pg. 78 + viii -- PDF - Gravitational and Space Research

Transcription

Gravitational and Space Biology Volume 25, Number 1
September 2011
Publication of the American Society for Gravitational and Space Biology
ISSN 1089-988X
ASGSB EDITORIAL BOARD
Luis Angel Cubano, Ph.D.
Uni. Central del Caribe
Ted A. Bateman, Ph.D.
University of North Carolina - Chapel Hill
Emily M. Holton, Ph.D.
John Z. Kiss, Ph.D.
NASA Ames Research Center
Miami University.
Dennis F Kucik, M.D., Ph.D.
University of Alabama at Birmingham
William J. Landis, Ph.D.
Gloria K. Muday, Ph.D.
Wake Forest Univ.
April E. Ronca, Ph.D.
Wake Forest Univ. Sch. of Medicine
Paul W. Todd, Ph.D.
Sarah Wyatt, Ph.D.
Techshot, Inc.
Ohio University
The University of Akron
ASGSB PUBLISHING STAFF Editor in Chief:
Copy Editor:
Publishing Editor:
Anna-Lisa Paul, Ph.D.
Janet V. Powers
Timothy J. Mulkey, Ph.D.
University of Florida
NASA Research & Technology Task
Book, NRESS/SDSE, LLC Indiana State University
From the cover: Hatchling Bobtail squid Euprymna scolopes used as a model organism to assess the role of
microgravity in symbiosis-induced animal development. From: “Potential of the Euprymna/Vibrio symbiosis as
a model to assess the impact of microgravity on bacteria-induced animal development.” J. Foster, et al., p. 45.
GENERAL INFORMATION
Gravitational and Space Biology (ISSN 1089-988X) is a journal devoted to research in
gravitational and space biology. It is published by the American Society for Gravitational and
Space Biology, a non-profit organization whose members share a common goal of furthering
the understanding of the biological effects of gravity and the use of the unique environment
of spaceflight for biological research. Gravitational and Space Biology is overseen by a
steering committee consisting of the Publications Committee, the Editor, the President, and
the Secretary-Treasurer of the ASGSB.
The American Society for Gravitational and Space Biology was created in 1984 to
provide an avenue for scientists interested in gravitational and space biology to share
information and join together to speak with a united voice in support of this field of science.
The biological effects of gravity have been acknowledged since Galileo’s time, but only
since the 1970s has gravitational biology begun to attract attention. With the birth of the
space age, the opportunity for experimentation over the full spectrum of gravity finally
became a reality, and a new environment and research tool became available to probe
biological phenomena and expand scientific knowledge. Space and spaceflight introduced
new questions about space radiation and the physiological and psychological effects of the
artificial environment of spacecraft.
The objectives of ASGSB are:
•
•
•
•
To promote research, education, training, and development in the areas of
gravitational and space biology and to apply the knowledge gained to a better
understanding of the effect of gravity and space environmental factors on the
flora and fauna of Earth.
To disseminate information on gravitational and space biology research and the
application of this research to the solution of terrestrial and space biological
problems.
To provide a forum for communication among professionals in academia,
government, business, and other segments of society involved in gravitational
and space biological research and application.
To promote the study of concepts and the implementation of programs that can
achieve these ends and further the advancement and welfare of humankind.
MEMBERSHIP: The American Society for Gravitational and Space Biology welcomes
individual, organizational, and corporate members in all of the basic and applied fields of the
space and gravitational life sciences. Members are active in the fields of space medicine,
plant and animal gravitational physiology, cell and developmental biology, biophysics, and
space hardware, and life support system development. Membership is open to nationals of
all countries. Members must have education or research or applied experience in areas
related to the Society’s purposes, and student members must be actively enrolled in an
academic curriculum leading toward a career related to the Society’s purposes. Membership
applications may be obtained at the society website (http://www.asgsb.org).
Copyright © 2011 by the American Society for Gravitational and Space Biology
ii Gravitational and Space Biology Volume 25 (1) Sept 2011
Gravitational and Space Biology Editorial Board
Editor in Chief:
Anna-Lisa Paul, Ph.D.
University of Florida
E-Mail: [email protected]
Expertise: plant molecular genetics, gene expression, plant space biology
Copy Editor:
Publishing Editor:
Janet V. Powers
Timothy J. Mulkey, Ph.D.
NASA Research & Technology Task Book,
NRESS/SDSE, LLC
Indiana State University
Expertise: plant growth, development, hormones, calcium
Expertise: plant physiology, gravitation & space biology,
informatics
Associate Editors:
Uni. Central del Caribe
Ted A. Bateman, Ph.D.
University of North Carolina - Chapel Hill
E-mail: [email protected]
Expertise: cell biology, cytoskeleton, gene expression
Expertise: radiation and bone loss
Emily M. Holton, Ph.D.
John Z. Kiss, Ph.D.
Dept of Botany , Miami Univ.
Luis Angel Cubano, Ph.D.
NASA Ames Research Center
Expertise: hindlimb unloading, calcium/bone metabolism
Expertise: Plant gravitational biology, tropisms, growth and
development
Dennis F Kucik, M.D., Ph.D.
University of Alabama at Birmingham
William J. Landis, Ph.D.
Expertise: cell adhesion and integrins, atherosclerosis &
immunology
Expertise: Bone structure & biochemistry, cartilage & tendon
biology
Gloria K. Muday, Ph.D.
Wake Forest Univ.
April E. Ronca, Ph.D.
Wake Forest Univ. Sch. of Medicine
Expertise: plant signaling mechanisms, molecular responses
Expertise: reproduction, development, micro- & hyper-gravity
Paul W. Todd, Ph.D.
Sarah Wyatt, Ph.D.
Techshot, Inc.
Ohio University
Expertise: Low-gravity physics, flight hardware, biotechnology,
cell biology, osteogenesis and microbiology.
Expertise: plant responses to gravity, genomics/gene expression
and proteomics
The University of Akron
Gravitational and Space Biology Volume 25 (1) Sept 2011
iii
Gravitational and Space Biology Instructions to Authors
Brief Overview:
The journal of the American Society for Gravitational and Space Biology (ASGSB), Gravitational and
Space Biology, publishes quality, peer reviewed manuscripts in several categories. Manuscripts should be selfcontained, and all conclusions substantiated and supported by results in the form of figures and/or tables.
Authors are held to standards of writing (American English) for clarity and material appropriate for the
Gravitational and Space Biology (GSB) journal. Subject matter can include any topic within the following
broad categories: the impact of gravity and changes in the gravity vector on biology, spaceflight research (ISS
and Shuttle), satellite payloads, advanced life support, planetary and orbital analog research, suborbital research,
parabolic flight, sounding rockets, high altitude balloons, astrobiology, plus hardware development,
mechanobiology, and other disciplines exploring the interface of biology and engineering technology. Brief
summaries of manuscript types and guidelines for each category are below; detailed instructions and templates
follow.
I.
Short Communications.
Short communications are submissions typically 2 - 3 formatted pages in length (1000 – 2000 words,
excluding references). These submissions are to be comprised predominantly of preliminary data for a larger
study or a brief report to support work of a larger nature. It may be beyond the scope of these submissions for
further experimentation, but a reviewer may request additional explanation of the presented data and hold the
authors to appropriate conclusions for those data.
II.
Methods papers.
Methods papers are manuscripts typically 3 - 6 pages in length. These manuscripts are comprised of data
and protocols that support flight experiments or ground control experiments, of protocols in support of
fundamental studies exploring biological responses to altered gravitational environments, and to biological
responses to space and planetary analogs. The manuscripts should contain sufficient detail to enable a reader to
replicate the protocol. Reviewers should particularly address shortfalls of detail, validation of protocols, and
inconsistencies in any aspect of presentation. Figures may include illustrations of procedures and set-up and
should include data that verify the efficacy of the procedures.
III. Research papers.
Research papers are manuscripts of typically 8 - 15 pages in length and, although there is no strict
limitation to size, a reviewer may address extremes of brevity or length as appropriate to conveying the
information. These manuscripts present original research of interest to the gravitational and space biology
community
IV. Review articles.
Review articles are typically 10 - 15 pages in length. These manuscripts are often solicited from
symposium speakers at the annual ASGSB meeting, but they are not limited to those solicitations. Any author
may approach the editorial board with a suggestion or request to submit a review article, to be peer-reviewed as
any other paper. A review article will be judged principally for accuracy of information and citation and
appropriate scope and relevance of the subject of the article.
Detailed Instructions:
Format
The same basic format is used for each type of article. Consult a current issue of Gravitational and Space
Biology, as well as the instructions below, for guidance on formatting, organizing, and preparing references,
figures, tables, and legends. An article must have a brief abstract that summarizes the principal conclusions of
iv Gravitational and Space Biology Volume 25 (1) Sept 2011
Instructions to Authors
the paper. Manuscripts are submitted electronically as single column, double spaced Word documents, and
figures as separate, individual documents. Details are provided below.
Arrangement
Arrange the manuscript in the following order, with all pages numbered consecutively in the footer of
the lower right corner. The last name of the first author should precede each page number.
Cover page – In a separate page, include the Title, suggested Running Head (not to exceed 60 characters,
including spaces), the full names and affiliations of all authors, and detailed Contact Information for the
Corresponding Author: name, address, e-mail, telephone number. Also, provide a list of about 10 Key
Words or short phrases; avoid generic terms, and terms already present in the Title.
The remaining sections proceed without page breaks
Title – Use a descriptive title (not to exceed 200 characters, including spaces).
Authors – Provide the complete names and affiliations of all authors; indicate the corresponding author.
Abstract – Summarize the principal approach and conclusions of the paper. Abstracts are not to exceed
150 words in Short Communications and 250 words in all others.
Body of paper – For Research Papers, the body of the paper should be arranged into subsections for
Introduction, Materials and Methods, Results, and Discussion. Review Papers should be organized in a
manner appropriate to the subject. Methods papers should include a short Introduction and also a
Discussion of the application addressing the significance of the method being described. The Short
Communication papers are not required to contain subdivisions, other than a short abstract, but may be
organized into subsections at the discretion of the authors.
References and Citations – Cite each reference in the text by author(s) name(s) and the publication date:
Examples: Smith, 1989 (one author) Smith and Jones, 2001 (two authors) Smith et al., 2010 (more than
two authors).
•
Alphabetize the reference list by authors' last names.
•
List only published or in-press articles. Unpublished results, including personal communications
and submitted manuscripts, should be cited as such in the text.
•
References formatted as follows: author(s): last name(s) comma followed by initial(s) and a
period comma before next author; year of publication followed by a period; article title in
sentence case, followed by a period; journal title (italicized), followed by volume number, issue
number in parenthesis (if applicable), a colon, and page numbers. Previous issues can be used as
a guide, and an EndNote™ style template can be downloaded at the website. Two examples are
provided below:
Journal Article:
Goodner, B. and Quatrano, R.S. 1993. Fucus embryogenesis: a model to study the establishment
of polarity. Plant Cell 5:1471-1481
Book:
Somero, G.N., Fields, P.A., Hofmann, G.E., Weinstein, R.B., and Kawall, H. 1998. Cold
adaptation and stenothermy in Antarctic notothenioid fishes: What has been gained and
what has been lost? In: Fishes of Antarctica: A Biological Overview. (di Prisco, G.,
Pisano, E. and Clarke, A., Eds.) Heidelberg: Springer-Verlag, pp. 97-109
Figures – Figures are submitted as separate graphic files. Resolution must be 300dpi
• Number Figures consecutively as they are used in the text.
• The first time a figure is discussed, refer to it actively rather than parenthetically.
• Provide enough information in the Figure Legend such that the reader can understand the figure
without significant input from the text. For submission, provide Figure Legends at the end of the
body of the manuscript, following the Reference section.
• Designate figure sections with letters and explain all symbols and abbreviations that are used in
the figure.
v
Instructions to Authors
Cover Art – GSB welcomes the submission of artwork for the cover of the issue in which the manuscript
will be published. Artwork must be submitted as a graphic file of 300 dpi or greater.
Tables – Tables are submitted on separated pages at the end of the manscript.
• Number Tables consecutively as they are used in the text.
• The first time a table is discussed, refer to it actively rather than parenthetically.
• Give each table a concise title, followed by a legend that makes the general meaning of the table
comprehensible without reference to the text. For submission, provide Tables and Table
Legends at the end of the body of the manuscript, following the Figure Legends.
• Tables should be constructed in Word or Excel with the general format:
Table 1
Atmospheric pressure relative to altitude.
Pressure (kPa)
101 – 70
Altitude (m)
0 – 3000
70 – 50
3000 – 5500
50 – 30
5500 – 9000
30 – 5
9000 – 27000
Comments
tropical / temperate / taiga biome - many
examples of human habitation
tundra / alpine biome - few examples of
human habitation
extreme terrestrial elevations - humans
require supplemental oxygen
plants can survive as long as temperature
is mediated and water is available
after: Paul A-L and Ferl R.J. 2006. Gravitational and Space Biology 19:3-17
Abbreviations or Other Standards
• Do not use abbreviations other than those that are standard for international usage.
• Use SI units as far as possible.
• Use g (italicized) for unit gravity, to distinguish it from the standard abbreviation g (not
italicized) for gram.
• Use spaceflight (one word) rather than space flight (two words).
• Any acronyms that are used in the manuscript must be defined at first mention.
Manuscript Review and Preparation of Final Version
Prior to publication, manuscripts are reviewed by the editor assigned to an author’s article and, generally,
by two scientific reviewers.
If reviewers recommend only minor textual changes, the editor may choose to make these changes and
accept the manuscript essentially as submitted. The editor then sends the accepted manuscript to the journal’s
publishing editor. Page proofs are provided to the authors for review prior to the journal going to press.
Deadlines
Editors will inform authors of their deadlines. Deadlines will have limited flexibility, but under no
circumstances will the publication of the journal be delayed to accommodate late manuscripts.
vi
Gravitational and Space Biology Journal Policies
Authorship Statement
Authorship of articles implies that an individual has made a substantial contribution to the article both in
terms of the design of the study or collection/evaluation of data and with regard to the intellectual content of the
manuscript.
Conflict-of-Interest Statement
Reviewers recruited for the evaluation of manuscripts being considered for publication in Gravitational
and Space Biology will be held to Conflict of Interest standards comparable to those required of NSF and
NASA panelists. You may be considered in conflict if:
You have an association with the submitting institution:
•
Employment as faculty at the submitting institution or as a consultant or advisor to the institution.
•
Previous employment with the institution within the last 12 months.
•
Being considered for employment at the institution.
•
Hold any office, governing board membership, or relevant committee chair in the institution.
•
Received and retained an honorarium or award from the institution within the last 12 months.
•
Ownership of investments in the research technology.
You have a relationship with an author on the manuscript:
•
A family relationship such as spouse, child, sibling, or parent.
•
Any affiliation or relationship of your spouse, of your minor child, of a relative living in your
immediate household or of anyone who is legally your partner.
•
A relationship, such as close personal friendship, that you think might tend to affect your judgment or
be seen as doing so by a reasonable person familiar with the relationship.
•
Business or professional partnership.
•
Past or present association as thesis advisor or thesis student.
•
Past or present association as post-doctoral advisor or postdoctoral student within the past 5 years.
•
Collaboration on a project or on a book, article, report, or paper within the last 2 years.
•
Co-editing of a journal, compendium, or conference proceedings within the last 1 year.
Published Statement of Human and Animal Rights:
Research involving Human and Animal Subjects must have been approved by the author’s institutional
review board. Authors must include in the Methods section a brief statement identifying the institutional and/or
licensing committee approving the experiments. For experiments involving human subjects, authors must also
include a statement confirming that informed consent was obtained from all subjects. All experiments involving
human subjects must have been conducted according to the principles expressed in the Declaration of Helsinki.
If doubt exists whether the research was conducted in accordance with the Helsinki Declaration, the authors
must explain the rationale for their approach, and demonstrate that the institutional review body explicitly
approved the doubtful aspects of the study. When reporting experiments on animals, authors should be asked to
vii
Journal Policies
indicate whether the institutional and national guide for the care and use of laboratory animals was followed.
For research using Recombinant DNA, physical and biological containment must conform to National Institutes
of Health guidelines or those of a corresponding agency.
Published Statement of Informed Consent:
The general requirements for informed consent conform to guidelines and requirements outlined by the
National Science Foundation http://www.nsf.gov/bfa/dias/policy/docs/45cfr690.pdf and Health and Human
services http://answers.hhs.gov/ohrp/categories/1566. No investigator may involve a human being as a subject
in research covered by this policy unless the investigator has obtained the legally effective informed consent of
the subject or the subject's legally authorized representative. An investigator shall seek such consent only under
circumstances that provide the prospective subject or the representative sufficient opportunity to consider
whether or not to participate and that minimize the possibility of coercion or undue influence. The information
that is given to the subject or the representative shall be in language understandable to the subject or the
representative. No informed consent, whether oral or written, may include any exculpatory language through
which the subject or the representative is made to waive or appear to waive any of the subject's legal rights, or
releases or appears to release the investigator, the sponsor, the institution or its agents from liability for
negligence.
Basic elements of informed consent.
In seeking informed consent the following information shall be provided to each subject:
•
A statement that the study involves research, an explanation of the purposes of the research and the
expected duration of the subject's participation, a description of the procedures to be followed, and
identification of any procedures which are experimental;
•
A description of any reasonably foreseeable risks or discomforts to the subject;
•
A description of any benefits to the subject or to others which may reasonably be expected from the
research;
•
A disclosure of appropriate alternative procedures or courses of treatment, if any, that might be
advantageous to the subject;
•
A statement describing the extent, if any, to which confidentiality of records identifying the subject
will be maintained;
•
For research involving more than minimal risk, an explanation as to whether any compensation and an
explanation as to whether any medical treatments are available if injury occurs and, if so, what they
consist of, or where further information may be obtained;
•
An explanation of whom to contact for answers to pertinent questions about the research and research
subjects' rights, and whom to contact in the event of a research-related injury to the subject; and
•
A statement that participation is voluntary, refusal to participate will involve no penalty or loss of
benefits to which the subject is otherwise entitled, and the subject may discontinue participation at any
time without penalty or loss of benefits to which the subject is otherwise entitled.
viii Gravitational and Space Biology Volume 25 (1) Sept 2011
Table of Contents
General Information ............................................................................................................................. ii
Editorial Board ................................................................................................................................ iii
Instruction to Authors .................................................................................................................... iv
Journal Policies ................................................................................................................................. vii
Table of Contents ...................................................................................................................................
1
In Memoriam
Remembering Mary .........................................................................................................................
3
Reviews
Autonomous Gravity Perception and Responses of Single Plant Cells.
Mari L. Salmi, Thomas J. Bushart, and Stanley J. Roux .....................................................................
6
Space Radiation and Bone Loss.
Jeffrey S. Willey, Shane A.J. Lloyd, Gregory A. Nelson, and Ted A. Bateman ................................ 14
Research Articles
Identification of Proteins Associated with Spatial-Specific Phytochrome-Mediated
Light Signaling in Arabidopsis thaliana by Liquid Chromatography-Tandem
Mass Spectrometry.
Sookyung Oh and Beronda L. Montgomery ...................................................................................
22
Short Communications
Bone Marrow Stem Cells Differentiated Into Cartilage by Manipulation
of Gas Phase.
Aileen Y. Wang, Heather Luong, Jerry English, William LeBoeuf, Quynh Diep,
Mihir Patel, Shiwei Cai, and P. Jackie Duke .................................................................................
33
Fluid Motion for Microgravity Simulations in a Random Positioning Machine.
Carole A. D. Leguy, Rene Delfos, Mathieu J.B.M. Pourquie, Christian Poelma,
Janneke Krooneman, Jerry Westerweel, and Jack J.W.A. van Loon ...............................................
36
Using Green Fluorescent Protein (GFP) Reporter Genes in
RNAlater™ Fixed Tissue.
Anna-Lisa Paul and Robert J. Ferl ................................................................................................
40
Potential of the Euprymna/Vibrio Symbiosis as a Model to Assess the
Impact of Microgravity on Bacteria-Induced Animal Development.
Jamie S. Foster, Krystal R. Kerney, Mirina L. Parrish,
Christina L.M. Khodadad, and Steven R. Ahrendt .........................................................................
44
Effects of Slow Clinorotation on Growth and Yield in Field Grown Rice.
Sagar S. Jatap, Kondiram N. Dhumal, and Pandit B. Vidyasagar ..................................................
48
1
A Retrospective Analysis on Gender Differences in the Arterial Stiffness
Response to Microgravity Exposure.
Eric C. Tuday, Steven H. Platts, Daniel Nyhan, Artin A. Shoukas, and Dan E. Berkowitz ............
51
Metabolic Control as a Strategy for Payload Cost Reductions and Mitigation
of Negative Space Environmental Factors.
Eugene Galicia, Ervin Palma, Florian Selch, David Gomez,
Richard Grindeland, and Yuri Griko ..............................................................................................
54
Iron Ion (56Fe) Radiation Increases the Size of Pre-Existing Atherosclerotic
Lesions in ApoE -/- Mice.
Tao Yu, Brian W. Parks, Shaohua Yu, Roshni Srivastava, Kiran Gupta, Xing Wu,
Saman Khaled, Polly Y. Chang, Janusz H. Kabarowski, and Dennis F. Kucik .............................
57
Cellular and Molecular Biology During Spaceflight.
Luchino Y. Cohen, Michel Fortin, Sebastien Leclair, Ozzy Mermut,
Genevieve Dubeau-Laramee, and Daniel Provencal ......................................................................
60
Differentiation of Dental Stem Cells in Simulated Microgravity.
Colleen Conlon, Vishnu S. Valluri, Jagan V. Valluri, and Pier Paolo Claudio .............................
63
Another Go-Around: Revisiting the Case for Space-Based Centrifuges.
Laurence R. Young, Erika B. Wagner, Joan Vernikos, Jessica E. Duda, Charles A. Fuller,
Kenneth A. Souza, Cynthia Martin-Brennan, and Christopher P. McKay ......................................
66
Informative Mapping by VESGEN Analysis of Venation Branching Pattern
in Plant Leaves such as Arabidopsis thaliana.
Patricia Parsons-Wingerter and Mary B. Vickerman ....................................................................
69
Attenuation of Muscle Atrophy by Triterpentene Celastrol.
Taesik Gwag, Haksub Shin, Takeshi Nikawa, and Inho Choi
......................................................
72
Rapid Phenotypic Response of LSMMG – “Primed” Candida albicans.
Stephen C. Searles, Linda E. Hyman, and Sheila M. Nielson-Preiss .............................................
75
Author Index ........................................................................................................................................
78
2 Gravitational and Space Biology Volume 25 (1) Sept 2011
In Memoriam
Remembering Mary
This August, the American Society of Gravitational and Space Biology lost one of its founding
members, Mary E. Musgrave. Members of the community lost a friend, a colleague, a mentor, a role
model and much more. But what we will never lose is her spirit; it infuses so many aspects of this
society, and has been shared and reflected on countless levels by us all. It has been said that “…Mary
never forgot that the real goal for space research was adventure.” Thank you Mary, we will
remember. – Anna-Lisa Paul
What follows is a set
of remembrances contributed by several who
knew Mary well. We
discussed as a group how
best to compile these
thoughts,
how
to
synthesize them into a
cohesive tribute that did
justice to her memory,
particularly in the context of the society. In the
end, it was clear that each tribute deserved its
own voice, and each was preserved as it was
contributed. Further, we beg the understanding
of all of you who would have liked to contribute
in kind … we know you share these thoughts.
Marshall Porterfield.
Every fall we in
academia welcome the start of a new academic
calendar, and the new freshmen who represent
rebirth in the next generation. This year those
feelings are tempered by the loss of a great
research leader, mentor, and teacher. Mary grew
up in an academic environment as her father was
a renowned professor at Cornell. She didn’t
stray far from her roots, and pursued graduate
degrees and an academic career track that
culminated in administration. During her career
Mary represented the pinnacle of success
because she mastered all aspects of teaching,
discovery, and service.
As my PhD advisor Mary had a tremendous
impact on my career, philosophical approach,
and personal outlook. As a mentor and role
model, I am extremely grateful and hold her in
my heart at the highest level of reverence,
respect and affection. Beyond my short time
with her in graduate school she continued to be
supportive
and
provide
advice
and
encouragement. She served as the shining
example of what could be achieved through hard
work, and innovative science. Leading by
example is the most powerful and direct way to
provide mentorship, and Mary provided this at,
all levels, to all she encountered and worked
with.
In the field of Gravitational and Space
Biology, there are only a handful of individuals
who have had as much impact as Mary. Her
research program innovated and pioneered the
application of stress physiology and fundamental
biophysical approaches in order to advance our
understanding, and ability to successfully grow
plants beyond the limits of earth. Her work
focused on plant reproduction, but has had
tremendous impact at all levels of the science as
well as current and future engineering efforts.
The impact of her work and pioneering efforts
will play a critical role in the development of
advanced biomimetic life support technologies
that will support the future of manned long
duration exploration.
Our society also continues to benefit from
the years of hard-work and dedication to the
society that she demonstrated. She served on
numerous committees, the governing board, and
eventually served as President of the society.
Her leadership and vision in these roles
continues to benefit and reverberate within our
community. She also served as Publications
Editor for the ASGSB, and dedicated countless
hours of work in that role. Beyond her scientific
impact we as a society owe Mary an incalculable
3
Remembering Mary
debt for her dedication and leadership to the
society.
In every conceivable way Mary exemplifies
the definition of scientist and professor. Beyond
her role as mentor, discoverer, colleague,
administrator, pioneer, advocate, and leader she
was also a dedicated mother and spouse. So in
our remembrance and in our sorrow and anguish
of our loss we should try to find solace in this
season of academic rebirth. Mary lives on in her
work but also in each of us that she touched. It is
now our obligation to pass that along to the next
generation in the classroom, laboratory, and
community. We all owe Mary a tremendous gift
of gratitude, and I am most thankful here today
standing on the shoulder of a giant… and the
view is pretty good up here.
Stan Roux. As a charter member of the ASGSB
I am keenly aware that Mary Musgrave was
among the top luminaries who guided and
contributed most to the Society during its first
three decades. She was one of those rare persons
who combined intelligence, eloquence, clarity of
vision, energy and loyalty into a force for good,
and it was by some remarkable good fortune that
she chose to channel so much of this force for the
benefit of ASGSB and for the gravitational and
space biology science the society promotes.
Many of her gifts to ASGSB were quietly given
and often unnoticed, but certainly her more
obvious gifts were most impressive: I would like
to especially note her classic, oft-cited
publications in the field and her invaluable
contributions as president of the Society and as
Editor of the ASGSB Newsletter and Science
issues. I and others in the Society sought her
counsel often and we were never disappointed.
Her passing is a great loss to the Society, but an
even greater loss to those who experienced the
impact of her gifts even more personally and
powerfully than we. Mary, we are grateful that
we knew you, and you will be sorely missed.
Joan Allen. I had the rare good fortune to work
with Mary on her NASA funded experiments on
seed development in hypogravity as a research
assistant from 2005 through 2008.
Her
professionalism, intelligence, kindness, sense of
humor and enthusiasm were outstanding traits
that come to mind. She was a mentor and a
friend. She was generous in sharing both her
knowledge and credit and recognition for our
work. The one thing I would like to share with
all those who knew, respected and loved Mary is
how apparent it was to me that she deeply cared
for and respected those she worked with and the
fine institutions that you all represent. At
UConn, she was a devoted advocate for
individuals, our department, and the university.
She was devoted to her family as well. I will
miss her very much.
Lanfang Levine. There are no words that can
adequately express my sorrow and the hole left
in my heart by the loss of Dr. Mary Musgrave.
There is no space that is sufficient to
accommodate my gratitude to her. The best I can
do is to relate some of my memories of her.
These are personal memories for me, but may
resonate with some of you.
I got to know Mary through her former
graduate, Lindsey Tuominen, who came to my
lab as a Planetary Biology Intern in 2004. Since
then, we started a productive collaboration. To
this date we still have an active project. I feel
privileged to have had the opportunity to work
with Mary, and I was constantly amazed at the
breadth and depth of her knowledge and
interests, clarity of her thoughts, lucidness of her
writing and contagiously sunny and positive
altitude. These traits and her sense of humor
were also displayed vividly in a feature article
titled “Reflections on Relevance” (Musgrave
2008).
Remembering Mary
Because she could seem reserved and
inaccessible at times, it was fun to discover the
personal side of her. It is not a stretch to call
Mary a champion of edible landscaping. If you
ever set foot on her property, you could feel the
life on every inch of the soil (corn, turnips,
onions, grains, black currants, red currants).
During one of my visits, she invited me to dine
with her and her husband John.
Almost
everything served came from their garden or was
raised by them. It was a surprise and delight to
find how tasty a simple free range oven baked
chicken could be.
It demonstrated that
something could be simple and exquisite at the
same time. Mary was a practitioner of many
causes, especially conservation. She was also a
great seamstress and used it to relax between her
research proposals.
I admired her most for her personal integrity
and was deeply touched by her thoughtfulness.
You could always count on Mary to deliver what
she said she’d do. Four months ago, I visited her
for the first time since her cancer diagnosis. I
arrived at Mary’s lab on a rainy April New
England day at 8:30 am. Knowing of her three
surgeries and months of chemotherapy, I did not
expect to see her at such an early time, yet she
was already in the lab to greet me and had
supplies autoclaved so that we could dive into
the experiment immediately. In spite of the toll
taken on her physical strength by the cancer and
medical procedures, she was still fully
committed to our research project. Literally
speaking, she was the honeybee, hand pollinating
hundreds flowers to ensure synchronized
development of siliques for our experiments.
She patiently explained the value of the
education aspect of the project and details of
experimental procedures under development.
