana G.M
Arun kumar R.C
Junior Research Fellow:
Manthosh Kumar
167
Biochemical Regulatory Mechanisms associated with
Synaptic Calcium Signaling
Archana G. M., Ramya.R.Prabhu.,Mayadevi M., and Omkumar R.V.
Calcium signaling at synapses that underlies
synaptic plasticity is regulated by molecules like
Calcium/calmodulin dependent protein kinase
II (CaMKII) and N-methyl-D-aspartate receptors
(NMDAR), which also play a role in excitotoxicity.
The phosphorylation status of NMDAR subunit
2B (NR2B) regulates its interaction with CaMKII.
The molecular events under stress conditions were
studied by monitoring the phosphorylation status
of NR2B-Ser1303 levels after glutamate treatment
in primary cortical neurons. There was reduction
in the phosphorylation level of NR2B-Ser1303 which
was blocked in the presence of MK801, the NMDAR
blocker. The reduction of phosphorylation was
found to be mediated by PP1. The action of purified
phosphatases upon NR2A was studied and it was
found that PP1, PP2A and PP2C but not PP2B
can cause dephosphorylation of phospho-GSTNR2A. Phospho-GST-NR2A was also found to be
dephosphorylated by PSD as well as cytosol and
it was found that only PP1 is responsible for this
dephosphorylation although PP2Band PP2C are
present in PSD and cytosol.
Bioprospecting for Neuroprotectants
Mathew Steephan, Soumya Paul, Arun kumar R.C., Mayadevi M., and Omkumar R.V.
The excess flow of calcium into neuronal cells
leads to excitotoxicity that underlies many
neurodegenerative diseases. The system that was
developed for activity assay of NMDA receptor
was used for screening plant extracts for agonists
and antagonists. Ethanolic extracts of two plants
were found to have antagonist properties against
calcium conducting NR1/NR2B channels.
A Novel Technology platform for the assay of Ca2+ channels
Mathew Steephan., Soumya Paul., Arunkumar R.C., Manthosh Kumar, Mayadevi,M. and
Omkumar R.V
Va r i o u s c a l c i u m c h a n n e l s c o n t r i b u t e
towards excitotoxic neuronal death in many
neurodegenerative diseases. One of the therapeutic
strategies is to block the calcium channels. We
have developed an assay system based on the
specific interaction between GFP--CaMKII and
MLS-NR2B to detect intracellular calcium release.
The other subunits of NMDA receptor, NR2A, NR2C,
NR3A failed to show any binding to GFP--CaMKII.
None of the NMDA receptor subunits could form
functional ion channels in the homomeric form
when tested by this assay method consistent with
earlier reports. Heteromeric NR1/NR2A, NR1/
NR2B, NR1/NR2C and NR1/NR2D form calcium
conducting channels. The autonomous T286DGFP--CaMKII mutant showed increased sensitivity
168
to calcium levels, as low as 10 nM, compared to
the wild-type form. The homomeric subunits of the
AMPAR, GluR1, GluR2 and GluR4 form homomeric
functional channels conducting calcium.
A calcium sensor cell line that stably expresses
GFP-α-CaMKII and mitochondrially localised NR2B
sequence (MLS-NR2B) has been developed. This
cell line responds to a calcium influx by forming
mitochondrially localised green fluorescent
punctae that represents the complex between
CaMKII and NR2B as a signal for calcium. Further
these cells were seeded in low density and different
clonal populations of the cells with MLS-NR2BIRES-GFP-α-CaMKII were developed. This cell
line can be used for observing the activity of any
calcium channel. transfect cDNAs of the calcium
channels, TRPV1 and P2X2. Both the channels
showed calcium and agonist dependent activity.
We have also made a vector for expressing MLSNR2B with a fluorescent tag, the MLS-NR2B-mRFP.
This vector can be used for co-transfection with
GFP-α-CaMKII so that calcium influx to the co-
transfected cells results in the formation of green
fluorescent punctae which would colocalise with
red fluorescent punctae formed due to MLS-NR2BmRFP.This would help in distinguishing the cells
which received a calcium influx from other cells. It
should also be possible to quantitate the calcium
entry to cells.
Modulators of Calcium/calmodulin dependent protein kinase II
Mayadevi M. and Omkumar R.V.
Calcium/calmodulin dependent protein kinase
II (CaMKII) is one of the kinases in calcium
signalling pathways. In many neuronal diseases
levels of CaMKII and its phosphorylation status
are known to undergo changes. Acute inhibition
of autonomous CaMKII was reported to be
neuroprotective. CaMKIIN is a natural inhibitor
of CaMKII present physiologically. However, the
function of, CaMKIIN is currently elusive. We are
exploring the interactions of CaMKIINα inhibitor
protein with CaMKII. We have identified curcumin
as an inhibitor of CaMKII. Ca2+dependent and
independent activities of CaMKII are inhibited
by curcumin. The inhibitory action was found to
be partially reversible. In addition to curcumin,
few analogues of curcumin were also found to be
inhibitors of CaMKII, the most potent inhibitor
being pyrazole curcumin. But curcumin minimally
affects CaMKII-NR2B interaction. We have initiated
a surface plasmon resonance (SPR) based study to
look into the interaction of CaMKII with various
substrates and ligands.
In primary neuronal cultures, phospho-T286CaMKII levels were found to be reduced after
treatment with high concentration of glutamate.
Such a system can be used to study the regulation
of CaMKII in primary neurons under conditions of
stress and upon treatment with various modulators.
Extra Mural Funding
Sl.No.
Name of Grant
Funding Agency
Total Amount
Duration
1
A Novel Technology Platform for Assay of
Calcium Channels
Department of
Biotechnology
Rs 81.05 lakhs
2011-2013
2
Molecular and functional characterization of
putative ZzR1 resistance gene from Zingiber
Zerumbet, a wild relative of ginger
Department of
Biotechnology
Rs 4.8 lakhs
2010-2012
169
Publications
»» Prabhu Ramya. R, Suma Priya, S., Mayadevi,
M. and Omkumar, R. V. (2012) Regulation of
phosphorylation at Ser-1303 of GluN2B receptor
in the post synaptic density. Neurochem.Int.
61(7):981-5. 10.1016/j.neuint.2012.08.016. IF
2.902
»» Mayadevi, M., Sherin, D.R, Keerthi, V.S.,
Rajasekharan, K.N., and Omkumar R.V. (2012).
Curcumin is an inhibitor of Calcium/calmodulin
dependent protein kinase II. Bioorg. Med. Chem.
20.6040-6047 IF 3.157
»» John Cheriyan, Archana G Mohanan, Pradeep
K K., Madhavan Mayadevi, Ramakrishnapillai
Vyomakesannair Omkumar (2012) Effect of
multimeric structure of CaMKII in the GluN2Bmediated modulation of kinetic parameters of
ATP PLoS One 7.9.e45064 IF 4.09
Book chapter
»» Mayadevi, M., Archana, G. M., Prabhu, R. R., and
Omkumar, R. V. (2012) Molecular Mechanisms in
Synaptic Plasticity, Neuroscience - Dealing With
Frontiers, Dr. Carlos M. Contreras (Ed.), ISBN:
978-953-51-0207-6, InTech, Available from:
http://www.intechopen.com/books/neurosciencedealing-with-frontiers/molecular-mechanisms-insynaptic-plasticity
170
Patents
»» US patent (US 8,304,198) granted for the
invention “Assay for Detection of Transient
Intracellular Calcium” by Omkumar, R. V.,
Rajeevkumar, R., Mathew Steephan, Mayadevi,
M. and Suma Priya. 2012
»» CIP No.13/661,787 on US Patent 8,304,198 was
filed.
»» A cell based assay system for L-type voltage gated
calcium channel. Archana, G.M., Mayadevi, M.
and Omkumar, R. V. XXX Annual conference
of Indian Academy of Neuroscience, October,
27-30, 2012,Amritsar, India
»» A stable calcium sensor cell line for calcium
channel assay. Arun Kumar R.C.., Mathew
Steephan., Mayadevi, M. and Omkumar, R. V.
XXX Annual conference of Indian Academy of
Neuroscience, October, 27-30, 2012, Amritsar,
India.
»» Inhibition of CaMKII functions by the aqueous
extract of Bacopa monnieri. Mayadevi M. and
Omkumar, R.V. XXX Annual conference of
Indian Academy of Neuroscience, October, 2730, 2012, Amritsar, India.
»» Regulation of phosphorylation at Ser 1303 of
GluN2B receptor- Identification of kinases and
phosphatases. Prabhu Remya R., Suma Priya S..,
Mayadevi M. and Omkumar, R.V. XXX Annual
conference of Indian Academy of Neuroscience,
October, 27-30, 2012, Amritsar, India.
NEURO-STEM CELL BIOLOGY LABORATORY
Jackson James, PhD
Scientist E1
[email protected]
Jackson James is a Ph.D in Molecular Neurobiology from Cochin University of Science &
Technology, India. He worked as Post-doctoral fellow at Lied Transplant Centre, Dept. of
Ophthalmology, University of Nebraska Medical Center, Omaha, USA, before joining RGCB
in April 2004.
PhD students:
Divya MS
Abdul Rasheed VT
Dhanesh SB
Divya TS
Subashini C
Technical Assistant
Biju S Nair
171
Transplantation of ES cell derived RGCs into NMDA injected
animal model and visual function analysis
Divya MS, Abdul Rasheed VT and Jackson James
Collaborator: Dr. Samer Hattar, Johns Hopkins University, Baltimore, USA
Glaucoma is one of the leading causes of irreversible
blindness affecting 60 million people worldwide,
with a projected increase of 80 million by 2020.
In India over 12 million people are affected with
glaucoma, accounting for 13% of India’s blindness.
Glaucoma is a chronic neuro-degenerative disease
characterised by the progressive, permanent visual
loss resulting from the degeneration of retinal
ganglion cells (RGCs). Cell replacement therapy
is one of the promising strategies for efficient
treatment for neuro degenerative disorders. We have
previously shown that RGCs could be generated
in vitro from ES cells (Jagatha et. al., Biochem.
Biophys. Res. Commun., 380, 2009; 230-235). The
inherent properties of the stem cells viz: self-renewal
and differentiation to multiple cell lineages, offers
a therapeutic hope for glaucoma, through selective
cell replacement of RGCs to restore lost vision.
However, the vision restoration at a translational
level has been hindered by several challenges
like source of the stem cells, differentiation into
appropriate cell type, transplantation, survival and
functional integration of cells in the target tissue. In
the current study, we
directed embryonic
stem cells towards
RGC lineage by using
a combination of
extrinsic and intrinsic
factors. Thus, the
conditions established
for the enhanced
RGC differentiation
and integration was
further evaluated for
functional integration
in NMDA (N-methylD-aspartate) injected
RGC ablated glaucoma
mice models. Visual
functions of these
animals were analysed
before and after
injection of NMDA
172
by behavioural experiments. Restoration of visual
behavioural functions in RGC depleted animals by
(NMDA)-induced excitotoxicity mice models were
evaluated. Visual acuity was analyzed by optometry
and light avoidance test. The NMDA induced RGC
depleted animals showed 50% reduction in Brn3a
positive cells in whole mount retinal staining as
well as a significant reduction in visual acuity.
Retrograde tracing showed significant reduction in
RGC projections in image forming centers in brain
like Lateral Geniculate Nucleus (LGN) and Superior
colliculus (SC) whereas projections into the nonimage forming brain targets like Suprachiasmatic
nucleus (SCN) and Olivery pretectal nucleus
(OPN) were not affected. A good number of EGFP
expressing cells were found integrated into GCL
layer in retinal sections as well as in whole mount
retinal staining (Fig-1). An improvement in visual
acuity was observed in animals after 3-4 weeks
of transplantation with EGFP expressing, ES cell
derived RGCs, compared to non-transplanted
NMDA injected animals.
Relevance of miRNAs in Retinal Ganglion cell fate specification
and differentiation
Abdul Rasheed VT and Jackson James
Collaborator: Dr. Ani Das V, Molecular Neurobiology Laboratory, RGCB, Trivandrum
The timing of RGC genesis, the first formed retinal
cell type among the seven, from its progenitors
and its maturation into a functional neuron is an
important aspect that still remains as a piece of
puzzle. Recent studies focus on understanding the
mechanisms involved in RGC fate specification
and differentiation so as to design therapeutic
strategies to prevent loss of vision due to retinal
degenerative diseases like glaucoma. Studies
suggest that miRNAs may play a vital role in the
regulation of eye development. Shedding light
into these complex molecular mechanisms, which
may involve microRNAs would be relevant in
understanding the molecular and cellular processes
involved in the development and differentiation
of RGCs. We have previously shown that miR-23a
and miR-374 has binding sites on 3’UTR of Brn3b
and can downregulate its expression. Temporal
expression analysis of miR-23a and miR-374
during different retinal developmental stages in
mice was done to assess the expression pattern
of these miRNAs using Taqman real time assay.
These analyses demonstrate that miR-23 and
miR-374 expressions were well orchestrated to
regulate the expression of Brn3b during mouse
retinal development. RT analysis of E14 primary
retinal culture and western analyses of RGC-5 cell
line showed that miR-23a and miR-374 by itself
could not significantly regulate the expression
of Brn3b but when expressed together could
precisely down regulate Brn3b expression and
also affect the patterning of the retina. Further,
we corroborated these finding in E14 retinal
explants after ex vivo electroporation where we
found altered patterning in Ganglion cell layer
with loss of Brn3b expressing cells upon combined
expression of miR-23a and miR-374 (Fig-2). Our
data confirms a post transcriptional regulatory
mechanism of Brn3b, an intrinsic factor involved
in RGC differentiation, survival and axonal growth
during retinal development.
173
Characterisation of neural progenitors having Notch independent
Hes-1 expression in developing neocortex
Dhanesh SB and Jackson James
Collaborators: Dr. Shubha Tole, TIFR, Mumbai, Dr. RV Omkumar, Molecular Neurobiology
Laboratory, RGCB, Trivandrum, Dr. Santhosh Kumar Shankaran, Animal Research Facility,
RGCB, Trivandrum
We have previously shown the existence of a Notch
independent Hes-1 activation in ES- cell derived
neural progenitors (Sanal et.al. J. Neurochem.,
113, 2010, 807–818). This alternative pathway will
ensure a constant expression of Hes-1 even in the
absence of canonical Notch mediated signaling,
thereby maintaining a pool of proliferating neural
progenitors required during development. We have
also shown the presence of Notch independent Hes1 activation in primary neural cultures. However,
Notch/CBF1 independent Hes-1 expression and
its role in developing neocortex are not well
characterized. In order to characterize the Notch
independent Hes-1 expressing neural progenitors
in vivo, we used the reporter plasmid, pmtCBFIEGFP in which EGFP is under the control of murine
Hes-1 promoter (2.5Kb) where in CBF1 binding
site in the Hes-1 promoter was mutated. Since
there is no marker available for the identification
and characterization of Notch
independent Hes-1 expressing
population, this reporter system
would be the ideal one to track
those unique population during
development. Hes1- EGFP, in
which EGFP is under the control of
wild type Hes-1 promoter (2.5Kb),
as a control. In order to check the
presence of this unique Notch
independent population having
differential Hes-1 expression in
developing neocortex, we first
in utero electroporated both
the reporter constructs, Hes1EGFP and mtCBFI-EGFP into
the cerebral cortex of mouse
E14 embryos. Electroporation
was done in different embryos
of the single mother to avoid the
developmental errors. The brains
were harvested two days post
in utero electroporation (2DPI)
and 14m cryosections were
immunostained with antibodies
against EGFP. Our results clearly
174
show that Notch independent Hes-1 expressing
neuronal precursors are present and restricted
to the Venticular Zone of the mouse neocortex
(Fig-3). In order to further characterize the Notch
dependent and independent Hes1 expressing
neural progenitors, in utero electroporated brains
with the reporter plasmids were immunostained
with neural progenitor cell marker, Nestin. As
shown in Fig-3, most of the EGFP expressing cells
both in control and Notch independent sections
were co-localized with Nestin indicating that they
are neural progenitors. Further characterization
of these unique progenitors during neo cortical
development needs to be carried out. We are
also trying to generate transgenic mice using the
mtCBF1-d2EGFP construct which will help track
the Notch independent Hes-1 activation during
development and its fate specification.
Transcriptional regulation of Tlx3 by Pax6 and its role on excitatory
neural fate specification in vitro and in vivo
Divya TS and Jackson James
Various transcription factors, signaling pathways
and environmental cues work together in a coordinated manner to direct the neuronal fate to either
glutamatergic or GABAergic. Tlx3, a homeobox
gene, was identified as a post-mitotic selector gene
involved in glutamatergic neurogenesis and is
found to be expressed in a subset of spinal neurons,
brainstem and cerebellum. Previous transcriptional
regulation studies of Tlx3 in our lab have proved
that Hes-1, a Notch target gene acts as a repressor
of Tlx3 expression (Indulekha et al, 2012; Cell Mol
Life Sci, 69;611-627). Currently we are looking
at activators of Tlx3 which could intern push
the neural progenitor towards an excitatory fate.
Bioinformatics analysis of Tlx3 promoter revealed
the presence of conserved regions upstream
and downstream of Tlx3 gene which may act as
possible enhancers. Further analysis revealed the
presence of conserved binding sites for Pax6 as
well as downstream effectors of Wnt signaling on
Tlx3 promoter. Pax-6 is found to have an evident
role in glutamatergic neuron fate determination
and it is expressed in various regions of brain
including granule neurons in cerebellum where
Tlx3 is also found to be expressed. Studies done
in cerebellar granule neuron cells (CGNs) proved
that Pax6 could act as a possible activator of Tlx3.
Immunohistochemical analysis in PN7 mouse
cerebellum with Ki67 showed that Tlx3 expression
starts at a pre-mitotic stage in CGNs and maintains
till those cells migrate from external granule
layer to internal granule layer and its expression
was specifically confined to the posterior lobes of
cerebellum (Fig-4). Further confirmation of Pax6/
Tlx3 interaction will be done in Pax6 knock out
mouse and the possible binding mechanism will
be proved using ChIP analysis.
175
Elucidation of role of Wnt signaling in neural subtype specification
Subashini C and Jackson James
Collaborator: Rejji Kuruvilla, Johns Hopkins University, Baltimore, USA
Wnt signaling exerts diverse role in
embryogenesis and development because of
its various ligands and respective downstream
target gene activation. One of the major noncanonical Wnt ligand, Wnt5a is known to
play pivotal role in development especially
in neurogenesis. It has been reported that
Wnt5a promotes dopaminergic neurogenesis
in midbrain and GABAergic neurogenesis in
forebrain but its expression in cerebellum has
not been reported. It is well known that signals
from isthmic organizer and roof plate, together
with various transcription factors co-ordinatively
regulate the process of cerebellum development.
Various studies demonstrated the role of other
signaling pathways such as Notch, Wnt and
Shh in neural progenitor maintenance and in
differentiation of cerebellar granule progenitor
cells (CGNP’s) but the role of non-canonical Wnt
signaling mediated by Wnt5a in the cerebellar
development is not yet known. Therefore, in
order to characterize the Wnt5a expression
pattern and its function in cerebellum, first we
analyzed the expression of Wnt5a in mouse
cerebellum during the postnatal stages PN1 to
PN7 using semi quantitative RT-PCR analysis.
We found Wnt5a to be consistently expressed in
the postnatal stages PN1-PN7 with no change
in the expression levels in any of the stage.
Next, we analyzed the role of Wnt5a signaling
in primary cerebellar culture using recombinant
Wnt5a (rWnt5a) protein and also by blocking
non-canonical Wnt signaling using Fumagillin.
The percentage of GABA positive cells increased
with Wnt5a treatment and decreased upon
treatment with Fumagillin (Fig-5). Preliminary
results indicated the role of non-canonical Wnt
signaling in regulation of cerebellar GABAergic
neuron development. These results are being
confirmed with conditional Wnt5a knockout
mouse during development.
176
Research Grants
No.
Investigator(s)
1.