She even arranged to have a key piece of
equipment loaned to me and seeds shipped. I
will never forget the last words she said to me
“This is such an interesting project, I just love
it.” She and those moments we shared will live
in my heart forever. As Helen Keller articulated:
"What we have once enjoyed we can never lose.
All that we love deeply becomes a part of us.”
scientist in her lab at UConn for eight months. I
was interested in doing a sabbatical in an area I
knew nothing about, and Mary kindly and
generously accepted me into her lab. I fully
expected that Mary would be very busy with
both her research and her administrative
responsibilities, and so was much surprised when
I arrived at her office the first day and she
personally escorted me to all the places I needed
to go to for my ID card, parking permit, library
privileges, etc. This generosity of spirit (and
time) set the tone for my working with Mary.
Mary was gentle, kind, respectful of others, and
ferociously passionate about science. We spent
several weeks working long hours together at the
NASA Ames Research Center and it was there I
really discovered that, along with all the other
wonderful attributes Mary exhibited, she had a
wonderfully low key sense of humor. I enjoyed
those trips immensely and will always be
grateful to Mary for allowing me to share her
world.
References and Links
Musgrave, M.E. 2008. Reflections on Relevance, ASPB
news, Women in Plant Biology Vol. 35 (5).
http://www.aspb.org/newsletter/septoct08/12wipb20
.cfm
Mary E. Blasiak. The Recorder. http://www.recorder.com/
article/mary-e-blasiak-0
Xerces Society http://www.xerces.org/give/
Rebecca Darnell. I didn’t know Mary through
the ASGSB Society, but rather, I was a visiting
5
Review
Autonomous Gravity Perception and Responses of Single Plant Cells
Mari L. Salmi1, Thomas J. Bushart2, and Stanley J. Roux2
1
Morehouse School of Medicine, 720 Westview Dr SW, Atlanta GA, 30301; 2The University of Texas at
Austin, Section of Molecular Cell and Developmental Biology, Austin, Texas 78712-0183
ABSTRACT
Although multicellular plants are favored
experimental subjects for most studies of gravitational
responses in plants, there are single cells that can both
sense and respond to gravity, independent of any
input from other cells. This review focuses on what is
known about these single-cell phenomena.
Highlighted are studies in algae and moss that relate
to the statolith hypothesis, studies in Euglena on
gravitaxis, and studies in fern spores on gravityinduced calcium signaling and gene expression
changes.
INTRODUCTION
Multicellular plants are favored experimental
subjects for most studies of gravitational responses in
plants, and the most studied of these responses is
gravitropism (Morita, 2010). In multicellular plant
gravitropisms, such as the downward curvature of
roots and upward curvature of shoots, diverse tissue
interactions are involved. Specifically in roots,
chemical communication between the cap and the
elongation zone, including the transport of the growth
hormone auxin, appears to be crucial for the gravity
signal to be transduced into downward growth
(Harrison et al., 2007). Of course, in all these
instances the initial response to gravity must occur in
individual cells, even though, as in the case of rootcap cells, the gravisensing cells themselves may not
show a growth change. However, there are single
cells that can both sense and respond to gravity,
independent of any input from other cells. This
review will focus on what is known about these
single-cell phenomena.
Correspondence to: Mari L. Salmi
Morehouse School of Medicine
720 WestView Dr. SW
Atlanta, GA 30301
Phone: 404-752-1727
Fax: 404-752-1064
Email: [email protected]
The simplicity of single-cell systems is
advantageous for studies of the early signaling steps
that link the gravity stimulus to biochemical or
growth changes. Do the results of these studies have
relevance or value for similar studies in multicellular
systems?
Gravity-induced membrane potential
changes observed in unattached single cells are also
observed in cells of multicellular plants (Weisenseel
and Meyer, 1997). More specifically, gravity-induced
rapid changes in calcium fluxes observed in single
cells (Chatterjee et al., 2000; Salmi et al., 2011) are
also observed as early steps in individual cells of
multicellular systems (Fasano et al., 2002; Plieth and
Trewavas, 2002; Toyota et al., 2008). Even at the
level of gene-expression changes, there are significant
similarities in the effects of gravity on single-cell and
multicellular systems (Salmi et al., 2005). Although
certainly there are major differences in how
gravitational stimuli are transduced in independent,
single cells compared to multicellular tissues, there
appears to be a fundamental conservation of
mechanisms in gravity responses that is maintained in
all responsive plant cells. This argues for the
significant value of single-cell systems as models for
understanding basic processes of gravity sensing and
responses.
Statolith Model of Gravity Sensing: Single-cell
Results
While perhaps not applicable to all plant species,
the statolith model of gravity sensing in multicellular
plants is well established. The model is based on the
observation that certain organelles move downward
(sediment) when the position of the cell is changed. In
Arabidopsis columella cells the statoliths are dense
starch-containing
amyloplasts.
Reduced-starch
mutants have both decreased statolith sedimentation
ability (MacCleery and Kiss, 1999) and reduced
gravitropic responses (Kiss et al., 1996). These and
subsequent studies favor the hypothesis that
sedimenting organelles help initiate gravity sensing in
multicellular tissues (Strohm et al., in press).
Evidence that sedimenting organelles initiate gravity
responses in single cells has also been published in
Salmi et al. -- Single Plant Cells and Gravity
multiple reports, most of which used cells of moss
and algae as experimental subjects.
In gametophytes of the moss Ceratodon
purpureus, amyloplast sedimentation results in
negative gravitropic curvature of the protonemata,
which act autonomously in their response to gravity.
Conveniently, there is a wrong way response (wwr)
mutant which exhibits positive gravitropic growth.
The kinetics and directions of growth of these wwr
mutant plants is exactly opposite that of the wild type.
The mutated gene product in wwr plants seems to
function downstream of amyloplast sedimentation
since the position of sedimenting amyloplasts is
unaltered in the mutant plants after gravity
reorientation (Wagner et al., 1997). Kuznetsov et al.,
(1999) utilized strong magnetic fields to manipulate
amyloplasts independent of the gravity vector to
cement the connection between statolith movement
and gravity response (Figure 1, A-D). In two
instances the protonemata were rotated in a clinostat
while exposed to a high-gradient magnetic field
(HGMF) while in the third case they were not rotated.
The clinostat served to nullify the sedimenting effects
of gravity, and indeed the untreated protonemata
showed neither sedimentation nor curvature (Figure
1D). However, the amyloplasts under clinorotation
would sediment with an applied magnetic vector. The
wild-type protonemata, as predicted, would grow
away from the HGMF vector forces (opposite the
direction of HGMF-induced sedimentation) as seen in
Figure 1A. This was true regardless of the shape of
the applied magnetic field. The wwr mutants showed
the expected inverted growth pattern in relation to the
HGMF displacement of their amyloplasts (Figure 1B).
The inclusion of wwr mutant plants in this study
demonstrates the importance of amyloplast
sedimentation location in this system, regardless of
differences in the signaling process downstream of
that sedimentation. The researchers also noted a
positive correlation between both field intensity and
amyloplast size and the degree of the curvature
response (Kuznetsov et al., 1999). They also noted
that the responses support the statolith hypothesis
over the pressure model. While these data directly tie
the location of sedimenting statoliths to the detection
of the vector of gravity, they do little to define the
nature of this receptor mechanism.
Still, this
dovetails neatly with their previous work in
Arabidopsis roots showing that magnetic field
alterations to amyloplast sedimentation alter
gravitropic curvature of the roots in a similar fashion
(Kuznetsov and Hasenstein, 1996).
Magnetic fields are not the only way of
experimentally manipulating statolith position in
single cells. Chara algae contain large (diameter up to
30 µm), tube-like rhizoid cells that exhibit positive
gravitropism (grow downward), and protonemata that
exhibit negative gravitropism (grow upward), offering
an elegant system for examining both gravitropic
responses in single cells within the same species.
Both of these gravity-sensing cell types contain
structures that have been reported to serve the role of
statoliths: i.e., vesicles filled with barium sulfate
crystals (BaSO4) that sediment in the direction of the
gravity vector. The rhizoids and protonemata of
characean algae have been manipulated using “optical
tweezer” laser trapping. Laser-assisted displacement
of 2-3 statoliths against the side of a horizontally
oriented rhizoid for at least 5 minutes was sufficient
to cause curvature towards that side (Figure 1, A’-D’).
Similarly, the same conditions in a protonema lead to
negative-gravitropic-like curvature away from the site
of the repositioned statoliths (Braun, 2002). The
manipulation of statoliths into various regions of
Chara cells was an especially valuable approach due
to two key observations. First, Chara cells appear to
have a limited, belt-like region where gravity
perception takes place (Braun, 2002) in both
protonemata and rhizoids. Other gravi-responsive
plant cell types are able to detect a change in
orientation in any direction. Second, the actual
movement of the statoliths does not appear to be the
key to responsiveness (Braun, 2002). Rather, a cell
requires contact, or pressure, from the statolith at the
regions of sensitivity.
Findings using the Chara model system to study
gravity perception in plant cells were recently
reviewed by Braun and Limbach (2006). As they
reiterate, it is clear that the position of the statolith
near the plasma membrane within the region of graviperception of the rhizoid or protonema is integral to
the cell’s gravity response and to its change in
direction of growth.
However, this positional
requirement could be acting through simple contact or
by secondary conditions of the contact (e.g. applied
pressure). While the contact model has strong
evidence, the pressure model cannot be discounted by
the data thus far.
In the first mechanism, the “gravity receptor” of
Chara cells could act as a classical hormone, lockand-key type receptor that specifically detects the
location of these statoliths and either directly or
indirectly opens ion channels. This model has some
of the strongest support in Chara rhizoids. Limbach
and colleagues observed that individual statoliths lose
contact with the cell cortex in as little as 2 s after
inverting C. globularis rhizoids.
Inverting for
repeated intervals as short as 10 s could impact
gravitropic growth (Limbach et al., 2005). Further,
the quantitative decrease in a rhizoid’s angle of
7
curvature appears to directly relate to the total time of
inversion, and logically, therefore, to the total time
interval statoliths were not in contact with the cell
cortex. Perhaps the most compelling data supporting
the contact model was that a decrease in gravitational
forces achieved during parabolic flight experiments
did not alter rhizoid curvature in any statistically
significant manner (Limbach et al., 2005). The
rhizoids were pre-stimulated by being rotated
horizontally for 10 minutes before the first parabola,
meaning that the statoliths would already have
sedimented and achieved cortex contact. Further, 2 g
conditions achieved through centrifugation did not
affect rhizoid curvature in any detectable manner
(Limbach et al., 2005). Therefore it appears that
graviperception in Chara rhizoids depends almost
entirely on physical contact between organelles.
Figure 1. Single-cell responses of single cells to various gravity-related manipulations.
A-D: Ceratodon
purpureus cells were exposed to various magnetic fields (directional force indicated by Fm) while rotated in a
clinostat to reduce the effects of gravity. Arrow heads indicate amyloplasts displaced by the High Gradient Magnetic
Field (HGMF). A. WT protonema after 12 h exposure to HGMF. B. wwr protonema after 6h exposure to HGMF. C.
wwr protonema exposed to uniform magnetic field (non-HGMF). D. WT protonema away from HGMF. Bars = 50
µm. (Adapted from Kuznetsov et al., 1999, Copyright American Society of Plant Biologists,
http://www.plantphysiol.org)
A’-D’: Growth changes over time of a single Chara globularis rhizoid manipulated
by laser-assisted (circle) displacement (arrows) of statoliths (st). Bars = 10 µm. (Adapted with kind permission from
Springer Science+Business Media: Protoplasma, 219, 2002, 150-159, Braun, M., figure 3, copyright Springer-Verlag
2002).
Circular histograms: Histogram of directional swimming of Euglena gracilis as determined by motion
analysis software. Upper left. Control cells. Lower right. RNAi against CaM.2, 15 days post-electroporation. Solid
black arrow (theta) represents main movement direction of cells in the culture. r value represents precision of
movement (0 = random, 1 = precise). (Adapted with kind permission from Springer Science+Business Media: Planta,
231, 2010, 1229-1236; Daiker, V. et al., figure 5, copyright Springer-Verlag 2010.)
Line graph: Representative
plot of calcium flux changes of a single Ceratopteris richardii spore in relation to changing gravity intensities from a
parabolic flight. Vertical grey bars represent time of transition between hyper- and micro-g conditions. Arrows
indicate time of change in calcium current (adapted with kind permission from Springer Science+Business Media:
Planta, 233, 2011, 911-920; Salmi, M. et al., figure 4, copyright Springer-Verlag 2011).
These results may be related to ultrastructural
observations in multicellular, statolith-dependent
gravitropic responses such as in Zea mays roots.
There, ER is largely absent in the central regions of
the cells but enriched at the cell cortex (Yoder et al.,
2001). A sedimenting statolith, then, would pass
through ER-devoid regions to settle at ER-rich points
at the cell membrane. It is likely not a coincidence
that the ER is a main storage organelle for Ca2+, an
ion implicated in gravity perception and responses
across multiple species. In animal systems, membrane
contacts between the ER and PM are important for the
phenomenon of store-operated calcium entry
mediated by certain calcium-release activated calcium
(CRAC) channels. To our knowledge, homologs of
the specific animal proteins involved in this
phenomenon (Orai1 and STIM1) have not been
identified in plant systems, but calcium-induced
calcium release has been documented with slow
vacuolar (SV) channels participating in calciuminduced calcium release (Bewell et al., 1999; Pottosin
et al., 2004; Scholz-Starke et al., 2006). It is therefore
possible that an analogous system involving contact
between transmembrane proteins could be at work in
gravity perception involving statoliths.
While the work of Limbach and colleagues
strongly supports the contact model in C. globularis
rhizoids, it does not completely rule out that statolith
deformation of the plasma membrane (or ER) could
result in the opening of a pressure-sensitive calcium
channel that is integral to the membrane. Considering
that the internodal cells of Chara lack sedimenting
organelles yet respond to position and differential
pressures, this second mode of gravity sensing is
likely to be active in this, and other, systems.
The research of Wayne, Staves, Leopold and
colleagues on Nitellopsis and on Chara intermodal
cells led to the hypothesis that the gravity sensor in
these cells was the mass of the protoplasm rather than
the movement of any specific subcellular organelle,
such as a statolith (Staves, 1997; Wayne et al., 1992).
The idea that pressure exerted by the entire cell is
what accounts for gravity perception was tested in
rice roots, and, consistent with the Chara results, high
density media which would diminish the force
generated by a falling protoplast but not prevent
amyloplasts from falling, significantly decreased the
rate of gravitropic curvature (Staves et al., 1997).
Thus, results from the Wayne laboratory both in
single cells and in multicellular tissues were
consistent with the authors’ gravitational pressure
theory of gravity sensing.
Schwuchow et al. (2002) tested this hypothesis
in single-cell protonemata of the moss C. purpureus,
which bend upward when reoriented horizontally.
Their results indicated that plastid-based sensing of
gravity, not pressure exerted by the entire cell
functioned to initiate gravitropism in these cells, a
conclusion also arrived at in the HGMF study by
Kuznetsov et al. (1999), noted earlier. Certainly the
statolith/plastid model of gravity sensing is the more
accepted one today (Blancaflor and Masson, 2003;
Strohm et al., 2011); but, as noted previously, certain
specific systems depend largely or singularly on
organelle contact rather than pressure. Further, the
participation of the whole protoplasm as a sensor,
integrated by the cytoskeleton to function as a single
mass, cannot be ruled out, particularly in systems
lacking sedimenting organelles or particles.
Single spore cells of the fern Ceratopteris
richardii offer another system for examining the
statolith hypothesis.
Here gravity directs the
downward migration of the nucleus, which occurs
about 24 h after the spore is induced to germinate by
light. That the downward migration of the nucleus is,
indeed, directed by gravity was demonstrated in
Shuttle mission STS93, during which video
microscopy revealed that the migration still occurred
in microgravity, but its direction was random instead
of being polarized toward one end of the cell (Roux et
al., 2003). The nuclear migration toward the bottom
of the cell at 1 g both localizes the site of the first cell
division and the subsequent downward emergence of
the rhizoid. These cytological effects of gravity on
cell polarity are already set about 15 h before the
nuclear migration begins, so the downward movement
of the nucleus is part of the response to gravity rather
than being part of the perception event (Edwards and
Roux, 1994; Edwards and Roux, 1998). Most of the
plastids in the spore are closely appressed around the
periphery of the nucleus, and separate movements of
these plastids prior to when the polarizing effects of
gravity are set were not observed. Thus a clear
statolith candidate has not yet been identified for the
fern spore response to gravity.
Single-cell Taxic Response to Gravity
Unlike the other plant systems discussed here,
the unicellular ciliate alga Euglena gracilis has a
slightly different relationship to gravity. Since they
are both aquatic and motile they exhibit a number of
taxic responses in order to optimize their ability to
carry out photosynthesis in a body of water. Light is
scattered by water so light intensities inversely relate
to depth. For proper photosynthesis E. gracilis needs
to remain near the surface of a water column. The
most obvious response, therefore, would be to light,
and they do exhibit strong, intensity-dependent
phototaxis. However, as light is not continuous
throughout a 24 hr cycle, gravity is also an important
Gravitational and Space Biology
Volume 25 (1) Sept 2011
9
stimulus for E. gracilis to remain near the surface. To
that end, E. gracilis exhibit a negative gravitaxic
response.
Spaceflight experiments have demonstrated that
the direction of swimming becomes random when
gravity is less than 0.16 of normal (Häder and
Hemmersbach, 1997). It is also thought that gravity is
mainly sensed through differential pressures (dictated
by the gravity vector) on cells. This idea is supported
in E. gracilis by experiments manipulating media
density. Directional movement was impaired without
affecting taxis rate by increasing the surrounding
densities. Even higher densities reversed the direction
of normal taxis, effectively resulting in a situation
where the algae appear to be positively gravitaxic
(Häder and Hemmersbach, 1997). These outcomes
parallel those we see in other plant systems. So while
these protozoan-like algae differ in response
(movement instead of asymmetric growth), they
appear to share a similar mechanism for detection of
the gravity vector.
More recent work with E. gracilis has expanded
the molecular understanding of the gravitaxic sensing
and response system(s). Like other systems, calcium
is thought to be a main component of the relay
between perception (orientation to gravity) and
response (flagellar beating). Specifically, the
gravitaxis of the ciliates Stylonychia mytilus (Krause
et al., 2010) and Euglena gracilis clearly involves
calcium signaling (Streb et al., 2001). To investigate
whether the calcium-binding protein, calmodulin, was
involved in the response, Daiker et al. (2010)
sequenced five distinct cDNAs of calmodulin family
proteins, designated CaM.1 through CaM.5. The
sequences were unique enough that specific dsRNAs
could be developed against each to knock them
out/down to examine the effects. RNAi against
CaM.3, CaM.4, and CaM.5 had no significant impact
on gravitaxis. RNAi against CaM.1 was shown to
have alterations in morphology and transient
alterations in gravitaxis. Early after treatment the
cells exhibited random, nonprogressive (euglenoid)
movement, but by 15 days post-electroporation
directed, negative gravitaxis was restored. It was
noted that the change in morphology did not include
noticeable alterations to the flagellum itself.
Suppression of CaM.2 through RNAi similarly had
alterations to morphology and induction of euglenoid
movement. In this case, free swimming behavior was
recovered within several days to a week. Also unlike
the suppression of CaM.1, RNAi against CaM.2 did
not show a return to normal gravitaxis for extended
periods post-electroporation (Figure 1, circular
histograms). The RNAi affects appear to be
maintained by these cells, as clonal E. gracilis out to
30 days post-electroporation still showed free
swimming, but in a generally randomized vector in
contrast to the strongly negative gravitaxis of the
control cells.
Taken in total, it appears that not only does E.
gracilis contain calmodulins with distinct functions,
but at least one of them, CaM.2, acts as a main
component in the gravitaxis response. The current
model positions CaM.2 as an intermediary between
gravity-induced [Ca2+] changes and alterations of
flagellar beating via the cAMP output of a
calmodulin-dependent adenyl cyclase. So while E.
gracilis exhibits an overall different end response to
the gravity vector when compared with other nonciliate plants, it utilizes similar transduction and/or
amplification steps involving calcium.
Modulating Responses in Single Cells by Changing
the g-Force
As described above, after light induces singlecell spores of Ceratopteris richardii to germinate,
gravity directs the polarization of these cells,
including the downward migration of the nucleus.
This migration occurs some 15 h after gravity has set
the polarity of the cell, so there must be some gravityinduced event that occurs earlier. Because trans-cell
calcium currents, driven by influx channels on one
end and pumps at the other end, were known to
induce cell polarization in tip-growing systems such
as pollen tubes (Weisenseel et al., 1975), Chatterjee et
al. (2000) used a self-referencing microelectrode to
test whether such currents occurred in C. richardii
spores, and, if so, whether gravity could influence the
direction of these currents. They found that within
two hours after the spores were irradiated, a calcium
influx occurred at the spore bottom, and a 20-fold
larger efflux emerged from the spore top. When the
spore was rotated, the bottom-to-top calcium current
rapidly realigned parallel to the vector of gravity.
This initial study could not resolve how quickly the
rotation-induced realignment of the current occurred,
but recently Salmi et al. (2011) answered this question
using a silicon microfabricated sensor array that could
simultaneously measure calcium currents in real time
from multiple cells arranged on a microchip. Their
assay showed that on earth rotating the spores on the
chip reverses the trans-cell calcium current within 25
sec.
Another key finding of Salmi et al. (2011) was
that a change in g-force could modulate the
magnitude of the current (Figure 1, line graph). When
the spores were assayed during parabolic flight on the
NASA C-9 aircraft, the trans-cell current increased in
hyper-g and decreased to near baseline in micro-g.
The current began to rise within 2 seconds as the g-
force increased during the aircraft’s transition out of
the micro-g segment of its flight. Very likely the
gravity-directed trans-cell calcium current, which
peaks about 9-10 h after light-induced germination
(Chatterjee et al., 2000; Salmi et al., 2011) is needed
for cell polarization, because at 1-g. the calciumchannel blocker nifedipine blocks the current and
randomizes the direction of rhizoid emergence
(Chatterjee et al., 2000, Salmi et al., 2011). In the
moss Funaria, the direction of nuclear migration is
also toward the site of calcium entry, and blocking
calcium entry also blocks this migration (Saunders
and Hepler, 1983). In contrast to the nifedipine
results, the calcium-pump inhibitor eosin yellow
suppressed the current, but not gravity-induced cell
polarization. Taken together, the results of this study
were consistent with the conclusion that gravityinduced calcium entry through channels at the bottom
of the spores is one of the earliest responses of C.
richardii spores to gravity, and that gravity can
rapidly (probably post-translationally) modulate the
activity of channels and pumps that drive a trans-cell
current in these cells.
Other studies in single-cell model systems also
show that gravity perception occurs in response to
gravity forces less than 1 g. Data from TEXUS
sounding rocket missions indicate that E. gracilis
responds to as low as 0.12 g (Häder et al., 1998) and
MAXUS-5 sounding rocket missions have provided
evidence that Chara rhizoids perceive 0.14 g
(Limbach et al., 2005). Although the precise gravity
force necessary to activate gravity receptors has not
been studied in the spores of C. richardii, an obvious
hysteresis of de-activation was observed on NASA
parabolic flight missions that may be explained by
cellular perception of sub-earth gravity (in the realm
of 0. 1 g) (Salmi et al., 2011). The evolutionarily
conserved role of calcium, as well as the kinetics of
gravity signaling in many cell types, suggests that a
likely candidate for the gravity receptor in plant cells
is a stretch activated calcium channel.
Gene-expression Changes That Occur in Single
Cells in Microgravity
Recently several genes have been added to the
list of those involved in various steps of the higher
plant gravitropism signaling cascade (Agee et al.,
2010; Fortunati et al., 2008; Nakamura et al., 2011;
Yang et al., 2011). Data from microarray analysis of
gene expression changes induced by changes in
orientation (Kimbrough et al., 2004; Moseyko et al.,
2002) or, perhaps even more significantly,
development in microgravity (Paul and Ferl, 2002;
Salmi and Roux, 2008) provide a very useful starting
point for a more targeted forward genetics approaches
to identify genetic components of gravity perception
signaling. Signaling components that have been
implicated in gravity perception and response in
several studies, in different plants, or even other
organisms are an obvious target for this type of
evaluation. For instance, those genes identified in the
study of root Arabidopsis gravitropism (Kimbrough et
al., 2004) that have likely homologs identified the
spore microgravity study (Salmi et al., 2008), such as
Catalase 3, Fibrillarin 2, and Protodermal factor 1,
could be evaluated in the commercially available
Arabidopsis insertional mutant lines.
A
comprehensive evaluation of knockout lines of these
genes might result in the identification of new and
valuable mutants in gravity perception and/or
gravitropism.
It is important to integrate these high throughput
data with previously identified aspects of gravity
perception and gravity directed polarity development,
like the well established involvement of calcium as a
component of the gravity response mechanism. In C.
richardii spores, numerous genes that likely encode
proteins involved in calcium mediated signal
transduction have been identified. A study of gene
expression changes during the germination and early
development of spores (Salmi et al., 2005) found
several genes which likely encode calcium binding
proteins that are up-regulated during early
development. None of these genes have changes
between microgravity and 1g development in any of
the developmental time points analyzed (Salmi et al.,
2008). Of course, the most important molecular
changes induced by gravity to alter the polarity of
growth could all be post-transcriptional, or even posttranslational. There is one gene however, likely to
encode a protein with a C2 domain, that is down
regulated during normal spore early development, and
is also up regulated in spaceflight samples at the time
point coincident with its highest abundance during
normal development. The C2 domain is a calcium
dependent, membrane binding feature of many
proteins involved in membrane trafficking. Proteins
with the C2 domain have recently been implicated in
the viability and elongation of tip growing pollen
tubes of angiosperms and seed germination (Lee et
al., 2009; Li et al., 2007; Yang et al., 2008), two plant
systems highly regulated by gravity. The expression
profile of this particular gene in C. richardii spores
indicates that it is more abundant early on in
germination, when the gravity-directed calcium
current of the spore is likely creating localized regions
of high cytosolic calcium (Chatterjee et al., 2000;
Salmi et al., 2011). As yet there has been no test to
determine whether this gene encodes a protein
essential for converting an elevated localized calcium
concentration into downstream cellular events to
11
establish polarity. In general all the data evaluating
gene expression changes should be translated into
systems where targeted genetic manipulations are
possible in order to directly evaluate the role of
candidate genes in the cellular perception and earliest
responses to gravity.
CONCLUSION
As indicated throughout this review there are
significant similarities between the gravity responses
found in single cells and those in other plant cells that
perceive gravity (i.e. angiosperm root columella
“statocytes”). Of course, there are other similarities
besides those highlighted in this review.
For
example, cytoskeletal elements significantly influence
gravity responses in single cells (e.g., in the stability
and movement of Chara statolith vesicles) (Braun,
2002; Limbach et al., 2005), and in multicellular
tissues (Blancaflor and Masson,2003). To the extent
that the cellular responses to gravity found in single
plant cells are fundamental to cellular gravity sensing
and responding, one could expect to find at least some
of them, such as the activation of mechanosensitive
channels and rapid calcium changes, also in animal
cells. Future studies will clarify the extent to which
the response mechanisms of cells to gravity are
evolutionarily conserved not only in single-cell and
multicellular plants, but also in animals.
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13
Review
Space Radiation and Bone Loss
Jeffrey S. Willey1, Shane A.J. Lloyd2, Gregory A. Nelson3, and Ted A. Bateman4
1
Department of Radiation Oncology, Comprehensive Cancer Center, and Section of Molecular Medicine, Wake
Forest School of Medicine, Radiation Biology 405 NRC, Medical Center Blvd., Winston-Salem, NC 27157;
2
Department of Orthopaedics and Rehabilitation, Division of Musculoskeletal Sciences, The Pennsylvania State
3
Department of Radiation
University College of Medicine, 500 University Drive, Hershey, PA 17033;
Medicine, Loma Linda University, 11175 Campus St., CSP A1010, Loma Linda, CA 92354; 4Departments of
Biomedical Engineering and Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC
27599
Keywords: osteoporosis, fracture, ionizing radiation, radiation therapy, spaceflight, microgravity, space radiation,
osteoclasts, bone, inflammation
ABSTRACT
Exposure to ionizing radiation may negatively
impact skeletal integrity during extended spaceflight
missions to the moon, Mars, or near-Earth asteroids.
However, our understanding of the effects of radiation
on bone is limited when compared to the effects of
weightlessness. In addition to microgravity, astronauts
will be exposed to space radiation from solar and
cosmic sources. Historically, radiation exposure has
been shown to damage both osteoblast precursors and
local vasculature within the irradiated volume. The
resulting suppression of bone formation and a general
state of low bone-turnover is thought to be the
primary contributor to bone loss and eventual
fracture. Recent investigations using mouse models
have identified a rapid, but transient, increase in
osteoclast activity immediately after irradiation with
both spaceflight and clinically-relevant radiation
qualities and doses. Together with a chronic
suppression of bone formation after radiation
exposure, this acute skeletal damage may contribute
to long-term deterioration of bone quality, potentially
increasing fracture risk. Direct evidence for the
damaging effects of radiation on human bone are
primarily demonstrated by the increased incidence of
fractures at sites that absorb high doses of radiation
during cancer therapy: exposures are considerably
higher than what could be expected during
Correspondence to: Ted A. Bateman
152 MacNider Hall
CB 7575
Chapel Hill, NC 27599-7575
Phone: 919-9660- 1174
Fax: 919-966-2963
spaceflight. However, both the rapidity of bone
damage and the chronic nature of the changes appear
similar between exposure scenarios. This review will
outline our current knowledge of space and clinical
exploration exposure to ionizing radiation on skeletal
health.
INTRODUCTION
The effects of microgravity and space radiation
on astronaut bone health represent two of the most
serious challenges present within the spaceflight
environment. Loss of bone strength resulting from a
reduction in bone mass or architectural stability can
increase the risk of a serious fracture, threatening
mission success. Countermeasures capable of both
limiting the loss of bone and permitting the eventual
recovery of bone strength post-flight are necessary to
ensure astronauts’ long-term skeletal health and
productivity of extended space missions.
Bone damage is a known chronic (late) effect of
exposure to therapeutic radiation. While animal
models have long documented a persistent decline in
bone volume after exposure, recent animal studies
have demonstrated an early and transient increase in
bone resorption followed by suppressed bone
formation following exposure to spaceflight-relevant
doses and qualities of radiation (Kondo et al., 2009;
Willey et al., 2010). This initial bone loss can be
prevented using the osteoporosis therapeutic
risedronate (Willey et al., 2010), further
demonstrating the role of osteoclastic bone resorption.
Skeletal unloading, due to extended periods of
bed rest (Spector et al., 2009) or the microgravity
environment of space (Lang et al., 2004), is a wellestablished cause of bone loss in both humans and
rodent models (Bikle and Halloran, 1999). The bone
Willey et al. -- Radiation and Bone Loss
loss that occurs as a result of unloading is
characterized by both an increase in bone resorption
and a decrease in bone formation. This mechanism
stands in contrast to traditional forms of bone loss,
such as post-menopausal osteoporosis, during which
bone resorption and formation move in parallel, albeit
with the former increasing to a relatively greater
degree. The combination of microgravity unloading
and spaceflight radiation may interact to enhance
bone loss (Alwood et al., 2010; Hamilton et al., 2006;
Kondo et al., 2010). As the mechanisms leading to
radiation-induced bone loss are less well established
than unloading-induced bone loss, this review will
focus on the current state of knowledge regarding the
influence of spaceflight and clinical radiation on the
development of osteoporosis and bone health in the
adult skeleton.
Radiation and Biological Damage
Energy absorbed from radiation sources, such as
from electromagnetic or charged particles, has the
ability to excite atoms to the point of generating an
ion. Non-ionizing radiation will not eject electrons
from an atom, while ionizing radiation can
sufficiently excite electrons enough to eject the
electron, creating an ionization event. The generation
of these charged particles can potentially break
molecular bonds and lead to biological damage, such
as DNA strand breaks or membrane damage. Bonds
can be broken directly by radiation, or indirectly
through radiation-generated reactive oxygen species
that result from the ionization of water molecules.
Cells have a remarkable ability to repair radiation
damage, but some cells inevitably die or propagate
damage to progeny. Humans on Earth are exposed to
natural, background sources of radiation (e.g.,
environment, space, medical imaging, internal
radioisotopes) at doses that do not affect long-term
health at a measurable level. However, as exposures
increase, damage can lead to greater levels of cell
death or radiation-induced phenotypic changes that
may be heritable.
Absorbed dose of radiation (D) is measured in
energy per unit mass with the SI unit being Gray (Gy
= Joule/kg). It is also common to see dose expressed
in the unit of rad, where 100 rad = 1 Gy (1 rad = 1
cGy). The degree of biological damage can vary
depending on the quality of radiation exposure, which
itself is influenced by factors such as linear energy
transfer (LET); type of radiation, such as alpha
particles, beta particles, gamma rays (X-rays),
neutrons, or heavy ions; and energy. For example,
exposure to 10 cGy of neutrons will be more
damaging than exposure to 10 cGy of X-rays. To
account for these varying degrees of damage to
humans, Sievert (Sv) is the SI unit for absorbed dose
equivalent (H), which is the quality factor (Q)
(relative biological effectiveness: RBE) times the
dose (H = D*Q). Generally, 1 Gy of X-rays is equal to
1 Sv (100 rad = 100 rem) (Hall, 2000).
Spaceflight and Clinical Radiation Environments
The space radiation environment consists of a
complex mix of ions from solar particle events
(SPEs), which are large mass ejections from the sun
commonly known as “solar flares,” and galactic
cosmic radiation (GCR), a type of background
radiation that originates from outside the solar system.
The majority of GCR flux is from protons. While only
1% of GCR is composed of ions heavier than helium,
due to the high LET of these high charge (Z) and
energy (E) particles (HZE), approximately 41% of the
dose equivalent is predicted to be from HZE particles
with approximately 13% being from iron alone
(Bandstra et al., 2009). LET is the rate of energy lost
per length of a particle track and varies as the charge
squared divided by the velocity squared. During
extended missions in space, estimated tissue doserates from GCR would be about 0.4-0.8 mGy/day and
1-2.5 mSv/day, respectively (NCRP, 2000). SPE
dose-rates may reach as high as 50 mGy/hr inside a
shielded vehicle and between 250 mGy/hr for an
astronaut exposed during extra-vehicular activity in
deep space. The cumulative GCR doses on a deep
space mission of 400 days to a near-Earth asteroid
would be 0.16 to 0.32 Gy or 0.4 to 1.0 Sv. For a large
SPE lasting 8-to-24 hours, the whole body cumulative
doses could reach the 1- to 2-Gy level for protons (1.0
to 2.0 Sv) depending upon the tissue site. For
comparison, cancer patients will receive daily doses
(fractions) of 1.8 to 2.0 Gy targeted locally to the
tumor and delivered over a period of a few minutes,
with the total dose to the tumor ranging from 50 to 80
Gy or more.
Radiation is also absorbed by normal tissues
surrounding a tumor during the course of radiation
therapy (RT) for the treatment of cancer. The
absorbed dose can be substantial. For example,
treatment regimens for gynecological or pelvic (e.g.,
anal) tumors commonly prescribe the administration
of thirty 1.8 Gy fractions over six weeks, for a total
dose of approximately 54 Gy to the tumor.
Throughout the course of treatment, normal skeletal
tissue constituting each “hip” (e.g., femoral neck,
sacrum, acetabular rim) can receive as much as half of
each fraction (0.9 Gy), totaling 27 Gy. While the
primary concern in radiotherapy has always been
curative treatment of the tumor, the incidental
irradiation of normal tissues is not insignificant.
Primary consideration has always been given to
radiation effects on normal nervous tissue,
reproductive structures, and hollow organs (i.e.,
15
esophagus, bowel), while concern for irradiation of
bone was not a priority. Advances in surgical
excision, concurrent chemotherapy, and modern RT
modalities have afforded greater freedom to focus on
minimizing the risk to bone and the potential for
increased fracture risk.
Fractures of Irradiated Bones After Clinical
Exposure
Ionizing radiation is an important and effective
modality for the treatment of malignancies and has
been a critical factor in the reduction of cancer
mortality rates. With an increase in long term
survivorship, however, the incidence of the long-term
side effects caused by radiation damage to normal
tissues near the tumor are of greater concern.
Fractures at irradiated skeletal sites represent one such
late effect. Bones within the irradiated volume
exhibit a substantially increased fracture risk (Baxter
et al., 2005; Brown and Guise, 2009; Florin et al.,
2007; Guise, 2006; Oeffinger et al., 2006). Rib
fractures have been documented in patients receiving
treatment for breast cancer, with fracture rates ranging
from 1.8% (Pierce et al., 1992) to as high as 19%
(Overgaard, 1988). Similarly, patients receiving
radiotherapy for various pelvic malignancies are at
increased risk for hip fracture at the aforementioned
pelvic skeletal locations that absorb dose (Baxter et
al., 2005; Mitchell and Logan, 1998; Williams and
Davies, 2006). A recent retrospective analysis of
more than 6,400 postmenopausal women receiving
RT for cervical, rectal, and anal cancers demonstrated
an increased relative risk for hip (primarily femoral
neck) fracture of 65%, 66%, and 214%, respectively,
compared to women receiving non-RT cancer
treatment such as surgery or chemotherapy (Baxter et
al., 2005). These fractures were localized to the area
that absorbed dose, and did not occur at distant
skeletal sites, such as the wrist. Therefore, while
systemic or non-targeted radiation effects cannot be
discounted, clinically, the response seems limited to
the irradiated volume.
Deterioration of Bone Quantity and Quality After
Irradiation
Deterioration of bone quantity and quality
following direct irradiation is thought to be associated
with traumatic and spontaneous fractures of bone
(Baxter et al., 2005; Ergun and Howland, 1980;
Howland, 1975). Reduction in bone mass and overall
bone quality is dependent on a variety of factors,
including the dose absorbed, the energy of the
radiation beam, the fraction size of the radiation dose,
and the age and developmental stage of the patient
(Mitchell and Logan, 1998; Overgaard, 1988;
Williams and Davies, 2006). For cancer patients
receiving doses considerably higher than spaceflight
exposures, osteopenia (defined as reduced bone
density) is frequently reported in patients one-year
post-therapy, although the observed timing and degree
of bone mass reduction can be variable (Hopewell,
2003; Mitchell and Logan, 1998). Overall,
demineralization of bone, thinning of bones, sclerosis,
and loss of trabecular connections has been
characterized as a consequence of radiotherapy
(Ergun and Howland, 1980; Hopewell, 2003;
Howland, 1975; Mitchell and Logan, 1998).
Thickening of trabeculae can be observed within the
irradiated volume (Williams and Davies, 2006). This
coarsening of trabeculae is also observed from the
marrow cavity of various animal models within weeks
of exposure (Furstman, 1972; Sawajiri and Mizoe,
2003). Quantitatively, an approximately 30%
reduction in bone mineral density from the third
lumbar vertebrae was observed among a group of
patients with uterine cervix carcinoma within five
weeks following irradiation with either 45 Gy or 22.5
Gy total dose of high energy photons (Nishiyama et
al., 1992). No subsequent recovery of bone mineral
density was observed twelve months after RT.
While late observed bone loss has been
documented for decades from irradiated animal
models, recent studies have quantified rapid loss of
bone that occurs following exposure to sub-clinical
doses of both photon and particulate radiation. In
particular, this bone loss can occur after exposure to
doses and qualities of radiation relevant to long
duration missions (Bandstra et al., 2008; Hamilton et
al., 2006) and confirms long-term suppression of bone
formation (Bandstra et al., 2008). A long-term
reduction in trabecular bone quantity and quality
occurs following a whole-body 2 Gy dose gammarays, protons, carbon, or iron ions (Hamilton et al.,
2006). Bone loss persists at four months after
irradiation with doses of protons as low as 1 Gy
(modeling a solar flare), and at nine weeks after
modeled galactic radiation of heavy ions <0.5 Gy
(Bandstra et al., 2008; Bandstra et al., 2009).
Functional bone loss has been identified as early as
three days after a 2 Gy dose of gamma-rays (Kondo et
al., 2009). In addition, an increase in osteoclast
activity when mice are irradiated with a 0.5 Gy dose
of iron ions during limb disuse has been observed,
modeling the reduced loading of the spaceflight
environment (Yumoto K, 2010).
Loss of Bone Strength in Animal Models After
Irradiation
The primary concern regarding radiationinduced loss of bone mineral content or architecture is
the weakening of the whole bone structure, leading to
fractures. Such changes in bone strength following
irradiation would account for the increased fracture
risk among RT patients. Animal models therefore
have been used to identify a reduction in bone
strength after exposure with time and at various
locations within the bone. These studies generally use
clinically-relevant, higher dose exposures, rather than
spaceflight-relevant scenarios. Radiation has been
shown to produce relatively greater damage to spongy
trabecular bone compared to dense cortical bone
(Bandstra et al., 2008), however, heavy ion radiation
at relatively low doses (50 cGy) does cause an
increase in cortical bone porosity, cortical area, and
polar moment of inertia (Bandstra et al., 2009).
Bone quality will be significantly compromised
with damage to both trabecular and cortical bone,
resulting in a cascade of changes throughout the
structure. For example, the loss of trabecular bone
will result in a greater proportion of the loads placed
upon the skeleton to be transferred to cortical bone.
The decline in polar moment of inertia represents a
reduced ability of this cortical bone to resist both
torsional and bending loads. Additionally, any defect
in the structure, such as a porous hole, will cause a
disruption of the load distribution resulting in a stress
concentration that further compromises structural
competency. These changes do not mean a fracture is
imminent, even when combined with microgravityinduced bone loss; however, the probability of
fracture later in life is greater, and depends upon
future regeneration or degeneration and the loads
placed upon the skeleton.
It is also important to comment on the effects
ionizing radiation has on the body mass of irradiated
subjects, as this can affect loading and therefore
skeletal strength. High doses can affect the health and
well-being of mice, causing them to temporarily or
permanently lose body mass and/or be inactive
(Willey et al., 2007). Chronic bone loss has been
reported in rats nine months after exposure to high
dose iron radiation (2 and 4 Gy), and these changes
were attributed primarily to a reduction in body mass
(Willey et al., 2008a). However, spaceflight relevant
doses of ionizing radiation (<2 Gy of X-rays or
protons; <1 Gy of heavy ions) do not change the body
mass, activity level, or nutritional status of mice
(Bandstra et al., 2008; Bandstra et al., 2009; Hamilton
et al., 2006; Lloyd et al., 2008; Willey et al., 2010;
Willey et al., 2008b).
A 2 Gy dose of iron ions causes a loss of
vertebral stiffness as tested by compression loading
and calculated by finite element analysis (FEA)
(Alwood et al., 2010). Compressive testing of mouse
distal femora showed a reduction of strength at twelve
weeks after 5 and 12 Gy acute doses of X-rays
(Wernle et al., 2010). Compressive strength at two
weeks was greater in the irradiated animals, which
corresponded with an acute increase in cortical bone
volume and bone mineral content, despite virtual
elimination of trabecular bone parameters (Wernle et
al., 2010). The subsequent loss of strength as
determined by compressive testing and estimated by
FEA occurred despite a sustained elevation of cortical
bone mineral content. Thus, the bone appeared to be
more brittle. Thus, strength changes in bone after
irradiation may be influenced by both architectural
and material properties.
Radiation and Bone Cells
Vasculature
Bone loss following radiotherapy has
traditionally been thought to be a result of
physiological changes within the vasculature and
bone cells (Bliss et al., 1996; Ergun and Howland,
1980; Gal et al., 2000; Hopewell, 2003; Konski and
Sowers, 1996; Mitchell and Logan, 1998; Rohrer et
al., 1979). The first report of radiation-induced bone
damage (termed “osteitis”) described reductions in
bone vasculature following obliterative endarteritis
(Ewing, 1926). Early loss of vascularization occurs as
a result of swelling and vacuolization of endothelial
cells within the vascular channels of the osteons
(Ergun and Howland, 1980; Hopewell, 2003; Rohrer
et al., 1979). Ultimately, this results in the deposition
of sclerotic connective tissue within the marrow
cavity. Fibrosis of the sub-intima and replacement of
vascular smooth muscle cells with hyaline-like
material occur as late injuries, resulting in constriction
of the vessel lumen. Bony elements such as the skull
and jaw are considered especially at risk for vascular
injury due in part to the inherent paucity of
vasculature and their rather superficial location
(Williams and Davies, 2006). Ablation of vasculature
has been identified within the bone, including marrow
cavity and Haversian systems, in a variety of animal
models following radiation exposure (Cao et al.,
2011; Furstman, 1972; Rohrer et al., 1979).
Osteoblasts and Osteocytes
Damage to osteoblasts and osteocytes is thought
to be a primary contributor to reduced bone mineral
density following irradiation (Ergun and Howland,
1980; Hopewell, 2003; Mitchell and Logan, 1998;
Sams, 1966). Most studies have examined high,
clinically-relevant doses of X-rays, but spaceflightrelevant doses and types of radiation have been shown
to negatively affect osteoblasts as well (Kondo et al.,
2009; Willey et al., 2010; Yumoto K, 2010). A
reduction in the overall number of osteoblasts occurs
following irradiation, along with reduced matrix
formation (Cao et al., 2011; Willey et al., 2010). Both
in vitro and in vivo data suggest that radiation can
impair bone formation by inducing a decrease in
17
osteoblast proliferation and differentiation, inducing
cell-cycle arrest, reducing collagen production, and
increasing sensitivity to apoptotic agents (Dudziak et
al., 2000; Gal et al., 2000; Sakurai et al., 2007;
Szymczyk et al., 2004). Radiation causes a decline in
RUNX2 levels in osteoblast cultures stimulated with
bone morphogenic protein-2 (BMP-2), indicative of
impaired osteoblast differentiation (Sakurai et al.,
2007). Receptor activator of nuclear factor kappa-B
ligand (RANKL) mRNA levels tended to increase in
osteoblasts following exposure to gamma rays, but
not to carbon ions (Sawajiri et al., 2006). Osteoblast
precursors are likely damaged by radiation (Kondo et
al., 2009). Mesenchymal stem cell (MSC) numbers
and colony forming ability under osteogenic
stimulation are also reduced in directly irradiated
bone after exposure, which would likely delay
recovery of osteoblast damage (Cao et al., 2011).
Oxidative stress appears to contribute to this early
damage to osteoprogenitors (Cao et al., 2011; Kondo
et al., 2009). However, others have indicated no loss
of MSC viability within irradiated bone, but rather
suggested a later effect, at the stage of terminal
differentiation into osteoblasts (Schonmeyr et al.,
2008).
The effect of radiation on osteocyte numbers and
health remain unclear. Several studies have identified
loss of osteocytes within irradiated bone following
exposure to high doses (Ergun and Howland, 1980;
Mitchell and Logan, 1998; Rohrer et al., 1979).
Osteocytes were shown to be killed within the
irradiated field inside cortical lamellar and Haversian
bone of monkey mandibles irradiated with 45 Gy,
although their numbers were not affected within
trabecular bone. Overall, however, reports suggest
that osteocytes are relatively radioresistant, remaining
viable for several months after a single high dose of
radiation in mice and rabbits (Jacobsson, 1985;
Rabelo et al., 2010; Sams, 1966; Sugimoto et al.,
1991).
Osteoclasts
Recent studies show that irradiation results in an
early increase in osteoclast number and activity,
which likely contributes to radiation-induced
osteoporosis (Willey et al., 2010). Serum tartrateresistant acid phosphatase (TRAP5b), a marker for
osteoclast activity, is elevated as early as twenty-four
hours after exposure to a whole body dose of X-rays.
An increase in osteoclast surface of >200% and
activity have been identified to occur within rodent
bones three days after exposure (Willey et al., 2008b),
with subsequent loss of bone within a week of
treatment (Kondo et al., 2009; Willey et al., 2010).
Serum chemistry and histological analyses show
significant increases in TRAP5b, as well as osteoblast
number and surface, during the first week of
treatment. However, in these rodent models, the
majority of the bone loss occurs during a time when
osteoblast numbers and bone formation are
unchanged relative to control (Willey et al., 2010).
Suppressing
osteoclast
activity
with
the
bisphosphonate antiresorptive risedronate has been
shown to completely block radiation-induced
increases in osteoclast activity and subsequent
deterioration of bone at multiple skeletal locations
(Willey et al., 2010). Decreased osteoclast activity
has been noted at time points greater than one week
post-exposure, with conflicting reports of recovery
(Margulies et al., 2003; Sawajiri et al., 2003). This
subsequent decline in both osteoclast and osteoblast
activity, if persistent, could sufficiently suppress bone
remodeling and turnover and result in impaired
material properties of bone tissue (Burr et al., 2003),
as described in rodents (Wernle et al., 2010). Taken
together, it can be seen that a combination of an early
and acute loss of bone via increased osteoclast
activity, as well as a persistent reduction in bone
formation, contribute to osteopenia and bone
deterioration following irradiation.
CONCLUSION
Radiation exposure represents a significant
concern for skeletal health. During long duration
spaceflight missions, astronauts could be exposed to
low doses of radiation from cosmic and solar sources.
These exposures could weaken bone and lead to
mission critical fractures, especially combined with
the significant bone loss associated with skeletal
unloading in microgravity. From the oncological
perspective, advances in cancer treatment, with
improvements in radiation, chemotherapy and
surgical modalities, have made concern for incidental
radiation damage to non-tumor tissues a growing
focus. Bone represents one such “normal” tissue
concern. Clinicians are now considering normal
tissue complication probabilities as part of the
treatment planning process. One must note that
radiation exposures outside the treatment volume may
be substantially greater than those predicted for
astronauts, and potentially at-risk volumes of bone
need to be considered as critical structures rather than
as neutral structures. Limited research has defined the
magnitude and mechanisms behind bone lossassociated with radiation exposure. An inflammatory
response does occur in response to radiation, and may
represent a mechanism that induces early activation of
osteoclast-mediated bone resorption.
Ultimately,
bone formation is also suppressed. Bone strength is
thus compromised, which could leave individuals at a
substantial risk for fractures, with accompanying
mortality as a late sequela, particularly in geriatric
patients.
A clear need exists for effective
countermeasures to radiation-induced bone loss, both
for the astronaut population and the ever growing
population of individuals taking advantage of highly
effective cancer radiotherapy. More work is required
to adequately define the molecular and cellular
mediators of this response and to define targets for
novel therapeutics.
ACKNOWLEDGMENTS
This research is supported by the National Space
Biomedical Research Institute through NASA NCC 958 (BL01302 TAB; PF01403 JSW), NASA
Cooperative Agreements NCC9-79 and NCC9-149
(GAN), and the National Institutes of Health (NIAMS
R21AR054889 TAB). Support was also provided by
the National Institutes of Health (T32 CA113267
JSW). We would also like to offer thanks to Procter
and Gamble Pharmaceuticals for providing an
unrestricted grant (TAB JSW) and to Dr. Henry J.
Donahue and the MD/PhD Program at The
Pennsylvania State University College of Medicine
for partial funding support (SAJL).
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21
Research Article
Identification of Proteins Associated with Spatial-Specific PhytochromeMediated Light Signaling in Arabidopsis thaliana by Liquid
Chromatography-Tandem Mass Spectrometry
Sookyung Oh1 and Beronda L. Montgomery1,2
1
2
Department of Energy–Plant Research Laboratory, Michigan State University, East Lansing, MI, 48824-1312;
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319
ABSTRACT
Photosensory phytochromes perceive mainly red
and far-red light and utilize a linear tetrapyrrole
chromophore,
phytochromobilin,
for
their
photoactivity in plants. Although phytochromes have
been extensively studied for light-dependent
regulation of numerous developmental processes, our
understanding of the molecular mechanisms
responsible for distinct organ- and tissue-specific
phytochrome responses is still limited. Recent studies
using transgenic Arabidopsis thaliana plants
expressing a gene that encodes the biliverdin IXα
reductase (BVR) enzyme, which reduces the
biosynthesis and accumulation of phytochromobilin,
and thus inactivates phytochromes, have led to
advances in probing tissue-specific roles of
phytochromes in plant development. We performed
one-dimensional SDS-PAGE, followed by protein
identification and peptide quantification using liquid
chromatography-tandem mass spectrometry (LC/MSMS) to identify proteins that accumulate differentially
in transgenic Arabidopsis lines with mesophyllspecific
phytochrome
deficiencies
(i.e.,
CAB3::pBVR2 plants) compared to wild-type (WT).
We identified the large subunit of Rubisco (RbcL)
and small subunit of Rubisco (RbcS), which
accumulated to lower levels in CAB3::pBVR2
relative to WT under continuous far-red light. We
found that Beta-glucosidase proteins (BGLUs)
Correspondence to: Beronda L. Montgomery
Michigan State University
Department of Energy—Plant Research Lab
106 Plant Biology Building
East Lansing, MI 48824-1312
Phone: 517-353-7802
Fax: 517-353-9168
accumulated highly in the CAB3::pBVR2 line under
these conditions. RT-PCR and microarray analyses
showed a positive correlation between the expression
of the target genes and the accumulation of their
products in BVR lines. We conclude that RbcL,
RbcS, and BGLU18 are targets of mesophyll-specific
phytochrome-mediated light signaling under far-red
conditions.
INTRODUCTION
As sessile organisms, plants have evolved
several photoreceptors to monitor and respond to
diverse light environments. Plants monitor several
different properties of available light, including
intensity, wavelength, duration, and direction.
Phytochromes
are
an
extensively-studied
photoreceptor family, which mainly perceive red (R)
and far-red (FR) light (Schepens et al., 2004; Franklin
and Quail, 2010; Kami et al., 2010). In the model
plant Arabidopsis thaliana, the phytochrome family
consists of five members, PHYA to PHYE (Sharrock
and Quail, 1989; Clack et al., 1994; Quail, 1994).
These proteins are encoded by individual nuclear
genes, but all utilize a single linear tetrapyrrole
chromophore phytochromobilin for the production of
photoactive phytochrome, i.e., holophytochrome
(Terry et al., 1993).
Phytochrome Functions in Plants
Phytochromes are involved in light-dependent
regulation of numerous developmental processes such
as seed germination, hypocotyl elongation responses,
hypocotyl gravitropism, shade avoidance, and
flowering (Franklin and Quail, 2010; Kami et al.,
2010). It has been demonstrated that the subcellular
localization of phytochromes is important for the
associated light-dependent responses (Nagy et al.,
2001; Nagatani, 2004). Phytochromes move from the
cytosol into the nucleus in a light-dependent manner
and in the nucleus they can interact with transcription
Oh and Montgomery -- Arabidopsis Phytochrome Protein
factors, including phytochrome-interacting factors
(PIFs), resulting in the regulation of light-responsive
genes (reviewed by Castillon et al., 2007).
Phytochromes function through PIFs to regulate a
number of light-dependent responses, including seed
germination, chlorophyll accumulation, hypocotyl and
cotyledon development and gravitropic responses
(Shin et al., 2009). Specifically, phytochromedependent regulation of PIFs is involved in the
control of hormone-dependent seed germination (Oh
et al., 2009). Also, it has been demonstrated that
phytochromes regulate hypocotyl gravitropism
through PIF-dependent effects on endodermal
amyloplast development (Kim et al., 2011).
Phytochromes also appear to have roles in the cytosol,
which include blue-light dependent negative
gravitropism and red-light enhanced phototropism
(Rösler et al., 2007).
The diverse roles of
phytochromes in various physiological responses in
plants recently have been reviewed extensively
(Franklin and Quail, 2010; Kami et al., 2010).
Phytochrome Accumulation in Specific Plant
Tissues
In addition to light-dependent differences in
subcellular phytochrome localization, the localization
of phytochromes at the tissue and organ level also has
been studied (for review see Nagatani, 1997). Early
reports established the differential accumulation of
phytochromes in distinct plant tissues, e.g.,
phytochromes accumulate differentially in the
epicotyl hook, cotyledons, and root tips in pea
seedlings (Furuya and Hillman, 1964).
Such
observations have been substantiated further by
phytochrome promoter fusion studies, which indicate
that distinct phytochrome isoforms display discrete
tissue- and organ-specific patterns of expression in a
range of plant species (Somers and Quail 1995a;
1995b; Adam et al., 1996; Goosey et al., 1997; Tόth
et al., 2001), as well as by immunolocalization of
phytochrome holoproteins (Sharrock and Clack,
2002).
These observations began to implicate
distinctive roles of phytochromes in specific tissues
and organs.
Phytochromes Regulate Discrete Responses in
Different Tissues and Organs
The
spatiotemporal
biochemical
and
physiological roles of phytochromes in lightdependent plant responses also have been explored
extensively (for review see Bou-Torrent et al., 2008;
Montgomery, 2008). At the physiological level, a
number of early studies utilized detached or foilcovered plant parts or site-specific microbeam
irradiation to photoactivate phytochromes in discrete
plant tissues and to assay for distinct phytochromedependent responses in distal sites (Piringer and
Heinze, 1954; Klein et al., 1956; De Greef et al.,
1971; De Greef and Caubergs, 1972a; 1972b; De
Greef and Verbelen, 1972; Tepfer and Bonnett, 1972;
Black and Shuttleworth, 1974; Oelze-Karow and
Mohr, 1974; Caubergs and De Greef, 1975; De Greef
et al., 1975; Lecharny, 1979; Powell and Morgan,
1980; Mandoli and Briggs, 1982; Nick et al., 1993).
Such studies, e.g., experiments using foil to cover
specific portions of cucumber seedlings during
irradiation (Black and Shuttleworth, 1974), led to the
observation that activation of phytochromes in
cotyledons could inhibit the elongation of hypocotyls
through interorgan signaling in response to light.
Other classical examples of cell- and tissue-specific,
as well as interorgan, phytochrome-mediated
responses emerged, including the perception of light
by leaf-localized phytochromes that results in the
regulation of the photoperiodic induction of flowering
that produces a transition from vegetative to
reproductive growth at the meristem (King and
Zeevaart, 1973). Although such early physiological
approaches have broadened our knowledge of spatialspecific phytochrome responses, our understanding of
the molecular mechanisms responsible for such
tissue-specific phytochrome responses is still limited.
These limitations persist partially due to a lack of
molecular tools for probing the role(s) of
phytochromes in a tissue-specific manner.
Light Perception by Photoreceptors Regulates
Gene Expression in a Tissue-Specific Manner
Through recent studies, insight into lightdependent regulation of gene expression at the organand tissue-specific level has increased. Tissuespecific expression of photoreceptors identified
distinct tissues in which cryptochromes (Endo et al.,
2007) and phytochromes (Endo et al., 2005) are
required for the regulation of specific responses.
Also, distinct subsets of genes have been shown to be
controlled by light in different tissues, e.g. cotyledon,
hypocotyls and roots in both rice and Arabidopsis
(Jiao et al., 2005; 2007). Thus, it appears that specific
factors are regulated in distinct tissues and organs
downstream of light perception by photoreceptors.