Dr. Jackson James (PI)
Dr. RV Omkumar
Dr. Santhosh Kumar SN
Characterization of Notch
independent Hes-1 mediated
maintenance and fate
specification of neural
progenitors
Dept. of
Biotechnology,
Govt. of India
20013 - 2016
Dr. Jackson James (PI)
Dr. RV Omkumar
Transcriptional regulation
of Tlx3(Hox11L2) by Notch
signaling and its involvement
in excitatory Vs. inhibitory
fate specification of neural
progenitors
Dept. of Science
& Technology,
Govt. of India
20013 - 2016
Dr. Sreekumar E (PI)
Dr. Jackson James
(Co-PI)
Characterization of
Neurovirulence of Chikungunya
virus in cellular and animal
models Dept. of
Biotechnology,
Govt. of India
20012 - 2015
2.
3.
Title
Funding Agency
Duration
(3Year)
(3Year)
(3Year)
Publications: 2011-2012
Sl. No.
Details
Impact Factor
1
Nishit Srivastava, Vijay Venugopalan, Divya MS, Rasheed VA,
Jackson James# and K. S. Narayan#, Neuronal differentiation of
embryonic stem cell derived neuronal progenitors can be regulated by
stretchable conducting polymers; Tissue Engineering (2013, In Press).
#
Corresponding Authors.
4.02
2
Divya MS, Roshin EG, Divya TS, Rasheed VA, Santhoshkumar TR,
Elizabeth KE, Jackson James# & Radhakrishna M Pillai. Umbilical Cord
blood derived mesenchymal stem cells consist of a unique population
of progenitors co-expressing MSC and neuronal markers capable of
instantaneous neuronal differentiation; Stem Cell Research & Therapy
2012, 3:57. #Corresponding Author.
3.21
3
Praveen K. Sobhan, Mahendra Seervi, Jeena Joseph, Saneesh Varghese,
Prakash Rajappan Pillai, Divya Mundackal Sivaraman, Jackson James,
Roshin Elizabeth George, K.E. Elizabeth, T.R. Santhoshkumar & M.
Radhakrishna Pillai. Immortalized Functional Endothelial Progenitor Cell
Lines from Umbilical Cord Blood for Vascular Tissue Engineering; Tissue
Engineering, 2012, Vol. 18, No. 11: 890-902
4.02
Abstracts published and
Conferences
»» 1. Abdul Rasheed VT, Divya MS and J James.
Functional relevance of miR-23a in Retinal
Ganglion Cell fate specification. Annals of
Neurosciences, 2012 19:33-34. Presented at the
XXXth Indian Academy of Neurosciences meet
and International Symposium, Guru Nanak
Dev University, Amritsar, October 27-30,
2012. (Awarded best poster presentation in the
category).
»» 2. S.B. Dhanesh, C. Subashini, T.S. Divya and J.
James. Maintenance, proliferation and fate specific
differentiat ion of neural progenitors having
Notch/CBF1 independent Hes-1 expression. .
177
Annals of Neurosciences, 2012 19:45. Presented
at the XXXth Indian Academy of Neurosciences
meet and International Symposium, Guru Nanak
Dev University, Amritsar, October 27-30, 2012.
»» 3. C. Subashini, S.B. Dhanesh, T.S. Divya and
J. James. Characterization of pax2 mediated
regulatory mechanisms involved in cerebellar
neurogenesis. Annals of Neurosciences, 2012
19:79. Presented at the XXXth Indian Academy
178
of Neurosciences meet and International
Symposium, Guru Nanak Dev University,
Amritsar, October 27-30, 2012.
»» 4. J James, T S Divya. Pax6 and Tlx3 mediated
regulation of granule neurogenesis in cerebellum.
.Annals of Neurosciences, 2012 19:7. Presented
at the XXXth Indian Academy of Neurosciences
meet and International Symposium, Guru Nanak
Dev University, Amritsar, October 27-30, 2012.
NEUROPHYSICS
Dr. Rashmi Mishra
Scientist C
[email protected]
179
Extra mural funding:3
Sl. No
Investigators
Title
Funding Agency
Duration
1
Rashmi Mishra
Ramalingaswami fellowship
DBT
5 years
2
Rashmi Mishra
Neuro TaskForce Grant
DBT
3 years
3
Rashmi Mishra
Rapid Grant for Young Investigator Award
DBT
3 years
Training programmes attended
»» ‘Leica Superresolution Microscopy Workshop’
at NCBS Campus, Bangalore, 4th October to 6th
October, 2012
180
»» ‘Mechanical manipulations on the scale of cells
and beyond’ MBI Singapore and NCBS workshop
and conference, NCBS Campus, Bangalore, 15th
April till 26th April 2013
HUMAN MOLECULAR GENETICS
Dr. Moinak Banerjee
Scientist E II
[email protected]
Moinak Banerjee received his Ph.D from M.L. Sukhadia University, Udaipur Subsequently
he worked as Post Doctoral fellow at AIIMS, New Delhi and CCMB, Hyderabad, before
joining RGCB in 1996.
Ph.D Students:
Lekshmy Srinivas
Sarada Lekshmi KR
Sanish Sathyan
Femina KMB Nair
Swathy B
Ann Mary Alex
Project Fellows:
Shabeesh Balan
Anaswara Ashok
Technical Support:
Veluthai G
Bindu Asokan
181
Genetics, Epigenetics and Pharmacogenetics of Complex diseases
Autoimmune Hypothesis in Schizophrenia
Lekshmy Srinivas, Chandrasekharan M.Nair*, Priya M. Allencherry**, S. Thara***,
Moinak Banerjee
*Nairs Hospital, Cochin, **Mental Health Centre, Thiruvananthapuram, *** SCARF, Chennai
Schizophrenia is complex disorder which may involve multiple genes with mild to moderate effect
and non-genetic risk factors like environmental
and psychological assaults. Numerous theories
have been proposed regarding the cause of
schizophrenia, ranging from developmental or
neurodegenerative processes or neurotransmitter
abnormalities to infectious or autoimmune
processes. Evidence from genetic linkage and
recent genome-wide association studies on
schizophrenia strongly suggest the involvement
of major histocompatibility complex (MHC) on
chromosome 6p21.3-22.1. This region has long
been proven to have significant role in infection and
autoimmunity, in the development of schizophrenia.
Genome-wide association studies in schizophrenia
have found significant associations with several
single nucleotide polymorphisms (SNPs) across
the major histocompatibility complex (MHC) region
reinforcing an immune component to schizophrenia
risk.
We performed a case-control association study
using 248 patients from Kerala, South India and
244 ethnically matched normal healthy controls.
For the MHC locus we investigated the association
of 15 SNPs from a 2.28 Mbp region spanning the
extended MHC in Chromosome 6p21.33 with
schizophrenia. Genotyping was carried out by
PCR-RFLP, Taqman allelic discrimination assay
and Kaspar assays. Data analysis including allelic,
genotypic, haplotypic and diplotypic frequencies
were calculated and compared using Unphased
v3.0.13 software. LD between SNPs was also
computed using Unphased. Hardy Weinberg
equilibrium (HWE) was calculated and LD plots
were generated using Haploview v4.1. Gene-gene
interactions associated with Schizophrenia were
detected by multifactor dimensionality reduction
(MDR) software
Within the HLA region, the most significant
association was observed for SNP rs3815087.
The TT genotype conferred a 7.9 fold increased
risk for schizophrenia. The T allele was also
strongly associated with the disease. A significant
association with schizophrenia was also observed
for SNP rs9267487. This SNP was in strong LD with
the associated TNFA SNP rs361525. Our findings
on association of MHC region with schizophrenia
support the hypothesis that genetically determined
changes in immune regulation may contribute to
the pathogenesis of schizophrenia confirming the
role of immune genes in disease susceptibility.
Pharmacoepigenomic Response of Antipsychotic Drugs
Swathy B, Sarada Lekshmi K R, Sanish Sathyan, Moinak Banerjee
Antipsychotic drugs are considered as the first
line treatment for psychotic disorders including
Schizophrenia. The patients receiving antipsychotic
medications show a wide variability in drug
response and drug induced side effects which
could be attributed to both genetic and nongenetic components influencing drug response.
Pharmacogenetic studies have identified the
potential involvement of polymorphic genes in
treatment response and drug-induced adverse
events in patients diagnosed with schizophrenia.
182
Epigenetic mechanisms can offer an alternate
mechanism for interindividual drug variability.
The present study aims to investigate the effect
of antipsychotic drugs on expression of genes
of pharmacogenetic interest and whether these
expressions are modulated by differential epigenetic
events in in-vitro model. The expression profile
of pharmacologically relevant genes including
drug metabolizing enzymes (CYP1A2, CYP2D6,
and CYP3A4), drug transporter ABCB1 and
neurotransmitter transporter SLC6A4 was studied
in Hep G2 cell line under treatment of haloperidol
(HLP), a typical antipsychotic drug.
Hep G2 was treated with varying concentration of
haloperidol (0-25µM) for different time interval (6,
12 and 24 hr) and gene expression was quantified
using real time PCR. Gene expression analysis
has shown that haloperidol induced significant
increase in ABCB1 and SLC6A4 gene expression
at 25 µM drug concentration at 24 hour.CYP1A2,
CYP2D6 and CYP3A4 mRNA level was increased
at 25uM haloperidol at all time points,24 hour
showing maximum increase in gene expression.
Results suggest that antipsychotic drugs can
modulate the expression of genes involved in
drug response. Also these gene expression
changes are dosage dependent or time dependent.
Further studies would focus on identifying
the epigenetic mechanisms
underlying variable gene
expression. Understanding
the expression profile of
pharmacokinetic and
pharmacodynamic genes
under the antipsychotic
drug treatment and
epigenetic mechanisms
underlying can account for
the better understanding of
antipsychotic drug response.
Genetics of Aneurysm
Sanish Sathyan, Linda Koshy, HV Easwera, S. Premkumarb, Jacob P. Alapattb, Suresh
Naira, RN Bhattacharya a Moinak Banerjee
a
Department of Neurosurgery, SCTIMST, Trivandrum
b
Department of Neurosurgery, Calicut Medical College, Calicut.
Intracranial aneurysms are pathological dilatations
of the arterial walls frequently located near
arterial bifurcations in the circle of Willis. The
pathogenesis of intracranial aneurysm is unknown.
Catastrophic hemorrhage is commonly first sign of
disease, early identification is essential. Besides
environmental risk factors, genetic factors also
play an important part in pathogenesis of SAH
and intracranial aneurysms. Of major types of
stroke, SAH from aneurismal rupture has the most
evidence in support of genetic cause. Five Genomewide association studies have been carried out in
intracranial aneurysm till now pointing towards
8q11.23-q12.1 region. Putative genes in the region
are involved in cell cycle progression and actin
remodeling. Two SNP’s rs10958409 and rs9298506
were found to be significantly associated with
intracranial aneurysm in European population,
while only rs10958409 was found associated in
Japanese population in the first genome wide
association study. rs10958409 and rs9298506 have
been used commercially for aneurysm detection
panel too. A recent GWAS also pointed towards
rs1072737 in this region. In the present study we
explored the role of 8q11.23-q12.1 region with
intracranial aneurysm in a south Indian population.
All the SNPs selected for the study were in HardyWeinberg equilibrium (p = >0.05). None of the
widely reported SNPs, rs10958409, rs9298506
and rs1072737 were not found to be associated
with intracranial aneurysm in our south Indian
population. We further investigated the role of
these allelic and genotypic variants in gender
stratification and hypertension to understand
whether the genetics of these confounding factors
act as modifiable risk factor. None of the alleles and
genotypes of these variants were found to influence
gender and hypertension in causing intracranial
aneurysm either directly or through modifiable
risk factor.
183
Pharmacogenetics of Epilepsy
Shabeesh Balan, Thashi Bharadwaj,
Sarada Lekshmi, Sanish Sathyan, K.
Radhakrishnan*, Moinak Banerjee.
Department of Neurology, SCTIMST,
Trivandrum, Kerala 695 011
*
Epilepsy constitutes a heterogeneous group of
disorders characterized by recurrent unprovoked
epileptic seizures due to widely different etiologies
that affects an estimated 50 million people
worldwide, of which 80% reside in resource-poor
countries. Although majority of patients with
epilepsy are responsive to presently available
antiepileptic drugs (AEDs), nearly one-third
of them continue to exhibit recurrent seizures
despite optimal AED therapy. Patients who are
unresponsive to the first and second prescribed
AEDs will often remain unresponsive to all other
AEDs, including the newer ones, and to multiple
AED combinations, indicating that they are multidrug resistant from the beginning.
The major targets of action of AEDs are voltage
gated sodium channels, calcium channels,
neurotransmitter systems viz: GABA and
glutamate. Many AEDs such as benzodiazepines,
phenobarbital, gabapentin and topiramate targets
GABA A receptor through positive allosteric
modulation of the receptors and thereby enhancing
GABAA receptor-mediated inhibition. Enormous
Publications
»» Balan S, Vellichirammal NN, Banerjee M,
Radhakrishnan K. Failure to find association
between febrile seizures and SCN1A rs3812718
polymorphism in south Indian patients with
mesial temporal lobe epilepsy and hippocampal
sclerosis. Epilepsy Res.101,3, 288-292, 2012
»» Shabeesh Balan, P. B. Sumitha, Thashi
Bharadwaj, Sarada Lekshmi, Sanish Sathyan,
Kurupath Radhakrishnan, Moinak Banerjee.
Role of ABCB1 variants on postoperative seizure
recurrence in patients with AED-Resistant
Temporal Lobe Epilepsy. Neurology, April 25,
2012; 78: P05.079
»» Neetha N. Vijayan, Anila Mathew, Shabeesh Balan,
Chandrasekhar Natarajan, Chandrashekharan M
Nair, Priya M. Allencherry and Moinak Banerjee.
184
diversity of GABAA receptors has been reported
in the central nervous system (CNS), which
is reflected by the fact that in each receptor,
at least three different subunits are present,
which derive from one of eight structurally and
genetically distinct families. Recent studies have
implicated the role of GABAA receptor variants
with epilepsy susceptibility and antiepileptic
drug (AED) resistance. We investigated the role of
SNPs in GABAA receptor subunit subtype genes
viz; rs2279020 (GABRA1) rs3219151 (GABRA6),
rs2229944 (GABRB2), and rs211037 (GABRG2), in
determining its role in predisposition to epilepsy
and AED response. This was assessed in three
cohorts of subjects with South Indian ancestry:
mesial temporal lobe epilepsy with hippocampal
sclerosis (MTLE-HS) (prototype of AED-resistant
epilepsy syndrome), juvenile myoclonic epilepsy
(JME) (prototype of AED-responsive epilepsy
syndrome) and ethnically matched non-epilepsy
controls.
We report a significant allelic and genotypic
association of a synonymous variant in GABRG2,
rs211037 (Asn196Asn) with epilepsy irrespective of
its phenotype i.e MTLE-HS or JME. However, this
association was not retained in epilepsy patients
with history of febrile seizure. The GABAA receptor
subunit subtype genes were not found to have
any role in susceptibility to drug resistance or
refractoriness.
Antipsychotic drug dosage and therapeutic
response in schizophrenia is influenced by
ABCB1 genotypes, a study from South Indian
perspective. Pharmacogenomics 13 (10), 11191127, 2012.
»» Isha Chauhan, Beena V.T., Lekshmy Srinivas,
Sanish Sathyan, and Moinak Banerjee Association
of Cytokine Gene Polymorphisms with Oral
Lichen Planus in Malayalam-Speaking Ethnicity
from South India (Kerala). Journal of Interferon
& Cytokine Research. May 7, 2013, doi:10.1089/
jir.2012.0115.
Conferences
»» Moinak Banerjee. How to dissect a complex
disorder. Academic Staff College, University of
Kerala. 19th July, 2012.
»» Moinak Banerjee. Challenges in Transplantation
a n d Tr a n s f u s i o n m e d i c i n e . S C T I M S T,
Thiruvananthapuram. 28th Sept, 2012.
»» Shabeesh Balan, P. B. Sumitha, Thashi
Bharadwaj, Sarada Lekshmi, Sanish Sathyan,
Kurupath Radhakrishnan, Moinak Banerjee.
Role of ABCB1 variants on post operative
seizure recurrence in drug resistant temporal lobe
epilepsy patients of South India. 137th Annual
Meeting American Neurological Association’s
Boston, MA October 7-9, 2012.
»» Moinak Banerjee. Key Note lecture during CDE
on Craniofacial anomalies Govt. Dental College,
Thiruvananthapuram 26th Oct., 2012.
»» Moinak Banerjee. Challenges in understanding
complex disorders. Academic Staff College,
Calicut University, Calicut, 14th Dec, 2012.
»» Moinak Banerjee. Impact of human genome
project in today’s science. AIR Trivandrum. 19th
Jan 2012
»» Moinak Banerjee. Is Pharmacogenomics a reality?
Workshop on Genetics & Genetic Disorders, St
Thomas College, Changanassery, 16th Feb, 2012.
»» Moinak Banerjee. Pharmacogenomics in Complex
disorders. International Conference on New
generation Bioinformatics ICNGB 13. 26th Feb.
2013
»» KR. Sarada Lekshmi, S Sanish, B Shabeesh,
B Swathy, NV Neetha, VN Indu, M. Banerjee
Global DNA methylation and mRNA expression
of MBD2 in peripheral blood leukocytes and
association of MBD2 polymorphisms with
schizophrenia in south Indian population “Joint
Conference of HGM 2013 and 21st International
Congress of Genetics 13 – 18 April 2013,
Singapore.
»» S. Sanish, KR Sarada Lekshmi, VH Linda
Koshy, S. Balan, B. Swathy, S Premkumar, JP
Alappatt, M. Banerjee. ELN-LIMK 1 locus in
7q11 and intracranial aneurysm –a south Indian
perspective. “Joint Conference of HGM 2013 and
21st International Congress of Genetics 13 – 18
April 2013, Singapore.
»» B. Swathy, KR Sarada Lekshmi, S. Sanish, B
Shabeesh, M. Banerjee. Effect of antipsychotic
drugs on global DNA methylation and DNA
methyltransferase expression in cell lines. Joint
Conference of HGM 2013 and 21st International
Congress of Genetics, 13 – 18 April 2013,
Singapore.
Workshop
»» Sanish Sathyan attended Wellcome trust Open
Door Workshop: Working with the Human
Genome Sequence 3-6, December 2012, Vietnam.
»» Swathy B attended “Workshop on Genomics &
Beyond”.16th ADNAT meeting .6-16th December,
2012
185
K. Santosh Kumar Ph.D
Scientist EII
[email protected]
Santhosh Kumar is a Ph.D in Chemistry from the School of Chemical Sciences, Mahatma
Gandhi University, Kottayam. He did his Postdoctoral training at the Department of
Biochemistry, University of Illinois, Urbana-Champaign, USA and joined RGCB in 1996.
Ph. D Students:
Parvin Abraham
Reshmi V.
Preethi. P. C
Asha. R
186
Project Fellow:
Smitha Devi
Structure function studies of an antimicrobial peptide BrevininCcuL
isolated from Clinotarsus curtipes of Western Ghats
Parvin Abraham & K. Santhosh Kumar
Cationic peptides with the ability to destroy a broad
spectrum of pathogens have been isolated from all
life forms and are considered as nature’s potential
antibiotics. These amphipathic antimicrobial
peptides (AMPs) are evolutionarily conserved
components of the innate immune response and are
produced as a part of the host defense mechanism.
Compared to conventional antibiotics, actions of
AMPs are rapid and therefore the possibility to
develop resistance by any microbial strain is in
the several orders of magnitude lower than that
of traditional antibiotics. Amphibian skins are
rich source of AMPs which are secreted by the
granular glands and released to the skin surface
upon different kinds of stimuli. A novel peptide
brevininCcuL (BCcuL) with potent antibacterial
activity was isolated from the skin secretion of the
frog Clinotarsus curtipes of the Western Ghats,
Kerala using RP-HPLC and characterized by amino
acid analysis, sequencing and MALDI TOF MS MS
techniques. It is active against gram negative and
certain gram positive bacteria. Several truncated
and substituted analogs of BCcuL were chemically
synthesized to analyse the importance of N and C
terminus amino acids on its activity. The results
showed that N-terminal amino acids, cationicity,
amphipathicity, hydrophobicity and helicity are
playing important role on antimicrobial character
and its specificity. Optimum balances between
these parameters are essential for antimicrobial
activity. BCcuL and its analogs showed a high
propensity to adopt α-helical structure in membrane
mimicking solvent. Dye release studies from
LUVs showed that peptides have the ability to
permeabilise the bacterial membrane.