Furthermore, reverse genetic studies of genes
regulated in response to light resulted in the
identification and characterization of factors
implicated in hypocotyl-localized phytochrome
responses (Khanna et al., 2006).
23
Probing
Tissueand
Organ-Specific
Phytochrome Regulation through Targeted
Phytochrome Depletion in Transgenic Plants
Identifying the Molecular Effectors that
Mediate
Tissueand
Organ-Specific
Phytochrome Responses
Recently, great advances have been made in the
elucidation of the molecular mechanisms responsible
for tissue- and organ-specific phytochrome responses
using transgenic plants expressing a gene that encodes
the mammalian enzyme biliverdin IXα reductase
(BVR). Constitutive expression of BVR in transgenic
Arabidopsis and tobacco plants using a strong plant
promoter, i.e., the promoter of the 35S RNA from
Cauliflower mosaic virus (i.e., CaMV 35S; Odell et
al., 1985), resulted in the inactivation of the
precursors of the phytochrome chromophore, and
alteration of light-dependent responses associated
with reduced phytochrome activity (Lagarias et al.,
1997; Montgomery et al., 1999; 2001). These
responses mirrored the responses that have been
observed in natural phytochrome chromophoredeficient mutants. Experiments with transgenic plants
selectively expressing the BVR gene using tissuespecific promoters, and consequently in which the
function of phytochromes have been inactivated in a
tissue-specific manner, have allowed successful
probing of the spatial-specific roles of phytochromes
in plant development (Montgomery, 2009;
Warnasooriya and Montgomery, 2009; Warnasooriya
et al., 2011; Costigan et al., submitted). BVR
expression in plastids of mesophyll tissues of
Arabidopsis was achieved using the CAB3 promoter
(i.e., CAB3::pBVR) and results in the disruption of
FR-dependent responses, including the inhibition of
hypocotyl elongation and the stimulation of
anthocyanin accumulation, suggesting roles for
mesophyll-localized phytochrome A (phyA) in the
regulation of FR-dependent responses (Warnasooriya
and Montgomery, 2009). CAB3::pBVR lines also
have disruptions in red- and blue-light-dependent
inhibition of hypocotyl elongation and anthocyanin
accumulation responses (Montgomery, 2009;
Warnasooriya and Montgomery, 2009; Warnasooriya
et al., 2011). Inactivation of phytochromes in the
shoot apical meristem in transgenic Arabidopsis
plants, i.e. plants expressing BVR under the control of
the MERI5 promoter, results in an enlarged leaf
phenotype and an increased leaf number specifically
under short-day photoperiods, suggesting a
photoperiod-dependent role of meristem-specific
phytochromes in the regulation of leaf initiation and
growth (Warnasooriya and Montgomery, 2009).
Using microarray analysis to identify genes
which were differentially expressed in FR-lightgrown 35S::pBVR seedlings relative to CAB3::pBVR
seedlings, we identified several genes involved in
mesophyll-specific
phytochrome
responses
(Warnasooriya, Oh, and Montgomery, unpublished
data). These studies are providing insight into the
molecular mechanisms underlying spatial-specific
phytochrome responses during Arabidopsis seedling
development. In this study, we report parallel
experiments to identify specific proteins that
accumulate differentially in No-0 wild-type (WT)
relative
to
35S::pBVR,
CAB3::pBVR,
or
MERI5::pBVR transgenic lines using SDS-PAGE
analysis followed by enzymatic digestion, liquid
chromatography-tandem mass spectrometry (LCMS/MS) and database searching. We found that the
large subunit of Rubisco (RbcL) and small subunit of
Rubisco (RbcS) accumulated to lower levels in
CAB3::pBVR lines than in No-0 WT or other BVR
lines, whereas Beta-glucosidase proteins (BGLUs)
accumulated highly in CAB3::pBVR lines relative to
No-0 WT or other BVR lines. Using microarray data
and RT-PCR analysis, we confirmed a positive
correlation of protein accumulation with the
accumulation of mRNA of the genes encoding the
identified proteins in CAB3::pBVR lines. These data
suggest that RbcL, RbcS, and BGLUs are targets of
mesophyll-specific, phytochrome-mediated light
signaling and potentially involved in tissue-specific
phytochrome responses during seedling development.
Identification of Proteins
Accumulated in BVR Lines
Differentially
To identify proteins responsible for tissue- and
organ-specific-phytochrome responses, we performed
profiling of proteins differentially accumulated in
BVR lines. Total soluble proteins were extracted from
No-0 WT (hereafter WT), 35S::pBVR3 (35S),
CAB3::pBVR2 (CAB3), and MERI5::pBVR1
(MERI5) seedlings grown on 1× MS medium
containing 1% sucrose at 22 oC in FR light at 5 µmol
m-2 sec-1 for 14 days. Soluble proteins (25 μg) were
resolved on 8 % or 12 % (w/v) polyacrylamide SDSPAGE gels. At least three distinctive protein bands
were detected visually: Proteins from band 1 or 2
accumulated to lower levels in CAB3 lines, whereas
proteins from band 3 accumulated highly in CAB3
lines (Figure 1). These altered patterns of protein
accumulation that were observed on gels were
reproducible in at least three biological replicates. The
Figure 1. SDS-polyacrylamide gel electrophoresis (SDSPAGE) of Arabidopsis total soluble proteins from wild-type
(WT), 35S::pBVR3 (35S), CAB3::pBVR2 (CAB3), and
MERI5::pBVR1 (MERI5) lines. Total soluble proteins
were extracted from 14 day-old entire seedlings of WT,
35S, CAB3, and MERI5 lines grown under constant far-red
light using 2× protein sample buffer. Proteins were
resolved on either (A) 8 % or (B) 12 % polyacrylamide gels
by SDS-PAGE electrophoresis. Protein bands (labeled 1, 2,
or 3) were excised from gels for LC-MS/MS analysis.
Reproducible SDS-PAGE gels were obtained from three
independent experiments.
three protein bands labeled 1 through 3 in the gels
from WT and CAB3 samples (Figure 1) were excised
and subjected to tryptic digestion followed by LCMS/MS and database searching for homology to
known proteins. All procedures for LC-MS/MS were
performed at the Research Technology Support
Facility (RTSF) at Michigan State University. The
Mascot search engine (Matrix Science, Inc., Boston,
MA) was used to match MS/MS spectra to peptide
sequences. Proteins identified with more than 99.7 %
of protein identification probability by using Scaffold
software (Proteome Software, Inc., Portland, OR) are
listed in Table 1. Spectral counts (i.e., number of
spectra) have been used as a good measure of relative
abundance among different samples (Liu et al., 2004).
We assessed the number of spectra, % of spectra, and
% of coverage for the comparison of candidate
proteins in WT relative to CAB3 (Table 1).
Consistent with data from SDS-PAGE gel
analyses, the large subunit of Rubisco (RbcL), which
is labeled protein band 1, was less abundant in CAB3
relative to WT when comparing spectral counts, i.e.,
106 vs. 176, respectively (Table 1). The LC-MS/MS
data for RbcL was reproducible when tested using
two biologically independent samples. As predicted
based on the protein gel, the molecular mass of RbcL
was 53 kDa (Figure 1). Proteins from band 2 were
identified as the small subunit of Rubisco (RbcS;
Table 1). Two RbcS proteins encoded by At1g67090
and At5g38410 accumulated to lower levels in CAB3
relative to WT – i.e. spectral counts for WT vs. CAB3
of 83 vs. 48 for At1g67090 and 13 vs. 8 for
At5g38410. Data from mass spectrometry analysis
for RbcS was reproducible in two biologically
independent replicates.
Whereas the predicted
molecular mass of RbcS was 20 kDa (Table 1), the
apparent molecular mass of RbcS on the protein gel
was approximately 10 kDa (Figure 1).
This
observation indicates post-translational modification
of Rubisco proteins, e.g. proteolytic processing of the
N-terminal portion as previously described (Houtz et
al., 2008).
Proteins from band 3, which were highly
accumulated in CAB3 relative to WT as observed on
SDS-PAGE gels (Figure 1), were identified as
BGLUs in our LC-MS/MS analysis (Table 1). The
Arabidopsis genome encodes at least 48 Betaglucosidase proteins (BGLUs) and BGLU23,
BGLU22, BGLU21, and BGLU18 are most closely
related (Xu et al., 2004; Ogasawara et al., 2009). All
of the closely related BGLU23, BGLU22, BGLU21,
and BGLU18 proteins were found to be more
abundant based on spectral count in CAB3 than in
WT—112 vs. 68 for BGLU23, 6 vs. 0 for BGLU22, 3
vs. 0 for BGLU21, and 5 vs. 2 for BGLU18 (Table 1).
25
Table 1. Identification of proteins differentially accumulated in wild-type (WT) and CAB3::pBVR2 (CAB3) lines
using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Proteins listed were identified with
probabilities of ≥ 99.7% after spectral searches with the Mascot database search engine (Matrix Science, Inc.) to
identify peptides and analysis with Scaffold software (Proteome Software, Inc.).
Band
No.
1
2
3
AGI No.
Samples
AtCg00490
(RBCL)
Annotation
(Mass)
Large subunit of Rubisco
(53 kDa)
WT
CAB3
No. of
spectra
176
106
% of
spectra
12.8
8.8
% of
coverage
49.9
55.7
At1g67090
(RBCS)
Small subunit of Rubisco
(20 kDa)
WT
CAB3
83
48
6.7
4.4
63.3
60.0
At5g38410
(RBCS)
Small subunit of Rubisco
(20 kDa)
WT
CAB3
13
8
1.7
1.3
55.8
45.3
At3g09260
(BGLU23)
Beta-glucosidase 23
(60 kDa)
WT
CAB3
68
112
3.9
6.3
52.9
55.5
At1g66280
Beta-glucosidase 22
WT
0
0
0
(BGLU22)
(60 kDa)
CAB3
6
0.3
14.1
At1g66270
(BGLU21)
Beta-glucosidase 21
(60 kDa)
WT
CAB3
0
3
0
0.2
0
12.0
At1g52400
(BGLU18)
Beta-glucosidase 18
(60 kDa)
WT
CAB3
2
5
0.1
0.3
10.0
7.2
Expression Patterns of Genes Encoding
Differentially Accumulated Proteins in BVR
Lines Are Also Altered
We previously performed comparative gene
expression profiling studies using BVR lines grown
on MS medium for 7 days in FR light (Warnasooriya,
Oh, and Montgomery, unpublished data). Using our
microarray data, which was produced in triplicate and
subjected to per chip normalization, the expression of
genes encoding the candidate proteins identified from
our LC-MS/MS analysis has been explored (Figure
2). AtCg00490 encoding RbcL was downregulated in
CAB3 lines compared to 35S and WT – i.e., 3.5- and
6.4-fold downregulated, respectively (Figure 2A). The
two genes encoding RbcS proteins, i.e., At1g67090
and At5g38410, were moderately downregulated by
1.6 fold in CAB3 compared to WT or 35S lines,
which were not significantly different from each other
(Figure 2B). Genes encoding the four identified
BGLUs tended to be upregulated in BVR lines
compared to WT (Figure 2C). Most strikingly,
BGLU18 was highly upregulated in CAB3 lines
relative to 35S and WT, i.e., 4.6- and 7.5-fold,
respectively.
To verify the expression of the genes queried in
our microarray data set, we performed RT-PCR
analyses (Figure 3). Also, in some cases microarray
data did not provide the expression of specific genes.
For example, the two RbcS-encoding genes and the
genes encoding BGLU21 and BGLU22 could not be
distinguished. Thus, it was very important to verify
the expression of those genes using RT-PCR analysis.
Primer pairs for candidate genes and a control gene
encoding an ubiquitin-conjugating enzyme 21
(UBC21) were designed using AtRTPrimer (Han and
Kim, 2006). RT-PCR analyses confirmed the
expression patterns of our candidate genes:
AtCg00490 and At1g67090 encoding RbcL and RbcS,
respectively, were downregulated in CAB3 lines
compared to WT or 35S lines; whereas At1g52400
encoding BGLU18 was highly expressed in CAB3
relative to WT (Figure 3). Altogether, the results from
microarray and RT-PCR analyses indicated a positive
correlation between gene expression and associated
protein accumulation in BVR lines.
Expression of Candidate Genes in Various
Tissues and Organs
To obtain more insight into the expression of the
candidate genes in different tissues and at distinct
developmental stages, we constructed heat maps using
AtGenExpress public Arabidopsis microarray data
set. RBCL was not represented in this data set.
Notably, the expression pattern of RBCS and BGLU18
in various tissues was quite similar (Figure 4). RBCS
and BGLU18 are expressed abundantly in aerial
tissues, including hypocotyls, cotyledons, and leaves.
Unlike BGLU18, BGLU23 and BGLU22/21 are
upregulated highly in roots (Figure 4). BGLU23 is
also abundant in hypocotyls and the shoot apex
(Figure 4). These observations correspond well to
BGLU18 being affected more significantly at the level
of expression than the other BGLU-encoding genes in
CAB3::pBVR lines where phytochromes are
inactivated in cotyledons and leaves—i.e. the site of
BGLU18 upregulation.
Figure 2. Selection of candidate genes differentially
expressed in BVR lines using microarray data.
Expression levels (Signal values) of genes encoding
RbcL, RbcS, and BGLU proteins identified from
liquid chromatography tandem mass spectrometry
(LC-MS/MS) analysis are shown. The data shown
are from Arabidopsis ATH1 (Affymetrix)
microarray experiments with three independent
replicates (Warnasooriya, Oh, and Montgomery,
unpublished data). A, RBCL: Large subunit of
ribulose 1,5-bisphosphate carboxylase (Rubisco); B,
RBCS: Small subunit of Rubisco; C, BGLU: Betaglucosidase. Note that for RBCS or BGLU22/21, a
single probe set from microarray data represents
multiple genes.
Potential Roles of RBCL, RBCS, and
BGLU18 in Mesophyll-Specific Phytochrome
Responses
In the stroma of chloroplasts, Rubisco catalyzes
the oxygenation of ribulose-1,5-bisphosphate in the
photorespiratory pathway and carboxylation of the
same substrate during the Calvin cycle (reviewed by
Jensen and Bahr, 1977). Rubisco is one of the most
abundant proteins comprised of RbcL and RbcS to
form a massive hexadecameric protein structure
(Baker et al., 1975). In Arabidopsis, RbcL protein is
encoded by a single chloroplast-localized gene,
AtCg00490 (Bedbrook et al., 1979), whereas RbcS
proteins are encoded by a nuclear multigene family,
including At1g67090 and At5g38410 (Dean et al.,
1989).
We found that the level of accumulation of RbcL
and RbcS proteins in CAB3 was lower than in WT,
35S, or MERI5 lines (Figure 1). In CAB3 lines, the
genes encoding those two proteins were also
downregulated, suggesting a role of mesophyllspecific phytochromes in the positive regulation of
RBCL and RBCS genes (Figures 2, 3). It has been
demonstrated that phytochromes are involved in the
induction of the expression of RBCL and RBCS at the
transcriptional level in several species (e.g. Sasaki et
Figure 3. RT-PCR analysis of candidate genes
encoding RBCL, RBCS, and BGLU18 proteins
identified from liquid chromatography tandem mass
spectrometry (LC-MS/MS) analysis. 7 day-old
seedlings of wild-type (WT), 35S::pBVR3 (35S), and
CAB3::pBVR2 lines (CAB3) grown under constant
far-red light were used. RT-, no reverse transcriptase
(RT) negative control. UBC21 gene encoding a
ubiquitin-conjugating enzyme was used as an internal
control to demonstrate relative quantity and quality of
the cDNA template for RT-PCR. Results shown are
representative of two independent biological replicates.
27
Figure 4. Heat map showing the expression of RBCS and BGLUs in different Arabidopsis tissues and organs at
various developmental stages.
Mean-normalized values from AtGenExpress microarray data set
(Hhttp://www.weigelworld.orgH)
and
BAR
Heatmapper
Plus
(Hhttp://esc4037shemp.csb.utoronto.ca/welcome.htmH) were used for heat map. Log-transformed microarray expression data is
displayed; Red represents high expression and yellow denotes low expression. The name of tissues/organs (tissue)
and age of samples (day) are indicated. Note that microarray data for RBCL was not available in AtGenExpress.
al., 1983; Thompson et al., 1983; Zhu et al., 1985;
Gilmartin and Chua, 1990; Silverthorne et al., 1990).
Furthermore, Antipova et al. (2004) demonstrated that
FR light induces the accumulation of RbcL protein
and antisense suppression of PHYA in transgenic
tobacco plants results in a reduced level of
accumulation of RbcL protein, indicating a role for
phyA in the FR light-mediated photoregulation of the
synthesis of RbcL proteins. Here, we further define
the mesophyll-specific pool of phyA as critical for
regulation of RbcL accumulation in Arabidopsis. It
also has been shown that At1g67090, which encodes a
RbcS protein, is highly expressed in mesophyll tissues
and upregulated in cotyledons and hypocotyls in
response to blue, red, and far-red light in Arabidopsis
(Sawchuk et al., 2008). Consistent with reduced levels
of RbcS protein in CAB3 lines (Figure 1), expression
of At1g67090 was downregulated in those lines
(Figures 2 and 3). Striking phenotypes observed in
CAB3 lines include defects in FR-dependent
inhibition of hypocotyl elongation and anthocyanin
accumulation, indicating roles of mesophyll-localized
phyA in FR responses (Warnasooriya and
Montgomery, 2009). Here, we highlight another
mesophyll-localized,
FR-dependent
phenotype
regulated by phyA—the regulation of expression of
RBCS and RBCL genes and accumulation of their
proteins.
We found that BGLU proteins were highly
accumulated in CAB3 lines and the expression of
BGLU18 gene was correlated with the observed
increased protein accumulation (Figures 1, 2 and 3),
suggesting a role of mesophyll-specific phytochrome
in the negative regulation of the BGLU18 gene.
BGLUs catalyze the hydrolysis of cellobiose, a unit of
cellulose, or other disaccharides with release of
glucose and play important roles in many biological
processes, including chemical defense against
pathogens (Morant et al., 2008), lignification
(Dharmawardhana and Ellis, 1998), degradation of
cell wall materials (Leah et al., 1995), and hydrolytic
hormone release (Lee et al., 2006). Interestingly, it
has been demonstrated that an oat BGLU protein, also
named Avenacosidase, co-purifies with phytochrome
(Gus-Mayer et al., 1994; Parker et al., 1995).
However, there has been no further evidence reported
of a functional role of BGLUs in phytochromemediated light signaling.
The Arabidopsis genome encodes 48 BGLUs and
eight of them, i.e., BGLU18 – BGLU25, are placed in
the same phylogenetic clade (Xu et al., 2004).
Identification of BGLU23, BGLU22, BGLU21, and
BGLU18 in our mass spectrometry analysis suggests
a functional redundancy of those proteins in spatialspecific phytochrome-mediated light signaling (Table
1). Among them, only the expression BGLU18 gene
was heavily upregulated (~5 fold) in CAB3 compared
to 35S lines (Figures 2 and 3), indicating a regulation
of BGLU18 by mesophyll-specific phytochromes.
Interestingly, the expression pattern of BGLU18 in
different Arabidopsis tissues was roughly correlated
with that of RBCS, i.e., RBCS and BGLU18 were
upregulated in mesophyll-abundant organs such as
cotyledons and leaves, whereas BGLU23, BGLU22,
and BGLU21 were abundantly expressed in roots
(Figure 4), suggesting a distinct mesophyll-specific
regulation of BGLU18 by phytochrome-mediated
light signaling.
It has been shown that BGLU18 is induced by
herbivory, methyl jasmonate, and wounding and its
protein accumulates in endoplasmic reticulum bodies
formed directly at the wounding site on cotyledons,
indicating a role of BGLU18 in plant defense
responses (Stotz et al., 2000; Ogasawara et al., 2009).
Since a positive correlation between the specific
activity of BGLU and the rate of cell wall elongation
has been observed in pea seedlings (Murray and
Bandurski, 1975), we propose that defense-related
BGLU18 could also be involved independently in the
regulation of cell elongation (e.g., inhibition of
hypocotyl elongation) during seedling development.
This involvement could be through the impact of
BGLU18 on the cell wall, or perhaps through lightdependent regulation of the hydrolytic release of a
hormone impacting cellular elongation. Interestingly,
inactivation of phytochrome in CAB3 lines resulted in
a defect in the inhibition of hypocotyl elongation
(Warnasooriya and Montgomery, 2009) and in the
present studies, BGLU18 proteins accumulated highly
in CAB3 lines, compared with other BVR lines
(Figure 1). Thus, it appears that an increased
accumulation of the BGLU18 enzyme is associated
with an elongation of hypocotyls – thus in normal
seedling development expression of BGLU18 may be
regulated
negatively
by
mesophyll-specific
phytochromes during the light-dependent inhibition of
hypocotyl elongation.
SUMMARY AND CONCLUSION
We applied SDS-PAGE gel analysis coupled
with mass spectrometry to identify key protein
components
regulated
by
mesophyll-specific
phytochromes. Our mass spectrometry-based protein
analysis identified three proteins differentially
accumulated in BVR lines. In CAB3, transcript
abundance of RBCS and RBCL and associated protein
abundance were low relative to WT and other BVR
lines with distinct tissue-specific disruptions in
phytochrome accumulation. However, BGLUs
accumulated to higher levels and transcript
accumulation of BGLU18 was upregulated in CAB3.
Since
mesophyll-specific
phytochromes
are
inactivated in CAB3 lines, we hypothesize that the
genes encoding RbcS, RbcL, and BGLU18 proteins
are targets of mesophyll-specific phytochrome
signaling pathways. Further investigations using
genetic approaches to characterize mutants for genes
encoding these proteins and/or two-dimensional gel
electrophoresis coupled with mass spectrometry
analysis to identify additional candidate proteins
differentially accumulated in various BVR lines will
allow a better understanding of the molecular
mechanisms underlying spatial-specific phytochromemediated light signaling and the associated regulation
of distinct phytochrome-dependent responses.
ACNKOWLEDGEMENTS
We would like to thank Dr. Sankalpi
Warnasooriya for conducting the microarray analyses
and for reading and critically commenting on the
manuscript. This work was supported by the National
Science Foundation (grant no. MCB-0919100 to
B.L.M.) and the Chemical Sciences, Geosciences and
Biosciences Division, Office of Basic Energy
Sciences, Office of Science, U.S. Department of
Energy (grant no. DE-FG02-91ER20021 to B.L.M.).
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Short Communication
Bone Marrow Stem Cells Differentiated Into Cartilage by Manipulation of
Gas Phase
Aileen Y. Wang1, Heather Luong1, Jerry English1, William LeBoeuf1, Quynh Diep1, Mihir Patel1,
Shiwei Cai2, and P. Jackie Duke1
1
Dept. of Orthodontics, 2Dept. of Endodontics, UT School of Dentistry, Houston TX 77030
INTRODUCTION
A continual need for bone in craniofacial repair
exists; one solution to the problem of procuring
enough bone for this purpose lies with tissue
engineering (Doan et al., 2010). In most bone tissue
engineering studies, osteoblasts are cultured on a
scaffold with or without growth factors and then
implanted (Doan et al., 2010). Because of concerns
about rejection of implants, embryonic stem cells and
stem cells from bone marrow have also been placed
on scaffolds, differentiated into bone cells and
implanted (Jukes et al., 2008). In a few instances,
stem cells have been differentiated into bone-forming
cartilage for bone repair (Oliveira et al., 2009; Jukes
et al., 2008; Doan et al., 2010).
The rationale for using cartilage for bone repair is
that during growth and development, the bony
skeleton forms primarily through endochondral
ossification,
involving
mineralization
of
a
cartilaginous template, then its vascularization and
replacement by bone (Duke et al., 1993). Fracture
healing also includes a cartilaginous stage.
Previously, mouse embryonic limb bud cells were
differentiated into cartilage in NASA’s rotating
bioreactor and used to heal defects in the skulls of
mice (Montufar-Solis et al., 2004; Doan et al., 2009).
The purpose of the current study was to isolate
mouse bone marrow stem cells (BMSCs), expand the
cells in culture and differentiate them into cartilage,
creating a flat cartilaginous implant. To grow
cartilage from BMSCs, we used hardware developed
for a 1992 space-flight, Flexcell (Flex I®) plates and
35 mm culture plates.
MATERIALS AND METHODS
Isolation of BMSCs was carried out according to
Masoud and Samad (2009) for isolation and culture of
MSC from mouse bone marrow. Isolated bone
marrow cells from 7-9 day old C57BL mice were
expanded in culture. After 2 passages, 0.5 ml
differentiation medium (see below) with 88,000 cells
was inoculated onto Silastic® membranes supported
by hardware casings or in assembled hardware units
that were placed in Petri dishes for culture. In other
experiments, Flex I® plates (Flexcell International) or
35 mm culture dishes (Falcon BD) were used. In
these experiments the cell number inoculated onto the
plate ranged from 7.55 x 106 cells to 34.2 x 106.
Cultures were incubated at 37oC with or without 5%
CO2 for two hours; 5% CO2 was used thereafter.
After medium addition, cells were cultured for 3-7
days with ½ the medium changed every other day,
then fixed with 10% buffered neutral formalin and
stained.
Medium for BMSC differentiation was BGJbFitton-Jackson modification (GIBCO) supplemented
with 10% fetal bovine serum (Fischer), 150 μg/ml
ascorbic acid, and 1% Penicillin-Streptomycin
solution (Sigma). BGJb-FJM is a chondrogenic/
osteogenic medium. In one experiment, DMEM
medium replaced the BGJb-FJM.
Cells were stained in situ with methylene blue and
cresyl violet, both of which stain neuronal Nissl
bodies, and either Toluidine blue, a metachromatic
dye specific for cartilage matrix, or Alcian blue at low
pH, again a cartilage specific stain (Huang et al.,
2010). Flex I® aggregates were sectioned and stained
with Toluidine blue.
Hardware used in these experiments was the BEX
unit designed for our CELLS experiment on the
International Microgravity Laboratory-1 mission in
1992 (Duke et al., 1995). The hardware (Figure 1)
consists of a two-welled polycarbonate chamber into
which is inserted a gas exchanging membrane of a
Silastic® compound (Dow-Corning 5150+360) in the
shape of two domes or “bubbles” which allow for gas
exchange, and inflate or collapse as medium is added
or removed. Deflector rings inside the bubble control
fluid forces related to medium removal or addition.
33
Wang et al. -- Bone Marrow Stem Cell Differentiation
The complete assembly also includes a silicon rubber
gasket, a polycarbonate bottom plate, and a stainless
steel support plate. For flight, cells were cultured on
a cover slip between the gasket and the bottom plate.
For some of these experiments, complete hardware
units were used, and for some, only Silastic
membrane bubbles were used. Cells were cultured on
the interior surface of the bubble and the units placed
in a Petri dish.
Figure 2. Neuronal like cells. A. Stained with
methylene blue. B. Stained with cresyl violet. Bars
= 50 µm.
Figure 1. BEX hardware. A. Position used to
culture prechondrocytes on coverslips for
spaceflight. B. Position used to culture BMSCs on
membrane.
To form a flat piece of tissue, cells were cultured
on Flex I® plates which look like regular culture
dishes, but have a bottom surface of Silastic. For
controls, 35 mm culture dishes were used.
RESULTS
Cells in units placed in CO2 for the attachment
period spread on the membrane assuming a neuronallike appearance.
Staining with methylene blue
revealed numerous dark bodies (Nissl bodies) in the
cytoplasm (Figure 2A). Cells in units not exposed to
CO2 during the attachment period did not spread, and
the cell layer consisted of rounded cells with areas of
aggregation typical of early cartilage formation
(Figure 3). When cells in these layers were stained
with Toluidine blue after 7 days, the entire layer was
metachromatic, indicating the presence of sulphated
glycosaminoglycans (data not shown). Alcian blue
staining identified the aggregated cells as
chondrocytes (Huang et al., 2010). When a nondifferentiating medium (e.g. DMEM) was used, only
neuronal-like cells formed (Figure 2B).
With
increased initial cell densities, the Flex I® group
formed aggregates that, when sectioned and stained
with Toluidine Blue, resembled hypertrophied
cartilage with metachromatic matrix (Figure 4).
Aggregates formed in the Petri dishes stained with
Alcian blue at low pH, confirming the presence of
cartilage matrix (Figure 5; Huang et al., 2010).
Figure 3. Aggregates of rounded cells interspersed with neuronal-like cells. Bar = 100 µm.
Figure 4. Aggregate from Flex I® plate, sectioned
and stained with Toluidine blue. Pink indicates
cartilage matrix. Bar = 50 µm.
Wang et al. -- Bone Marrow Stem Cell Differentiation
offering a promising alternative approach in the realm
of bone regeneration and reconstruction.
ACKNOWLEDGEMENTS
We thank Wei Chen, Department of Endodontics,
Dental Branch, for stem cell preparation and culture,
Dr. Zhang and Jun Ju, Dept of Diagnostic Sciences,
Dental Branch, who supplied the mice. Support:
UTHSC Office of Biotechnology (AW, PJD, HL,
WL, QD); UT Dental Branch startup funds (SC).
REFERENCES
Figure 5. Aggregate of cells on culture dish
stained with Alcian blue. Bar = 100 µm.
DISCUSSION
This study shows that BMSCs can be
differentiated into cartilage or neuronal-like cells by
manipulating the gas environment during the initial
attachment phase. The exposure to CO2 during this
initial stage of culture changed the pH of the medium,
making it more alkaline as indicated by the phenol red
indicator in the medium. The exposure to CO2 using
a non-differentiating medium (e.g. DMEM) did not
result in chondrocyte-like cells being present; instead
all the cells were spread with numerous processes.
The same result was seen in all culture vessels.
To advance these studies toward the ultimate goal
of developing a cartilage implant for humans, the
ability to grow more cartilage must be developed.
One way this could be done is by the use of TGFβ.
The medium used for human studies must be
serumless, so that the implant is not exposed to
animal products during culture. First, BMSCs must
be grown in serumless medium, differentiated into
cartilage which will be implanted into defects in the
skulls of mice to determine its bone-forming
capabilities.
Another major problem arising in this study was
the tendency of the cartilage to come off the plate
because of the cartilage’s loss of contact with the
surface as matrix develops. Using Cell-Tac as in the
spaceflight experiment could solve this problem.
The phenomenon of forming bone from a
cartilaginous template continues to ignite hope for
patients with skeletal and craniofacial disorders,
Doan, L., Kelley, C., Luong, H., English, J., Gomez, H.,
Johnson, E., Cody, D., and Duke, P.J. 2010.
Engineered cartilage heals skull defects. American
Journal of Orthodontics and Dentofacial
Orthopedics 137: 162-163.
Duke, P.J., Daane, E., and Montufar-Solis, D. 1993.
Studies of chondrogenesis in rotating systems.
Journal of Cellular Biochemistry 51:274-282.
Duke, P.J., Doan, L., Luong, H., Kelley, C., LeBoeuf, W.,
Diep, Q., Johnson, E., and Cody D.D. 2009.
Correlation between micro-ct sections and
histological sections of mouse skull defects
implanted with engineered cartilage. Gravitational
and Space Biology 22: 45-50.
Duke, P.J., Montufar-Solis, D., and Daane, E. 1995.
Chondrogenesis in cultures of embryonic mouse
limb mesenchyme exposed to microgravity. In:
Biorack on Spacelab IML-1. Mattok, C., ed. ESA
Publications Division, Noordwijk, The Netherlands.
pp.115-127.
Huang, A.H., Farrell, M.J., and Mauck, R.L 2010.
Mechanics and mechanobiology of mesenchymal
stem cell-based engineered cartilage. Journal of
Biomechanics 43: 128–136.
Jukes, J.M., Both, S.K., Leusink, A., Sterk, L.M., van
Blitterswijk, C.A., and de Boer, J.
2008.
Endochondral bone tissue engineering using
embryonic stem cells. Proceedings of the National
Academy of Sciences USA 105:6840-45.
Masoud S. and Samad N. 2009. A protocol for isolation
and culture of mesenchymal stem cells from mouse
bone marrow. Nature Protocols 4: 102-106.
Montufar-Solis, D., Nguyen, H.C., Nguyen, H.D., Horn,
W.N., Cody, C.D., and Duke, P.J. 2004. Using
cartilage to repair bone: an alternative approach in
tissue engineering. Annals of Biomedical
Engineering 32:1-6.
Oliveira, S.M., Mijares, D.Q., Turner, G., Amaral, I.F.,
Barbosa, M.A., and Teixeira, C.C.
2009.
Engineering endochondral bone: in vivo studies.
Tissue Engineering Part A 15: 635-643.
35
Short Communication
Fluid Motion for Microgravity Simulations in a Random Positioning
Machine
Carole A. D. Leguy1, Rene Delfos1, Mathieu J.B.M. Pourquie1, Christian Poelma1, Janneke
Krooneman2, Jerry Westerweel1 , and Jack J.W.A. van Loon3
1
3
Laboratory for Aero and Hydrodynamics, Delft University of Technology, (NL);
DESC @ OC, Academic Centre for Dentistry Amsterdam (NL)
BACKGROUND
To understand the role of gravity in biological
systems one may decrease inertial acceleration by
going into free-fall conditions such as available on
various platforms.
These experiments are
cumbersome and expensive.
Thus, alternative
techniques like Random Positioning Machines (RPM)
are now widely used to simulate the micro-gravity
environment (Yuge et al., 2003; Borst and van Loon,
2009; Pardo et al., 2005). These instruments generate
random movements so that cumulative gravitational
effects cancel out over time. However, comparative
studies performed with the RPM and culture cells
were unable to reproduce the spaceflight results
(Hoson et al., 1997). These differences may be
explained by stresses acting on the culture cells in an
RPM whereas these stresses are not present in
microgravity conditions. They may be caused by
internal fluid motion, originating from instationary
rotation. The aim of this study is to quantify fluid
flow behavior and wall shear stresses (as they are
relevant to cells cultured at the flask wall), and
internal shear stresses as they are relevant to
suspended (free-floating) cells in an RPM container.
We do this both experimentally using Particle Image
Velocimetry (PIV) and numerically using 3D Direct
Numerical Simulation (DNS) of the flow.
METHODS
A dual-axis rotating frame machine is used to
reproduce the motion of a real RPM in a controllable
way according to a pre-programmed motion protocol.
A flask filled with fluorescent tracer particles (d=13
μm, ρ=1150 kg/m3) and water with sodium chloride
of the same density as the particles is positioned at the
center of rotation, where it is surrounded by a corotating 2-component planar PIV system (Adrian and
Westerweel, 2011), see Figure 1. A green light dye-
2
Bioclear, Groningen (NL);
laser of 534 nm wavelength is used to create a light
sheet inside the flask. We use a double-frame camera
to record the particles inside the flask. The motor
motion is generated with a servo-control system up to
a maximum angular speed of 30º/s, and acceleration
up to 60º/s2. Images are recorded at a maximum
frequency of 15 Hz for a duration of typically 30s
corresponding to 6 motion cycles.
Figure 1. Left: Particle Image Velocimetry system
mounted on a two-axis rotating frame. Right:
Detailed view of the container. A light sheet is
created in the XY plane at a distance ∆z from the
container wall.
The same experiment is simulated numerically
using an existing DNS model (Pourquié, 2009) to
which the effect of rotation is added by implementing
extra body forces (angular acceleration, centrifugal,
Coriolis) to the momentum equation, including the
motion protocol as applied to the experiment. The
simulation has sufficient spatial and temporal
resolution to fully resolve the flow field. The
(rectangular) flask geometry equals the one used for
the experimental study (65 x 40 x 20 mm3).
Leguy et al. -- Microgravity Random Positioning Machine
Figure 2. Simulated velocity field in the y direction (t-6.3 s) with slice view of the XY, YZ and ZX planes.
RESULTS
For a periodic and sinusoidal rotation around a
single axis, fluid motion induced by inertia is
observed parallel to the wall; see Figure 2 and 3. The
RMS-average difference between the simulated and
measured velocity field is equal to 0.5 mm/s, or 6% of
the maximum velocity.
The quantitative result
demonstrates the good agreement between the
simulated and the measured velocity profiles.
In Figure 3, measured and simulated velocities
at the center of the measurement plane are depicted as
a function of time. It can be seen that a periodic state
is reached after only one period. The time evolution
of the velocity profiles is shown on Figure 4 for the
velocity in the y direction Vy. The increase and
decrease of Vy along the y direction due to the
circulation in the container can be seen. Along the xaxis of rotation, the profile is flat (i.e. 2D flow),
except for end-effects (boundary layers) near the flask
wall. Since the PIV measurements provided the
velocity field only over the XY plane at a (fixed)
distance of 3.3 mm from the wall, the 3-D simulated
results are used to study the velocity profile along the
z axis. The evolution of the boundary layer can be
seen near the flask wall. A maximum wall shear stress
of 6.2 mPa is obtained with an angular phase shift of
54 degree with respect to the angular velocity of the
rotating axis.
Figure 3. Velocity in the Y direction at the center
of the z = 3.3 mm plane obtained for the
measurements (in blue) and from simulated data
(in red).
For spin-up about 2 axes according to the
instationary angular velocity used in a commercial
RPM (see Figure 5), more complex patterns are
obtained in the DNS. A maximum velocity up to 70
mm/s near the wall and a maximum stress at the
container wall of 62 mPa are derived.
37
Figure 4. Comparison between the simulated and measured velocity profiles at several time steps as a
function of Y position, at the intersection of the Sxy and Syz plane (left), and as a function of the x position
at the intersection between the Sxy and Szx planes (right).
Figure 5. Angular velocity patterns for both axes (top), maximum wall shear stress (bottom) according to
the angular velocity used in an RPM. Box-size: 6 × 4 × 2 [cm].
CONCLUSION
For sinusoidal rotation, quantitative agreement
between simulated and measured velocity field has
been found which is considered as a validation of the
numerical model. Considering motions as are
employed for an RPM, the numerical study suggests
that stresses induced by the fluid are particularly high
during changes of rotation velocity, i.e. acceleration.
In the near future, we aim to provide a quantitative
comparison between experimental and numerical
results for dual axis motion. Furthermore, we are
developing a user protocol for the RPM users.
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Buchen B.
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Evaluation of the treedimensional clinostat as a simulator of
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Sorescu G.P., Xu M., van Loon J.J., Wang M.D.,
and Jo H. 2005. Simulated microgravity using the
Random Positioning Machine inhibits differentiation
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preosteoblasts. American Journal of Physiology.
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Yuge L., Hide I., Kumagai T., Kumei Y., Takeda S., Kanno
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Anim. 39:89-97
39
Short Communication
Using Green Fluorescent Protein (GFP) Reporter Genes in RNAlater™
Fixed Tissue
Anna-Lisa Paul1 and Robert J. Ferl1,2
1
Department of Horticultural Sciences and 2Interdisciplinary Center for Biotechnology Research,
University of Florida, Gainesville, FL 32611 USA
Plants in an orbital environment experience
conditions that are distinctly unlike the earth-bound
environments that have directed their evolution on
Earth. This presents a unique opportunity to examine
biological responses, particularly those involved in
integrating gravity as a force shaping biological
systems. One means of measuring these adaptive
responses is to monitor the expression of genes that
allow survival in those peculiar environments. In a
recent series of spaceflight experiments conducted on
the International Space Station (ISS) we utilized
Arabidopsis thaliana (Arabidopsis) plants engineered
with specific gene promoters driving to Green
Fluorescent Protein (GFP). These biological sensor
plants are referred to as TAGES, an acronym for
Transgenic Arabidopsis Gene Expression System.
One component of the TAGES Spaceflight
experiment was the use of several different GFP
reporter gene constructions to record tissue-specific
changes in the patterns of gene expression in real
time. Another component was the evaluation of
genome-wide changes in these plants, a goal that
required the use of the nucleic acid preservative
RNAlater™ (Ambion).
The utility of GFP gene reporters is well
established (Haseloff & Amos, 1995; Manak et al.,
2002; Paul et al., 2003; Paul et al., 2004; Sheen et al.,
1995). The application to spaceflight is especially
appealing as gene expression data can be collected
telemetrically, thereby placing minimal demands on
crew time.
The use of RNAlater™ also offered the
opportunity to evaluate the preservation of GFP for
postflight analysis. In a series of preliminary
experiments prior to launch, the TAGES plants were
utilized in ground-based studies as part of a Payload
Verification Test (PVT) to test the operations and
science before the experiment was launched. One
aspect that was evaluated was the responsiveness of
the biosensor plants to environmental stress and the
efficacy of using RNAlater™ to preserve evidence of
GFP expression long after the stress response was
incurred, in addition to its general use as an effective
means to preserve the integrity of RNA.
In the PVT, plates of TAGES plates were prepared
with two or three genotypes and a variety of methods
used to collect GFP expression data. Figure 1 shows
GFP reporter gene plants grown vertically on nutrient
agar plates. The TAGES ISS experiments utilized a
variety of plant lines engineered to report on several
aspects of the spaceflight environment, and among
the plants shown in Figure 1, the left-hand side of
each plate contains Adh::GFP plants, which respond
to reduced levels of oxygen. The plants on the far
right-hand side of the plate are used as positive
controls (CaMV35s::GFP) and continuously express
GFP throughout their cells. In the experiment shown
A
B
C
Figure 1. 100mm2 Petri plate containing TAGES
seedlings from the Payload Verification Test. The
photograph on the left (A) was taken in white light, (B)
shows the same plate photographed through a 510nm
long pass filter in 488nm blue light with an Illumatool™
device. The native red fluorescence of chlorophyll is
prominent in the Adh::GFP plants on the left side of the
plate, and intense GFP expression can be seen in the
positive control plants (CaMV35s::GFP) on the right
side of the plate. The image in C was captured during
PVT with the GFP Imaging System that is currently
installed aboard the ISS.
Paul and Ferl -- GFP Reporter Genes and RNAlater™
in Figure 1A and 1B, the roots of the plants were
covered with a thin layer of solid agar, which reduced
the flow of oxygen to those tissues. As seen from the
faint green glow in the roots of those plants, that thin
blanket of agar was sufficient to induce the sensitive
Adh::GFP reporter gene.
Figure 1A was
photographed with a standard digital camera in white
light, and that same camera was used to photograph
1B through an Illumatool™ (Light Tools Research)
GFP imaging device. The image in Figure 1C was
captured by the TAGES GFP Imaging System (GIS)
developed by Kennedy Space Center, which was later
launched on STS-129 and installed on the ISS for the
subsequent TAGES spaceflight experiments.
Figure 2. GFP expressing in plants preserved in
on
orbit.
A
representative
RNAlater™
CaMV35s::GFP plant photographed in white light
(A) with the corresponding fluorescent image (B).
Panel (C) shows roots from a GFP expressing plant
on the background of non-GFP roots.
Although the resolution of the GIS is excellent for
general patterns of gene expression, it was our hope
that RNALater™-preserved material returned from
the ISS could be utilized to capture higher resolution
tissue-specific data. Figure 2 shows images of a
representative plant returned from orbit in TAGES
experiment 2B, which was launched on STS-131, and
spent several weeks on the ISS as part of the APEXTAGES payload. The plants were grown for 12 days
on the ISS, and then harvested to RNAlater™ in a
Kennedy Space Center Fixation Tube (KFT) (Ferl et
al., 2011). The KFT was allowed to equilibrate at
ambient temperature for 12-18 hours, and then placed
in the MELFI freezer on the ISS. The samples were
recovered on the return flight of STS-132,
photographed and then prepared for subsequent
biochemical assays (analyses in progress). The
RNAlater™ fixed plant in Figure 2 is a constitutive
GFP line (CaMV35s::GFP). This line was used as a
positive control for GFP expression in the imaging
portion of the TAGES experiment. Figure 2A shows
a white light image 2A and 2B provides the
corresponding fluorescent image of the plant. 2C
shows a tangle of roots from the collection of
harvested material before the individual plants were
separated. The GFP expressing root can be seen
clearly on the backgrounds of the non-GFP roots.
Ground-based experiments were also conducted to
calibrate GFP reporter gene utility for gene
expression assays in RNAlater™ fixed material.
Figure 3 shows the hypocotyl-root junction of
Adh::GFP plants. The top row shows un-stressed
plants, while the bottom row shows the pattern of
GFP expression in hypoxic plants (flooding for 48
hours). Panels 3A and 3D are white light image, 3B
and 3E are the corresponding fluorescent microscope
images, and panels 3C and 3F are fluorescent
microscope images of the same regions of plants after
preservation in RNAlater™. The exposure times for
3B and 3E is 5 seconds (Olympus S2X12, GFP longpass filter); however, since preservation in
RNAlater™ diminishes the fluorescence of both
chlorophyll (red) and GFP (green), the exposure time
for 3C and 3F was extended to 10 seconds. In 3C the
autofluorescence of plant cell wall is noticeable, and
in 3F it can be seen that the GFP fluorescence that
was masked by chlorophyll in the hypocotyl can now
be seen with better clarity. The phenomenon of
diminished GFP fluorescence in RNAlater™
preserved samples has also been demonstrated in
flow cytometry applications (Zaitoun et al., 2010).
Figure 3. The top row shows un-stressed plants, while
the bottom row shows the pattern of GFP expression in
hypoxic plants. Panels 3A and 3D are white light images,
3B and 3E are fluorescent images, and panels 3C and 3F
show the same sections of plants after preservation in
RNAlater™.
41
Cell and subcellular resolution of GFP is possible
in RNAlater™ preserved tissue. Figure 4 shows the
fluorescent microscopy used to collect subcellular
data from a plant preserved in RNAlater™. The white
light photograph in 4A shows the surface of the leaf,
venation and a trichome. Figure 4B shows the same
section of the leaf under fluorescent illumination to
visualize GFP expression and 4C shows the same
section after preservation in RNAlater™. Again, an
extended exposure time was required to obtain
similar signal strengths as seen in fresh tissue. In this
case, the image in 4C was also subjected to postcapture processing. Minor adjustments of brightness
and contrast can enhance the visualization of GFP. A
comparison of the positions of the guard cells
fluorescing in 4B compared to those in 4C confirm
that the green fluorescence Fluorescent microscopy
shows that GFP is distributed primarily among the
guard cells of the epidermis, and is especially
prominent in the nuclei of the guard cells and the
trichome.
Figure 5. Other methods of subcellular labeling are
also effective in RNAlater™ fixed tissue. The top row
shows DAPI staining in fresh (A) and RNAlater™
fixed tissue (B and C). The photo in D shows cell wall
staining with calcofluor white in RNALater™ fixed
Arabidopsis roots.
DAPI and calcofluor white are both fluorescent stains
(e.g. Kamo & Griesbach, 1993; Zhou et al., 2009).
As seen in the figure, both stains are very effective in
RNAlater™ preserved material. Figure 5A shows a
freshly stained root next to a root stained after
fixation in RNAlater™ (Figure 5B). Figure 5C shows
the nucleus of a trichome stained with DAPI after
Figure 5D shows an
fixing in RNAlater™.
RNAlater™ fixed root stained with calcofluor white.
Figure 4. A section of a flooded leaf photographed in
white light (4A) with fluorescent microscope before
preservation in RNAlater™ (4B) and after
RNAlater™ preservation (4C).
RNAlater™ preservation does not seem to be an
impediment to at least a few other standard methods
of sub-cellular labeling techniques.
Figure 5
provides examples of DAPI nuclear staining (Figures
5A, 5B, 5C) and calcofluor white cell wall staining.
In conclusion, plants containing GFP reporter
genes can still be evaluated after being preserved in
This method of preserving GFP
RNAlater™.
analyses may be important in situations where
sophisticated imaging technology is not immediately
available on orbit. It does appear that the level of
GFP visualization is lessened in RNAlater™, but that
the location of the signal is maintained even at
subcellular levels. In addition, it is clear that plant
material preserved in RNAlater™ can also be
effectively used for other traditional sub-cellular
labeling techniques. Given that RNAlater™ is a welldocumented on-orbit fixative with a repeated and
extensive deployment history within KFTs, it is
likely that RNAlater™ could serve space biology
science well beyond its originally intended purposes
of nucleic acid preservation.
ACKNOWLEDGEMENTS
This work was supported by NASA grants
NNX07AH27G and NNX09AL96G to ALP and RJF.
The GFP imaging system was developed at Kennedy
Space center, primarily under the direction of Dr.
Trevor Murdoch. The use of RNAlater™ and KFTs,
together with the manifesting, on-orbit deployment,
and sample return, were all made possible by the
dedicated payload developers and managers at KSC –
including David Reed, Kelly Norwood, April
Spinale, David Cox, and Howard Levine.
REFERENCES
Ferl, R.J., Zupanska, A., Spinale, A., Reed, D., ManningRoach, S., Guerra, G., Cox, D., and Paul, A.-L.
2011. The performance of KSC Fixation Tubes
with RNALater for orbital experiments: a case
study in ISS operations for molecular biology.
Advances in Space Research 48:199-206.
Haseloff, J. and Amos, B. 1995. GFP in plants. Trends
Genet 11: 328-329.
Kamo, K.K. and Griesbach, R.J. 1993. Evaluation of DAPI
as a fluorescent probe for DNA in viable Petunia
protoplasts. Biotech Histochem 68: 350-359.
Manak, M.S., Paul, A.L., Sehnke, P.C., and Ferl, R.J.
2002. Remote sensing of gene expression in
Planta: transgenic plants as monitors of
exogenous stress perception in extraterrestrial
environments. Life Support Biosph Sci 8: 83-91.
Paul, A.L., Murdoch, T., Ferl, E., Levine, H.G., and Ferl,
R.
2003. The TAGES Imaging System:
Optimizing a green fluorescent protein imaging
system for plants. SAE Technical Paper 2003-012477.
Paul, A.L., Schuerger, A.C., Popp, M.P., Richards, J.T.,
Manak, M.S., and Ferl, R.J. 2004. Hypobaric
biology: Arabidopsis gene expression at low
atmospheric pressure. Plant Physiol 134: 215223.
Sheen, J., Hwang, S., Niwa, Y., Kobayashi, H., and
Galbraith, D.W. 1995. Green-fluorescent protein
as a new vital marker in plant cells. Plant J 8:
777-784.
Zaitoun, I., Erickson, C.S., Schell, K., and Epstein, M.L.
2010. Use of RNAlater in fluorescence-activated
cell sorting (FACS) reduces the fluorescence
from GFP but not from DsRed. BMC Res Notes
3: 328.
Zhou, J., Lee, C., Zhong, R., and Ye, Z.H. 2009. MYB58
and MYB63 are transcriptional activators of the
lignin biosynthetic pathway during secondary cell
wall formation in Arabidopsis. Plant Cell 21:
248-266.
43
Short Communication
Potential of the Euprymna/Vibrio Symbiosis as a Model to Assess the
Impact of Microgravity on Bacteria-Induced Animal Development
Jamie S. Foster1, Krystal R. Kerney2, Mirina L. Parrish2, Christina L.M. Khodadad2, and Steven R.
Ahrendt2
1
Department of Microbiology and Cell Science, University of Florida;
Space Center, FL 32899 USA
Long-duration spaceflight imposes numerous
physiological challenges to astronauts working in the
space environment. Some of these challenges have
been well-documented including bone and tissue loss
(Smith and Heer, 2002), dysregulation of the immune
system (Crucian et al., 2008), and increased risk to
infectious diseases (Wilson et al., 2007). However,
relatively little is known regarding the impact of the
space environment, specifically microgravity, on
those mutualistic bacteria that interact directly with
animal cells.
To understand the effects of
microgravity on beneficial microbes we propose using
the model system between the bobtail squid
Euprymna scolopes and the luminescent bacterium
Vibrio fischeri strain ES114. For over 20 years the
Euprymna/Vibrio system has provided key insight
into the role that symbiotic bacteria play in the normal
development of animal tissues (McFall-Ngai and
Ruby, 1991; Weir et al., 2010). This mutualistic
association has several advantages that render it
highly amenable to experimental manipulation in the
space environment.
First, the symbiosis is
monospecific with only one host and one symbiont
making it easier to discern the effects of the symbiont
on host cells. Second, both partners can be cultured
independently in the laboratory and the symbiosis can
be initiated at any point after the juvenile animal
hatches. Lastly, the symbiosis can be activated and
monitored using currently available flight hardware
such as the Fluid Processing Apparatus (FPA;
BioServe Space Technologies, Boulder, CO). Here,
we present evidence from a pilot study that
demonstrates the overall potential of the
Euprymna/Vibrio model system to examine the
impact of microgravity on bacteria-induced
development of host animal tissues.
In the Euprymna/Vibrio model system the
mutualism is initiated when symbiosis-competent
strains of V. fischeri colonize a specialized host
2
Space Life Science Lab, Kennedy
structure called the light organ (Figure 1A). On either
side of the incipient light organ there are two
appendage-like structures comprised of ciliated
epithelial cells (Figure 1B). The function of these
ciliated cells is to entrain bacteria from the
environment into the vicinity of pores found on the
surface of the light organ (Figure 1C). If the
symbiosis-competent strain of V. fischeri is not
present in the environment then these ciliated
epithelial appendages are retained and the light organ
does not undergo morphogenesis (Figure 1C, D).
However, if symbiosis-competent V. fischeri is
present then the bacteria enter the light organ through
one of the surface pores and travel to an epitheliallined crypt space where the bacteria then begin to
divide. Upon reaching a critical cell density the
bacteria begin to luminesce within the light organ. A
few hours after initial colonization the bacteria trigger
a series of developmental remodeling events that
includes an apoptotic cell death event of the ciliated
epithelial appendages (Figure 1E). Over a period of 4
d the light organ undergoes complete morphogenesis
resulting in the loss of the ciliated appendages (Figure
1F). Several of these bacteria-induced events, such as
the onset of luminescence, occur at discreet time
intervals within the first 24 h after the initiation of
symbiosis. These early developmental events can be
readily monitored to assess the progression of the
natural symbiosis. To assess the tractability of the
Euprymna/Vibrio symbiosis in the space environment,
we first determined whether the animals could survive
under microgravity conditions.
To simulate
microgravity we used eight 10-ml volume high aspect
ratio vessels with a rotary culture system (HARV;
Synthecon, Houston, TX) at 13 rpm. These HARV
have been successfully used in numerous
microgravity studies to reproduce space-like
conditions (Hammond and Hammond, 2001) and have
been shown to be directly comparable to real
Foster et al. -- Euprymna/Vibrio Symbiosis Model
Figure 1. The morphology of the Euprymna scolopes light organ. A. Juvenile animal with light organ (arrow) in the
center of mantle cavity. Bar = 0.5 mm. B. Micrograph of nascent light organ depicting fields of ciliated epithelial
appendages (cea) Bar = 100 μm. C. One half of 24 h aposymbiotic light organ with arrow pointing to pores. Bar = 50
μm. D. Micrograph of 96 h aposymbiotic light organ showing no signs of morphogenesis in the absence of competent
bacteria. E. Symbiotic light organ at 14 h stained with acridine orange, a fluorescent molecule that intercalates into
cells undergoing cell death. F. Symbiotic animal at 96 h showing full morphogenesis of the light organ and the loss
of the cea.
Figure 2. Effects of simulated microgravity on Euprymna/Vibrio symbiosis. A. Comparison of the survivability of
aposymbiotic (Apo) and symbiotic (Sym) host squid under gravity (+) and simulated microgravity (-). B. Impact of
microgravity (-) on the luminescence of host squid compared to gravity (+) conditions.
45
microgravity conditions (for review Nickerson et al.,
2004). Animals were collected within 1 h of
hatching, rinsed in filtered seawater (FSW) and either
maintained aposymbiotically (i.e., without V. fischeri
ES114) or were rendered symbiotic by inoculating
with V. fischeri at a concentration of 1 x 105 cells per
ml of aerated FSW. Animals were placed in the
HARV chambers and incubated for up to 72 h at
23°C. A subset of control animals was maintained
under normal gravity conditions in 5 ml of aerated
FSW in borosilicate vials at 23°C. The results of five
replicate experiments (n = 8 animals per treatment)
are depicted in Figure 2A. All of the animals
maintained aposymbiotically under both normal
gravity (n = 40) and simulated microgravity (n = 40)
conditions survived to 72 h. However, in the
symbiotic animals there was a difference in the
survival rate between gravity and simulated
microgravity treatments. In normal gravity conditions
all symbiotic animals survived throughout the
experiment, whereas in symbiotic animals exposed to
simulated microgravity there was a gradual increase
in animal death rate over the course of the three-day
experiment. At 24 h, an average of 98% of the
animals exposed to symbiotic bacteria survived in
each of the five replicate experiments. However, the
mean survival rate decreased to 90% and 75% by 48
and 72 h, respectively. The variability between
experiments also increased over time (Figure 2A) and
may be the result of differences between host squid
size (2-3 mm), or may reflect changes in the symbiont
under microgravity conditions. Despite the gradual
decrease in survival, most symbiotic squid survived
the HARV experiments during the critical time
window (0 - 24 h) when many of the early symbiosis
phenotypes are being initiated. Additional testing of
symbiotic animals using the HARV chambers
perpendicular to the axis of rotation in the
microgravity treatments showed that the survival rate
was 100% (n = 15) after 72 h. These results coupled
with the 100% survival of the aposymbiotic controls
in the HARV chambers under microgravity conditions
indicate that the HARV chamber itself has a minimal
impact on the squid survival. Lastly, animals tested
with flight approved FPA hardware under normal
gravity conditions indicated that the animals survived
up to five days in aerated FSW (data not shown); well
beyond the four-day time window of light organ
morphogenesis.
In addition to basic survival under microgravity
conditions we examined the onset of luminescence, an
early phenotype of the symbiosis and bacterial growth
rate in the HARV chambers. In the experiments
described above, the luminescence of the host squid
was checked by temporarily removing the
aposymbiotic and symbiotic animals from the HARV
chamber for ∼5 min, placing them in borosilicate vials
and inserting the vial into a luminometer to measure
luminescence (GloMax 20/20, Promega, WI). The
results indicated that at 24 h there was no statistical
differences between luminescence levels between
symbiotic animals maintained under normal gravity
and simulated microgravity (Figure 2B). However,
by 48 h there was a significant decrease in the
luminescence output of microgravity-exposed
symbiotic animals. In addition to luminescence there
were also differences detected in the growth rate of
the symbiont under microgravity conditions. V.
fischeri cultures within the HARV were examined and
shown to have a 2-fold increase in growth rate at 24 h
compared to gravity controls (data not shown). These
results indicate that although microgravity does not
impact the initiation of the symbiosis, microgravity
does affect the persistence and maintenance of the
symbiosis compared to the normal gravity controls.
These results may reflect the health of the host
organism, or the flow of oxygen into the light organ
crypt spaces, as luminescence requires aerobic
conditions. However, the bacteria were viable despite
the loss of luminescence. Plating of dissected,
homogenized light organs on sea water tryptone agar
plates revealed no significant loss in cell numbers
between animals exposed to microgravity and gravity
conditions (data not shown).
Together these results suggest that the
Euprymna/Vibrio model system can be successfully
manipulated under simulated microgravity conditions
and that the symbiotic association may be negatively
impacted by microgravity. Further work is required to
elucidate the precise effects that microgravity has on
the symbiotic partners and the overall developmental
time line of the host squid. Understanding the effects
that spaceflight has on the animal/human microbiome
may provide critical insight into maintaining the
health of crew members and decrease their potential
risk during the exploration of space.