Fig1. Helical wheel representing the spatial arrangement of hydrophobic and hydrophilic amino acids in BCcuL
and BCcuL-LIA.
Calculated
Observed
Net
PI
peptide amide peptide amide
charge (theoretical)
mass (Da)
mass (Da)
No of
residue
Amino acid sequence
GRAVY
BCcu L
21
LIAGLAANFLPQILCKIARKC
1.067
+3
9.50
2255.84
2255.57
BCcu K
21
KIAGLAANFLPQILCKIARKC
0.700
+4
9.85
2270.86
2270.90
BCcu W
21
WIAGLAANFLPQILCKIARKC
0.843
+3
9.50
2328.89
2329.94
BCcu LGG
21
LGGGLAANFLPQILCKIARKC
0.728
+3
9.50
2185.71
2188
BCcu -L
20
_ IAGLAANFLPQILCKIARKC
0.930
+3
9.50
2142.68
2143.87
BCcu -LI
19
_ _ AGLAANFLPQILCKIARKC 0.742
+3
9.51
2029.52
2030.8
BCcu -LIA
18
_ _ _ GLAANFLPQILCKIARKC
0.683
+3
9.50
1958.45
1958.09
BCcu -LIAG
17
_ _ _ _ LAANFLPQILCKIARKC
0.747
+3
9.50
1901.39
1902.6
Peptide
Table 1. Structural parameters of BCcuL and analogs using ProtParam tool.
187
200
% (Brevinin Ccu )
% (Brevinin Ccu LGG)
% (Brevinin Ccu W)
% (Brevinin Ccu K)
150
100
50
0
50
0
-50
-50
-100
190
% (Brevinin Ccu I)
% (Brevinin Ccu G)
% (Brevinin Ccu A)
% (Brevinin Ccu L)
100
Molar Ellipticity (θ)
Molar Ellipticity (θ)
150
200
210
220
230
240
-100
190
250
200
Wavelength (nm)
210
220
230
240
250
Wavelength (nm)
Fig 2. CD spectra of the peptides in 30% TFE.
Peptides
Minimum Inhibitory Concentration (µg/ml)a
E. coli
MG1655
V.cholerae
S.aureus
25293
S.
epidermidis
12228
B.subtilis
14416
Percentage
Hemolysis
BCcu L
7
15
50
>200
>200
37.5
BCcu K
20
50
>100
>200
>200
13.7
BCcu W
8
25
>100
>200
>200
35.24
BCcu LGG
60
>100
ND
>200
>200
23.4
BCcu -L
20
35
>100
>200
>200
32.5
BCcu -LI
100
>100
>100
>200
>200
26.8
BCcu -LIA
18
70
>100
>200
>200
28.2
BCcu -LIAG
50
>100
ND
>200
>200
26.9
Concentrations represent mean values of three independent experiments performed in triplicates.
Table 2. Antibacterial and hemolytic activity of BCcuL and analogs.
Fig 3. Release of calcein from LUV upon treatment with BCcuL and analogs.
188
Structure- function studies of Esculentin-2 peptide amides from the
skin secretion of Hylarana temporalis.
Reshmy V and Santhosh Kumar K
Over usage and misuse of antibiotics lead to
the emergence of resistant microorganisms to
clinically used drugs has resulted severe crisis
in the treatment and management of infectious
diseases. This initiated the quest to identify new
classes of antibiotics that can display diverse
mode(s) of action. Host defense peptides (HDPs) are
endogenous antibiotics secreted by the endocrine
glands, play a multifunctional role in the innate
immunity of vertebrates that permit all multicellular
organisms to live in harmony with microbes. They
are considered as the most promising candidates for
the development of novel anti-infective agents. The
anuran (frog) skin is the most generous sources for
these polycationic peptides which are vary in size,
sequence and conformation. Analysis of the c-DNA
isolated from the skin secretion of Indian bronzed
frog Hylarana temporalis lead to the identification
of two novel peptides Esculentin-2HLte1(E2HLte1)
and Esculentin-2HLte2 (E2HLte2). These peptides
and their N-truncated analogs were chemically
synthesised by solid-phase synthesis (SPPS) using
Fmoc amino acids to understand the role of each
amino acids and the secondary structure of the
peptide on bio-activity. Synthetic peptides were
then purified by RP-HPLC and their purity and
identity were confirmed by RP HPLC and MALDITOF-MS. Analysis showed that parent peptides has
better MIC against both gram positive and gram
negative clinical strains compared to the truncated
peptides and are least hemolytic and cytotoxic
towards eukaryotic cells.
Fig.1: 37-residue peptide esculentin-2HLte1 (a) α-helical structure (b) RP-HPLC profile (c) mass spectrum.
Fig.2:
32-residue truncated esculentin-2HLte1 a) α-helical structure b) RP-HPLC profile c) mass spectrum.
189
Fig.3: 22-residue truncated esculentin-2HLte1 peptide a) α-helical structure b) RP-HPLC profile c) mass spectrum.
Fig.4: 37-residue esculentin-2HLte2 peptide a) RP-HPLC profile b) α-helical structure c) mass spectrum.
Micro organism
Gram positive
S.aureus (MTCC
9542)
Antibiotic (μM)
MIC of Peptides (μM)
Kanamycin Ampicillin E2HLte1 E2HLte1 E2HLte1 E2HLte1 E2HLte2 E2HLte2
-
0.7
1
>50
100
25
>100
>100
P.Aeurogenosa
(MTCC 8076)
50
-
25
100
>100
>100
>100
>100
Mc Vo9-clinical
3
-
3
25
50
50
100
>100
ETEC
1
-
3
25
>25
100
>100
>100
Gram negative
Table 1: Activity of mature peptides and their truncated sequences.
190
Fig.5: Hemolytic effect of mature peptide
Fig.6: Effect of peptides on viability of HEK cells
Fig.7: Graphs representing MICs of mature peptides along with control antibiotics.
Arginine Rich Cell Penetrating Peptides as
Efficient Antimicrobial Agent
Asha R and K. Santhosh Kumar
Cell Penetrating Peptides (CPP) has the ability to
penetrate into the cell membrane and translocate
across it without causing toxicity to the cell. This
type of molecules can be used as a delivery vehicle
to deliver macromolecules into cells. A thorough
understanding of their primary and secondary
structural requirements and mechanism of action
can help to design short peptides as carriers for site
specific drug delivery, gene delivery and molecular
transporters for controlling and regulating various
biochemical processes within the cell.
Arginine rich peptides (10-mer) having
XRXRXRXRXR template (‘R’ is arginine and ‘X’ is a
natural amino acid) were designed and synthesized
to analyse the importance of their primary and
secondary structure on function. A judicial selection
of the amino acids and their sequential connectivity
191
in the peptide could provide a clear understanding
about CPPs, their specificity in selectivity, how
they interact differently with various biological
membranes and perform different functions such
as antibacterial, hemolytic or cell penetration
without causing cytotoxicity. This could also help
to understand how a peptide can achieve selectivity
between microbial membranes and mammalian
membranes. This could also help us to understand
various parameters that helped to change the cell
disruption property of AMPs to cell penetration
property of CPPs without causing cytotoxicity to
the cell. Peptides R5V5, R5M5 and R5Y5 were
manually synthesized by solid phase synthesis,
Fig 1.
Fig 2.
Fig 3.
Fig 1, 2, 3. Mass spectra and HPLC profiles of the peptides
192
purified by RP-HPLC and characterized by MALDITOF-MS. The minimum inhibitory concentrations
(MICs) of the peptides were determined using broth
dilution method. R5V5 and R5Y5 showed good
activity against tested gram negative and positive
bacteria. The peptide was labeled with fluorescein
isothiocyanate to study the cell penetration ability.
The fluorescence microscopic images of labeled
the labeled R5Y5 showed that they can penetrate
in to the MCF7 cells without causing cytotoxicity.
Presence of five arginine residues in alternate
position with tyrosine can show cell penetration
ability.
Peptide
MIC(µM)
E.coli (MTCC 4315)
S.aureus (MTCC 9542)
R5V5
6.25
12.5
R5M5
25
>100
R5Y5
25
6.25
R5I5
50
100
Kanamycin
12.5
-
Ampicillin
-
0.7
Table 1. Antibacterial activity of the peptides
Fig. 4 Cellular uptake of R5Y5 peptide by MCF7 cells
Fmoc synthesis of peptide thioesters by Double linker strategy for the
synthesis of proteins by native chemical ligation
Preethi. P. C and K. Santhosh Kumar
Chemical ligation is a technique used for the
chemical synthesis of mini-proteins and their
analogs to study the structure function analysis of
proteins. It can be carried out by joining two peptide
fragments via a peptide bond. One fragment
should be a C-terminal peptide thioesters and the
other fragment should be a peptide containing
an N-terminal cysteine residue. Trans-thioesterification takes place with the side chain thiol
of the N-terminal cysteine residue and the resulting
intermediate spontaneously rearranges to a fivemembered ring. The intramolecular nucleophilic
attack by the cysteine R-amino group results the
formation amide bond at the ligation site.
Synthesis of peptide thioester using Fmoc amino
acid is problematic because the thioesters undergo
cleavage during the piperidine cleavage of Fmoc
deprotection. Stability of the thioester can be
enhanced by introducing a spacer between the
resin and the alkane sulfonamide. The polymer
support also plays a very important role in
determining the yield and purity of the thioester.
A double linker strategy was used to increase
the stability of the thioester by introducing a rink
amide linker and 3-carboxy propane sulfonamide.
The C-terminal amino acid incorporation required
24h stirring at -20oC. 1,8-Diazabicyclo[5.4.0]undec7-ene (DBU) for the removal of Fmoc groups. The
purity was analysed by RP-HPLC and MALDI-TOF
MS. A comparative study showed that the peptide
thioester synthesized without activation with
iodoacetonitrile is more pure than that synthesised
with activation.
193
mV
UV Detector Ch1:214nm
175 UV Detector Ch2:280nm
mV
800
150
700
125
600
500
100
400
75
300
50
200
25
100
0
0
-100
-25
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
32.5
min
Fig 1. RPHPLC profile of peptide thioester synthesized
on CLEAR resin by double linker Fmoc strategy (with
activation)
-200
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
32.5
min
Fig 2. R HPLC profile of peptide thioester synthesized on
CLEAR resin by double linker Fmoc strategy (without
activation)
mV
mV
600
550
125
500
450
100
400
350
75
300
50
250
200
25
150
100
0
50
0
-25
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
32.5
min
on Tentagel resin by double linker Fmoc strategy (with
activation)
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
32.5
min
Fig 4. RPHPLC profile of peptide thioester synthesized on
Tentagel resin by double linker Fmoc strategy (without
activation)
mV
mV
1100
1000
1500
900
1400
800
700
1300
600
1200
500
400
1100
300
200
1000
100
900
0
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
32.5
min
on Merrifield resin by double linker Fmoc strategy (with
activation)
Publications
»» Won-Tak Choi, Santhosh Kumar, Navid
Madani,Xiaofeng Han,Shaomin Tian,† ChangZhi Dong,Dongxiang Liu, Srinivas Duggineni,Jian
Yuan,Joseph G. Sodroski, Ziwei Huang and Jing
An “A Novel Synthetic Bivalent Ligand To Probe
Chemokine Receptor CXCR4 Dimerization and
Inhibit HIV1 Entry” Biochemistry. 2012, 51 (36),
pp 7078–7086
194
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
32.5
min
Fig 6. RPHPLC profile of peptide thioester synthesized on
Merrifield resin by double linker Fmoc strategy (without
activation)
»» Dong CZ, Tian S, Choi WT, Santhosh Kumar,
Liu D, Xu Y, Han X, Huang Z, An J. Critical Role
in CXCR4 Signaling and Internalization of the
Polypeptide Main Chain in the Amino Terminus
of SDF-1α Probed by Novel N-Methylated
Synthetically and Modularly Modified Chemokine
Analogues. Biochemistry. 2012, 51 (30), pp
5951–5957
CHEMICAL BIOLOGY – 2
Sanil George, PhD
Scientist EII
[email protected]
Sanil George received his Ph.D in Zoology at Mahatma Gandhi University, Kerala and
joined RGCB in 1992.
Project Fellows:
Mr. Sujith V.G
Miss Julie P.T
Ph.D Students
Vineethkumar T.V
195
Studies on the Host defence peptides isolated of frog skin secretions
Vineethkumar T.V and Sanil George
Co evolution with microbes in the habitat lead
to the development of an array of host defence
peptides, which provide the first line of defence
against invading microbes. Host defence peptides
are secretary peptides that serve universally in all
living organisms for both offensive and defensive
purposes. They can rapidly kill a broad range
of microbes and have additional activities that
impact on the quality and effectiveness of innate
responses and inflammation. Furthermore, the
challenge of bacterial resistance to conventional
antibiotics and the unique mode of action of
host defence peptides have made such peptides
promising candidates for the development of
a new class of antibiotics. The unique diverse
function and architecture of these peptides
has opened new vistas in designing molecular
template for new anti-infective drugs.
Amphibian skin is a rich source of host defence
peptides. Their amphibious nature enabled the
immune system to defend against the invasions
from both terrestrial and aquatic environment.
Anuran skin secretary peptides are classified
under Frog skin active peptide family (FSAP).
Peptides of this family are classified on the basis
of their biological activity as (a) Antimicrobial
peptides(AMPs) (2) Smooth Muscle active peptides
(3) Nervous system active peptides. Hylarana
malabarica, an endemic frog species were
collected from different parts of Western Ghats,
India. The skin secretions were obtained by mild
transdermal electrical stimulation and collected by
washing the dorsal region with nuclease-free water.
The frogs were then released immediately to the
same habitat. The collected solutions were snap
frozen with liquid nitrogen and were lyophilized.
Peptidomic Approach: Lyophilized skin
secretions were dissolved in 1ml (TFA)/ water.
The sample was purified by reverse phase highperformance liquid chromatography (250x4.60mm
phenmenex C18 RP-HPLC) column. It was
eluted with a gradient formed from TFA/water:
0.1:99.9(v/v) (Buffer A) to TFA/water/ acetonitrile:
0.1/19.9:80 (v/v/v) (Buffer B) for 130 min at a flow
rate of 1ml/min. Absorbance of the column effluent
was monitored at 214 nm and 280nm (Fig.1). The
peak fractions of different time intervals were
collected and subjected to the antimicrobial
activity test. The fractions showing antimicrobial
activity were further purified using the same RPHPLC column but with revised programs to a final
purity higher than 95%, and were lyophilized. The
lyophilized samples were used for subsequent
structural characterization including de novo
sequencing and MS analysis.
mV
200 Detector A Ch1:214nm
Detector A Ch2:280nm
MPa
175
35.0
150
30.0
125
25.0
100
20.0
75
15.0
50
10.0
25
5.0
0
0
10
20
30
40
50
60
70
80
min
0.0
Fig. 1 RP-HPLC profile of lyophilized crude skin secretion of Hylarana malabarica. Lyophilized skin secretion
reconstituted in 1ml (TFA)/water, injected onto 250x4.60mm phenmenex C18 RP-HPLC column, eluted with a gradient
formed from TFA/water:0.1:99.9(v/v)to TFA/water/acetonitrile: 0.1/19.9:80(v/v/v) in 130 min at flow rate 1ml/min.
196
Transcriptomic Approach: Polyadenylated
mRNA was isolated using magnetic oligo (dT) beads
and PCR-based cDNA library was constructed
using SMART- cDNA Amplification Kit (Clontech,
UK). RACE product obtained (Fig.2) was purified
and cloned into pGEM-T easy vector system
(Promega Corp.). Plasmid isolated from each
positive clone, was further purified and sequenced
using automated sequencer. The primary structure
of deduced peptides was subjected to homology
searches using BLAST (NCBI) and Antimicrobial
peptide database was used for peptide prediction.
Theoretical molecular weight, net charge,
hydrophobicity of the peptides was computed using
ProtParam (http://expasy.org/tools/protparam.
html). The peptides obtained was then be named
Details of Isolated peptides
30 novel host defence peptides were isolated by
transcriptomic approach. Of the 30 peptides 21
Fig.2. Gel image showing RACE product (1-3)
based on the accepted nomenclature system for
antimicrobial peptides.
peptides belong to 10 known families and 9 belong
to new families (Fig.3). The isolated peptides were
again subjected to secondary structure prediction
using PEP-FOLD software (Fig.4).
Fig. 3 Peptides isolated by trascriptomic approach. The numbers in parenthesis denoted the paralogs obtained in
each family.
Fig. 4 Secondary structures of the peptides isolated from Hylarana malabarica
197
Molecular Phylogeny and Population Genetics of Amphibians Endemic
to Western Ghats Biodiversity Hot spot.
Sujith V Gopalan and Sanil George
Collaborator: Dr. Juha Merilla, Ecological Genetics Research Unit, Department of Biosciences,
University of Helsinki, Finland
Amphibians are facing a global decline, with 41%
of known amphibian species threatened by the
risk of extinction. Many causes are suggested to
contribute to the observed declines, including
increased levels of UV-B radiation and pollution,
climate change, emerging infectious diseases,
habitat loss and fragmentation. On the other hand,
recent studies have identified many new cryptic
amphibian lineages and species from different
biodiversity hotspots, indicating that the amphibian
diversity within these hotspots may be heavily
underestimated. Biodiversity hotspots are regions
rich in endemic species diversity, but at the same
time, are also known to experience exceptional
habitat losses. An increasing number of reports
about amphibian extinctions from biodiversity
hotspots – even before their scientific description–
have generated much concern regarding efforts of
their conservation. Therefore, there is an urgent
need to increase efforts towards studying diversity
and identifying the potential genetic problems that
local amphibian populations are likely to be facing
in these hotspots.
Fig. 5. Map showing the collection localities of Indirana
beddomii frog populations from Western Ghats, India
198
The Western Ghats-Sri Lanka biodiversity hotspot
is one of the world’s recognized biodiversity
hotspots. The Western Ghats are comprised of
mountain chains running parallel to the west coast
of India for over 1600 km (Fig. 1). Along its entire
length, there is only one major discontinuity – the
Palghat Gap of Kerala – which is a low mountain
pass at an elevation of only 100 m asl and about
30 km in width. Another smaller (7.5 km) gap – the
Shencottah Gap – is present at 9° N (Fig. 1). These
mountain chains harbour diverse endemic flora
and fauna. The endemic diversity is particularly
pronounced for amphibians, as many new families
and genera have recently been discovered from
these mountain ranges. In general, the amphibian
fauna of southern India is one 8 of the most diverse
– and poorly known – in tropical Asia. Presently,
about 132 species are known to be endemic to this
region. Current knowledge of the amphibian fauna
of the Western Ghats is scant and fragmented,
Fig. 6. Unrooted neighbour joining tree based on Nei’s
DA distances estimated from the microsatellite data.
The nodes indicate the bootstrap values over loci (1,000
replicates). The population numbers correspond to
localities in Fig 5. Colour of the population abbreviations
correspond to Fig. 7.
Fig. 7. Population clustering as indicated by structure analyses. The vertical column numbers correspond to
populations in Fig 5. The populations are partitioned into three (K = 3) clusters.
but it is known to be unique with a high degree of
endemism (Inger 1999; Biju 2001). There are three
families (viz. Micrixalidae, Nasikabatrachidae and
Ranixalidae) and 10 genera which are endemic to
the Western Ghats
The goal was targeted to resolve the phylogenetic
relationships between species of frogs endemic
to Western Ghats, including identification of yet
unknown (i.e. cryptic) species. The secondary aim
was to study genetic variability and differentiation
within and among different populations of these
endemic frogs, with the aid of novel microsatellite
markers developed specifically for this purpose. In
addition, we also investigated the possible presence
of amphibian diseases (cf. Chytrid and Ranavirus
infections), known to have caused amphibian
declines, in the Western Ghats biodiversity
hotspot. The broader aim of the present work was
to contribute to the understanding of taxonomic
and genetic biodiversity of the poorly studied
amphibian fauna of the Western Ghats biodiversity
hotspot, as well as to produce information useful
for delimiting management and conservation units
in endemic frogs.