ACKNOWLEDGEMENTS
This project was supported by NASA through a
University of Central Florida’s Florida Space Grant
Consortium award to J.F. The authors would also like
to thank Margaret McFall-Ngai, Edward Ruby, and
Nell Bekiares for providing the V. fischeri and egg
clutches, which were made available by an NSF IOS
0817232 award to M.M-N. and E.R.. We also thank
Wayne Nicholson for the use of his HARV chambers;
Louis Stodieck and Mark Rupert of BioServe Space
Technologies for the use of their FPA hardware; and
the Promega Corporation for their donation of the
GloMax 20/20 luminometer to this pilot study.
REFERENCES
Crucian, B. E., Stowe, R. P., Pierson, D. L., and Sams, C. F.
2008. Immune system dysregulation following shortvs long-duration spaceflight. Aviat Space Environ Med
79(9): 835-843.
Hammond, T. G. and Hammond, J. M. 2001. Optimized
suspension culture: the rotating-wall vessel. Amer J
Physiol Renal Physiol. 281(1): F12-25.
McFall-Ngai, M. J. and E. G. Ruby. 1991. Symbiont
recognition and subsequent morphogenesis as early
events in an animal-bacterial mutualism. Science.
254(5037): 1491-1494.
Nickerson, C. A., Ott, C.M., Wilson, J.W., Ramamurthy, R.,
and Pierson, D.L. 2004. Microbial responses to
microgravity and other low-shear environments.
Microbiol Mol Biol Rev. 68(2): 345-361.
Smith, S. M. and Heer, M. 2002. Calcium and bone
metabolism during spaceflight. Nutrition. 18(10): 849852.
Wier, A. M., Nyholm, S.V., Mandela, M.J., MassengoTiasséc, P.R., Schaefera, A.L., Korolevad, I., SplinterBonDurante, S., Brown, B., Manzellad, L., Snir, E.,
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M., Casavantf, T.L., Soares, M.B., Cronanc, J.E.,
Reed, J.L., Rubya, E.G., and McFall-Ngaia, M.J.
2010. Transcriptional patterns in both host and
bacterium underlie a daily rhythm of anatomical and
metabolic change in a beneficial symbiosis. PNAS.
107(5): 2259-2264.
Wilson, J. W., Ott, C. M., Höner zu Bentrup, K.,
Ramamurthy, R., Quick, L., Porwollik, S., Cheng, P.,
McClelland, M., Tsaprailis, G., Radabaugh, T., Hunt,
A., Fernandez, D., Richter, E., Shah, M., Kilcoyne, M.,
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Stafford, P., Catella, L., Schurr, M.J., Buchanan, K.,
Morici, L., McCracken, J., Allen, P., Baker-Coleman,
C., Hammond, T., Vogel, J., Nelson, R., Pierson, D. L.,
Stefanyshyn-Piper, H. M., and Nickerson, C. A. 2007.
Spaceflight alters bacterial gene expression and
virulence and reveals a role for global regulator Hfq.
PNAS. 104(41): 16299-16304.
47
Short Communication
Effects of Slow Clinorotation on Growth and Yield in Field Grown Rice
1
2
1
Sagar S. Jagtap , Kondiram N. Dhumal , and Pandit B. Vidyasagar
1
Biophysics Laboratory, Department of Physics, University of Pune, Pune, MH 411007 INDIA;
of Botany, University of Pune, Pune, MH 411007 INDIA
Several studies concerning with the effects of
microgravity conditions on plant growth have already
been carried out using spacecrafts (Abilov et al.,
1986; Aliyev et al., 1987; Kordyum, 1994).
Clinostats have been used to compensate for the
unilateral influence of gravity for a long period of
time in the field of plant physiology. Several
experiments have been carried out on a slow rotating
(ranging from 0.25 – 3 rpm) clinostat to study the
effects of clinorotation also termed as simulated
microgravity on plant growth and development.
Plants, especially cereal crops, are the basis for
human being to accomplish the food requirement;
hence, it is of great importance to study the influence
of microgravity on growth and development in food
grain crops. Earlier studies carried out on the effects
of clinorotation on plants showed that plant growth,
morphology, chlorophyll content was affected by
slow clinorotation (Sievers et al., 1996; Sack 1997;
Jagtap et al., 2010). Several reports have described the
effects of clinorotation on physiology of plants and
cell biology (Hilaire et al., 1995a), protein expression
(Piastuch and Brown, 1995), carbohydrate
metabolism (Brown and Piastuch, 1994, Obenland
and Brown, 1994), calcium distribution (Hilaire et al.,
1995b) and the cell cycle (Legue et al., 1992). Colla
et al. (2007) showed that morphological and growth
characteristics such as growth, yield, and quality of
the dwarf tomato variety ‘Micro-Tom’ were modified
during clinorotation treatment. They also showed that
clinorotation-exposed seeds were viable and
subsequent generations might be obtained in
microgravity (Colla et al., 2007). In most of the
studies, seeds were germinated and grown under
clinorotation. As per our knowledge, there are no
reports on the effects on changes in growth, yield, and
soluble protein content of seeds from plants that were
grown from clinorotated seeds. In the present
investigation, effects on growth, various yield
attributes as well as seed nutrient contents such as
starch, carbohydrates and protein content in plants
raised from rice seeds (Oryza sattiva L) exposed to
2
Department
slow clinorotation (2 rpm) for 12 days and grown in
the field at normal (1 g) condition were studied.
Authentic seeds of rice (variety PRH-10) were
procured from National Seeds Corporation, New
Delhi, India. Total 600 mature seeds of uniform size
were selected and surface sterilized by using 0.5%
fungicide (Uthane M-45® manufactured by United
Phosphorus Limited), washed 4-5 times with distilled
water (D/W) to remove traces of fungicide, and
soaked in D/W for 24 h. After 24 h soaking, seeds
were removed from D/W and blotted dry with blotting
paper to prevent germination during clinorotation. For
clinorotation treatment, 300 seeds were placed in
cavities made in disc shaped thermacol on a periphery
of a circle of radius 1.5 cm. On each piece of
Figure 1. Plant growth in control and clinorotated
samples. The Control plants are shown on the left and
the plants grown from clinorotated seeds on the right.
Jagtap et al. – Slow Clinorotation on Growth and Yield of Rice
content of rice seeds of plants raised from seeds
exposed to slow clinorotation (2 rpm) for 12 days and
grown in field which is highly important and having a
dietary significance (Table 1). However, there was ~
1% and ~ 5 % reduction in carbohydrate and starch
content respectively. It is well documented that plants
with high chlorophyll concentration exhibit higher
rates of photosynthesis under low light relative to
plants with less chlorophyll pigment concentration
(Bjorkman, 1981; Boardman, 1977). Therefore
increase in growth and various yield attributes in this
study may be due to an increase in chlorophyll
content in clinorotated sample.
Figure 2. Number of panicles in control (left, blue
bowl) and the plants grown from clinorotated seeds
(right, red bowl).
thermacol 25 seeds were placed. About 12 such
thermacol discs containing 300 seeds separated with
cotton were placed in two Perspex® beakers and then
clinorotated for 12 days continuously at 2 rpm
resulting in g ~ 7x10-5 (Jagtap et al., 2010) in dark
under ambient conditions of temperature (23±5 °C)
and humidity (50±10 %). Remaining 300 seeds at
same environmental conditions but without
clinorotation were used as control.
After the
treatment, clinorotated and control seeds packed in
polythene bags were stored at room temperature, and
sent to Agricultural Research Station, Shirgaon,
District-Ratnagiri (Maharashtra) India. On 15th day
from the date of clinorotation treatment, seeds were
sown in the field at Agricultural Research Station,
Shirgaon. Seedlings were transplanted on 40th day
after sowing (DAS). All the measurements such as
plant height, number of productive tillers per plant,
length of panicle, number of tassels, number of filled
grains per panicle, weight of 1000 filled grains,
biological yield per plant, and harvest index were
recorded at the time of harvest, i.e. on 80th day after
transplanting. The results of present study revealed
that the exposure of seeds to slow clinorotation for 12
days had caused significant improvement in paddy
yield and protein content over control.
The appreciable changes were observed in various
yield attributes. Number of productive tillers per
plant, height of tiller from soil level (Figure 1), length
as well as number of panicle (Figure 2), number of
tassels, number of filled grains, total grain weight per
plant, weight of 1000 grains, and economic yield per
plant was significantly increased with clinorotation in
rice (Table 1).
Observations indicate that number of productive
tillers per plant, height of tiller from soil level, length
as well as number of panicles, and also number of
tassels increased in clinorotated samples. Total
chlorophyll content was also enhanced by ~ 28% in
clinorotated samples. Results of present study for the
first time revealed about ~ 6 % increase in protein
Table 1. Effects of clinorotation on different yield
attributes in field grown rice
Attribute
No. of productive tillers /
plant
Control
Clinorotated
14.5±0.7
18.0±0.7*
Height of tiller from soil (cm)
40.0±1.7
42.4±1.3**
Number of tassels
8.0±0.7
10.4±0.6*
Length of panicles (cm)
18.7±0.3
23.3±0.3*
Number of grains per panicle
71.6±0.5
105.4±0.6*
Filled grains per panicle
Number of filled grains per
plant
Total grain weight per plant
(g)
34.6±0.8
46.2±0.1*
493.6±12.9
828.5±17.3*
14.4±0.2
23.3±0.1*
Weight of 1000 grains (g)
20.4±0.01
20.8±0.02*
24.3±0.9*
Biological yield per plant (g)
18.0±0.6
Harvest Index
80.1±3.4
95.9±3.3*
Total Chl content (3rd leaf)
5.71±0.9
7.97±1*
Protein content
11.10%
11.80%
Carbohydrate (Total sugars)
66.90%
66.40%
Starch
Embryo viability (harvested
seeds)
54.60%
51.60%
~ 97%
~ 97%
In comparison with these observations the earlier
studies have shown that the ‘Micro-Tom’ plants
grown under simulated microgravity exhibited a
spreading growth and an increase in internodal length.
The number of flowers per plant was significantly
increased by 32% in clinorotated plants in comparison
to those grown in absence of clinorotation. However,
total fruit yield, small fruit yield, leaf area, leaf dry
weight, fruit dry weight, total dry weight were
decreased significantly in the clinorotated tomato
plants than those grown in the control treatment
(Colla et al., 2007). Also the studies carried out by
Hilaire et al. (1996) confirmed that slow clinorotation
had influenced morphology and ethylene production,
i.e. metabolic process in soybean seedlings.
However, the present study differs in one respect; that
49
Jagtap et al. – Slow Clinorotation on Growth and Yield of Rice
is, the seeds were exposed to clinorotation and plants
were grown under normal (1 g) condition. Hence the
observations in the present study show a marked
difference from the studies reported earlier.
Also the results of tetrazolium test performed on
rice seeds from harvested plants showed ~ 97 %
embryo viability in control as well as clinorotated
seeds suggesting that these seeds have mature embryo
and can be used further for sowing to raise new crop.
Similar results were obtained by Colla et al. (2007)
showing that seeds formed under simulated
microgravity proved to be biologically and
functionally complete, showing that ‘Micro-Tom’
plants could realize complete ontogenesis, from seed
to seed in microgravity (Colla et al., 2007).
In the present investigation seeds exposed to
clinorotation before germination were sowed under
normal (1 g) conditions fifteen days after the
exposure, and compared directly to seeds with
identical heritage that had not been subjected to
clinorotation. Comparisons of the development of
seeds exposed to clinorotation to the development of
the control seeds showed statistically significant
differences in the two sets as the plants matured.
These data suggest that the physiological differences
between the two sets of mature plants are related to
the exposure of the seeds to clinorotation, as all other
aspects of the seed treatment and culture were
controlled between the two sets. This is a novel
observation and may have future benefit for
improving crop productivity of various cereals,
including rice.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge authorities at
the Agricultural Research Station, Shirgaon, DistrictRatnagiri (Maharashtra) India for performing field
trials of our experiments. Financial support was
provided by the Department of Science and
Technology (DST), Govt. of India under the scheme
(GoI-A-563).
REFERENCES
Abilov, Z. K., Alekperov, U. K., Mashinskiy, A. L., and
Aliyev, A. A.
1986.
The morphological and
functional and functional state of the photosynthetic
system of plant cells grown for varying periods under
spaceflight conditions. USSR Space Life Sci Digest, 8:
15-18.
Aliyev, A. A., Abilov, Z. K., Mashinskiy, A. L., Ganiyeva,
R. A., and Ragimova, G. K. 1987. The ultrastructure
and physiological characteristics of the photosynthesis
system of shoots of garden pea grown for 29 days on
the ‘Salyut-7’ space station. USSR Space Life Sci
Digest, 10: 15-16.
Bjorkman, O. 1981. Responses to different quantum flux
densities.
In: Physiological Plant Ecology I.
Responses to the Physical Environment. Encyclopedia
of Plant Physiology New Series, (O. L. Lange, P. S.
Nobel, C. B. Osmond & H. Ziegler, Eds.), Vol. 12A.
pp. 57-107
Boardman, N. K. 1977. Comparative photosynthesis of sun
and shade plants. Ann Rev of Plant Physiol. 28: 355377.
Brown, C.S. and Piastuch, W.C. 1994. Starch metabolism
in germinating soybean cotyledons is sensitive to
clinorotation. Plant Cell Environ. 17: 341-344.
Colla, G., Rouphael,Y., Cardarelli, M., Mazzucato, A., and
Olimpieri, I. 2007. Growth, yield and reproduction of
dwarf tomato grown under simulated microgravity
conditions. Plant Biosystems 141(1): 75 – 81.
Hilaire, E., Paulsen, A.Q., Brown, C.S., and Guikema, J.A.
1995a. Effects of clinorotation and microgravity on
sweet clover columella cells treated with cytochalasin
D. Physiol Plant. 95: 267-273.
Hilaire, E., Paulsen, A.Q., Brown, C.S., and Guikema, J.A.
1995b.
Microgravity and clinorotation cause
redistribution of free calcium in sweet clover columella
cells. Plant Cell Physiol. 36: 831-837.
Hilaire, E., Peterson B. V., Guikema J. A., and Brown C. S.
1996. Clinorotation affects morphology and ethylene
production in soybean seedlings. Plant Cell Physiol.
37(7): 929-934.
Jagtap S. S., Awhad R. B., Santosh B., and Vidyasagar P. B.
2010.
Effects of clinorotation on growth and
chlorophyll content of rice seeds. Microgravity Sci
Technol. DOI 10.1007/s12217-010-9222-9.
Kordyum, E. L.
1994.
Biology of plant cells in
microgravity and under clinostating. Int Rev Cytol.
171: 1-78.
Obenland, D.M. and Brown, C.S. 1994. The influence of
altered gravity on carbohydrate metabolism in excised
wheat leaves. J Plant Physiol. 144: 696-699.
Piastuch, W.C. and Brown, C.S. 1995. Protein expression in
Arabidopsis thaliana after chronic clinorotation. J
Plant Physiol. 146: 329-332.
Sack, F. D. 1997. Plastids and gravitropic sensing. Planta,
203: S63-S68.
Sievers, A., Buchen, B., and Hodick, D. 1996. Gravity
sensing in tip-growing cells. Trends Plant Sci. 1: 273279.
Yoshioka R., Soga K., Wakabayashi K., Takeba G., and
Hoson T. 2003. Hypergravity-induced changes in
gene expression in Arabidopsis hypocotyls. Adv Space
Res. 31(10): 2187-2193.
Short Communication
A Retrospective Analysis on Gender Differences in the Arterial Stiffness
Response to Microgravity Exposure
Eric C. Tuday1, Steven H. Platts3, Daniel Nyhan2, Artin A. Shoukas1, and Dan E. Berkowitz1,2
1
Department of Biomedical Engineering and 2Department of Anesthesiology and Critical Care Medicine, Johns Hopkins
University School of Medicine, Johns Hopkins Hospital, 600 N Wolfe Street, Baltimore, MD, USA, 21287. Email:
[email protected] ; 3Human Adaptation and Countermeasures Division, National Aeronautics and Space Administration
Johnson Space Center, Houston, TX
BRIEF SUMMARY
Several studies indicate that the incidence of
orthostatic intolerance (OI) among female
astronauts is significantly higher than compared to
their male counterparts. Our lab has previously
demonstrated
that
astronauts
who
are
orthostatically tolerant after spaceflight develop an
increase in central arterial stiffness during
spaceflight. We hypothesize that the arterial
stiffness of female astronauts does not increase, or
increases insufficiently, during spaceflight. This
may explain, in part, the gender difference in the
incidence of OI. We explored this hypothesis by
using previously collected hemodynamic data from
astronauts as well as from subjects subjected to -6°
head-down tilt bedrest. Female astronauts had a
non-significant decrease in arterial stiffness
following spaceflight (0.08 ± 0.066 mmHg/ml
N=11, P=0.2405). In contrast, male astronauts
demonstrated a significant increase in arterial
stiffness (0.10 ± 0.04 mmHg/ml N=46, P=0.0145).
Female bedrest subjects had an insignificant
increase in arterial stiffness during bedrest,
(0.17±0.059 mmHg/ml, p=0.0973, N=3); however,
it was not as great as the increase in arterial
stiffness developed by the male subjects
(0.33±0.056 mmHg/ml, p=0.01, N=4). Thus, it is
apparent that there is a gender difference in the
arterial stiffness response to microgravity
exposure. This gender-based differential arterial
stiffness response may explain, in part, the
disparity of microgravity-induced OI incidence
between the genders.
INTRODUCTION
It has been reported that the incidence of
post-spaceflight orthostatic intolerance (OI) is
between 35-100% for women compared to 7-20%
for men (Fritsch-Yelle, et al., 1996; Waters, et al.,
2002). There is a large disparity between males
and females in terms of microgravity induced OI.
It is well known that OI is due to a collection of
maladaptive responses to spaceflight involving the
cardiovascular system. These responses have been
noted in both men and women. However, gender
studies among the astronaut population have
highlighted that female astronauts have further
depressed adrenergic responsiveness, a higher
dependence on plasma volume status, and a
decreased ability to increase total peripheral
resistance, in response to a gravitational stress
following microgravity
compared to male
astronauts (Meck, et al., 2004; Waters, et al.,
2002). Although these findings are all likely
significant contributors to the gender disparity in
OI, they alone may not entirely account for the
difference.
We have recently demonstrated that
orthostatically tolerant (OT) astronauts had an
increase in central arterial vascular stiffness
following
spaceflight while orthostatically
intolerant (OI) astronauts showed either no change
or a slight decrease in this parameter (Tuday, et al.,
2006) We hypothesized that the increase in
vascular stiffness was beneficial for the astronauts
in terms of OI based on the inverse relationship of
arterial stiffness and the resistance to venous return
(RVR). The OT astronauts were able to decrease
their RVR through an increase in arterial stiffness
which allowed for the maintenance of cardiac
output and arterial blood pressure during an
orthostatic stress.
We contend that females
experience little to no change in arterial stiffness
while males exhibit large increases in vascular
stiffness as a result of microgravity exposure. This
would predispose females to OI, and may represent
an underlying cause in the gender disparity in
microgravity induced OI.
51
Tuday et al. – Arterial Stiffness and Microgravity
mmHg/ml on landing day, N=11). Both male and
female blood pressures and stroke volumes are
presented in Table 1.
0.75
0.50
METHODS
RESULTS
Male astronauts demonstrated a significant
increase in arterial stiffness after spaceflight
(0.57±0.024 mmHg/ml pre-flight vs. 0.67±0.032
mmHg/ml at landing day, p=0.0145 N=46) (Figure
1).
Female astronauts did not respond in the same
manner to microgravity as did males despite the
fact they both had similar pre-flight values (Figure
2). The female astronauts demonstrated a trend
toward a significant decrease in arterial stiffness
(0.56±0.060 mmHg/ml pre-flight vs. 0.48±0.027
0.25
0.00
Preflight
Landing Post Landing
Figure 1. Estimated arterial stiffness of male
astronauts ten days before, immediately after, and
three days following a mission. *p= 0.0145. Data are
reported as means +/- SE.
0.75
Stiffness (m m H g/m l)
Human astronaut data was obtained and
analyzed as previously described (Tuday, et al.,
2006). Briefly, all human astronaut data was from
previously published studies, with permission from
the lead researcher, conducted by Meck, et al.
(2004)
The data set consisted of supine
hemodynamic measures taken from 57 short
duration flight astronauts (11.5 +/- 1 days for
females and 12.1 +/- 0.4 days for males, range 5-18
days; p=0.22) usually ten days before launch, on
landing day, and three days after landing. Detailed
methods are available from Meck, et al. (2004) and
Fritsche-Yelle, et al. (1996).
Briefly, blood
pressure was measured using an automated arm
cuff. Aortic cross-sectional area, determined using
two-dimensional echocardio-graphy, and ascending
aortic flow, sampled with pulsed-Doppler, were
used to determine the stroke volume. Arterial
stiffness was estimated as the arterial pulse
pressure (systolic minus diastolic blood pressure)
divided by the stroke volume. The data was then
pooled into male and female groups.
The human bedrest protocol was approved by
the Johnson Space Center Institutional Review
Board, and all subjects gave their written informed
consent. Seven healthy subjects (four males, three
females) underwent 45-60 days (49.5 +/- 3.6 days
for males and 57.7 +/- 2.3 days for females;
p=0.5039) of –6° head-down bed rest.
Hemodynamic data was collected in the same
manner as was done for the astronauts. Arterial
stiffness was estimated as described.
*
S t if f n e s s ( m m H g /m l)
The goal of this study was to determine
whether there is a gender difference in the vascular
stiffness response to microgravity. We hypothesize
that female vascular stiffness does not increase to
the same degree as men in response to actual
(spaceflight) or simulated (bedrest) microgravity.
We have undertaken this study utilizing previously
collected astronaut and bedrest data.
0.50
0.25
0.00
Preflight
Landing
Post Landing
Figure 2. Estimated arterial stiffness of female
astronauts ten days before, immediately after, and
three days after a mission. Data are reported as
means +/- SE.
Table 1. Astronaut mean arterial pressure (MAP)
and stroke volumes (SV) ten days before, immediately
after, and three days after a mission. Data are
reported as means (+/- SD). * p<0.02 compared to
preflight.
Preflight
Landing
Post
Landing
Male
MAP
SV
89 (7)
86.6 (20.4)
94 (10)*
78.8 (21.4)
90 (9)
84.9 (24.9)
Female
MAP
75 (6)
82 (6)*
77 (6)
79.3 (15.7)
77.1 (10.6)
79.2 (17.8)
SV
Tuday et al. – Arterial Stiffness and Microgravity
Male bedrest subjects had a significant
increase in arterial stiffness following bedrest
(0.61±0.11 mmHg/ml pre-bedrest vs. 0.94±0.10
mmHg/ml post-bedrest, p=0.01, N=4) (Figure 3).
Female bedrest subjects had an small increase in
arterial stiffness, though it was less than that of the
male subjects (0.61±0.055 mmHg/ml pre-bedrest
vs. 0.78±0.10 mmHg/ml post-bedrest, p=0.0973,
N=3). Both male and female blood pressures and
stroke volumes are presented in Table 2.
Male
1.25
Arterial Stiffness
(mmHg/ml)
*
Female
1.00
0.75
0.50
0.25
0.00
Pre BR
Pre BR
Post BR
Post BR
Figure 3. Estimated arterial stiffness of bedrest (BR)
subjects. *p= 0.01. Data are reported as means +/SE.
Table 2. Bedrest (BR) mean arterial pressure (MAP)
and stroke volumes (SV) before and after bedrest.
Data are reported as means (+/- SD).
males, the failure of females to increase vascular
stiffness during spaceflight impairs their ability to
lower their RVR. This in turn makes it more
difficult to maintain adequate venous return,
cardiac output, and arterial blood pressure during
an orthostatic stress. Finally, our data present a
strong case for acutal vessel stiffness changes
rather than simply vessel distension; though the
mean arterial pressure was elevated in both male
and female subjects at landing, stroke volume was
decreased (though not significantly) indicate a
greater pressure response to less volume.
Furthermore, both groups had parallel changes
which would not explain the gender discrepancy in
stiffnes. However, caution must be used in
interpreting these results as they are estimates and
not direct measures of stiffness. In conclusion, the
observed gender-based differential stiffness
response may explain, in part, the large disparity of
OI incidence between the genders. The physiologic
and molecular mechanisms underlying this
observation remain to be explored.
ACKNOWLEDGEMENTS
We appreciate the help of the NASA
cardiovascular team. This research was supported
in part by a grant from the NSBRI (CA00405), by a
NIH grant (ROI HL105296), and by a grant from
NASA (BRC-2003-0000-0543).
REFERENCES
Pre BR
Post BR
MAP
SV
86 (8.5)
80.2 (31.4)
96 (4.8)
68.4 (17.3)
MAP
80 (5.8)
76.7 (3.1)
59.7 (4.5)
57.7 (3.6)
Male
Female
SV
DISCUSSION
It is apparent from the data that there is
indeed a gender difference in the arterial stiffness
response to microgravity exposure. This was
evidenced by the significant increases in male
vascular stiffness during both actual and simulated
spaceflight compared to unmatched changes in
their female counterparts. As compared with
Fritsch-Yelle, J.M., Whitson, P.A., Bondar, R.L., and
Brown, T.E. 1996. Subnormal norepinephrine
release relates to presyncope in astronauts after
spaceflight. J Appl Physiol 81: 2134-2141
Meck, J.V., Waters, W.W., Ziegler, M.G., deBlock, H.F.,
Mills, P.J., Robertson, D., and Huang, P.L. 2004.
Mechanisms of postspaceflight orthostatic
hypotension: low alpha1-adrenergic receptor
responses before flight and central autonomic
dysregulation postflight. Am J Physiol Heart Circ
Physiol 286: H1486-1495
Tuday, E.C., Meck, J.V., Nyhan, D., Shoukas, A.A., and
Berkowitz, D.E. 2006. Microgravity Induced
Changes in Aortic Stiffness and its Role in
Orthostatic Intolerance. J Appl Physiol 101:
2751-1759
Waters, W.W., Ziegler, M.G., and Meck, J.V. 2002.
Postspaceflight orthostatic hypotension occurs
mostly in women and is predicted by low vascular
resistance. J Appl Physiol 92: 586-594
53
Short Communication
Metabolic Control as a Strategy for Payload Cost Reduction and
Mitigation of Negative Space Environmental Factors
Eugene Galicia1, Ervin Palma2, Florian Selch3, David Gomez2, Richard Grindeland1, and Yuri Griko1
1
National Aeronautics and Space Administration, Ames Research Center, Moffett Field, CA 94035 USA; 2San
Jose State University, San Jose, CA 95192; 3Carnegie Mellon University Silicon Valley, Moffett Field CA
94035
As human presence in space will likely extend to
the Moon and beyond, such exploration missions will
involve significant risks of accidents, system failures,
and various life-threatening emergency situations.
The ability to put space explorers into a temporary
hypo-metabolic state using metabolic control
technologies would enhance our ability to manage
these risks. Reversible arrest of essential life
processes would radically reduce astronaut needs for
life support and produce extraordinary resistance to
environmental stress, such as radiation exposure and
low-gravity. It can also solve many medical problems
including diseases and physical injury over a long
period in space.
Despite promising avenues of these enquiries,
the mechanism of inducing such reversible
hypometabolic state in non-hibernated animals is not
yet fully understood (Blackstone, et al., 2005; Tøien,
et al., 2011; Haouzi, et al., 2008). We developed the
methodology to achieve metabolic control, which
allows the metabolism of non-hibernated animal
model system (mice) to be reduced to a minimum
level for a significant time and subsequent restoration
to normal level with no apparent effect on physiology
or neurological function. In order to demonstrate the
potential application of the metabolic control
technology we have engineered a hypometabolic
chamber with a range of life-monitoring equipment
Figure 1. Schematic presentation of
the hypometabolic system used in this
study.
Galicia et al. -- Metabolic Control and Space Environmental Factors
for high-throughput testing of hypometabolic
parameters and conditions that enable reversible
induction of a state of suspended animation in mice
(Figure 1). The automated monitoring system is
responsible for initiation, maintenance, and
termination of the stasis through the administration of
hypometabolic agents and providing immediate shortterm monitoring and control.
C57BL/6J male mice from Jackson Laboratories
were used in this study. The animals were 6 weeks of
age at the time of experimentation and weight 20-30
g. Two groups of 5 animals (control and
experimental) were implanted with biotelemetry
transmitters to continuously measure body
temperature and cardiac activity. Induction of the
hypometabolic state in mice was carried out by using
a custom designed atmospheric chamber. Following
short adaptation in air atmosphere mice was exposed
to 80 ppm H2S air mixture at a constant pressure and
flow rate 1L/min to induce hypometabolic state
(Blackstone et al., 2005).
At this point the
environmental temperature was decreased gradually
using programmable temperature control software
from ambient temperature to 15oC. Hypometabolic
state was evident by reduction in metabolic rate and
drop in oxygen consumption and carbon dioxide
output. After 10 hours of exposure to H2S the
atmosphere in the chamber was substituted with air
and mice were returned to room temperature. The
recovery to the normal state was monitored by
metabolic rate and core body temperature returning to
normal. After this stage animals were placed in a
standard vivarium cage for 30 days observation
period. All animal experiments were performed in
strict accordance with National Institutes of Health
guidelines, and animal protocols were approved by
the IACUC at Ames Research Center.
After exposure to the hypometabolic mixture,
core body temperature drops from 37oC to 15oC over
8.5 hours of the gas exposure (Figure 2) leading to
substantial reduction in rate of all biochemical
reactions in organism.