Biodiversity, DNA barcoding and Phylogeny of commercially valuable
marine molluscs of India
Julie P.T and Sanil George
Collaborators: *Dr. Bjukumar A, Department of Aquatic Biology and Fisheries, University of
Kerala.**Dr. Patterson Edward, Suganthi Devadason Marine Research Institute, Tuticorin,
India
Mollusca represent one of few phyla that are
of India based on conventional and molecular
routinely considered in marine biodiversity surveys
taxonomy methods (using mitochondrial genes
and often considered as “indicator
group” for rapid assessment of
biodiversity. The 3 years multiinstitutional project involving Rajiv
Gandhi Centre for Biotechnology,
Thiruvananthapuram, Department
of Aquatic Biology and Fisheries,
University of Kerala and Suganthi
Devadason Marine Research
Institute, Tuticorin, Tamil Nadu
was implemented in September
2011 with the major objective to
prepare biodiversity database and
Amphioctopus neglectus
Limaria (Limaria)fragilis
DNA barcode data of commercially marine molluscs of and to prepare
a database on marine molluscs Fig. 8 Molluscs reported for the first time from India
199
such as cytochrome oxidase subunit 1, cytochrome
b, 12S and 16S genes). The project recorded the
presence of 183 (west coast of India) and 172
species (east coast of India) of molluscs during the
first year of survey. All the specimens collected will
be geo-referenced and photographed using digital
camera in standard data sheets for beta biodiversity
studies. Collections were made from the east and
west coasts of India, covering Kerala, Karnataka,
Goa, Maharashtra, Tamil Nadu and Odisha states.
Extra-Mural Grants
Sl. No
1
Investigators
Sanil George
K. Santhosh Kumar
Ajay Kumar
Title
Funding Agency
Duration
Antimicrobial peptides from the skin
secretion of fungoid frog Hylarana
malabarica of Western Ghats, India
KSCSTE
2012-2015
GENBANK SUBMISSIONS
JX185406-JX185412
»» Vineeth Kumar T V and Sanil George (2013) Host
Defence Peptides from the Skin Secretion of
Endemic Frog of Western Ghats. 4 th Indian
peptide symposium organized by Indian Peptide
Society (IPS) Feb 16-22, 2013, Saha Institute of
Nuclear physics, Kolkata.
PUBLICATIONS
»» Bijukumar A., Kate l. Sanders, Sanil George &
John C Murphy. 2012. The status of Eurostus
dussumierii and Hypsirhina chinensis (Reptilia,
Squamata, serpentens): with comments on the
origin of salt tolerance in homolopsid snakes.
Systematics and Biodiversity 10(4):479-489
»» Ramachandran K., Wilkinson, M Oommen, V.O.,
George, S., Nussbaum R.A and D.J. Gower 2012.
On the systematics, distribution and conservation
status of Ichthyophis longicephalus Pillai, 1986
(Amphibia: Gymnophiona: Ichthyophiidae).
J.Nat.Hist. 46 (47-48), 2935-2959
»» Suresh Kumar U, Ratheesh R.V, George Thomas
and George S. 2012. Use of DNA Barcode
Sequences in Wildlife Forensics – A Study of
200
Sambar Deer. Journal of Forest Science and
Technology. 8 (4), 224–226.
»» A. Nair, S. V. Gopalan, S. George, K. S. Kumar,
A.G.F Teacher & J. Merilä 2012.Endemic
Indirana frogs of western Ghats biodiversity
hotspots. Ann.Zool.Fennici 49(5-6): 257-286.
»» A. Nair, S. V. Gopalan, S. George, K. S. Kumar,
Takahito Shikano & J. Merilä 2012. Genetic
variation and differentiation in Indirana beddomii
frogs endemic to the Western Ghats biodiversity
hotspot. Conserv Genet. 13(6): 1459-1467.
»» A. Nair, S. V. Gopalan, S. George, K. S. Kumar &
J. Merilä 2012. Cross-species testing and utility
of microsatellite loci in Indirana frogs. BMC
Research Notes 5:389.
»» Sujith V. Gopalan, Abhilash Nair, K. Santhosh
Kumar, Juha Merilä, Sanil George 2012.
Morphology of Indirana semipalmata (Boulenger,
1882) (Amphibia;Anura) adults and tadpoles
from the Western Ghats, India. Herpetology
Notes, 5: 263-273
»» K.U. Suresh , R.V. Ratheesh, R. Chungath, G.
Thomas , S. George 2012: Co-Inheritance Of
Y-Chromosome Haplogroups And Lineages In
‘Parayi Petta Panthirukulam’: An Evaluation
Of Human Motifs In A Popular Folktale In
Kerala, India.. The Internet Journal of Biological
Anthropology. 5:1-7
CHEMICAL BIOLOGY -3
G.S. Vinod Kumar, PhD
Scientist EII
[email protected]
Vinod Kumar received his Ph.D in Polymer Chemistry from School of Chemical Sciences,
Mahatma Gandhi University, Kottayam and joined RGCB In 2004.
PhD Students:
Mithun V Varghese
Ashwani Kumar. N
Amritha Vijayan
Meenu Vasudevan. S
Project Fellows:
Parvathi Nair
Devika Nandan. C
Tech Assistant:
S.Jannet Binu
201
Methacrylic Based Nanogel for the pH Sensitive Delivery
of 5-Flourouracil
Ashwani Kumar N, Nisha Asok Kumar*, S. Asha Nair* and G.S. Vinod Kumar
Collaborator: * CRP-4, Rajiv Gandhi Centre for Biotechnology
The pH sensitivity of the polymeric drug delivery
vehicle is an inevitable factor in the field of oral
drug delivery systems in the treatment of colon
cancer. Even though 5-Flourouracil (5-FU) was
proved to be the most effective drug employed
for the treatment of colon cancer; the inefficacy is
due to several factors such as short biological half
life, poor absorption due to dihydropyramidine
dehydrogenase enzyme and nonselective action
against healthy cells of gastrointestinal tract and
bone marrow. In addition to these; there exist
variations in the transit time throughout the
colon, the drug release can be incomplete from
the polymer coated tablet when the colon specific
tablet matrix does not readily disintegrate and
the treatment with 5-FU remains inadequate. To
overcome these limitations and to improve the
targeted delivery of 5-FU; the drug needs to be
given in nano sized formulation to the colon with
a pH sensitive polymer matrix. Methacrylic based
copolymers are known to be showing the property
of pH sensitive swelling behaviour and also form
Figure.1 1H NMR spectra of Methacrylic acid-co-2-Ethyl hexyl acrylate (MAEHA)
copolymer. Methyl (-CH3) and methylenic (-CH2-) and 30 methyl groups are shown as
(Peak A, B, C) and methylenic group adjacent to the carboxyl group of EHA as peak D.
Figure.2 Differential Scanning Calorimetry of Methacrylic acid-co-2-Ethyl hexyl acrylate
(MAEHA) copolymer
202
Figure.3 Cellular uptake studies of MAEHA nanogel, Confocal images of HCT116 cells with Rhodamine-entrapped
MAEHA nanogel and Blank MAEHA nanogel
the hydrogel matrix. Hydrogels
are hydrophilic polymeric three
dimensional networks which
exhibit a semi-solid morphology
and can absorb large amount
of water. The hydrophilicity is
due to the presence of ionisable
functional groups which accounts
for the characteristics of the
hydrogel such as permeability,
mechanical stability and
biocompatibility.
Figure.4 The MTT analysis of MAEHA nanogels with 5-FU on HCT116
cells at 24, 48 and 72 h
Biodegradable hydrogels
have been represented as an
attractive dr ug for mulation
because of their advantages
such as biocompatibility, high
responsibility for specific
degradation, and a feasible
approach to incorporate drug into
matrices. Hydrogel- based devices
belong to the group of the swelling
controlled drug delivery systems.
The swelling of the hydrogel
depends on the pH of the medium.
In case of methacrylic based
hydrogels the swelling is observed
at pH 7.2-7.8. So in the colonic
environment they are assumed
to exhibit a dynamic swelling
behaviour and sustained release
of the entrapped drug molecules.
The design of an amphiphlic
hydrogel based drug delivery
systems (DDS) by incorporating
a hydrophobic moiety to the
methacrylic based copolymer will
203
serve as an efficient system with better loading of
hydrophilic drug and high mechanical stability. In
oral route of drug administration to colon the (DDS)
should be able to bypass the high acidic pH of the
stomach (pH=1.5-2.5), pH of duodenum (pH=6)
and needs to reach the colon having pH=7.6-7.8.
The proposed polymer in the work having ionisable
–COOH group shows pH sensitive swelling
behaviour in basic pH than acidic environment.
In the present study we have synthesized a novel
copolymeric pH sensitive DDS for the delivery of
5-FU using Methacrylic acid (MA) and 2-Ethyl
hexyl acrylate (EHA). The synthetic Methacrylic
acid-Ethyl hexyl acrylate (MAEHA) copolymer
was characterized by using different techniques
like Fourier Transform Infrared spectroscopy
(FT-IR), Proton-Nuclear Magnetic Resonance
Spectroscopy (H1 NMR) (Fig.1) and Differential
Scanning Calorimetry (DSC) (Fig.2). The nano sized
hydrogel containing the 5-FU was prepared using
the MAEHA copolymer by solvent evaporation
technique. The size of the nanogel and morphology
was characterized using Transmission Electron
Microscopy (TEM) and Atomic Force Microscopy
(AFM). The thermal behaviour of the polymer was
also monitored using DSC. In-vitro drug release
behaviour was assessed to study the pH sensitive
drug release behaviour of the nanogel. For biological
evaluation HCT-116, colon cancer cell line was
selected. The cellular uptake of these nanogels
was studied with a fluorescent dye using confocal
microscopy (Fig.3). Controlled release of the drug
from the nanogel and its in-vitro cytotoxicity when
compared to free 5-FU was analyzed using MTT (3[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium
bromide) assay (Fig.4). In order to confirm the
induction of apoptosis in the tumor cell line, Poly
(ADP-ribose) Polymerase (PARP) cleavage was
detected by Western Blot analysis. The alteration
in cell cycle was investigated by Flow Cytometer.
Dry Powder Cationic Lipopolymeric Nanomicelle Inhalation for
Targeted Delivery of Antitubercular Drug to Alveolar Macrophage
Mithun V Varghese, Annapoorna K* , K.C. Sivakumar**, Sathish Mundayoor* and
G.S.Vinod Kumar
Collaborators: *Mycobacterium Research Group, RGCB,**Bioinformatics Facility, RGCB
After the approval of first inhalable antimicrobial
agent (Nebupent®) by US FDA; researchers realized
the potential of inhalation route, especially for
treating the ailments affecting lung. Compared
to conventional oral therapy, inhalation drug
delivery proved to be superior in delivering high
pay load of drug to deep of lung (alveoli). The
limited clearance of deposited materials from the
lung is the greatest deterrent in the development
of inhalable formulations. Pressurized inhalers
are the first generation portable inhalation dosage
forms. They need patient co-ordination and it is
also a fact that loss of drugs in oropharyngeal
region by impaction is more when compared to dry
powder inhalers (DPI). In recent years importance
of pressurized inhalers are observed to be waning
due to environmental problems and huge loss of
administered dose.
Excipients having self assembling properties are
less explored in the field of dry powder inhalation
(DPI) technology. An amphiphilic lipopolymer
204
system was developed using stearic acid (SA) and
branched polyethyleneimine (BPEI) (1800 Dalton)
at different proportions by covalent conjugation.
Molecular dynamic (MD) simulation tool was
employed for predicting the carrier behaviour
in polar in vivo condition (Fig.5). The structural
characterization was carried using nuclear
magnetic resonance spectroscopy (NMR) and
fourier transform-Infrared spectroscopy (FTIR). The
physical nature of the lipopolymer was analysed by
differential scanning calorimetry. Determination of
zeta potential and diameter of the micelles showed
existence of cationic particles in the nano size
range when less number of primary amino group of
BPEI are grafted with stearic acid. The rifampicin
(RIF) loaded lipopolymer was also formulated
further in to spray dried microparticle. Powder
X-ray diffraction (PXRD) (Fig.6) studies revealed
that the rifampicin API (Active pharmaceutical
ingredient) exists as molecular dispersion in
spray dried microparticles. Topological analysis
of the spray dried nano micelle was carried out
Figure.5 MD simulation folding of SABPEI (5050 & 7030) in aqueous
environment. (1a, 1b, 1c, 1d, 1e) and (2a, 2b, 2c, 2d, 2e) represent the
conformation of SABPEI 5050 and SABPEI 7030 at (0, 50, 100, 150, 200) ps
Figure.6 PXRD pattern of spray dried drug loaded (a) nanomicelle (b)
Rifampicin API
205
Fig.7 Confocal microscopy images of THP-1 macrophage differentiated cells infected with GFP tagged Mycobacterium
smegmatis after incubating with Rhodamine entrapped SABPEI 50:50 nanomicelle. (a, e) and (i) are the bright field
images of cells fixed after 30min, 1hr and 2 hr incubation. (b, f) and (j) are the fluorescent images of GFP tagged
M.smegmatis after 30min, 1hr and 2 hr time intervals after incubating with nanomicelle. Rhodamine entrapped
nanomicelles taken by the cells at 30min (c), 1hr (g) and 2hr (k). Colocalisation Nanomicelle and M.smeg inside
THP-1 cell at the end of 30 min (d), 1hr (h) and 2hr (l).
using scanning electron microscopy (SEM). A
large population of the drug carrying particles
was found to be under inhalable size rage (Fine
particle fraction 67.88±3%). In-vitro drug release
kinetics from spray dried nanomicelle was carried
out at lung fluid pH. Cell uptake was studied in
macrophage differentiated THP-1 cell lines after
infecting with green fluorescent protein (GFP)
tagged Mycobacterium smegmatis (M.smeg)
(Fig.7) .
Research Grants
Title
Investigators
Funding Agency
Duration
Novel polymer nanoparticles
based drug releasing systems
for improving the efficacy of
drug administration in cancer
chemotherapy
Ruby John Anto (Co-PI)
Department of
Biotechnology,
Govt. of India
2008-12
A novel site specifically targeting
nanoparticle based oral - drug and
siRNA releasing polymer systems
for colon cancer
Prof. M.Radhakrishna Pillai
(Project Coordinator)
Hari Krishnan (Co-PI)
Asha Nair.S (Co-PI)
Bramanandam Manavathi (Co-PI)
Department of
Biotechnology,
Govt. of India
2010-13
206
Publications
»» Deepa G, Ashwanikumar N, Pillai JJ, Kumar
GSV. Polymer nanoparticles-a novel strategy
for administration of Paclitaxel in cancer
chemotherapy. Current Medicinal Chemistry
2012;19(36): 6207-6213.
»» Deepa G, Thulasidasan AK, Anto RJ, Pillai JJ,
Kumar GSV. Cross-linked acrylic hydrogel for
the controlled delivery of hydrophobic drugs
in cancer therapy. International Journal of
Nanomedicine 2012;7: 4077-4088.
»» Ashwanikumar N, Kumar NA, Nair SA, Kumar
GSV. Methacrylic-based nanogels for the pHsensitive delivery of 5-fluorouracil in the colon.
International Journal of Nanomedicine 2012;7:
5769-5779.
»» Siyad MA, Kumar GSV. Synthesis, characterization,
and evaluation of PS-PPDC resin: a novel flexible
cross-linked polymeric support for solid-phase
organic synthesis. Biopolymer: Peptide Sciences
2012;98(3):239-248.
»» Simon AM, Jagadeeshan S, Abraham E,
Akhilandeshwaran A, Pillai JJ, Kumar NA,
Sivakumari AN, Kumar GSV. Poly (D,L-lacticco-glycolide) nanoparticles for the improved
therapeutic efficacy of all-trans-retinoic acid:
a study of acute myeloid leukemia (AML) cell
differentiation in vitro. Medicinal Chemistry
2012;8(5):805-810.
»» Nair KL, Thulasidasan AK, Deepa G, Anto
RJ, Kumar GSV. Purely aqueous PLGA
nanoparticulate formulations of curcumin exhibit
enhanced anticancer activity with dependence on
the combination of the carrier.
International
Journal of Pharmaceutics 2012;425(1-2):44-52.
»» Siyad MA, Kumar GSV. SPED -(styrene polyethyleneglycol diacrylate -9- decen-1ol): a novel resin for solid phase peptide
synthesis; synthesis and characterization of
biologically potent endothelin classes of peptides.
Combinatorial Chemistry & High Throughput
Screening 2012;15(5):386-394.
»» Nair KL, Vidyanand S, James J, Kumar GSV.
Pilocarpine-loaded poly(DL-lactic-co-glycolic
acid) nanoparticles as potential candidates for
controlled drug delivery with enhanced ocular
pharmacological response. Journal of Applied
Polymer Sciences 2012;124(3) 2030-2036.
»» Siyad MA Kumar GSV. Poly(ethylene glycol)
grafted polystyrene dendrimer resins: Novel class
of supports for solid phase peptide synthesis.
Polymer 2012;53, 4076-4090.
»» Siyad MA, Kumar GSV. PEGylated dendrimer
polystyrene support: synthesis, characterisation
and evaluation of biologically active peptides.
Amino Acids 2013;44(3):947-959.
»» S i y a d M A , Ku m a r G S V. S y n t h e s i s ,
Characterization, and Application of Bisphenol
A Glycerolate Dimethacrylate Cross-Linked
Polystyrene (PS-BGD): A Novel Support for
Gel Phase Peptide Synthesis. Current Organic
Synthesis. 2013;10 (2) 318-327.
»» Ashwani Kumar.N, Nisha Asok Kumar,
K.C.Sivakumar, Asha.S.Nair and GS Vinod Kumar.
Self assembling peptide nanofibrous scaffold for
the controlled delivery of 5-Fluorouracil. 6th
International meeting on Halogen Chemistry
(HALCHEM-6).Indian Institute of Science,
Bangalore; 2012.
»» Mithun V. Varghese, Annapoorna K, Sivakumar
K C, Satish Mundayoor and G S Vinod Kumar.
“Cationic lipopolymer micelle as an efficient
carrier for delivery of xenobiotics to human
macrophage cells.” INDO-US symposium on
structure dynamics and mechanics of biological
membranes-2012. Department of Chemical
Engineering, Indian Institute of Science,
Bangalore. 2012.
Asha.S.Nair and GS Vinod Kumar. Facile
synthesis and biological evaluation of a polymeric
nanogel for the pH responsive delivery of
5-Fluorouracil, National Seminar on Emerging
Trends in Chemical Science, St Aloysius College
Mangalore; 2013.
K.C.Sivakumar, Asha.S.Nair and GS Vinod
Kumar. Peptide based nanomaterials:-A
promising tool for the controlled delivery of
5-Fluorouracil. International Conference on
Recent Advances in Material Science and
Technology (ICRAMST-13). National Institute
for Technology Surathkal Karnataka; 2013.
Asha.S.Nair and GS Vinod Kumar. A novel
207
pH responsive polymeric drug delivery system
for colon cancer. 25th Kerala Science Congress.
Techno Park, Trivandrum; 2013.
K.C.Sivakumar, Asha.S.Nair and GS Vinod
Kumar. Peptide based nanomaterials:-A promising
tool for the controlled delivery of 5-Fluorouracil
5th International Conference on Current Trends
in Drug Discovery and Research (CTDDR-2013).
Central Drug research Institute, Lucknow; 2013.
»» Mithun V. Varghese, Annapoorna K, Sivakumar
K C, Satish Mundayoor and G S Vinod Kumar.