The mouse initially started at a heart rate of 762
beats/minute, then dropped to as low as 120
beats/minute after 10 hours (Figure 3) – a 75% drop
in heart rate.
Figure 3. Heart rate recorded at different body
temperatures. (a) 37oC; (b) 15oC; (c) 37oC, after
recovery from the stasis.
Figure 2. Core body temperature of mice exposed to
H2S using the same protocol and recovered from the
stasis at different times. Control mice were not exposed
to H2S. Each data point is average of three experiments.
Our results show that the hypometabolic stasis
can be significantly extended from 4 hours as
demonstrated in previous study (Blackstone et al.,
2005), up to 11, or even 18 hours (not shown) by
optimization of the transform kinetics and
environmental parameters. Under this condition the
respiratory rates dropped from132 breaths per minute
55
Galicia et al. -- Metabolic Control and Space Environmental Factors
to less than 5 breaths per minute, demonstrating
reduction in metabolism more than 94%. After
recovery from the stasis, none of the hypometabolic
animals displayed any behavior deficits over 3 months
of observation. We have shown that recovery from
the intentionally induced hypometabolic state may be
30% better than in natural hibernators (~47%
lethality) when the kinetics of induction, extension
and recovery from this state are optimized.
Our data provide basis for subsequent animal
studies and novel approaches in further extension of
this state and development of continuous external
monitoring and modification of metabolic conditions.
Despite the obvious limitations of H2S for induction
of hipometabolism in large animals, such as sheep
(Haouzi et al., 2008), its application on small animals
allows investigation of key underlying mechanisms
and consequences of metabolic alteration in nonhibernated animals regardless of stasis protocol.
Some other potential beneficial outcomes of
themetabolic reduction, such as increase in life span,
resistance to ionized radiation, low gravity and many
pathogenisities are currently under investigation.
Investment in a program of research to define
the optimum methods for achieving metabolic control
in larger animals is needed at this time, in order to
demonstrate application of a metabolic control system
within deep space mission architecture as a potentially
enabling technology.
REFERENCES
Blackstone, E., Morrison, M., and Roth, M.
2005.
Hydrogen Sulfide Induces a Suspended AnimationLike State in Mice. Science. 308(5721):518.
Tøien, Ø., Blake, J., Edgar, D., Dennis A., Grahn, H.,
Heller, C., and Barnes, B. 2011. Hibernation in
Black Bears: Independence of Metabolic Suppression
from Body Temperature. Science. 331: 906 -909.
Haouzi, P., Notet, V., Chenuel, B., Chalon, B., Sponne, I.,
Ogier, V., and Bihain, B. 2008. H2S induced
hypometabolism in mice is missing in sedated sheep.
Respiratory Physiology & Neurobiology. 160: 109115.
Short Communication
Iron Ion (56Fe) Radiation Increases the Size of Pre-Existing Atherosclerotic
Lesions in ApoE-/- Mice
Tao Yu1, Brian W. Parks2, Shaohua Yu2, Roshni Srivastava2, Kiran Gupta1, Xing Wu1, Saman
Khaled1, Polly Y. Chang3, Janusz H. Kabarowski2, and Dennis F. Kucik1,4,5
Departments of 1Pathology, 2Microbiology and 4Biomedical Engineering, University of Alabama at
Birmingham, Birmingham, AL; 3SRI International, Menlo Park, CA; 5VA Medical Center, Birmingham, AL
Epidemiological studies have demonstrated that
radiation exposure from a number of terrestrial
sources is associated with an increased risk for
atherosclerosis. For example, for women with early
breast cancer, the benefit of radiotherapy can be
nearly offset by the increased risk of mortality from
vascular disease (Early Breast Cancer Trialists'
Collaborative, G., 2000). In fact, head and neck
cancer patients who undergo radiation treatment are at
significantly elevated risk of stroke, even in a
relatively young patient population. Similarly, atomic
bomb survivors have an increased incidence of
coronary artery disease and stroke. Even radiation
technologists
working
before
1950
(when
occupational exposure was higher) had increased
mortality due to circulatory diseases (Hauptmann et
al., 2003).
The risk of radiation during deep-space travel,
however, is difficult to predict. To date, the only
travel beyond the protection of the Earth’s magnetic
field by humans has been the moon missions. Since
so few astronauts have been exposed to these high
levels of radiation, there is a lack of epidemiological
data available to estimate the cardiovascular risks
involved.
Furthermore, studies of the effects of
cosmic radiation using animal atherosclerosis models
have been lacking.
In addition, astronauts will encounter not only Xrays and γ-rays, which are major components of most
terrestrial radiation, but also cosmic radiation, which
differs from radiation encountered on Earth in
important ways.
Cosmic radiation includes
accelerated ions, which interact with tissues
differently than photons. A small percentage of these
ions are heavier elements such as 56Fe. This small
component of cosmic radiation is particularly
damaging, however, due to its high energy and
propensity to interact with shielding to generate
secondary particles.
Recently, we examined the effect of 56Fe on 10week old apolipoprotein-E deficient (apoE-/-) mice, a
well-established atherosclerosis model (Yu et al., in
D
E
F
Figure 1. Radiation increases atherosclerotic plaque
development in the aortic arch of apoE -/- mice. ApoE -/mice were anesthetized by intraperitoneal injection of
ketamine and xylazine (150 mg/kg and 15 mg/kg body weight
respectively), immobilized in custom-built mouse holders,
and irradiated with 2 or 5 Gy of 56Fe. Thirteen weeks later,
aortae were dissected out, cut open, pinned flat, and stained
with Sudan IV. Images were acquired with a digital camera
attached to a dissecting microscope. A-C are images of the
aortic arch, cut open and pinned flat so that the entire inner
surface is visible. Red = atherosclerotic plaques. (D)
Experimental setup showing mouse in a holder.
A
collimator was used to restrict radiation exposure to a
targeted area slightly smaller than the cutout in the holder.
White boxes over image of dissected mouse (E) and en face
aorta preparation (F) delineate the portion of the aortic tree
exposed to radiation.
57
Yu et al. -- Iron Ion Radiation and Atherosclerotic Lesions
press). These mice spontaneously develop
atherosclerotic lesions while on a normal diet, as do
humans. Radiation was targeted to the aortic arch,
avoiding exposure of other major organs. Thirteen
weeks later, irradiated mice had significantly greater
atherosclerosis than un-irradiated controls. It was not
determined, however, whether this was due to
increased production of new atherosclerotic plaques
or accelerated development of existing lesions.
The current study was undertaken to address this
question. Ten-week-old apoE -/- mice were irradiated
at Brookhaven National Laboratory with 2 or 5 Gy of
600 MeV 56Fe, restricted to the area of the aortic arch.
The mice were then shipped to the University of
Alabama at Birmingham and maintained on a normal
diet. At 13 weeks post-irradiation, the mice were
euthanized and dissected to analyze atherosclerotic
plaque development (Figure 1).
A
% lesion area
4
3
2
1
0
0Gy
2Gy
5Gy
0Gy
2Gy
5Gy
Number of lesions
B
7
6
5
4
3
2
1
0
Therefore, exposure to 56Fe accelerates
development of existing plaques to increase lesion
size. This radiation-induced vessel wall damage might
have serious consequences years after deep space
exploration.
Since asymptomatic atherosclerotic
lesions are common in young, healthy adults (Velican
and Velican, 1980), it is expected that exacerbation of
pre-existing plaques may pose a significant threat for
astronauts.
In this study, a brief exposure to radiation caused
irreversible damage to the artery, resulting in
atherosclerotic progression as the animal aged. This
is consistent with human epidemiologic data, where a
brief exposure to radiation from an atomic blast is
known to increase cardiovascular risk years later.
Interestingly, protracted exposure to relatively low
level radiation, such as that experienced by early
radiation technologists, carries similar risks.
*
5
Images similar to those in Figure 1A-C were then
analyzed to quantify the number and size of the aortic
lesions for each radiation dose (Figure 2). The lesion
size in mice exposed to 5 Gy 56Fe was significantly
greater than in un-irradiated controls (measured as a
percentage of total vessel surface area to control for
differences in stretching of the tissue during
preparation). This difference was not present in unirradiated portions of the aorta. The number of
atherosclerotic lesions, however, was not affected.
Figure 2. Radiation increases lesion area but not lesion
number. 10-week old apoE -/- mice were irradiated once
with the indicated dose of 56Fe, then fed a normal diet
for 13 weeks. Images of dissected aortas (as in Figure 1)
were then analyzed to determine the area of
atherosclerotic lesions as a percent of total vessel area.
While the percent of the irradiated aorta occupied by
atherosclerotic lesions (A) was significantly increased at
5 Gy (p = 0.012 by Mann-Whitney non-parametric test),
the number of lesions (B) was not significantly different.
N = 10 animals in each group.
The dose required to produce this effect was
somewhat higher than what astronauts would
encounter, for example, on a Mars mission. It has
been estimated that astronauts would absorb a
cumulative dose of 0.42 Gy of accelerated ions during
a 1000-day mission to Mars and back (Cucinotta, F.
and Durante, M., 2006). The mice in this study,
however, were exposed to an acute dose of 56Fe over
less than ten minutes, whereas astronauts will be
exposed to a mixture of accelerated ions and highenergy photons over a prolonged period. It is likely
that all components of radiation in the galactocosmic
radiation spectrum, both photons and particles, will
contribute to atherosclerosis.
In addition, mice are known to be relatively
resistant to radiation-induced atherosclerosis. For
example, in a similar study (in which radiation was
similarly restricted to the upper aorta and parts of the
carotid arteries), atherosclerotic changes in apoE -/mice were seen following 14 Gy of X-rays (Stewart,
F.A., et al., 2006). It is known from epidemiologic
studies, however, that X- and gamma rays can be a
risk factor for humans at doses as low as 1Gy. It is,
therefore, quite possible that much lower doses of
56
Fe might have significant effects on atherosclerosis
in humans.
Yu et al. -- Iron Ion Radiation and Atherosclerotic Lesions
We conclude that the potential for 56Fe radiation to
exacerbate
early,
subclinical
atherosclerosis
constitutes a major concern for astronaut health on
prolonged deep-space missions. Further studies will
explore the underlying pathological mechanisms in
order to better estimate risk and develop appropriate
countermeasures.
ACKNOWLEDGEMENTS
This work was supported by the National Space
Biomedical Research Institute through NASA NCC 958.
REFERENCES
Cucinotta, F. A. and Durante, M. 2006. Cancer risk from
exposure to galactic cosmic rays: implications for
space exploration by human beings. Lancet Oncol,
7(5).
Early Breast Cancer Trialists' Collaborative, G. 2000.
Favourable and unfavourable effects on long-term
survival of radiotherapy for early breast cancer: an
overview of the randomised trials.
355(9217): 1757-1770.
The Lancet,
Hauptmann, M., Mohan, A. K., Doody, M. M., Linet, M. S.,
and Mabuchi, K. 2003. Mortality from Diseases of
the Circulatory System in Radiologic Technologists
in the United States.
American Journal of
Epidemiology, 157(3): 239-248.
Stewart, F. A., Heeneman, S., Poele, J. T., Kruse, J.,
Russell, N. S., Gijbels, M., and Daemen, M. 2006.
Ionizing radiation accelerates the development of
atherosclerotic lesions in ApoE(-/-) mice and
predisposes to an inflammatory plaque phenotype
prone to hemorrhage.
American Journal of
Pathology, 168(2): 649-658.
Velican, D. and Velican, C. 1980. Atherosclerotic
involvement of the coronary arteries of adolescents
and young adults. Atherosclerosis, 36(4): 449-460.
Yu, T., Yu., S., Parks, B.W., Gupta, K., Wu, X., Khaled, S.,
Chang, P.Y., Srivastava, R., Kabarowski, J.H.S., and
Kucik, D.F.
(in press).
Iron-ion irradiation
accelerates athero-genesis in the Apo-E mouse model.
Radiat Res.
Gravitational and Space Biology Volume (25) Sept 2011
59
Short Communication
Cellular and Molecular Biology During Spaceflight
1
2
2
2
1
Luchino Y. Cohen , Michel Fortin , Sebastien Leclair , Ozzy Mermut , Genevieve Dubeau-Laramee ,
1
and Daniel Provencal
1
Canadian Space Agency, St-Hubert, Qc, Canada; 2Institut National d’Optique, Quebec City, Qc, Canada
While the International Space Station (ISS) offers
the possibility of maintaining microorganisms, cells,
and tissues, it does not possess the instrumentation
and facilities required to perform modern (analytical)
molecular biology techniques. In the near future and
due to the lack of sample return capacity following
shuttle retirement, state-of-the-art instrumentation
enabling onsite acquisition of molecular data will be
critically needed. Space-adapted technologies will
also serve to develop in situ medical diagnostic tools
for future manned spaceflights. The Canadian Space
Agency has selected a few innovative technology
platforms intrinsically endowed with a very high
potential for supporting in situ biomolecular analysis
and biodiagnostics. These systems were selected for
their versatility, their potential as robust, portable and
compact instruments, and their capacity to detect and
quantify a large spectrum of macromolecules and
biomarkers.
This paper will focus on the
development of a miniaturized fiber optic flow
cytometer for space life science research and on-board
medical diagnostic.
One of the most versatile analytical instruments in
molecular biology is a flow cytometer. By using
fluorescent dyes or fluorochrome-coupled antibodies,
a flow cytometer can be used to perform
immunophenotyping, intracellular staining, gene
expression studies, signal transduction studies, and
many assays such as calcium flux, proliferation,
apoptosis, cytokine secretion, etc. (Bonetta, 2005).
Once cell suspensions are labeled with fluorescent
markers, they are excited by one or several light
sources at specific wavelengths and the emitted light
is collected and analyzed to perform quantitative
molecular assessment.
Alternatively by using
microbead-coupled antibodies, concentration of
multiple soluble analytes (hormones, microbial
molecules, biomarkers) can be quantified. The
availability of such an instrument in Low Earth Orbit
(LEO) would significantly accelerate space life
science research, reduce the requirement to bring
samples back to investigators, decrease sample
storage needs on the ISS and support crew medical
monitoring (i.e. immunology, hematology sampling
amongst others). A first attempt to determine whether
a flow cytometer could function in microgravity was
performed using NASA KC-135 reduced gravity
research aircraft (Crucian and Sams, 2005).
However, even if they could withstand launch,
conventional flow cytometers would be restricted for
use in ISS due to their need for manipulation by
highly qualified personnel, and cumbersome optical
and fluidic management requirements. The current
flow cytometers available on the market, with their
footprint, complexity and fragility are not compatible
with the constraints of human spaceflight. Moreover,
experience has shown that in particular, fluidic
interactions in microgravity are highly affected. For
instance, tiny air bubbles that naturally move to the
top of a ground-based apparatus can coalesce in a
side-mounted pump cavity in microgravity,
preventing it to prime. In consideration of capillary
and/or centrifugal fluidics forces, which are foremost
at play, a miniaturized fiber-based and sheathless flow
cytometer, would have substantial advantages in its
acceptance as standard crew equipment. Another
advantage of a small, portable system is that it can be
battery operated, simplifying the interface restrictions
and handling, while increasing the flexibility for
operation locations within the ISS. A cytometer that
can be independently used by the crew, without
requirements for call down, specific time-dependant
data transfers or long and complex operational
procedure, for instance, will also gain valuable time
as well as maximum utilization efficiency with the
crew.
As shown in Figure 1, a new compact and portable
flow cytometer (Microflow-1) is under development
based on a fiber-optic flow cell (FOFC) technology
designed at the ‘Institut National d’Optique’ (INO) in
Canada. A special optical fiber has been designed and
engineered, through which a square hole is
transversally bored by laser micromachining. A
capillary is fitted into that hole to flow analyte within
Cohen et al. -– Cellular and Molecular Biology and Space Flight
the fiber square cross-section for detection and
counting (Figure 2).
Figure 3. Comparison between FACSarray (right panel)
and Microflow-1 (left panel) using LinearFlow Deep Red
beads.
Figure 1. Fiber optic flow cytometer: Microflow-1.
Figure 2. Fiber-optic flow cell (FOFC) concept.
With demonstrated performance benchmarks
potentially comparable to commercial flow
cytometers, this novel FOFC technology provides
several advantages compared to classic free-space
configurations, mainly, sheathless flow, low cost,
reduced number of optical components, no need for
alignment (occurring in the fabrication process only),
ease-of-use,
miniaturization,
portability,
and
robustness. This sheathless configuration, based on a
fiber optic flow module, renders this fiberized
cytometer amenable to space-grade microgravity
environments.
Laboratory tests performed at the Canadian
Space Agency (CSA) have confirmed that using
LinearFlow Deep Red fluorescent beads (Molecular
Probes) as well as anti-CD4-labeled Jurkat T cell
lines, performance of the Microflow-1 are similar to
the one of the FACSarray system (Becton-Dickinson)
in terms of Coefficient of Variation (CV), detection
efficiency, sensitivity, and background.
A micro-flow cytometer would strongly enhance
the capabilities of a diagnostic system designed to
monitor spacecraft environment as well as crew health
status (Cohen, 2008). Flow cytometers are now
routinely used in clinics to perform cell-based assays
for real-time determination of leukocyte counts,
immune system activation, identification of abnormal
DNA content due to the presence of tumor cells, as
well as HIV infection based on CD4 T cell counts
(Brown and Wittner, 2000). Beside medical
diagnostic applications for astronauts, such an
instrument will be invaluable to collect real-time data
from gravitational biology experiments for instance
using GFP reporter genes in transformed cell lines.
The possibility to use microbead assays such as the
Cytometric Bead Array (CBA) assay (BD
Biosciences) further broadens the application
potential of this platform to molecular detection. The
principle of the CBA assay is to mix several
populations
of
antibody-coupled
fluorescent
microbeads to a sample of 25 µl to 50 µl of biological
fluid (lysate, serum, or cell supernatant), the beads
playing the role of solid support in the suspension.
Microbeads are linked to specific molecules within
the sample through antibodies, and fluorochromeconjugated antibodies are added to the sample to form
complete sandwich complexes. For instance, the
Human Th1/Th2 Cytokine assay (BD Biosciences)
can be used to simultaneously quantify the
concentration of six cytokines. Such assay can be
customized by using antibodies against soluble factors
of interest for space life science experiment and can
then be performed in situ on the ISS or future
spacecrafts.
The key feature sets of portability, affordability,
and inherent ruggedness of the micro-flow cytometer
concomitantly render it ideal as a point-of care (PoC)
tool. This is mainly attributed to the usage of the core
fiber-optic flow cell technology, and compatibility
with low-cost optical sources (i.e. diode lasers,
Gravitational and Space Biology Volume 25(1) Sept 2011
61
Cohen et al. –- Cellular and Molecular Biology and Space Flight
LEDs). It also has the ability to translate to a handheld
device for both absolute count and assessment of
cellular and molecular biomarkers. One of the major
commercial drivers for the development of such a
device is to enable regular and continuous monitoring
of immunologically compromised individuals. For
example, patients requiring partial white blood cell
differentials analysis, HIV and acute leukemia testing,
or sepsis monitoring, can be accommodated within
access restricted areas (i.e., war zones, economically
challenged countries, where resources are scarce and
test simplicity is highly valued). To this aim, we have
conducted initial pilot benchmarking studies of our
Microflow technology (comparing to commercial
cytometers) for CD45 and CD4 cell staining in the
context of PoC HIV testing and T-cell monitoring
applications. Using standard methods and protocols in
lysed whole blood samples, the outcome was very
promising, indicating the ability to clearly distinguish
monocyte, lymphocyte and granulocyte populations,
based on CD45 expression and light scattering
channels (Figure 4). The results compared well in
terms of sensitivity and CV performances parameters
to current commercial cytometer technologies.
The development of biomedical devices for PoC
testing is a growing worldwide trend and is now a
focus of the Canadian program in Space Life
Sciences. Such instrumentation will support state-ofthe-art experiments and accelerate research in
gravitational biology by providing real-time data
while decreasing the requirement for sample return.
Figure 4. Discrimination of leukocyte populations in whole blood using a Partec Cyflow (left panel) and the
Microflow-1 (right panel) based on light scatter and CD45 expression.
REFERENCES
Bonetta, L. 2005. Flow cytometry smaller and better.
Nature Methods, 2 (10): 785-794.
Brown, M, and Wittner, C. 2000. Flow Cytometry:
Principles and Clinical Applications in Hematology.
Clinical Chemistry 46: 1221-1229.
Cohen, L.Y., Vernon, M., and Bergeron, M.G. 2008. New
molecular technologies against infectious diseases
during spaceflight. Acta Astronautica 63: 769-775.
62 Gravitational and Space Biology Volume (25) Sept 2011
Crucian, B. and Sams, C. 2005. Reduced Gravity
Evaluation of Potential Spaceflight-Compatible
Flow Cytometer Technology. Cytometry B (Clinical
Cytometry) 66B:1-9.
Short Communication
Differentiation of Dental Stem Cells in Simulated Microgravity
Colleen Conlon1,2, Vishnu S. Valluri1, Jagan V.Valluri1, and Pier Paolo Claudio2,3
Marshall University, Joan C. Edwards School of Medicine, 1Department of Biological Sciences, 2Department of
Biochemistry and Microbiology, 3Department of Surgery, One John Marshall Drive, Huntington, WV 25755,
USA
Stem cell niches are present in many different
tissues and organs, however, there are currently no
reliable methods to induce stem cells to differentiate
into specific cell types or to generate enough cells for
transplantation, limiting their application in clinical
therapy. Among these tissues, dental pulp, entrapped
within the 'sealed niche' of the pulp chamber, is an
extremely rich site for collecting stem cells. These
stem cells are called DPSCs (dental pulp stem cells),
which can differentiate into various cytotypes such as
neural progenitors, adipocytes, myotubes, and preosteoblasts under particular conditions. The focus of
this study was to optimize conditions for adult stem
cell purification, proliferation, and differentiation.
The first two of these objectives were
accomplished by culturing either stem cells isolated
from dental pulp (DPSCs) or HEPM cells (preosteoblasts) purchased from ATCC in 2D monolayer.
The second objective was accomplished by culturing
the DPSCs and HEMP cells in the rotary cell culture
(RCC) bioreactor (Synthecon, Houston, TX) for 72hours to investigate the potential of osteogenic
differentiation.
The RCC provides a 3D environment with
simulated microgravity, which is optimal for stem cell
differentiation. It has been previously shown that the
RCC environment induces differentiation (Facer et
al., 2005; Ko et al., 2007). Successful differentiation
of adult stem cells was determined by investigating
several osteogenic differentiation markers using flow
cytometry. This novel technique for stem cell
differentiation can potentially provide new therapeutic
option for bone tissue regeneration.
INTRODUCTION
As the aging population increases, more people
will become reliant on regenerative medicine. Likely
situation of necessary regenerative medicine is bone
augmentation for partially edentulous jaws, followed
by the use of dental implants. For the best biologic,
biomechanical, and aesthetic results of these implant
rehabilitations, appropriate quality and quantity of
bone available at the surgical site is essential for
stable implant placement. The placement of an
implant into a defective osseous site not only prevents
adequate positioning of the final prosthetic
restoration, but also results in poor osseointegration
and subsequently, a poor prognosis for the therapeutic
outcome. Current approaches in bone reconstruction
use biomaterials, autografts, or allografts, although
restrictions on all these techniques exist (Papa et al.,
2005; Papa et al., 2009). These restrictions include
donor site morbidity and donor shortage for
autografts, immunologic barriers for allografts, and
the risk of transmitting infectious diseases.
Artificial bone substitutes containing metals,
ceramics, and polymers have been introduced to
maintain bone function; however, each material has
specific disadvantages, and none can adequately
substitute for current autograft therapies.
A viable alternative to current autograft
procedures is osseous tissue engineering for tissue
regenerative medicine. Our laboratory (as well as
other researchers) has been investigating the effects of
simulated microgravity on osteoblast differentiation
as a means to study cellular and molecular
mechanisms associated with 3D osseous tissue growth
(Ko et al., 2007). Several studies have analyzed the
effect of simulated microgravity on osteoblast
differentiation using established osteoblast cell lines
(Ko et al., 2007). These studies demonstrated
increased cellular aggregation and enhancement of
osteoblast differentiation using the RCC bioreactor.
A theoretical way to circumvent problems
associated to currently used autologous or allogeneic
grafting materials could be to grow adult stem cells
such as stromal cells isolated from adult human dental
pulp (DPSCs) in a microgravity environment to
enhance the rate of osteoblast differentiation and
mineralization.
Thus, the objective of this current study was to test
the hypothesis that simulated microgravity
environments created by RCC would enhance
osteoblast differentiation and osteogenic gene
expression.
63
Conlon et al. -– Differentiation of Dental Stem Cells in Simulated Microgravity
METHODS
Dental pulp extraction and digestion
Human dental pulp was extracted from teeth of
healthy adult subjects aged 21–45 years. Before
extraction, each subject was checked for systemic and
oral infection or diseases. Only disease-free subjects
were selected for pulp collection. Each subject was
pretreated for a week with professional dental
hygiene. Before extraction, the dental crown was
covered with a 0.3% chlorexidin gel (Forhans, New
York, NY), for 2 min. Dental pulp was obtained by
means of a dentinal excavator or a Gracey curette.
The pulp was gently removed and immersed in a
digestive solution: penicillin 100 U/ml/streptomycin
100 mg/ ml, 0.6 ml claritromycin 500mg/ml in 4ml
PBS 1M, with 3 mg/ml type I collagenase, 4 mg/ml
dispase for 1 h at 37°C. Once digested, the solution
was filtered through 70 mm Falcon strainers (Becton
& Dickinson, Franklin Lakes, NJ) (Graziano et al.,
2008).
Cell culture
After filtration, the DPSCs were placed in DMEM culture medium supplemented with 10% heat
inactivated FBS, 2mM glutamine, 100 U/ml
penicillin, 100 mg/ml streptomycin (all purchased
from Invitrogen, Carlsbad, CA) and placed in 75 ml
flasks with filtered caps. Flasks were incubated at
37ºC in a 5% CO2 and the medium changed twice a
week. As soon as cells became confluent, they were
subdivided into new flasks. Several passages were
performed in total. Stem cells were sorted using a
FACsorter (Becton & Dickinson) and the mouse antihuman CD117 (c-kit) and CD34 antibodies.
10758), Osteocalcin (#sc-30044), and Osteonectin
(#sc-25574) purchased from SantaCruz (Santa Cruz,
CA), and against Osteopontin (#2671-1) purchased
from Epitomix (Burlingame, CA), following standard
protocols. Briefly, cells were detached using 0.02%
EDTA in PBS and pelleted (10 min at 1,000 rpm),
washed in 0.1% BSA in PBS at 4°C, and incubated in
a solution of 1µg antibody + 9 µl 0.1% BSA in 1X
PBS. Cells were washed in the same solution once
and were processed for sorting (FACS Aria, BD
Bioscience, San Jose, CA).
RESULTS
Compared with pre-osteoblasts cultured in 2D
tissue culture plastic environments, cells grown in 3D
simulated microgravity environments (Figure 1) show
a very distinctive pattern of cell growth. By 24-hours,
cells cultured in the RCC started to aggregate. These
3D small aggregates coalesced into larger aggregates
of approximately 7 mm in size by 72-hours (Figure
1).
Gross light microscopic analysis of aggregates
isolated from 3D RCC revealed a mass that appeared
tissue-like with a white, shiny, translucent surface,
similar to that of cartilage or bone. The harvested 3-D
cells were analyzed by flow cytometry to characterize
osteogenic markers. The expression of Osteocalcin
and RUNX-2, which have been described as
osteogenic markers, were increased following culture
of the dental stem cells in the RCC bioreactor as
observed by flow cytometric analysis (Figure 2).
As a control, HEPM 1486; ATCC pre-osteoblasts
were cultured in EMEM supplemented with 10% FBS
2mM glutamine, 100 U/ml penicillin, 100 mg/ml
streptomycin (all purchased from Invitrogen,
Carlsbad, CA) and placed in 75 ml flasks with filtered
caps.
Simulated microgravity cultures
Cells were counted and 2x107 cells were placed in
a 50 mL Synthecon rotating vessel RCC for 72-hours.
Rotation was set at 10 RPM for the first 24-hours and
then was increased to 30 RPM for the next 48 –hours.
Cells were then removed, and dissociated from the
formed 3D aggregate. Cells were counted again using
trypan blue to determine cell viability and reacted
with fluoresceinated antibodies for flow cytometric
analysis.
Flow cytometry analysis
Cells were analyzed by flow cytometry using
antibodies against Stro-1 (#sc-47733), RunX-2 (#sc-
Figure 1. 3-D RCC chamber aggregate at 72-hours
showing a cell aggregate measuring 7 mm across.
Conlon et al. -– Differentiation of Dental Stem Cells in Simulated Microgravity
Figure 2. Expression of osteogenetic markers in dental stem cells cultured in simulated microgravity condition for
72-hours.
Interestingly, the expression of Stro-1, a marker of
stemness, dramatically decreased, indicating that the
dental stem cells underwent differentiation following
the RCC treatment. The decrease of other markers of
early differentiation such as osteopontin, also
indicates that the cells were committed to differentiate
toward the osteogenic lineage. The RCC derived 3D
specimens will be further characterized by
immunohistochemistry analysis and morphological
staining.
REFERENCES
Facer, S.R., Zaharias, R.S., Andracki, M.E., Lafoon, J.,
Hunter, S.K., and Schneider, G.B. 2005. Rotary
culture enhances pre-osteoblast aggregation and
mineralization. J Dent Res, 84: 542-7.
Graziano, A., d'Aquino, R., Cusella-De Angelis, M.G., De
Francesco, F., Giordano, A., Laino, G., Piattelli, A.,
Traini, T., De Rosa, A., and Papaccio, G. 2008.