“Dry powder inhaler formulation of an anti TB
drug for treating pulmonary tuberculosis.” 25th
Kerala science congress-2013. Technopark,
Thiruvanathapuram. 2013.
»» Mithun V Varghese, Annapoorna K, Sivakumar
K C, Satish Mundayoor and G S Vinod
Kumar. “Development of cationic amphiphilic
lipopolymer as an efficient carrier for anti-TB
dry powder inhalation formulation.” Current
Trend in Drug Discovery Research (CTDDR)
- 2013, Central drug research institute,
Lucknow. 2013.
208
Awards and Honours
»» Nair KL received the Dr.MR Das career award for
best outgoing Ph.D student, March 2013.
»» Siyad MA was awarded first prize for the poster
presentation at Kerala Science Congress held
Techno park, Thiruvananthapuram, on January,
2013.
»» Ashwani Kumar.N was awarded Best paper award
for the oral presentation at National Seminar
on Emerging Trends in Chemical Science, St
Aloysius College Mangalore; Feb 14-16, 2013
»» Ashwani Kumar.N was awarded second prize
for the poster presentation at International
Conference on Recent Advances in Material
Science and Technology (ICRAMST-13).
National Institute for Technology Surathkal
Karnataka; Jan 17-19, 2013.
Ph.D Awarded
»» Lekha Nair. K and Siyad M.A successfully
defended their Ph.D thesis and were awarded
the degree in March and April 2013 respectively.
PLANT BIOTECHNOLOGY
V.V Asha
Scientist E1
[email protected]
VV Asha has a PhD in Botany from University of Kerala, Trivandrum, and joined RGCB
in 1997.
PhD students:
Krishna Radhika N
Krishnakumar K.A
Praseeja R.J
Sreejith P.S
Greeshma Tom
Sheena Philip
Technical Assistants:
Gayathri L.T
209
Screening of anti-HCC activity of Cuscuta reflexa Roxb
Praseeja RJ and Asha VV
Hepatocellular carcinoma (HCC) is the fifth most
common neoplasm and the third most common
cause of cancer related mortality worldwide. HCC
usually develops in cirrhotic liver and cirrhosis
is the strongest predisposing condition for HCC.
HCC is usually diagnosed at a late stage and
only 10 % to 20% of HCC patients are eligible for
surgical resection with curative intent at the time
of diagnosis. The development of an effective
therapeutic agent to increase the life span of HCC
Figure 1: Annexin V-FITC apoptotic assay a. Control
cells without treatment b. Silymarin 50 µg/ml
c. CRCE 20 µg/ml, 36 hr d. CRCE 20 µg/ml, 48 hrs
Figure2: Effect of CRCE on the expression of proteins
involved in apoptotic process (Western blotting).
210
patients is very essential. Tumorigenesis is not
merely the result of excessive proliferation due
to the activation of oncogenes but to the same
extent depends on the impairment of apoptosis
checkpoints. Apoptosis is a type of programmed cell
death that involves a series of biochemical events
leading to a variety of morphological changes
including membrane blebbing, cell shrinkage,
nuclear fragmentation and chromatin condensation.
Many of the morphological and structural changes
in apoptotic cells like degradation
of PARP, DNA – PK and chromatin
cleavage occurs by the activation
of caspases. In the course of
carcinogenesis, cancer cells
usually develop the ability to
evade apoptosis. Hence induction
of apoptosis in tumor cells is an
important strategy for treating
cancer.
Cuscuta reflexa Roxb (CR) is a
perennial parasitic herb commonly
found in plains of India, Malaya
etc. Paste from the whole plant
of C. reflexa is taken orally to
cure jaundice. Folk people of
Bangladesh use this plant for the
treatment of tumors. It is also
used the in treatment of various diseases like
epilepsy, arthritis, jaundice and weakness of liver,
stomach and spleen. Several compounds isolated
from plants have been found to reduce
the risk of developing cancer and many of
these have the ability to induce apoptosis
in neoplastic cells. Studies have reported
the presence of scoparone, melanettin,
quercetin, hyperoside, phenolic compounds,
and caffeoylquinic acids in C. reflexa.
Chloroform extract of CR (CRCE) was found
to have antiproliferative effects on HCC cell
lines Hep3B and HepG2. CRCE was found to
cause G1 arrest in both Hep3B and HepG2
cells. The extent of G1 arrest was found to
be more in HepG2 compared to Hep3B. TLC
and HPTLC analysis resolved CRCE into 10
bands of which two were found to be active.
Further characterisation of these bands is
needed for their proper identification of
active components. The apoptosis inducing
effect of CRCE was revealed by various assays
including Annexin V-FITC apoptotic assay, Hoechst
staining etc. Effect of CRCE on the expression of
apoptosis related genes at transcriptional level
was studied by SQ-RT PCR. Western blotting
was done to find out how CRCE treatment affects
protein level expression of various apoptosis related
genes. Total protein was isolated from Hep3B after
treatment with CRCE for different time periods.
Changes in the expression of Bax and Bcl2 from
0 hr to 48 hr with 12 hr interval were checked.
Expression of Bax was found to be higher at 48 hr
whereas Bcl2 showed slight reduction.
Poly (ADP) ribose polymerase (PARP) is a nuclear
enzyme involved in DNA repair process and the
intact 116 kDa PARP protein is cleaved into 85 kDa
fragment by caspase-3 during apoptosis. Treatment
with CRCE caused the PARP cleavage in a time
dependent manner. But the cleaved fragments
were not persistent as seen in the positive control.
Caspase-3 expression was found to be higher in
the CRCE treated group. After 24 hr incubation, an
increase in the caspase-3 expression was observed.
Caspase 9, the initiator caspase is involved in
the intrinsic mitochondrial apoptotic pathway.
The expression of caspase 9 was also found to be
increasing from 12 hr onwards as compared to the
normal control. β-actin was used as the loading
control. This proves that CRCE treatment causes
caspase activation, resulting in PARP cleavage and
induces apoptosis using intrinsic mitochondrial
apoptotic pathway.
Evaluating the synergistic effect of common chemotherapeutics with
pharmacologically active molecule from Glycosmis pentaphylla
in Human HCC cell line.
Sreejith PS and Asha VV
Natural products, unlike conventional drugs,
provide a complex mixture of bioactive entities,
which may or may not provide therapeutic activity.
Often a complete characterization of all the
chemical constituents from a natural product is
unknown. Unknown chemical compounds in plants
may cause side effects. So we need a scientific
approach to overcome these difficulties. It has been
observed that the active ingredients of a whole
herb, when extracted and isolated do not have
Figure1. Percentage of cell death (MTT assay)
after 48 hrs treatment of Hep3B cells with different
chemotherapeutics, isolated compound and alcohol
extract of GP.
the same effect as the whole plant preparation.
This is due to the effect of complex synergies
within whole plant preparation which lead to
nonlinear interaction between the constituents.
The evidence of interactions between some
commonly used herbal products and other dietary
supplements and drugs is usually based on
known or suspected pharmacological activity.
Herbs are often administered in combination with
therapeutic drugs, raising the potential of herbdrug interactions. Many medicinal
herbs and pharmaceutical drugs are
therapeutic at one dose and toxic at
another. Interactions between herbs
and drugs may increase or decrease
the pharmacological or toxicological
effects of either component.
Glycosmis pentaphylla (Retz) Correa
belongs to Rutaceae family. G.
pentaphylla (GP) has a long history of
usage in traditional medicine against
various ailments around the world.
G. pentaphylla is considered as one
of the most important alternative
medicines against various liver
ailments by the traditional people
of India. But a scientific validation regarding the
active compound identification, mechanism of
action, safety and efficacy is still not completely
carried out. In the first part of this work, we have
211
identified the active extract with anti HCC activity
and its mechanism of action. In our study, we
have isolated the most active compound from the
crude alcohol extract of GP by TLC method and the
process of compound identification is in progress
by NMR, IR, LC-MS methods. Protein expression
studies with crude alcohol extract shows that the
extract time dependently induces apoptosis by
regulating expression of pro and anti-apoptotic
proteins. In order to reduce the side effects of
currently used anti-cancer drugs we have assessed
the synergistic efficacy of isolated active molecule
with already approved chemotherapeutics like
doxorubicin, silymarin and sorafenib. We have
tried several combinations to get the better one.
By using Chou-Talalay method we identified the
better combination of compound and drug. In the
present study we have observed that the isolated
active compound from G. pentaphylla effectively
shows synergistic apoptosis induction and cell
cycle regulation activity with sorafenib in human
HCC cell line Hep3B.
Studies on the comparative and synergistic effects of isolated plant
active molecules with known chemotherapeutic drugs
Greeshma Tom, Sheena Philip and Asha VV
Due to the extensive range of vital functions being
performed by liver in the body, it is the major target
of damage by several factors which can weaken
the liver. This can eventually lead to progressive
damage of the liver beginning from acute or
chronic inflammation, hepatitis, fibrosis, cirrhosis
and ultimately liver cancer. Preliminary studies
from our lab have established the liver specific
pharmacological properties of certain traditionally
used medicinal plants. The lab has isolated active
principles from such plants and characterized
them. We have determined the IC50 values of these
molecules in liver cell lines which are indicative of
their liver ailment specific activities. This suggests
the progression of the studies towards determining
their potentiality as future therapeutic agents. Our
aim is to evaluate and compare the synergistic
effects of the isolated active plant molecules with
currently used chemotherapeutic drugs and to
elucidate the mechanistic factors in the synergism.
Extra-Mural Grants
Sl. No
1
Investigator
V V Asha
Title of project
Funding agency
Duration
Isolation of active fraction having antihepatocellular carcinoma activity from
Lygodium flexuosum and analysis of its
synergistic effect with sorafenib, the drug
currently used for treatment of HCC
Kerala Forest
Department
January 2010 December -2013
PUBLICATIONS
»» PS Sreejith, RR Mascarenhas, RJ Praseeja, VV
Asha. The apoptosis inducing effect of Glycosmis
pentaphylla (Retz.) Correa and its influence on
gene expression in hepatocellular carcinoma
cell line, Hep3 B. J Ethnopharmacol. 2012: 139:
359-365.
»» Sreejith PS, Praseeja RJ, Asha VV A review
on the pharmacology and phytochemistry of
traditional medicinal plant, Glycosmis pentaphylla
(Retz.) Correa. j pharm res. 2012. 5:2723-2728.
»» PJ. Wills,VV. Asha, Lygodium flexuosam
212
extract down regulates the expression of
proinflammmatory cytokines in CCl4 –induced
hepatotoxicity. Asian Pacific Journal of Tropical
Medicine. 2012. 412-420.
»» Sreejith PS and Asha VV “Identification of
pharmacologically active anti-Hepato Cellular
Carcinoma (HCC) compounds from medicinal
plant, Glycosmis pentaphylla (Retz.) Correa and
find out its mechanism of action” International
conference conducted by Sree Narayana College
of Arts and Science, Chempazhanthy. 2012
Plant Molecular Biology
George Thomas
Scientist E II
[email protected]
George Thomas received his PhD in Life Sciences from University of Hyderabad and joined
RGCB in 1997.
PhDs:
Dinesh Raj, R
Kiran, A. G
Mariet Jose
Geethu Elizabath Thomas
Smini Varghese
Lesly Augustine
Technical:
George Varghese
Project Fellow:
Vinitha M. R
213
Molecular basis of incompatible interactions in Zingiber-Pythium
pathosystem.
Kiran, A.G., Geethu Elizabath Thomas, Smini Varghese, Lesly Augustine and George
Thomas
We were engaged in comparative gene expression
analysis in Pythium susceptible cultivated ginger
(Zingiber officinale) and its resistant wild congener,
Z. zerumbet. We followed two approaches. The
first approach involved real time quantification of
transcripts of those genes that are known to be
involved in host defense in other plant species. In
the other approach, in order to gain a global view of
expression reprogramming, we performed RNA-Seq
quantification in ginger and Z. zerumbet before
and after inoculating with Pythium.
Altogether 60 key genes belonging to major defense
pathways were analyzed in the first approach
in both ginger and resistant Z. zerumbet at
different time period following inoculating them
with Pythium. Our study revealed differential
induction of genes involved in the biosynthesis of
major plant defense hormones in both ginger and Z.
zerumbet. Genes involved in phenolic compound
production had a higher transcript accumulation
in Z zerumbet. Reactive oxygen species (ROS)
generating genes showed similar levels of induction
in both the resistant and susceptible pathosystems
whereas the genes responsible for ROS scavenging
showed differential modulation between resistant
214
and susceptible species. Differential modulation
was recorded in hypersensitive response related
genes also. The study strongly suggested that
the incompatible response of Z. zerumbet to
be hypersensitive response mediated involving
phenolic compound production.
In the other approach, whole genome transcriptome
sequencing was done on Illumina GA-IIx platform
and the high quality reads were assembled using
Velvet 1.1.05. The transcripts were generated
using Oases and CAP3. The fold changes (digital
gene expression) in transcripts were determined
after Pythium inoculation. The gene ontology
categorization and pathway mapping was also
performed using the transcripts. RNA-Seq data
revealed significant upregulation of genes mediating
hormone signalling, hypersensitive response and
pathogen recognition in Z. zerumbet, whereas the
induction of genes involved in oxidative stress and
general metabolism were found in ginger. Robust
induction of the defence related genes seem to play
a key role in Pythuim resistance in Z. zerumbet.
In ginger, the transcriptome reprogramming was
targeted mainly to protect vital physiological
activities.
Gene ontology classification of transcripts in Z. zerumbet and ginger
We were also interested in understanding the
genetic basis of Pythium resistance in Z. zerumbet
and susceptibility in cultivated ginger. Red Queen
Hypothesis assumes that pathogen maintains sex
in host. Sexual reproduction generates genetic
variability, the raw material for selection and
evolution whereas the asexual reproduction
hinders evolutionary forces to act on a system,
thereby preventing co-evolution between host and
pathogen, resulting in susceptibility. Zingiber spp.
represents a valuable system to study the effect
of mode of reproduction on co-evolution between
host and pathogen. While cultivated ginger, the
Z. officinale is obligatory asexual, propagates
exclusively through rhizome, Z. neesanum and
Z. cernuum are predominantly sexual, and Z.
zerumbet follows a mixed breeding system
consist of both the methods. Earlier we found a
concordance between mode of reproduction and
genetic variability in the four species of Zingiber
as expected, but discordance between genetic
variability and Pythium resistance. The Z.
cernuum and Z. neesanum populations recorded
high rate of seed set and correspondingly high
rate of genetic variability, but showed complete
susceptibility to Pythium. No seed set was
observed in obligatory asexual ginger and no
resistance as well as expected. However, in Z.
zerumbet two different types of populations were
found. Contrary to the expectations, the seed
producing populations, which recorded relatively
high within population genetic variability, were
highly susceptible to the pathogen, while the
non-seed setting populations, which recorded
less intra-population variability were highly
resistant. The data indicates that the variability
with adaptive significance generated by occasional
sexual reproduction could be fixed in a population
by vegetative mode of reproduction. We further
analyzed Z. zerumbet and ginger populations.
Different genomic segments, amounting to a total
length of 5 kb, were sequenced in different varieties
of ginger and Z. zerumbet populations. The
emerging results attribute a role for heterozygosityfitness correlations in explaining resistance in Z.
zerumbet and the work is underway.
Icrosatellite assisted genetic resources characterization and
circumscription of the medicinal rice ‘Njavara’
George Varghese, Dinesh Raj, R., Mariet Jose, and George Thomas.
Njavara belongs to the Shashtika group of
medicinal rice described in ancient Sanskrit
Ayurveda treatises. From the earlier studies, we
suggested that the stabilizing selection performed
by farmers over centuries using short duration as
a selection criterion has helped to maintain the
genetic purity of Njavara. In this programme our
interest was to locate the phylogenetic position
of Njavara in global rice gene
pool. This programme witnessed
building up of microsatellite
data over a period of time from
different samples including 109
traditional rice cultivars from
Kerala state, 235 traditional
cultivars from other parts of
India, 63 traditional cultivars from
different rice growing countries,
23 improved rice varieties and 295
samples of Oryza rufipogon, the
progenitor of rice from different
parts of the world, especially
India. Altogether 70 microsatellite
215
markers were used for genotyping. This year we
concluded the phylogenetic analysis of Njavara rice.
The results highlighted the distinctness of Njavara
rice gene pool and helped us to locate it in the
global rice gene pool. The data has also provided
vital insight into the genetic characteristics and
evolution of cultivated rice in India.
Another important component of this programme
was to develop molecular markers diagnostic
to Njavara. We achieved this component to a
greater extent this year. Predominantly Njavara
specific amplified fragment length polymorphisms
(AFLPs) identified earlier was analyzed towards
the development of Njavara specific markers.
Sequence characterization of amplicons yielded
by primers designed to the AFLP sequences
revealed Njavara specific profiles by one of the
primer pairs. This amplicon was validated by
sequencing in additional cultivars and varieties and
finally converted into a marker, yielding Njavara
specific allele, amenable for allele detection on an
automated DNA sequencing machine.
Phylogenetic affinity of Njavara revealed by microsatellite markers
This year we further characterized the hybrids
between a Njavara accession N8 (N8) and the
high yielding improved cultivar Jyothi (J) in order
to assess the value of Njavara in contributing
to the genetic improvement of rice. Of the 308
F2 progenies developed earlier, 53 randomly
selected F2s were raised to F5 by single seed
descendence. Thirteen yield related traits were
scored in progenies and parents, and the F5s were
genotyped using 68 SSR markers. Of the 68 loci, 49
loci were QTLs controlling different quantitative
traits in other reports and the 19 were unlinked. Of
the 53 F5s, 49 individuals retained transgressive
phenotypes in most of the traits for which they
showed transgression in F2. Regression analysis
detected 49 QTLs controlling different traits, of
which 14 QTLs were reported earlier. Contribution
of N8 allele to the transgressive phenotypes was
also evaluated.
Extramural Funding
Investigator
George Thomas
Title
Prospecting Zingiber zerumbet for
molecular resources
»» Geethu, E. T., Kiran A. G., Augustine, L, Sabu,
M and Thomas, G. Revisiting Red Queen
Hypothesis using Zingiber spp. with contrasting
breeding system provides evidence of better
adaptive value for a mixed breeding system. Paper
presented at 6th International Symposium on the
Family Zingiberaceae, September 10 – 13, 2012,
University of Calicut, Kerala, India.
»» Vinitha, M.R., Suresh Kumar, U., Sabu,
M and Thomas, G. DNA barcoding and
molecular identification in Zingiberaceae – less
216
Funding Agency
Duration
Department of
Biotechnology (DBT)
2010-2013
success and more problems. Paper presented
at 6th International Symposium on the Family
Zingiberaceae, September 10 – 13, 2012,
»» Kiran A. G., Augustine, L., Smini, V and Thomas
G. Reactive oxygen species sequestration,
hypersensitive response and salicylic acid
signalling together hold a central role in Pythium
resistance in Zingiber zerumbet. Paper presented
at 6th International Symposium on the Family
Zingiberaceae, September 10 – 13, 2012,
PLANT BIOTECHNOLOGY
Dr. EV Soniya
Scientist E II
evsoniya@rgcb,res.in
Soniya received her Ph.D in Botany from Department of Botany, University of Kerala.
She was a Research Associate at Central Tuber Crops Research Institute, Sreekaryam,
Thiruvananthapuram before joining Rajiv Gandhi Centre for Biotechnology in 1997.
Technical support:
Dr. Manoj P. Kumar
PhD Students:
Vivek PJ
Resmi MS
Tara G Menon
Asha S
Aiswarya G
Mallika V
Project fellows:
Sweda Sreekumar
Lovely Jael P
217
Identification of novel forms of Type III polyketide synthases for
exploiting its potential use in metabolic engineering
Resmi MS, Mallika V, Aiswarya G and EV Soniya
Polyketide synthases (PKSs) are family of multifunctional enzymes catalyzing the biosynthesis
of structurally diverse natural products with
remarkable biological and pharmacological
properties. Among the three different form of
PKSs (type I, II and III), type III PKS is structurally
and mechanically distinct from the other PKSs
and can be easily distinguished by their physical
composition. In plants, type III polyketides
synthase are two types, chalcone synthase (CHSs)
and non- chalcone synthase (non-CHSs). CHSs
are the most widely studied protein among the
type III PKSs. Non-CHS proteins include 2-pyrone
synthase, stilbene synthase, benzalacetone
synthase, biphenyl synthase etc.
Our present study describes cloning and
characterization of a novel type III PKS from Aegle
marmelos (rutaceae), which is named quinolone
synthase (QNS) on the basis of its activity. The
reaction involves decarboxylative condensation
of malonyl-CoA with N-methylanthraniloyl-CoA
to form an intermediate, which spontaneously
cyclise by amide formation to yield 4-hydroxy2(1H)-quinolone. Kinetic analysis indicated that
the catalytic efficiency of QNS protein to accept
larger acyl-CoA substrate is several fold higher than
that for smaller substrates. Molecular modelling
studies suggested that QNS might have emerged
by the gain of function (by the substitution of
simply two active site residues) mutation from a
structurally homologous CHS type III protein. The
catalytic and structural importance of active site
residues, as predicted by our structural model,
was investigated by performing site-directed
mutagenesis. The modelling and mutagenesis
studies provide an insight into the structural
mechanism for the enzyme that could be used to
generate pharmaceutically important products.
Based on these studies, we are currently using
computational structural biology aspects to
elucidate the structural mechanisms behind the
functional behaviour of the system.
In addition to that, we have isolated chalcone
synthase (CHS) from the Indian medicinal plant
Emblica officinalis (Gooseberry). The plant is
known for its pharmacological applications and
is found beneficial in the treatment of various
ailments like cancer. Medicinal properties of E.
officinalis are imparted by the presence of high
level of secondary metabolites like flavonoids,
tannins and Vitamin C. Initially we have
isolated and cloned two isoforms of chalcone
synthase from E. officinalis. Further with the
aid of RACE PCR, we have successfully isolated
and cloned a full length EoCHS1 cDNA sequence
from E. officinalis. Southern blot analysis with
EoCHS1as probe confirmed the existence of CHS
as multigene family in E. Officinalis. In order to
understand the functional activity of EoCHS1
we are currently doing in vitro enzymatic
studies which will reveal the role of CHS in the
biosynthesis of flavonoids in E. officinalis.
Docking studies of QNS wild-type and mutant proteins, MSD1 and MSD2 with ligands.
A, QNS WT with N-methylanthraniloyl-CoA; B, QNS WT with p-coumaroyl-CoA; C and D, MSD1 (S132T/A133S)
and MSD2 (S132T/A133S/V265F) with p-coumaroyl-CoA. Substrates were shown in yellow. Some of the crucial
active site residues along with the catalytic triad Cys, His, and Asn were shown in red.
218
TLC-based analysis of radiolabeled products of QNS wild-type. The starter substrates used in the assay are
indicated above each lane. Chemical structures of the major products obtained by N-methylanthraniloyl-CoA and
p-coumaroyl-CoA primed reactions are indicated next to the radioactive bands.
Identification of QNS wild-type products. A and B illustrate the fragmentation pattern observed by tandem
mass spectrometry for N-methylanthraniloyl-CoA-primed products, 4-hydroxy-1-methyl-2(1H)-quinolone (M-H)
− m/z 174.08 (A) and 1,3-dihydroxy-N-methylacridone (M-H)− m/z 240.09 (B). C illustrates the fragmentation
pattern for p-coumaroyl-CoA-primed product, p-hydroxybenzalacetone (M-H) − at m/z 161.08. Fragment ions are
indicated above the corresponding peaks. The structure of the molecule with the expected fragmentation profile
is drawn next to its MS/MS spectra.
219
Molecular Characterisation of an Abiotic Stress Inducible Calcium
Dependent Protein Kinase gene from Zingiber officinale
Vivek PJ and EV Soniya
Calcium dependent protein kinases (CDPKs) play
important roles in plants by decoding Ca2+ signals
in diverse phosphorylation-dependent calcium
signaling networks in plants. They are having a
calmodulin-like domain (CaM-LD) and a kinase
catalytic domain and hence functions as sensorresponder proteins. The precise role of specific
CDPK forms in plants especially in abiotic/
biotic stress signaling is largely unknown.
photosynthetic efficiency and other photosynthetic
parameters. Also, transgenic tobacco subjected to
salinity/drought stress exhibited 50% more growth
during stress conditions as compared to wild type
plant during normal conditions.
Figure 1
ZoCDPK1 is salinity and drought inducible
CDPK identified from ginger and it shows its
abundance in rhizome than leaves. ZoCDPK1
is a cytosolic protein with potential nuclear
localization as it is having nuclear localization
sequence (NLS) which was proved through
yeast two hybrid experiments. Over-expression
of ginger CDPK1 gene in tobacco conferred
tolerance to salinity and drought stress as
reflected by the high percentage of seed
germination(Fig 1), higher relative water
content, expression of stress responsive genes,
higher leaf chlorophyll content, increased
Figure 2
Fig 2. Physiological assessment of transgenic plants (T1) and wild type plants. (A) Net CO2 uptake rate measured
at a PPFD of 20 and 2000 umol quanta m-2 sec-1 and 400 p.p.m. CO2. Effect of salinity and drought stress on the
net photosynthetic rate (B), Transpiration rate (C), inter-cellular CO2 concentration (D), leaf conductance (E) and
photosynthetic efficiency (F) of transgenic and wild type plants
220
Isolation and characterisation of stress responsive miRNAs and other
small functional non coding RNAs from black pepper
Asha S, Sweda Sreekumar and EV Soniya
Small RNAs (17-28nt) are the regulatory, noncoding RNAs controlling gene expression of target
genes at transcriptional or post transcriptional
level in a sequence dependent manner and has
pivotal roles in development and stress response
of eukaryotic organisms. Among this, micro RNAs
are small, endogenous RNAs generated from the
stem region of the hairpin-shaped precursors (premiRNAs) by RNase-III-type Dicer endonuclease.
miRNA guided gene regulation plays a crucial
role in the plant defence against pathogens and
abiotic factors. Our study focused to trace out stress
responsive miRNAs and other functional small
RNAs from black pepper (Piper nigrum), widely
known as ‘King of spices’. We isolated and cloned
small RNAs (sRNAs) enriched for miRNAs (1727nt) from the leaves and roots of Phytophthora
capsici infected black pepper plants. Sequencing
of the small RNA fractions revealed diverse set of
non-coding RNAs. Among the small RNAs, micro
RNAs were identified based on their size, typical
stem loop precursor structure, MFE values etc.
The expression profiling of these putative miRNA
candidates by the stem loop qRT-PCR experiment
is in progress. Two conserved miRNAs, miR166
and miR171 and their targets were identified
and validated from the black pepper. Along with
miRNAs, tRFs were also identified and their varied
expression during stress conditions points towards
the biological role of these functional small RNA
candidates in plant stress. The functional validation
of targets of these putative miRNAs will help in
elucidating their exact regulatory mechanisms.
We carried out the de novo sequencing using
illumine HiSeqTM 2000 to generate the first leaf
transcriptome of black pepper. The 55 million raw
reads obtained, when assembled using Trinity
program generated 2, 23, 386 contigs and 1,28,157
unigenes. The analysis identified 3913 different
types of SSR motifs with an average of one SSR
per 3.04 MB of the transcriptome. About 0.033%
of the transcriptome constituted ‘pre-miRNA
candidates bearing SSRs’. The abundance, type
and distribution of SSR motifs studied across the
hair-pin miRNA precursors, showed a significant
bias in the position of SSRs towards the downstream
of predicted ‘pre-miRNA candidates’.
A
B
221
C
D
Predicted Stem-loop structures of A) pni-miR166 B) putative stress responsive miRNA identified from black pepper
C) Mapping of the miRNA cleavage sites on the targets. D) Relative position of microsatellite motifs with respect
to potential ‘pre-miRNA candidates’.
222
Identification of genes responsive to salt (abiotic) stress in a mangrove
Tara G Menon and EV Soniya
Salinity stress is a major impeding factor to the
growth of agricultural economy, worldwide.
Halophytes are plants that are naturally tolerant
to salinity as high as that present in sea water.
Mangrove, as extreme halophytes, is also believed
to possess several gene or gene combinations that
may have played a significant role in the evolution
of salt tolerance. Rhizophora apiculata Blume
belongs to the category of true mangroves, which
are found only in the coastal ecosystems and are
non secretor types which accumulate the excess
salt in the old leaves, which are later shed.
In the present study, we have identified about 66
unique genes that were differentially upregulated
in response to salt stress, through Suppression
subtractive hybridization. The differential
expressions of twelve of these transcripts were
confirmed by real time RT PCR analysis. Fig 1
shows the expression levels of the genes studied at
three timepoints viz, 6hr,12hr and 24hr of salinity
stress.
The gene that showed maximum upregulation in
the real time PCR analysis was Cytochrome P 450/
(S)-N-methylcoclaurine 3’-hydroxylase.The full
length cDNA of the gene was isolated by RACE
PCR and sequenced. The gene was further cloned
into a binary vector for its expression in planta.
Work is in progress to obtain stable transformant
containing the transgene.
Ongoing Projects
Sl No.
Title of Project
Funding Agency
Duration
1.
Evaluation of differentially expressed miRNAs during biotic
stress in black pepper
CSIR
2011- 14
2.
Identification and functional validation of Type III PKS from
A. marmelos involved in anthranilic acid derived alkaloid
biosynthesis
DST
2012-2015
Publications
»» Asha S, Nisha J, E. V. Soniya (2012) In
silico characterisation and phylogenetic analysis of
two evolutionarily conserved miRNAs (miR166
and miR171) from black Pepper (Piper nigrum L.)
Plant Mol Biology Rep. DOI 10.1007/s11105012-0532-5.
»» Asha Poorna C, Resmi M. S and E. V. Soniya
(2012). In Vitro Antioxidant Analysis and the
DNA Damage Protective Activity of Leaf Extract
of the Excoecaria Agallocha Linn Mangrove Plant.
International Journal of Agricultural Chemistry
DOI: 10.5772/51895.
223
»» Sudha C. G, T. V. Sherina, V. P. Anu Anand,
J. V. Reji, P. Padmesh & E. V. Soniya (2012).
Agrobacterium rhizogenes mediated
transformation of the medicinal plant Decalepis
arayalpathra and production of 2-hydroxy-4methoxy benzaldehyde. Plant Cell Tiss Organ
Culture. DOI 10.1007/s11240-012-0226-6.
»» Resmi MS, Verma P, Gokhale RS, Soniya EV
(2013).Identification and characterization of a
type III polyketide synthase involved in quinolone
alkaloid biosynthesis from Aegle marmelos Correa.
J Biol Chem. 288(10):7271-81.
»» Nisha Joy, Asha S, Mallika V, Soniya EV (2013)
De novo Transcriptome Sequencing Reveals a
Considerable Bias in the Incidence of Simple
Sequence Repeats towards the Downstream of
‘Pre-miRNAs’ of Black Pepper. PLoS ONE 8(3):
e56694.
»» Mallika V, Resmi M S and E V Soniya presented a
poster “Structure based analysis of Non-chalcone
forming type III polyketide synthase proteins
and docking studies of putative ligands” in the
International Conference on Biomolecular Forms
and Functions (ICBFF 2013), held at Indian
Institute of Science, Bangalore, 8 - 12th January,
2013.
»» Tara G Menon and E V Soniya presented a
paper titled “Rhizophora apiculata Blume, a true
mangrove as a potential reservoir of genes involved
in salt stress tolerance” in 25th Kerala Science
Congress held at Technopark, Trivandrum during
29th January - 1st February,2013.
»» Asha S and EV Soniya presented a paper titled
“The spatio-temporal expression variation of
miR171 under different environmental stress
and its potential application in stress tolerance
in black pepper (Piper nigrum L.)”, in the
National Symposium on ‘Innovative Approaches
and Modern Technologies for Crop Productivity,
224
Food Safety and Environmental Sustainability’
held at Thrissur, Kerala during 19 – 20 th ,
November 2012.
»» Aiswarya G, Mallika V and EV Soniya presented
a paper titled “In silico analysis and homology
modelling of chalcone synthase, a key enzyme
involved in the biosynthesis of flavonoids from
Emblica officinalis Gaertn.” in International
Conference on Advances in Plant Sciences
(ICAPS2012), held at Chiang Mai, Thailand
during 14-18th, November 2012.
»» Tara G Menon and E V Soniya presented a paper
titled “Identification and cloning of Cytochrome
P450/(S)-N-methylcoclaurine 3’-hydroxylase in
mangrove Rhizophora apiculata Blume and its
role in salt stress adaptation” in International
Conference on Advances in Plant Sciences
(ICAPS2012), held at Chiang Mai, Thailand
during 14-18th, November 2012.
»» Aiswarya G and E V Soniya presented a paper
titled “Type III Polyektide synthase gene from
Emblica officinalis Gaertn with potential role in
secondary metabolite synthesis” in the International Seminar on Botanica 2012 held
at SN College, Chempazhanthy during 7th – 8t,
September, 2012.
»» E V Soniya, Nisha Joy and Asha S “a significant
increase of microRNAs indicates its regulatory
role during pathogen attack in black pepper”. 77th
Cold Spring Harbor Symposium on Quantitative
Biology. The Biology of Plants. May 30 - June 4,
2012 at Cold spring Harbour, NewYork.
Awards and Honours
»» Aiswarya G and EV Soniya (Best Paper Award)
titled “Type III Polyketide synthase gene from
Emblica officinalis Gaertn with potential role
in secondary metabolite synthesis” in the
International Seminar Botanica 2012 held at
SN College, Chempazhanthy during 7th – 8th,
September 2012.
PLANT BIOTECHNOLOGY: Laboratory - 4
Manjula.S, PhD
Scientist EI
[email protected]
Manjula is a Ph.D. in Botany from University of Kerala. She joined RGCB in 2000.
PhD students:
Nisha Nair
Jisha.S
Anu K
Chidambareswaran M
225
RNAi methods for functional validation of defense related genes
from Piper colubrinum
Anu K, Chidambareswaren M , and Manjula. S
Optimization of Virus Induced Gene Silencing
(VIGS) in P.colubrinum
from the cultures. The purified vectors were
used for further studies with VIGS for functional
characterisation in Piper sps. The TRV vector,
Piper colubrinum is highly resistant to
pTV00 is designed to contain a multiple cloning
Phytophthora capsici, the oomycete fungus which
site enabling to subclone desired gene fragments
is a devastating pathogen on cultivated Piper
for generation VIGS vector containing a gene
nigrum. We had earlier identified some candidate
of interest. Agroinfiltration experiments were
defense related genes in P.colubrinum , which need
performed to optimize VIGS in P.colubrinum using
to be functionally validated in planta. Our ongoing
PDS reporter gene silencing constructs. Phytoene
experiments prove that Tobacco Rattle Virus (TRV),
desaturase (PDS) is a key regulator of carotenoid
which is widely used as a VIGS vector, successfully
biosynthesis and its silencing in the plant produce
infects and multiplies in the host (P.colubrinum)
a typical bleached appearance in the leaves
plants.
in the presence of light. PDS was cloned from
P.colubrinum using gene specific primers. TRV:PDS
The bipartite vector pBIN (harbouring RNA1 of
constructs were transformed into Agrobacterium
TRV) and pTV00 (containing RNA2 of TRV) were
GV3103. Equal amounts of TRV1 (RNA1) and TRV2
propagated in E.coli strain DH5α and purified
or TRV2-PDS (RNA2) cultures were combined.
The solutions were then
incubated at room temperature
for 3–4 hours and used for
infiltration. Young leaves were
selected for infiltration. Mock
(infiltration buffer alone) and
TRV empty vector infiltrations
were maintained as control.
The TRV-PDS infiltrated plants
showed typical appearance
of yellow patches at sites of
Fig.1: Typical symptoms of PDS silencing observed 21 days post inoculation PDS silencing after 21 days
(Fig.1). Real Time PCR analysis
using PDS gene primers
confir med the significant
down regulation of the gene
in TRV:PDS Agroinfiltrated
plants (Fig.2). RT- PCR analysis
confirmed the presence of virus
particles (Fig.3) in the newly
emerged leaves. Typical visual
manifestation of PDS silencing
marked as mottled pale green
areas on the infiltrated leaves
and newly emerged leaves
was obser ved even after 5
months of infiltration (Fig.4).
A VIGS –mediated gene
silencing strategy is thus
established in P.colubrinum for
rapid functional evaluation of
Fig.2: Real Time PCR analysis of PDS –silenced leaf
candidate defense genes.
226
Fig.3: RT-PCR analysis for viral movement and replication
C
S
Fig.4: PDS-silenced (S) and control (empty vector inoculated) (C) leaves 5 months post inoculation
Optimization of Human recombinant Granulocyte Colony Stimulating
Factor production in tobacco.
Nisha Nair.R and S. Manjula
Human granulocyte-colony stimulating factor
(h-GCSF) belongs to a group of colony stimulating
factors that play an important role in stimulating the
differentiation and proliferation of hematopoietic
precursor cells and activation of mature neutrophils.
We tried to express hGCSF protein in tobacco BY-2
cells by stable transformation method. For this, the
plant binary vector pMDC 83 containing hGCSF
ORF with a C-terminus GFP fusion, was used to
transform tobacco BY-2 suspension cells. Another
vector, pMDC 43 was generated with an ER
extention signal (KDEL) added at the C-terminus
of rhGCSF protein in addition to an N-terminal GFP.
The binary vectors pMDC83 and pMDC43 were
mobilized into Agrobacterium strain EHA105,
and used to transform tobacco BY-2 suspension
cells, which were plated on selection medium.
The positive clones grew into calli and were
subjected to Southern and RT-PCR analysis, which
confirmed the stable integration and expression
of GCSF transcripts (Fig. 5). Confocal imaging of
callus transformed with pMDC 83 showed GFP
localization mainly in the apoplast (Fig. 7). Calli
transformed with pMDC43 were stained with ER
Tracker Red (Invitrogen) prior to confocal imaging.
Co-localization of red and green fluorescence in
transgenic cells confirmed that GCSF was retained
in the ER (Fig. 8).
50µg of total soluble protein from putative
transformants were analysed by Western blot.
All the transgenic lines transformed with pMDC
83 showed the expected band size of 48 KDa
corresponding to GCSF-GFP fusion protein
when probed with primary monoclonal mouse
anti human GCSF antibodies. In the callus lines
transformed with pMDC83, band size of 46 KD
was obtained (Fig.6), confirming the expression of
recombinant GCSF.
227
A
B
Fig. 5: Souther n blot analysis
of control and transgenic BY-2
cells. (A) Southern blot of cells
transformed with pMDC83
(B) Southern blot of cells
transformed with pMDC 43.
Lane1 - positive control,
Lane 2 - empty well,
Lane - 3 untransformed control
calli,
Lane 4 -7 - transgenic calli. All
the analysed transgenics showed
single transgene integration.
Fig.6: Western blot analysis of BY-2
cells transformed with pMDC 83.
Lane P - Positive control;
Lane E - empty well,
Lane WT - non transformed
control,
Lane 1-3 - transgenic lines showing
a protein band of size 46 kDa
Fig.7: Tobacco BY-2 cells transformed
with pMDC83 showing apoplast
localized GFP expression.
Fig.8: Cells transformed with pMDC43 showing ER-targeted expression of GCSF.
A) bright field image, (B) at an excitation of 543nm & emission of 589-650nm specific for ER tracker Red (C) at an
excitation of 488nm and emission between 500-530nm for GFP (D) overlay image of (1) - untransformed control
cells, (2) - transgenic cell.
228
Molecular analysis of mutualistic interaction of Centella asiatica and
Piriformospora indica and its effects on secondary metabolite production
Jisha S and S.Manjula
Piriformospora indica, a mutualistic Basidiomycete
which exhibits a broad host range has evolved
efficient strategies for improved nutrient acquisition.
Centella asiatica (L.) Urban is widely acclaimed
in traditional systems of medicine owing to the
presence of pharmaceutically relevant triterpenoids,
specifically the asiaticosides. We examined the
effect of P. indica colonization in C. asiatica plants
Fig.9: HPLC analysis of Asiaticoside content in C.
asiatica. Maximum asiaticoside production was
recorded in plants maintained in MS medium containing
the lowest level (LP5) of phosphate.
LP5-Low phosphate (5% of normal MS)
LP10-Low phosphate (10% of normal MS)
NP- normal phosphate as in MS medium (1250 µM)
HP2- High phosphate (2X normal P)
HP4- High phosphate (4X normal P)
maintained in vitro at varying phosphate levels in
the growth medium. At a phosphate concentration
of 125 µ M (10% of normal), significant stimulation
of asiaticoside production (Fig.9) and host plant
growth (Fig.10) were observed in colonized
plants, which also induced an overproduction
of auxin (IAA). The presence of P. indica has a
protective role in alleviating the effects of P stress,
as evidenced by decreased H2O2 production, acid
phosphatase activity and increased SOD activity
in colonized plants. Induction of asiaticoside
production in P. indica colonized plants under low
P conditions coincided with a significant transcript
up regulation of β-Amyrin Synthase (BAS), a key
gene in the asiaticoside pathway (Fig.11).
Fig. 10: Effect of phosphate concentration on plant
growth. FW- fresh weight
LN- Leaf number; SL- shoot length; RN- root number;
RL- root length.
** denotes significance at p< 0.05
Fig. 11: Real-time PCR analysis of BAS expressed as fold expression in leaves of Centella asiatia. Relative
quantification represents the fold expression levels of BAS relative to the level of 5.8S rRNA gene expression, which
was constant in all RNA samples. Number of replications (n) =3
*** depicts significance at p<0.001
229
Extramural Funding
No.
Investigator(s)
Title
Duration
1
S.Manjula (PI)
(Muti-institutional
project)
RNAi approaches for validation of
defense related genes from resistant
wild pepper-Piper colubrinum Link
DBT
2010-2013
2
S.Manjula (Co-PI
(Muti-institutional
project)
Prospecting Zingiber zerumbet for
molecular resources
DBT
2010-2013
Publications (2012-2013)
»» Jisha S, Anith KN and S.Manjula.2012. Induction
of root colonization by Piriformospora indica leads
to enhanced asiaticoside production in Centella
asiatica. Mycorrhiza 22: 195-202.
230
Funding Agency
Mass Spectrometry and Proteomic Core Facility
Abdul Jaleel K.A, Ph.D
Scientist E-2 and Senior Consultant in Proteomics
Technical Officers:
Mr. M. Saravanakumar (M.Sc)
Mr. Arun Surendran (B.Tech)
Biotechnology Trainee Engineer:
Mr. Srikanth Jandhyam (M. Tech)
It has been one year since the Mass Spectrometry
and Proteomic Core Facility is being established at
Rajiv Gandhi Centre for Biotechnology. The purpose
of these state-of-the-art new technology platforms
in the Proteomic Core is to make the cutting edge
mass spectrometry technology available to the
research infrastructure of RGCB. A major goal of
the facility is to become a research environment
for multidisciplinary research that utilizes mass
spectrometry as the key technology and other
proteomics technologies for the qualitative and
quantitative characterization of proteins. While
the primary emphasis of the core is geared toward
supporting proteomics research, the facility also
provides basic MS support for a broad range of
research and sample types, such as polymers,
natural products, small synthetic molecules, and
large intact proteins and nucleic acids.
Infrastructure
The MS and Proteomics Core Facility is equipped
with couple of new high-performance mass
spectrometers, including a state-of-the-art Q-TOF
(Synapt G2 HDMS, Waters) and a MALDI/TOF/TOF
(UltrafleXtreme, Bruker Daltonics). Besides, mass
spectrometers the facility has a Surface Plasmon
Resonance (SPR) System for interaction studies
231
(ProteOn™ XPR36, Bio-Rad) as a major equipment.
These instruments are operated by the MS Core
Facility personnel only. The instrumentation details
are as follows.
Synapt G2-HDMS
(Waters Corporation)
Synapt G2 HDMS is a hybrid, quadrupole time-offlight (Q-TOF), ion mobility, orthogonal acceleration
mass spectrometer with electrospray (ESI)
ionization and MS/MS capabilities controlled by
MassLynx software. The installation of this LC/
MS/MS system got completed in April 2012. The
system combines exact-mass quadrupole and high
resolution time-of-flight mass spectrometry with
Triwave technology, enabling both TOF mode and
high-efficiency ion-mobility-based measurements
and separations (IMS-MS). The types of acquisition
modes present in Synapt G2 HDMS are (1) MS, (2)
MS/MS, (3) MSE, (4) HDMSE (High Definition MSE),
and (5) DDA (Data Dependant Analysis) mode.
In addition to the standard NanoLockSpray dual
electrospray ion source, we also have a TRIZAIC
UPLC source with nanoTile technology as the
electrospary source for the Synapt G2. Both features
LockSpray system for the optimized co-introduction
of analyte and lock mass compounds directly into
the ion source offering a superior alternative for the
acquisition of exact mass data.
The Synapt G2 HDMS is equipped or supplemented
with two nano LC systems. A 1D NanoACQUITY
UPLC and the other a 2D NanoACQUITY UPLC,
both from Waters Corporation. The nanoACQUITY
Ultra Performance LC (UPLC) System is designed
for nano-scale, capillar y, and nar row-bore
232
separations to attain the highest chromatographic
resolution, sensitivity, and reproducibility. These
systems provide the best technology for the
separation and delivery of peptides to the MS for
proteomics applications, for protein identification
and characterization. The system’s 10,000 psi
operating pressure capability allows for superior
high-peak capacity separations by operating longer
columns packed with sub-2 micron particles. It is
optimized for high-resolution identification and
2D-LC separations at precise nano-flow rates.
This innovative 2D system effectively uses twodimensional (2D) UPLC for better chromatographic
resolution of complex proteomic samples by using a
dual reversed-phase (RP) approach. The entire LC/
MS/MS system is operated my MassLynx software
and data analysis is performed by ProteinLynx
Global Server (PLGS) version 2.5.3.
UltrafleXtreme (Bruker Daltonics)
In May 2012, a new MALDI/TOF/TOF mass
spectrometer (UltrafleXtreme, Bruker Daltonics)
was installed. The instrument has MS and MS/
MS capability with high resolution and high
mass accuracy. The instrument has laser-induced
dissociation (LID) fragmentation for denovo peptide
sequencing, top-down protein sequencing, and
other applications, as well as high-energy CID
fragmentation. The UltrafleXtreme is a highperformance instrument well suited for a wide
range of applications, including polymers, proteins,
peptides, carbohydrates, and other biomolecules.
UltrafleXtreme is supplemented with a nano LC
(EASY-nLC II, Bruker) and MALDI spotter (Proteineer
fcII, Bruker). The EASY-nLC II is a nano-flow HPLC
system tailored to the requirements
of proteomics applications. This LC
system is used to separate protein and
peptide mixtures for MALDI MS. The
system is used in combination with
a MALDI spotter. Proteineer fc II is a
MALDI spotter which enables automatic
liquid handling for MALDI preparation
of LC separated peptide fractions. It is
designed to deposit fractions eluting
from a NanoLC column (Easy-nLC II )
onto a MALDI plate, with the MALDI
matrix automatically being added for
offline MS and MS/MS analysis. MALDI
system is operated by Flex Control and
Flex Analysis software, and the search
engine Mascot is used for proteomics
analysis.
ProteOn™ XPR36 Surface Plasmon
Resonance System (Bio-Rad)
The ProteOn™ XPR36 protein interaction array
system monitors in a label-free manner the
interaction of biomolecules in real-time using
surface plasmon resonance (SPR) technology.
Interactions are monitored over time by detecting
the binding of an analyte flowing in a microfluidic
channel to a ligand immobilized on a sensor chip.
This system generates a 6 x 6 interaction array for
the simultaneous analysis of up to six ligands with
up to six analytes. Interactions such as Proteinprotein, Protein –peptide, Protein- small molecule,
& Protein – DNA can be carried out using specific
chips. The system applications are primarily
analyte screening, kinetic analysis, equilibrium
analysis, and concentration determination.
Services:
The primary focus of the facility is to provide mass
spectrometry services and consultation for the
research and academic community at RGCB. The
facility started accepting a broad range of research
applications and sample types for analysis. The
details of the services provided at present are given
in the table below.
Services offered at the MS & Proteomics Core Facility of RGCB. The analyses are
categorized according to equipment employed.
S. No
Analysis / Description
WATERS Synapt G-2 HDMS (LC-ESI Q-TOF (LC/MS/MS)
1
In gel (1D & 2D gel) trypsin digestion & protein identification by MSE
2
protein identification by MSE
3
Protein Profiling for simple mixture using 1D nano-LC.
4
Protein Profiling for complex mixture using 2D nano-LC.
5
Relative protein quantification or protein expression by Label-free method 1D
6
Relative protein quantification or protein expression by Label-free method 2D
7
De-novo sequencing of Peptides using BioLynxs software
Bruker – UltrafleXtreme (MALDI TOF/TOF)
8
In gel (1D & 2D gel) trypsin digestion & protein identification by PMF
9
In gel Trypsin digestion & Protein profiling using nanoLC-Proteineer Fc II.
10
Molecular weight confirmation/accurate mass determination.
233
11
De-novo sequencing of Peptides using BioTools software.
12
N-terminal sequencing of Peptides
13
In Source Decay (ISD) for top down sequencing of pure protein (up to 60 Kda).
14
Polymer analysis
15
Oligo-nucleotides Molecular weight determination
ProteOn XPR36 Protein Interaction Array System
16
Protein- protein interaction
17
Protein –peptide interaction
20
Protein-DNA interaction
21
Protein- small molecule interaction
Agilent - Liquid Phase IEF system
22
1. Off-gel, liquid phase fractionation of proteins by iso-electric focussing (IEF).
23
2. In-gel IEF for 2D gel electrophoresis.
Number of Samples
Since June 2012, the facility started offering various
proteomics analysis services for the scientists
within the institute. Altogether the proteomics
facility analyzed a total of six hundred (600) samples
from June 2012 to April 2013. The samples analyzed
234
were; 244 for proteomics protein profiling and
relative protein quantification, 56 were gel bands/
spots for protein identification, 255 samples were
natural or synthetic peptides for peptide analysis
and 5 SPR assays for protein interaction analysis.
LABORATORY MEDICINE AND MOLECULAR DIAGOSTICS
(LMMD)
Dr. Radhakrishnan Nair
Scientist E1
[email protected]
Radhakrishnan received his PhD from the University of Kerala working at the Regional
Cancer Centre on Non Hodgkins Lymphoma.He subsequently saved as senior Faculty at
various medical colleges including Manipal college of Medical Sciences, Oman University
Medical College (Affiliated to the West Virgenia University, USA) Sparten University,
Newyork and the Atlantic university, Newyork. He joined RGCB in October 2011
Senior Manager
(Technical Services):
Sanjai D
Senior Research Fellow:
Sanughosh K
Junior Research Fellow:
Sreeja.S
Anjana.S.S
Research Assistant:
Karthika V
Technologist:
Binu Kumar D
Heera Pillai
Vineetha P T
Sreeja S
Antony K.P
Junior Technical
Assistant:
Rintu Varghese
Helper:
Sumaja V
235
Laboratory Medicine and Molecular Diagnostics
(LMMD) was instigated as a Special purpose
vehicle with the desire of being a comprehensive
solution provider in Molecular Diagnostic support,
for all branches of diagnostic and prognostic
medical practice, additionally being a technology
translator for diagnostic aids, inclusive of near
patient monitoring.
The laboratory ensures quality assurance by
participating in the external quality control program
of Quality Control for Molecular Diagnostics
(QCMD) of the United Kingdom, the only service
provider for molecular diagnostics. The Division
is also accredited by the National Accreditation
Board for Testing and Calibration Laboratories
(NABL), Dept. of Science and Technology, Govt. of
India under the ISO 15189 standards. This realizes
LMMD as the first DBT molecular testing lab in
India to be accredited for viral testing platform.
Currently LMMD has two mandates. The first
being molecular diagnostic support to the hospitals,
thereby assisting the clinicians in maintaining the
health of general public. The second mandate
being development of simplified point-of-care
testing platforms. To realize the first objective
LMMD provides molecular diagnostic services for
numerous viral infections including Dengue, viral
hepatitis, influenza viruses, etc. The list is given
in Table 1. The volume based list for 2012-2013
is depicted in Table 2. Dengue sub-type 4 was
isolated from clinical samples and characterized
for the first time in Kerala State by LMMD.
The cardiovascular damage and risk assessment
is done by screening mutations in atherosclerotic
cardiovascular disease, Br ugada/long QT,
Congenital long QT syndrome (LQT), hemorrhagic
telangiectasia, hereditary (HHT), primary pulmonary
hypertension (PPH1), hypercholesterolemia and
familial hypertrophic cardiomyopathy.
which is portable, stable and with more than 90%
sensitivity (Image 1, 2 & 3). Two lateral flow devices
were developed for Classical Swine Fever (CSF)
detection. The first one an antibody based device
and the second one, worlds’ first antigen based
lateral flow device. The patent application for
the same is filed. Both the devices are tested and
validated by external research and testing agencies
of the North-eastern states of India, where CSF is
endemic. A completely transportable test based
on Loop mediated isothermal amplification (LAMP)
assay for both CSF and Dengue is being developed
and it is in the final testing phase (Image 4). Inhouse multiplex PCR for detection and subtyping of
Dengue infection is developed at LMMD. (Image 5)
Ongoing research activities
Hepatitis B virus characterized from HIV-HBV
co-infection cases showed evolutionary distance
from the ones that were characterized from monoinfection cases. This modulation of the virus could
be attributed to the suppression of the immune
system due to external factors. This hypothesis
remains to be tested in Hepatitis B virus isolated
from other cases of induced immune suppression
like the ones on Chemotherapy and Hemodialysis.
A step in this direction has already been taken
by collaborating with Regional Cancer Center,
Thiruvananthapuram, to obtain samples for the
project ‘Determination and Characterization
of Mutations in Hepatitis B in patients with
Hematological Malignancies’ and collaborating
with Department of Nephrology, Medical Collegiate
Hospital, Thiruvananthapuram, to obtain samples
for the project “Evolutionary trend of Hepatitis B
virus in Patients on Hemodialysis, a case of immune
suppression”.
TABLE 1
SlNo
Name of the test
Method
1
Dengue IgG
Elisa
2
Dengue IgM
Elisa
3
Chikun IgM
Elisa
4
HSV IgM
Elisa
5
Measles IgM
Elisa
Prediction of a novel vaccine candidate that
offers protection over all four dengue serotypes, is
undergoing at LMMD.
6
Lepto IgM
Elisa
7
Dengue NS1 Ag
Elisa
The second mandate is fulfilled by in-house
development of lateral flow testing platform,
8
CMV IgM
Elisa
9
JEV IgM
Elisa
Pharmacogenomic analysis of the impact of
CYP3A5 genetic polymorphism on Tacrolimus to
predict the optimal initial dose requirements in
renal recipients is initiated. In addition the Division
has optimized Chimerism Testing/Engraftment
Analysis, Cytochrome P-450, TPMT Genotype and
Warfarin sensitivity in transplant recepients.
236
10
EBV IgM
Elisa
11
RSV IgM
Elisa
12
Entero IgM
Elisa
13
Scrub Typhus IgM
Elisa
21
TB Qualitative
PCR
Inhouse
22
HAV Qualitative
PCR
Inhouse
23
Parvovirus Qualitative
PCR
Inhouse
SLNo
Name of the test
Method
1
HEV Qualitative
PCR
Inhouse
24
Influenza A virus
Qualitative
PCR
Inhouse
2
Measles
PCR
Inhouse
25
Influenza B virus
Qualitative
PCR
Inhouse
3
Hanta virus Qualitative
PCR
Inhouse
26
Parainfluenza virus 1
Qualitative
PCR
Inhouse
4
Scrub Typhus
Qualitative
PCR
Inhouse
27
Qualitative
PCR
Inhouse
5
HBV Qualitative
PCR
Inhouse
28
Qualitative
PCR
Inhouse
6
HBV Quantitative
qPCR
29
RSV Qualitative
7
HCV Qualitative
PCR
Inhouse
PCR
Inhouse
30
Human Meta pneumo
virus Qualitative
PCR
Inhouse
8
HCV Quantitative
qPCR
9
BK JC Qualitative
PCR
Inhouse
31
Rota virus Qualitative
PCR
Inhouse
10
BK JC Quantitative
qPCR
32
Noro virus Qualitative
PCR
Inhouse
11
Dengue Qualitative
PCR
Inhouse
33
Adeno virus Qualitative
PCR
Inhouse
12
Chikun Qualitative
PCR
Inhouse
34
Acanthamoeba
Qualitative
PCR
Inhouse
13
HSV Qualitative
PCR
Inhouse
35
Mumps Qualitative
PCR
Inhouse
14
WestNile Qualitative
PCR
Inhouse
36
Varicella Zoster Virus
Qualitative
PCR
Inhouse
15
Enterovirus Qualitative
PCR
Inhouse
37
Rubella Qualitative
PCR
Inhouse
16
Leptospira Qualitative
PCR
Inhouse
38
HHV-6 Qualitative
PCR
Inhouse
17
CMV Qualitative
PCR
Inhouse
39
HHV-7 Qualitative
PCR
Inhouse
18
CMV Quantitative
qPCR
40
HHV -8 Qualitative
PCR
Inhouse
41
Corona Qualitative
PCR
Inhouse
42
Rubella
PCR
Inhouse
19
20
EBV Qualitative
PCR
Inhouse
JEV Qualitative
PCR
Inhouse
237
TABLE 2
Tests
Samples
Serology IgM
Serology IgG
Conventional PCR
Quantitative PCR
Sequencing
Total
6646
565
1146
3033
1698
121
Image 1
Image 2
Image 3
Image 4
Image 5
238
Regional Facility for DNA Fingerprinting (RFDF)
Cancer Research Program:Laboratory 1
Chief Scientific Officer:
George Thomas
Case Receiving Officer:
Dr. Sanil George
Case Registrant:
Ambili S. Nair
DNA Examiner:
Suresh Kumar U.
Laboratory Technician:
Ratheesh R.V.
239
RFDF offers DNA finger printing service to legal
bodies, crime investigating and law enforcing
agencies. The samples analysed at RFDF relate to
maternity/paternity disputes, crime, rape incidents
and cases involving man missing. CO1-based
molecular identification and DNA bar-coding of
fauna especially for species identification in wildlife
forensics is yet another service offered by RFDF.
Other services offered by this facility include DNA
fingerprinting of plants and animals in case-bycase manner using RAPD, AFLP or microsatellite
markers and DNA bar-coding of animals using
CO1 gene and plants using matK and rbcL. The
240
facility also offers “hands on” training in DNA finger
printing and DNA bar-coding techniques.
In 2012-2013 the facility analysed more than
170 samples related to identification, maternity/
paternity and relationship disputes forwarded
by courts from different districts of Kerala and
Kerala Women’s Commission. In addition more
than 70 samples related to animal poaching were
also analysed for species identification. Fourteen
candidates were given training in DNA finger
printing/bar coding during the year.
Instrumentation Engineering and Information
Technology Division
Shaj Upendran
General Manager
General Manager:
Shaj Upendran
Staff Members:
Rajasekharan K.
Mr. Rahul C.S. Nair
Sajan I.X.
S. Rajeev
S. Ajith Kumar
K. Manoj Kumar
V. Prem Kumar
V. Shaji
V.S. Vijaykumar
241
Rajiv Gandhi Centre for Biotechnology has a large
number of instruments used for Cell biology,
Molecular biology and biotechnology research. The
Instrumentation Engineering Division is responsible
for installation, maintenance and repair of these
sophisticated research instruments in RGCB as
well as the maintenance of Central instrumentation
facility. Some of the new instruments procured
during the year includeWaters make SYNAPT G2
HDMS System, 1D Nano UPLC, 2D Nano UPLC &
HPLC, Bruker Daltonics make MALDI TOF TOF
System with Nano LC and Robotic Spotter, Biorad
SPR based Protein Array Interaction System, Agilent
Liquid Phase IEF System, ABI Real Time PCRs &
96 Well thermal cyclers, Echo Cardiography system
from Phillips, Biorad Protein Purification System,
Ultra deep freezers, Refrigerated Centrifuges etc.
The Division also maintains a well equipped
engineering workshop with facilities required for
the repair and calibration of the sophisticated
instruments as its part. Repair up to the PCB
level is done here reducing the downtime and
repair costs. By attending to many essential repair
works of instruments, dependence on expensive
maintenance contracts with dealers has been
reduced. The Instrumentation Division also
carries out design, modification, and fabrication of
research instruments. During the year, problems of
BioradiQ5 real time PCR, Biorad IEF, Liquid Nitrogen
Plant, FACS ARIA Flow Cytometer, Confocal Laser
Scanning Microscope, Spectrophotometers, Protein
sequencer ,Amino acid analyser, Ultra Centrifuges,
High speed Centrifuges, Table Top Centrifuges, Gel
Documentation systems, Transmission Electron
Microscope, Upright and Inverted Microscopes,
242
PCR machines, Electronic balances, Speed Vac
Concentrator, CO2 Incubators, HPLC, Freeze
Dryers, Microplate Washer etc. have been effected
successfully.
The Instrumentation Division also maintains the
Centralized Instrumentation facilities, Computers,
PC based security surveillance system, Biometrics
Time Attendance recorders, Conferencing facilities,
Communication systems, Liquid Nitrogen Plant,
Incinerator, Auditoriums, Convention Centre etc. It
also carries out the supervision of 11KV electrical
substation, 340 ton AC plant, 750 KVA & 1010KVA
DG sets.
Centralized Instrumentation Facility
In addition to the basic facilities available in all
the research laboratories, we have a centralized
core facility equipped with several minor and
major equipments to cater the requirements of our
research personnel. The following are the facilities
available in the core facility:
Spectroscopy
•
Spectrophotometer - Perkin elmer, Thermo,
Labomed, Shimadzu
•
•
•
•
Luminescence Spectrometer - Perkin Elmer
Multimode Plate Reader - Tecan, PE
FTIR Spectrometer - Thermo, PE
Nano Spectrophotometer - GE, Eppendorf,
Thermo
Library and Information Services
Lathika K.
Librarian
Staff Members:
Meera N.V.
Gopakumar G.
S. Vijayakumar
Lekshmi Sree
243
RGCB Library is deeply committed to catering
to emerging needs of the scientific community
and R & D activities of RGCB. The library is
equipped with the state of the art facilities and
maintains a place of distinction as a premier
library.The Library has a collection of more than
7500 documents and subscribes a large number of
reputed journals mostly in the electronic version.
In addition, being a member of DeLCON, Delnet,
Biomed Central etc. RGCB library can access a
wide range of online journals, e- books etc. Apart
from books on life science, the library also has a
good collection of books on protocols, standards,
manuals, PhD theses, conference proceedings,
general reading books etc. There is also a wide
collection of audio -visual materials. The library
has a separate news paper reading section where
18 popular magazines and 10 daily news papers
are made available. DelCON membership enables
the accession of international e- journals and at
present there is access to more than 900 journals
of twenty international publishers. Usage statistics
for the year under review reveals a definite increase
in the usage of articles on a comparison with the
previous year.Updating of in house databases of
books, periodical, bound volumes of journals, PhD
thesis, reports, conference proceedings, CD Rom
etc. were carried out and uplinked to the website.
Database of PhD these and publications of RGCB
covering the period 2012-2013 were updated.
The membership in Biomed Central facilitated
publication of transcripts by scientists, in addition
to the special facility of getting access to selected
e- resources not available elsewhere.
244
Library services
•
Document Lending service: Each scientist
member is entitled to borrow 4 books at a time,
while the number is restricted to two books in
the case of students and other users.
•
OPAC - Library offers Online Public Access
Catalogue (OPAC) which allows user to browse
library collection through the web.
•
E- Resources and Internet Facility: The Library
is well equipped with a good number of
computers with internet connectivity through
LAN. The Library is having access to a very
good number of electronic journals, e- books,
journals archives etc. Users are having full
access to e- resources.
•
Bibliometric services: Library helps to prepare
various bibliometric reports specially usage
statistics, citation analysis, Impact factor of
journals etc.
•
Resource Sharing activities: The library shares
its resources with other special libraries of
India.
•
Electronic Document Delivery Service is yet
another service available.
•
Reprographic services: Facilities provided with
photocopiers, printers, scanners, including
colour printer.
Animal Research Facility
Santhosh Kumar. S
Veterinarian & Officer-in-Charge
Veterinarian:
Dr. R. Rajagopal
Supporting staff:
Vinod. V.M.
G. Vinod
K.Y. Anwar
Pradeep Kumar S.
Lalkumar C.
Ratheesh Kumar
G. Thankamany
245
Laboratory animals play a quintessential role in
biomedical research. The Animal Research Facility
(ARF) at RGCB has been growing and evolving over
the years as a well equipped and dedicated facility
whose primary objectives are to provide animals
for researchers and facilitate the animal related
research. ARF functions in conjunction with an
efficient Institutional Animal Ethics Committee
(IAEC) and thereby ensures ethical and humane
treatment of animals by all means.
There are separate breeding rooms for each
species of animals such as mice, rat and rabbits.
Presently, ARF has conventional rooms for small
rodents, two transgenic rooms for mice (transgenic
experimental room and transgenic breeding room),
rabbit room, procedure room for conducting minor
procedures and euthanasia. In the Transgenic
facility, valuable and immune-compromised
animals are housed in Individually Ventilated
Caging System (IVC) with all the necessary adjunct
facilities including animal changing station, air
handling unit, air conditioning, dehumidifier etc.,
to maintain optimum conditions for the animals.
246
These are bio security type II cabinet for doing
procedures involving infectious agents. In addition
to these, ARF also has many equipment required
for animal works such as Non-invasive blood
pressure monitor, small animal ventilator, inhalant
anaesthesia machine etc. This year ARF upgraded
its infrastructure with new equipment such as
Non-Invasive Multimode Small Animal imaging
system, Tread mill for cardiovascular studies,
Stereomicroscope etc. A small laboratory was also
set up for preliminary disease investigations which
will be expanded in the future so that serological/
molecular diagnosis of diseases and other breeding
related procedures like genotyping/SNP testing
can be performed in-house. Entry to the facility
is restricted by Biometric Access System and
fair practice/compliance with the ARF policies is
monitored by the surveillance cameras installed
at various locations inside ARF. The strains of
animals currently maintained in ARF are BALB/c,
Swiss Albino, NOD SCID, BALB/c SCID, pHesld2EGFPTg,C57BL/6J, Nude mice, Wistar rats, SHR
rats and New Zealand White rabbits.
Distributed Information Sub-centre
Sathish Mundayoor
Co-ordinator
Technical Officer:
Sivakumar K.C.
247
The Bioinformatics Centre (DISC) at RGCB funded
by Department of Biotechnology, Govt of India,
under the National Bioinformatics Network
program started functioning from May 2002 with
a view to catering to the needs of the scientific
community. The main function of the Centre is
to act as a member of Bioinformatics Network
System for providing information and technology
inputs to the interested users on topics pertaining
to the relevant areas of Biotechnology, especially
genomics and proteomics.
Infrastructure facilities
Computer hardware/ Communication
facilities
Cn3D, Rasmol, SOAP, Bowtie, HMMER, MEGA4,
XMGRACE, GROMACS, FTDOCK, PyMOl, Z-dock
etc.
Websites/Web servers
The Centre is actively involved in creating software’s
and integrated knowledgebases for those who are
working in the areas of plant bioinformatics. Some
of the databases and web servers developed at the
centre are: PKSIIIexplorer[http://type3pks.in/tsvm/
pks3/], PKSIIIpred [http://type3pks.in/prediction/],
TypeIII Polyketide Synthase Database [http://
type3pks.in].
Services provided
To keep in pace with the developments in
bioinformatics field, impetus is given to set up
the necessary computational infrastructure and
resources for the research community. The centre
has upgraded its computational resources by
setting up high performance computing facility.
Twelve Intel Core-i7 processors (customized for
cluster computing), three Dell PowerEgde T300
Workstations and five Dell Vostro 400 computers
are available for computational research activities.
The centre has 10 Mbps managed leased line
connectivity and computers are well connected
through LAN.
Data retrieval, Next-Generation Sequencing
analysis, Molecular modelling, Drug design and
Virtual screening services are offered. This facility
provides project work to students undergoing
B.Tech/M.Tech/M.Phil/M.Sc courses from a large
number of universities/institutes.
Scientific Software packages
•
•
•
•
The molecular modelling package Accelrys
Discovery Studio 2.5 was purchased on DBT
grand, is used for molecular modelling, docking
and simulation studies. Apart from commercial
packages, the centre maintains latest version of
free bioinformatics software’s such as: - EMBOSS,
Autodock, WHATIF, MODELLER, ClustalW, Phylip,
Publications
»» Deepa Rajan S, Rejimoan R, Sivakumar KC,
Sathish Mundayoor.,An SVM based Tool for
the Prediction of Nitrogen Fixing Proteins.
International Journal of Computer Applications, June
2012. 0975 – 8887.
»» Anitha Jose, Rejimoan R, Sivakumar Kc,Sathish
Mundayoor., Prediction of Extracellular Matrix
Proteins using SVMhmm Classifier. International
Journal of Computer Applications ,June 2012,
0975 – 8888.
»» Thasni KA, Ratheeshkumar T, Rojini G,
248
Major Activities
Development of Centre to cater the needs for the
researchers, students related to biotechnology,
bioinformatics and computer technology. Initiated
and enriched the research environment which
resulted in:
•
•
Publications (peer reviewed): 03
Poster Presentations: 02
Training: B.Tech: 04, M.Sc: 07, M.Tech: 04,
Development of database archives and
softwares.
R&D activities in Bioinformatics
Hypotheses generation and validation of
possible outcomes
Sivakumar KC, Rakesh Sathish N, Srinivas G,
Asoke B, Veena S, Priya S., Structure activity
relationship of plumbagin in BRCA1 related
cancer cells. Molecular Carcinogenesis 2012 Jan
30. doi: 10.1002/mc.21877.
Presentations
»» Mallika V, Aiswarya G, Sivakumar KC, Soniya EV.
“Structural analysis of chalcone synthase proteins
from selected members of Zingeberabceae: An
insilico approach” In the International Conferences
on Advances in Biological Sciences (ICABS 2012)
held at Kannur, Kerala, March 15-17, 2012.
Administration Division
Mr. Rajan Panicker IA&AS (Retd.)
Mr. S. Mohanan Nair
Controller of Administration
Senior General Manager
Although modest in size, the RGCB Administrative
Service provides efficient support for all scientific
and technical activities of the institute in addition
to making and balancing the budget in these
difficult times. The administration ensures that
all meetings of statutory committees such as the
RGCB Society General Body, Governing Council,
Scientific Advisory Committee and Building
Committee were promptly convened and the
effective decisions arrived at so as to the pay
for progress of Institute.RGCB has effectively
used and implemented the official language.
The Department of Biotechnology, Government
of India accorded to RGCB second position
among all its autonomous institutes for effective
implementation of Official Language Act and
Policy for the Year 2011-12. A key achievement of
the RGCB Institute Development Division led by
the Senior General Manager was obtaining free of
cost the ownership title of both the present campus
and the 20 acres allotted for the second campus
which was till now allotted on lease basis by the
Government of Kerala. The process of building
the Bio Innovation Centre (BIC) in the Second
Campus commenced with selection of architects
after elaborate proceedings of with the building
committee and the specially appointedgroup
of experts. This consultancy contract has been
awarded and an ambitious master plan, to be
implemented over a total construction period
of 10 years has been finalized after repeated
discussions at various Governing & Research
Councils. This master plan was presented at
RGCB Society General Body, which approved
the same.Planning action for buildings/facilities
under Phase-I of Project has been completed. Site
investigation works for facilitating detailed design
is in progress. Preliminary work of soil testing, site
clearance etc is currently on. A site Office will be
erected, facilitating further construction of Phase
- 1 of BIC. Equipment and instruments needed in
the First Phase were identified and arrangements
for their procurement have been done. To prevent
any delay to planned scientific work action has
been initiated for setting up of a Transit Campus
as approved by the Governing Council. The
Contributory Health Plan introduced during 2011
is operating to comfort and complete satisfaction
of employees.
249
Staff List
SCIENTIFIC CADRE
1.
Professor and Director
Dr. M. RadhakrishnaPillai
17. Scientist E II
Dr. AbdulJaleel
2.
Professor of Eminence
Dr. C.C. Kartha
18. Scientist E I
Dr. V.V. Asha
3.
Scientist G
Dr. Sathish Mundayoor
19. Scientist E I
Dr. Jackson James
4.
Scientist F
Dr. G. Pradeep Kumar
20. Scientist E I
Dr. Sabu Thomas
5.
Scientist F
Dr. R.V. Omkumar
21. Scientist E I
Dr. Radhakrishnan R Nair
6.
Scientist F
Dr. Malini Laloraya
22. Scientist E I
Dr. Sanil George
7.
Scientist E II
Dr. Moinak Banerjee
23. Scientist E I
Dr. S. Sreeja
8.
Scientist E II
Dr. K. Santhosh Kumar
24. Scientist E I
Dr. E. Sreekumar
9.
Scientist E II
Dr. Ruby John Anto
25. Scientist E I
Dr. G.S. Vinod Kumar
10. Scientist E II
Dr. George Thomas
26. Scientist E I
Dr. S. Manjula
11. Scientist E II
Dr. R. Ajay Kumar
27. Scientist C
Dr. K. Harikrishnan
12. Scientist E II
Dr. E.V. Soniya
28. Scientist C
Dr. Rashmi Mishra
13
29. Scientist C
Dr. K.B. Harikumar
Scientist E II
Dr. Suparna Sengupta
14. Scientist E II
Dr. T.R. Santhosh Kumar
30. Scientist C
Dr. Debasree Dutta
15. Scientist E II
Dr. Priya Srinivas
31. Scientist C
Dr. Rakesh Singh Laishram
16. Scientist E II
Dr. S. Asha Nair
32. Scientist C
Dr. M. Maya Devi
250
TECHNICAL CADRE
33. Senior General Manager
Mr. S. Mohanan Nair
53. Technical Officer Grade I
Mr. I.X. Sajan
34. General Manager
(Instrumentation Engineering.)
Mr. Shaj Upendran
54. Assistant Engineer
Mr. C. Durga Prasad
35. Deputy General Manager ( Estates)
Mr. R. Jayachandran Nair
36. Deputy Librarian
Mrs. K. Lathika
37. Senior Manager (Technical Services)
Mrs. V. Jiji
Mr. George Varghese
Mr. D. Sanjai
40. Manager (Technical Services)
Mr. P. Manoj
Mr. K. Rajasekharan
42. Veterinarian & Animal House-in-Charge
Mr. S. Santhosh Kumar
43. Manager(Technical Services)
Mr. M. Saravana kumar
44. Senior Technical Officer
Ms. Sandhya C. Nair
Ms. Indu Ramachandran
Ms. Laiza Paul
Ms. Sudha B.Nair
Ms. Bindu Asokan
Ms. Ciji Varghese
50. Manager (Technical Services.)
Ms. Ambili S. Nair
51. Technical Officer
Mr. K.C. Sivakumar
52. Technical Officer Grade I
Mr. Rahul C.S. Nair
251
55. Technical Officer
Mr. Arun Surendran
56. Technical Assistant Group III
Mr. K. Deepu
57. Deputy Engineer (Electrical)
Mr. S. Ajith Kumar
58. Technical Assistant Gp – III
Mrs. Rintu T. Varghese
59. Technical Assistant Group I
Mr. V. Amal
60. Technical Assistant Group I
Mr. R. Dileep Kumar
61. Technical Assistant Gp.I Gr.I
Mr. G. Johny
Ms. G. Sheela
Mr. V. R. Unnikrishnan
Mr. K. P. Antony
Mr. S. Edwin
Mrs. G. Velthai
Mr. S. Vijayakumar
68. Technical Assistant Gp.I Gr.II
Mr. S. Rajeev
69. Technical Assistant Gp.I Gr.II
Mr. Biju S. Nair
Mr. S. Santhosh
71. Technical Assistant Gp.I Gr.I Ms. N.V. Meera
72. Technical Assistant
Mr. Aswani Kumar
73. Technical Assistant
Ms. Reena Prasad
76. Helper
Mr. K.A. Vinod Lal
74. Helper
Mr. G. Gopakumar
77. Helper
Mr. J. Jayanandan
75. Helper
Mr. J. Venugopalan.
78. Helper / Laboratory Helper
Ms. V. Sumaja
ADMINISTRATIVE CADRE
79. Chief Controller
Mr. K.M. Nair
87. Manager (Accounts & Audit)
Mr. R. Kumar
80. Registrar
Dr. R. Ashok
88. Senior P.S. to Director
Ms. U.S. Jayalakshmi
81. Finance Officer Mr. M. Babu
89. Senior P.S. to Director
Ms. R. Priya
82. Chief Manager (Purchase)
Mr. Jeevan Chacko
90. Assistant Administrative Officer
Ms. Asha R. Nair
83. Accounts Officer
Ms. K.K. Jayasree
91. Private Secretary Ms. O. Girija Kumari
84. Administrative Officer
Ms. S. Suthakumari
92. Management Assistant
Ms. J. Preetha
85. Manager (Purchase)
Mr. N. Jayakrishnan
93. Office Assistant
Mr. K. Subash
86. Asst. Accounts Officer
Ms. Usha Devi
94. Junior Office Assistant
Mr. R. Anil Kumar
SUPPORT STAFF
95. Driver Grade III
Mr. S. Harikumar
99. Attendant Grade I
Mr. Thapasi Muthu
96. Driver Grade I
Mr. V.M. Manukumar
Ms. B. Usha
97. Attendant Grade III
Mr. T. Wilson
Mr. S. R. Vinod Kumar
98. Attendant Grade - III
Ms. B. Chandrika Devi
Ms. R. Thankamani
252
Invited Speakers at RGCB 2012-13
Sl No
1
2
SPEAKER
Dr. Nixon M. Abraham
Laboratory of Sensory
Perception and Plasticity
Department of Fundamental
Neuroscience,
University Medical Centre,
University of Geneva,
1 Rue Michel
Anup Gopalakrishna Pillai PhD, Prof. Marian Joels Laboratory
Brain Center, University
Post-doctoral fellow Medical Center, Utrecht
3
Dr. Vijai Joseph, PhD
4
Rejji Kuruvilla, Ph.D,
Associate Professor
5
Dr. Aryan Namboodiri, Ph.D.
6
Seetha Krishnan, PhD,
Senior research investigator
253
ADDRESS
Clinical Genetics Research
Labs, Cancers Sloan Kettering
Institute for Cancer Research
New York, The Netherlands.
Department of Biology,
Johns Hopkins University,
3400 N. Charles St,
Mudd 224, Baltimore,
MD 21218
Department of Anatomy,
Physiology and Genetics,
Uniformed Services, University
of Health Sciences, Bethesda,
Maryland, USA
Scientific Manager, Biology
Division, Syngene
TITLE OF THE TALK
Temporal aspects of
information processing in
olfactory system
Impact of stress, its
duration and timing:
from single neurons to
behavior
Germline Susceptibility
to Cancers in the
Personalized Genomics
Era
Target-derived
neurotrophins: signaling,
trafficking and functions
N-Acetylaspartate in the
CNS: From Canavan
Disease to Cancer
Application of Molecular
Biology in Drug
Discovery

Annual Report 2012 - Rajiv Gandhi Centre for Biotechnology

Transcription

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