Scaffold's surface geometry significantly affects
human stem cell bone tissue engineering. J Cell
Physiol, 214: 166-72.
Ko, Y.J., Zaharias, R.S., Seabold, D.A., Lafoon, J., and
Schneider, G.B. 2007. Osteoblast differentiation is
enhanced in rotary cell culture simulated
microgravity environments. J Prosthodont, 16: 4318.
Papa, F., Cortese, A., Maltarello, M.C., Sagliocco, R.,
Felice, P., and Claudio, P.P. 2005. Outcome of 50
consecutive sinus lift operations.
Br J Oral
Maxillofac Surg, 43: 309-13.
Papa, F., Cortese, A., Sagliocco, R., Farella, M., Banzi, C.,
Maltarello, M.C., Pellegrini, C., D'Agostino, E.,
Aimola, P., and Claudio, P.P. 2009. Outcome of 47
consecutive sinus lift operations using aragonitic
calcium carbonate associated with autologous
platelet-rich plasma: clinical, histologic, and
histomorphometrical evaluations. J Craniofac Surg,
20: 2067-74.
65
Short Communication
Another Go-Around: Revisiting the Case for Space-Based Centrifuges
Laurence R. Young1, Erika B. Wagner1, Joan Vernikos2, Jessica E. Duda3, Charles A. Fuller4,
Kenneth A. Souza5, Cynthia Martin-Brennan6, and Christopher P. McKay5
1
Massachusetts Institute of Technology; 2Thirdage, LLC; 3Aurora Flight Sciences Corp.; 4U. CaliforniaDavis; 5NASA Ames Research Center; 6American Society for Gravitational and Space Biology
Please note that the following paper reflects the
personal opinions of the listed co-authors. These
opinions and recommendations are based on our
discipline expertise in the areas of gravitational
biology and artificial gravity. None of the following
comments are official NASA or U.S. government
positions.
The need for space-based centrifuges for both
research applications and astronaut countermeasures
has been articulated for decades. Key reviews and
reports from NASA and the space life sciences
community have long identified artificial gravity
(AG) facilities as a top priority for the gravitational
biology and aeromedical communities:
There was unanimity of opinion that
any major adverse effects of spaceflight
could probably be prevented by creating
an artificial gravitational field within the
spacecraft… The need for a centrifuge on
future flights is of the highest priority
(NRC, 1979).
Variable Force Centrifuge…is the
single most important facility in any life
sciences program…[and] should increase
the scientific return from space
experiments by orders of magnitude…A
VFC is an essential instrument for the
future of space biology and medicine
(NRC, 1987).
Whether used in the near-term to
facilitate human missions to Mars, or put
off until developing missions to
destinations farther away, artificial
gravity will eventually be required to
protect humans exploring space (IAA,
2009).
The
cancellation
of
the
Centrifuge
Accommodation Module planned for ISS left life
sciences researchers with no rotational or glovebox
facilities for flight investigations, and reopened the
call for such a core capability. As recently as 2009,
approximately 10 percent of the nearly 150 position
papers publicly submitted to the ongoing National
Academies’ Decadal Survey on Biological and
Physical Sciences in Space included some mention of
centrifugation or artificial gravity (National Research
Council, 2010), including both a key position paper
from the Aerospace Medical Association and a highly
focused white paper representing the views of 18
senior investigators and engineers in the field.
Together,
these
documents
make
specific
recommendations for developing an effective artificial
gravity regimen to protect astronaut health through a
combination of ground and flight resources for
studying cells, animals, and humans.
Although free-flyers, such as the Russian
Bion/Biocosmos, can test some animals and provide
for centrifugation, only the ISS will currently permit
both animal and human testing of artificial gravity.
Once the ISS ceases operation, our opportunities to
explore the efficacy of AG as a human
countermeasure will be limited to yet undefined
opportunities on commercial or other spacecraft.
Clinical
deconditioning
associated
with
microgravity exposure is dramatic and progressive,
with changes markedly faster than age-related
declines and recovery in some systems incomplete
after as long as a year postflight (Buckey, 2006).
Serious commitment to exploration-class missions,
whether or not they involve surface stays, requires
more effective and efficient countermeasures, with
better knowledge of gravity threshold requirements
and clearer prescription of how much, how often, how
long such loads must be applied. This requires
understanding the role of gravity in biology and
physiology, and will not be adequately met by current
approaches based on ground-based trials. To do so
requires the ability to test a range of living organisms,
including humans, using space-based rotating
facilities. Importantly, such experiments are also
relevant to understanding the role of gravity in
Young et al. -– Case for Space-Based Centrifuges
maintaining health on Earth, as well as the nature of
life elsewhere in the Universe.
The needs of the life sciences community with
respect to artificial gravity systems are not
monolithic, nor is the appropriate solution likely to
be. Core themes that could be addressed with flight
centrifuges are enumerated below:
1. Countermeasure development and testing
(a) Efficacy: Artificial gravity is the only known
integrated countermeasure designed to address
all physiological systems affected by
microgravity exposures. However, while the
efficacy of very large rotating vehicles operating
at 1-g is generally accepted by the community,
the effectiveness of short-radius intermittent
exposures still needs substantial research
(International Academy of Astronautics, 2009).
Successful ground studies (Young and Paloski,
2007; Warren, et al., 2006) require flight
validation.
(b) Responses to rotation: Numerous studies have
shown that short-radius centrifugation up to 30
rpm is acceptable on the ground (e.g. Iwasaki, et
al., 2001); however, an in-flight human
countermeasure feasibility test is needed, which
can also answer fundamental questions about the
role of gravity in vestibular function, serving the
basic research community.
2. Fundamental gravitational biology
(a) 1-g control: Among the greatest criticisms of
microgravity research efforts to date has been
the inability to accurately distinguish the effects
of reduced gravity from other factors in the
flight environment, including launch and entry
loads, radiation, stress, etc. An appropriate 1-g
control environment would substantially
improve the quality of research conducted on
ISS.
(b) Partial-g responses: Understanding biological
responses to partial gravity environments
between microgravity and 1-g is a key
unexplored area of both basic and applied
research. Partial gravity data is vital for
understanding the fundamental mechanisms of
mechanosensing, and for designing potential
countermeasures appropriate for Lunar and Mars
exploration. Research into plant and animal
response at partial gravity will be essential to
planning long-term life support systems to
support lunar and Mars bases, and animal results
will provide an early indication of the magnitude
of any problems for long-term human bases. This
information is needed as early as possible in the
design and planning for extended human stays on
the Moon and Mars.
3. Systems architecture trades
Despite dozens of white papers and trade studies
since the earliest days of human spaceflight, the
concept of a large-radius rotating vehicle has yet
to be demonstrated in flight. Hardware designs
abound, from deployable tethers and booms to
more traditional assembled structures. If such an
architecture is to be seriously considered for
exploration-class missions, a successful flight
with living organisms using a free-flyer platform
would be a critical first step towards future
crewed flights.
Table 1. Alignment between research themes,
model organisms and flight platforms
*Free-flyers do not at this time permit human testing.
1-g control
Partial-g
responses
Responses
to rotation
Efficacy
Systems
architecture
trades
Cells
Animal
X
X
X
X
X
ISS
Free
flyer*
X
X
X
X
X
X
X
X
X
X
Human
X
X
Interest from Japan, ESA, Russia, and China has
been growing; we have recently visited ground
centrifuges and discussed AG research needs with
colleagues in Nagoya, Xi’An, Toulouse, Cologne, and
Moscow. Separate ESA Topical Teams are at work on
flight animal centrifuges and large radius ground
centrifuges. Furthermore, JAXA, ESA, and NASA
are reviewing the AGREE project (Iwase, et al., 2010)
studying the requirements and practicality of the
placement of a short-arm human centrifuge on the
ISS. All participants appear convinced of the
importance of international cooperation in the further
research on both the scientific and countermeasure
aspects of AG. This is consistent with NASA’s
renewed commitment to ISS research, transformative
technology and international cooperation.
We strongly recommend that NASA fund
immediate technical evaluation of the
readily available flight technologies, with
the aim of a rapidly deployed flight system
or systems designed to deliver near-term
science results that can clearly
recommend
for
or
against
the
development of more expensive and
complex systems.
67
Young et al. –- Case for Space-Based Centrifuges
A subset of such approaches, any of which we
estimate could be readied for flight in less than three
years, given appropriate resources, include:
• Requalifying the Neurolab Rotator previously
flown for human research on STS-98;
• Design of a small, human-powered portable
centrifuge for ISS, drawing on substantial
ground experience (e.g. Clément and Buckley,
2007);
• Adaptation or reflight of the Russian Biocosmos
centrifuge being readied for animal flights in
2013;
• Possible salvage of elements of the Japanese
CAM rotor and supporting hardware;
• Leveraging existing CAM experience and
designs to prepare a double rack-scale small
animal centrifuge for use on ISS or a free-flyer
platform.
REFERENCES
Buckey, J.C. 2006. Space Physiology. New York, NY:
Oxford University Press.
Clément, G.A. and Buckley, A. 2007. Artificial Gravity.
New York, NY: Springer.
International Academy of Astronautics, 2009. Study on
Artificial Gravity Research to Enable Human Space
Exploration.
Iwasaki, K.I., Sasaki, T., Hirayanagi, K., and Yajima, K.
2001.
Usefulness of daily +2Gz load as a
countermeasure against physiological problems
during weightlessness. Acta Astronautica. 49:227-35.
Iwase, S., Sugenoya, J., Nishimura, N., Paloski, W., Young,
L., van Loon, J., Wuyts, F., Clément, G., Rittweger,
J., Gerzer, R., and Lackner J. 2010. Artificial
Gravity with Ergometric Exercise on International
Space Station as the Countermeasure for Space
Deconditioning in Humans.
Life Sciences
Symposium, Trieste.
Depending on upmass, downmass, and volume
availability, systems proposed for use on ISS could be
flown in the existing Station volume, or mated to a
docked transfer module such as an ATV or COTS
platform. Free-flyer approaches could similarly
leverage either a dedicated platform, or commercial
orbital access, e.g., SpaceX DragonLab or Bigelow
inflatable platforms. While it might be possible to
mount biology research payloads to a human
centrifuge, or even to mate a human platform to a
biology centrifuge rotor, we see this added
complexity as a barrier to rapid progress and support
parallel evaluation of the strategic and scientific value
of independent human and animal system designs.
National Research Council Space Studies Board, 2010.
Decadal Survey on Biological and Physical Sciences
in Space.
We believe firmly that there is an engaged
community, both domestically and across our ISS
partner nations, ready to respond to opportunities
aligned with any of the options presented above. A
coordinated, collaborative approach could provide
answers to pressing scientific and operational
questions. Success is crucial to safe planning and
execution of exploration programs, and holds
substantial promise for basic research. Space agencies
interested in human space exploration should not let
this opportunity pass by again.
Young, L.R. and Paloski, W.H. 2007. Short radius
intermittent centrifugation as a countermeasure to
bed-rest and 0-G deconditioning: IMAG pilot study
summary and recommendations for research. Journal
of Gravitational Physiology 14:P31-3.
National Research Council Space Science Board, 1979.
Life beyond the Earth’s Environment. The Biology of
Living Organisms in Space.
National Research Council Space Science Board, 1987. A
Strategy for Space Biology and Medical Science for
the 1980s and 1990s.
Warren, L.E., Paloski, W.H., and Young, L.R. 2006.
Artificial gravity as a Multi-system countermeasure to
Bed Rest Deconditioning:
Preliminary Results
(Abstract).
20(1):47.
Short Communication
Informative Mapping by VESGEN Analysis of Venation Branching
Pattern in Plant Leaves Such as Arabidopsis thaliana
1*
Patricia Parsons-Wingerter and Mary B. Vickerman
1
1
John Glenn Research Center, National Aeronautics and Space Administration, Cleveland, OH 44135 USA
*Corresponding Author
Humans face daunting challenges to successful
extra-terrestrial exploration and colonization,
including adverse alterations in gravity and radiation.
The Earth-determined biology of plants, animals and
humans is significantly modified in space
environments. One physiological requirement shared
by larger plants with humans and animals is a
complex, highly branching vascular system. Genetic
programs for these vasculatures respond sensitively to
alterations in cellular metabolism, immunological
status, and other specialized cellular/tissue signals
resulting from environmental effects.
VESsel GENeration Analysis (VESGEN) software
is being developed at NASA Glenn as a mature, userinteractive research computer code for mapping and
quantification of the fractal-based complexity of
vascular branching. Change in vascular branching
pattern provides an informative read-out of alterations
in complex regulatory signaling pathways. VESGEN
has provided novel insights into the cytokine,
transgenic, and therapeutic regulation of pathological
and physiological angiogenesis, lymphangiogenesis,
and other microvascular remodeling phenomena (Liu
et al., 2009; McKay et al., 2008; Parsons-Wingerter et
al., 1998, 2000a&b, 2006a&b, 2010). Vascular
morphology is mapped and quantified by Userspecified selection from the VESGEN Tree, Network
and Tree-Network Composite Analysis Options.
Innovative applications described in these studies
include disease progression from ophthalmic clinical
images of the human retina, experimental regulation
of vascular remodeling in the mouse retina, avian and
mouse coronary vasculature, and other experimental
models in vivo reviewed recently by Vickerman et al.
(2009).
As a feasibility study, we demonstrate here that
alterations of venation pattern in the leaves of plants
flown on ISS such as Arabidopsis thaliana can be
Figure 1.
Mapping of Developing Leaf Venation
Pattern by VESGEN. Binary vascular patterns (a, d)
extracted from microscopic images of terrestrially
grown Arabidopsis thaliana leaves at Day 2 (D2) and Day
8 (D8) were analyzed by VESGEN software. To provide
useful quantification (Table 1), the binary vascular
patterns (a, d) were mapped as large structural (1°-2°)
veins and small reticular veins (≥ 3°, b, e) according to
established rules of leaf venation architecture. 10 Other
venation groupings determined by branching orders are
available to the User in VESGEN. Note the large
increase in leaf size from Day 2 to Day 8 (e). By an
alternative VESGEN representation (c, f), for example,
distance mapping displays the local thickness of vessel
diameter throughout the vascular tree-network
composite. Black indicates avascular spaces enclosed
by, and quantified for, the tree-network structures.
69
Parsons-Wingerter and Vickerman –- VESGEN Analysis of Venation Pattern
analyzed successfully by VESGEN.
Although
venation patterning in plants such as A. thaliana has
not yet been characterized in ISS experiments, such
patterning is clearly important for plant health and
applications in space. More generally, terrestrial leaf
venations display valuable, genetically determined
‘signature’ patterns that, when mapped and
quantified, would contribute greatly to fundamental
and applied botanical systematics (Ellis et al., 2009).
Finally, the richness of terrestrial biodiversity was
attributed recently to the sudden, increased
complexity in leaf venation pattern during the Early
Cretaceous Period (Pennisi, 2010).
We therefore assessed the suitability of VESGEN
analysis for plant leaves (Fig. 1) by analyzing
venation patterns at Day 2 and Day 8 from A. thaliana
seedlings grown terrestrially (Kang and Dengler,
2004). Leaf size increased greatly during maturation
from Day 2 to Day 8 (Fig. 1e). Venation patterns
within the leaves were binarized by semi-automatic
image processing (Fig. 1) described previously for
human ophthalmic clinical images and laboratory
animal experiments (Parsons-Wingerter et al., 2010;
Vickerman et al., 2009). The black/white vascular
pattern and its region of interest (ROI) were analyzed
automatically by VESGEN using the Vascular TreeNetwork Composite option. Vessels within the
branching tree-network composites were grouped into
successively smaller generations of venous branching.
VESGEN further mapped the vascular patterns into
two groups of large structural veins (1°-2°) and small
reticular veins (≥ 3°, Fig. 1 and Table 1).
Vascular groups were determined by VESGEN
according to established rules for leaf venation
patterning, in which the large structural, branching
veins are of primary (1°) and secondary (2°) order and
the smaller net-worked veins forming the reticulum,
of tertiary or greater (≥ 3°) order (Ellis, et al., 2009).
In the ordering of large structural vessels for pinnate
leaves such as A. thaliana, 2° (costal) veins branch
from the single, central 1° vein. Veins of order ≥3°
branch primarily from 2° veins to fill intercostal gaps
between the roughly parallel 2° veins. Venation
patterns in dicot leaves (pinnate and palmate) are
therefore organized as tree-network composites, in
which large structural veins form the hierarchical
vascular branching tree and small reticular veins form
the intercostal vascular network or net.
By VESGEN analysis, leaf venation pattern
matured considerably in branching complexity during
leaf development (Table 1). As a sensitive measure
of branching complexity and space-filling capacity
(Mandelbrot, 1983; Parsons-Wingerter et al., 1998),
the fractal dimension (Df) of the entire skeletonized
branching pattern increased from 1.32 at Day 2 to
1.47 at Day 8. The ratio of vessel density (Av) for
small reticular vessels to large structural vessels
increased from 1.16 at Day 2 to 1.51 at Day 8. From
Day 2 to Day 8, the average diameter (Dv, also termed
vein gauge) of large structural vessels increased from
14.0 µm to 100.9 µm and Dv of small reticular
vessels, from 9.3 µm to 70.0 µm. Other venation
parameters measured by VESGEN but not reported
here include vessel branch point density, vessel end
point density, vessel length density and network
analysis of avascular spaces (Vickerman et al., 2009).
Table 1. Quantification of Venation Pattern in A.
thaliana Leaves by VESGEN Analysis.
Large =
Structural Vein Orders 1°-2°; Small = Reticular Vein
Orders ≥ 3°. Symbols and units: Fractal Dimension (Df),
unitless; Vessel Area Density (Av), µm2/µm2; Vessel
Diameter (Dv), µm
Day
Vessel Group
Df
Av
Dv
2
All
Large
Small
1.32
–
–
0.208
0.096
0.111
–
14.0
9.3
8
All
Large
Small
1.47
–
–
0.276
0.110
0.166
–
100.9
70.0
As a feasibility study, results of the VESGEN
analysis generally correspond to the botanical rules
for large structural (1°-2°) and small reticular (≥3°)
vessels described above (Ellis et al., 2009).
Discrepancies between these rules and our
quantitative assessment may result from: 1) the
immature state of the developing A. thaliana venation
patterns, which is consistent with pattern irregularities
in developing animal vasculature, 2) insufficient
image resolution, and 3) current VESGEN mapping
limitations. In the future, VESGEN mapping
capabilities will be optimized for improved detection
of specific dicot leaf venation attributes such as
branching angle and vessel tapering that differ
somewhat from human and animal vascular
branching.
In conclusion, we recommend that mapping and
quantification of leaf venation patterns by VESGEN
of plants flying as experiments on ISS would provide
new, helpful insights for successful space exploration
and colonization.
Interesting questions to be
addressed include:
What are the effects of
microgravity on leaf venation pattern? Of radiation?
What genetically engineered modifications in leaf
venation pattern improve plant growth and survival in
space environments?
Parsons-Wingerter and Vickerman -– VESGEN Analysis of Venation Pattern
REFERENCES
Ellis, B., Daly, D.C., Hickey, L.J., Mitchell, J.D., Johnson,
K.R., Wilf, P., and Wing, S.L. 2009. Manual of
Leaf Architecture. Ithaca, NY: Cornell University
Press.
Kang, J. and Dengler, N. 2004. Vein pattern development
in adult leaves of Arabiodopsis thaliana.
International Journal of Plant Science 165(2): 231242.
Liu, H., Yang, Q., Radhakrishnan, K., Whitfield, D.E.,
Everhart, C.L., Parsons-Wingerter, P., and Fisher,
S.A. 2009. Role of VEGF and tissue hypoxia in
patterning of neural and vascular cells recruited to
the embryonic heart. Dev Dyn 238(11): 2760-9.
Mandelbrot, B. B. 1983. The Fractal Geometry of Nature.
San Francisco: W. H. Freeman.
McKay, T. L., Gedeon,D.J., Vickerman, MB., Hylton, A.G.,
Ribita, D., Olar, H.H., Kaiser, P.K., and ParsonWingerter, P.
2008.
Selective inhibition of
angiogenesis in small blood vessels and decrease in
vessel diameter throughout the vascular tree by
triamcinolone acetonide. Invest Ophthalmol Vis Sci
49(3): 1184-90.
Parsons-Wingerter, P., Chandrasekharan, U.M., McKay,
T.L., Radhakrishnan, K., DiCorleto, P.E., Albarran,
B., and Farr, A.G. 2006a. A VEGF165-induced
phenotypic switch from increased vessel density to
increased vessel diameter and increased endothelial
NOS activity. Microvasc Res 72(3): 91-100.
Parsons-Wingerter, P., Elliott, K.E., Clark, J.I., and Farr,
A.G. 2000a. Fibroblast growth factor-2 selectively
stimulates angiogenesis of small vessels in arterial
tree. Arterioscler Thromb Vasc Biol 20(5): 1250-6.
Parsons-Wingerter, P., Elliott, K.E., Farr, A.G.,
Radhakrishnan, K., Clark, J.I., and Sage, E.H.
2000b. Generational analysis reveals that TGFbeta1 inhibits the rate of angiogenesis in vivo by
selective decrease in the number of new vessels.
Microvasc Res 59(2): 221-32.
Parsons-Wingerter, P., Lwai, B., Yang, M.C., Elliott, K.E.,
Milaninia, A., Redlitz, A., Clark, J.I., and Sage, E.H.
1998. A novel assay of angiogenesis in the quail
chorioallantoic membrane: stimulation by bFGF and
inhibition by angiostatin according to fractal
dimension and grid intersection. Microvasc Res.
55(3): 201-14.
Parsons-Wingerter, P., McKay, T.L., Leontiev, D.,
Vickerman, M.B., Condrich, T.K., and Dicorleto,
P.E. 2006b. Lymphangiogenesis by blind-ended
vessel sprouting is concurrent with hemangiogenesis
by vascular splitting. Anat Rec A Discov Mol Cell
Evol Biol 288(3): 233-47.
Parsons-Wingerter, P., Radhakrishnan K., Vickerman,
M.B., and Kaiser, P.K. 2010.
Oscillation of
Angiogenesis with Vascular Dropout in Diabetic
Retinopathy by VESsel GENeration Analysis
(VESGEN). Invest Ophthalmol Vis Sci 51(1): 27609.
Pennisi, E. 2010. On rarity and richness.
327(5971): 1318-9.
Science
Vickerman, M. B., Keith, P.A., McKay, T.L., Gedeon, D.J.,
Watanabe, M., Montano, M., Karunamuni, G.,
Kaiser, P.K., Sears, J.E., Ebrahem, Q., Ribita, D.,
Hylton, A.G., and Parsons-Wingerter, P. 2009.
VESGEN 2D: automated, user-interactive software
for quantification and mapping of angiogenic and
lymphangiogenic trees and networks. Anat Rec A
Integ Anat Evol Biol 292(3): 320-32.
71
Short Communication
Attenuation of Muscle Atrophy by Triterpentene Celastrol
1
1
2
Taesik Gwag , Haksub Shin , Takeshi Nikawa , and Inho Choi
1
1
Division of Biological Science and Technology, Yonsei University, Wonju, Gangwon-do, Republic of Korea;
Department of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima Graduate
School, Tokushima, Japan
2
Heat stress has been found to attenuate atrophy of
skeletal muscle in vivo and in vitro (Naito et al., 2000;
Westerheide et al., 2004). This anti-atrophy effect is
known to involve induction of heat shock proteins
(HSPs) like HSP72, an important molecular
chaperone for protein quality control. For instance,
hindlimb-unloaded rats whose body temperature was
raised to 41.6oC by mild heat showed a significant
retention of muscle mass, concurrently with
significant upregulation of HSP72 expression,
compared to the vivarium control. Muscle atrophy
occurs when the proteoytic rate exceeds the anabolic
rate (Hoffman and Nader, 2004). Because HSPs play
a critical role in folding and repair of proteins as well
as cytoprotection against external stresses (e.g.,
hyperthermia and physical stress) (Naito et al., 2000),
it is reasonable to consider that upregulation of HSPs
aid not only to elevate protein anabolism but also to
reduce protein degradation. Moreover, since these
proteins are crucial for organization and protection of
myofilaments (Srikakulam and Winkelmann, 2004),
structural integrity of myofibrils can be stabilized by
upregulation of HSPs even in the unloaded state.
Muscle atrophy is an inevitable phenomenon in
space microgravity where mechanical loading is
nearly absent. Previous reports demonstrate that the
astronauts lost muscle force by about ~ 3% per week
despite continuous daily exercise (Adams et al.,
2003). As upregulation of HSPs seems to parallel
anti-atrophy effect on the skeletal muscle, it is
therefore worth investigating an alternative means of
countermeasure such as natural compounds that
stimulate HSP expression. Celastrol (CEL), a quinone
methide triterpene, was revealed to activate heat
shock transcription factor 1 (HSF1) in various cell
types (e.g., cancer and kidney cells) (Westerheide et
al., 2004). This compound was also reported to have
many functions including antioxidant activity,
neuroprotection, and inhibition of proteasomal
activity (Walcott and Heikkila, 2010). However,
despite significant researches on these multiple
functions of CEL, its anti-atrophy effect on muscle
cells has not been elucidated to date. Here, we present
our results of CEL effect on C2C12 muscle cell size
in the presence or absence of dexamethasone (DEX),
a synthetic form of glucocorticoid that is known to
induce muscle atrophy (Zheng et al., 2010).
C2C12 mouse muscle cells were cultured in 100mm culture dishes containing Dulbecco’s modified
Eagle’s Medium (DMEM) (Welgene, Daegu, South
Korea) supplemented with 10% fetal bovine serum
(FBS) (Hyclone, Logan, UT) and 1% antibioticantimycotic (Gibco, Burlington, Ontario, Canada)
until the cells reached 90% confluence. The
confluenced myoblasts were then differentiated to
myotubes by differentiation media (2% horse serum
in DMEM) for five days. All cells were maintained at
37oC in 5% CO2. Fresh medium was replaced every 2
days.
CEL (Cayman Chemical; Ann Arbor, MI) and
DEX (Calbiochem; San Diego, CA) were dissolved in
dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St.
Louis, MO) at a stock concentration of 25 mM and
200 mM, respectively. To elucidate potency of CEL
on HSP72 expression, myotubes were treated with
0.5, 1.0, 1.2, and 1.5 μM of CEL and incubated for 24
hr. To determine dose-dependent effects of DEX on
the myotube diameter, the cells were treated with 10,
25, 50, 100, 200, 400 μM of DEX and incubated for
24 hr. To test the anti-atrophy efficacy of CEL, the
cell diameter was also determined under treatments of
the following conditions: (1) vehicle (DMSO), (2)
200 μM DEX, (3) 1.0 μM CEL, and (4) both 200 μM
DEX and 1.0 μM CEL (DEX+CEL). Images of cells
were photographed after 24 h of the treatments under
a phase contrast microscope at 100X magnification.
The diameters of the cells were quantified for each
group with Image J software (NIH, Frederick, MD).
To gauge HSP72 expression, the cells were
harvested after 24 h of the treatments, and
homogenized in RIPA buffer containing the protease
inhibitor Complete Mini and phosphatase inhibitor
Gwag et al. -– Attenuation of Muscle Atrophy
cocktail (Roche, Mannheim, Germany). The samples
were sonicated for 3 sec on ice and centrifuged for 15
min at 13,000 rpm at 4oC. The supernatants were
collected as the whole-cell soluble lysate, and its
protein concentration was determined using a
Bradford assay kit. The protein samples were subject
to SDS-PAGE to detect HSP72 bands, and were
electrophoretically transferred from a gel to a
nitrocellulose membrane. The membrane was
incubated in a blocking buffer for 1 h at room
temperature. The membrane was subsequently
incubated overnight at 4oC with primary antibodies.
The membrane was then incubated with HRPconjugated secondary antibody for detection in 10 ml
of blocking buffer with gentle agitation for 1 h at
room temperature. Immune complexes were then
detected with the ECL system (GE Healthcare,
Fairfield, CT). The bands were quantified using a
densitometer (Bio-Rad, Hercules, CA). Densities of
the proteins were normalized to mouse monoclonal
GAPDH (ab8245, Abcam, Cambridge, UK). The
primary antibody used for the assays were mouse
anti-HSP72 from Stressgen (C92F3A-5, Victoria, BC,
Canada). The secondary antibody was HRPconjugated anti-mouse IgG from Cell signaling
Technology (#7076, Beverly, CA).
Data are presented as mean ± SEM (n = 3).
Significance of differences in protein quantity or cell
diameter among groups was examined by KruskalWallis test and post-hoc comparisons as in Langley
(1971).
almost abolished when the cells were treated together
with CEL.
Figure 1. CEL treatment significantly upregulated the
HSP72 expression in a dose-dependent manner in the
C2C12 myotubes. Data: mean ± SEM (n = 3). a, b, and c:
significantly different from each other at P < 0.05.
Our data demonstrate that HSP72 expression levels
increased 6-, 21-, 35-, and 42-fold at CEL
concentrations of 0.5, 1.0, 1.2, and 1.5 μM,
respectively, in comparison to the vehicle control
(Fig. 1). As in other cell types (Westerheide et al.,
2004), this data indicate that CEL functioned as a
potent HSP inducer in the muscle cell.
Dexamethasone is a synthetic member of
glucocorticoid that induces cell death and protein
degradation via the proteasomal pathway (Zheng et
al., 2010). Our result showed that the diameter of the
C2C12 cells gradually decreased with the increase in
dexamethasone concentrations (Fig. 2). At DEX
concentrations of 50 – 400 μM, the diameter was 0.7~ 0.77-fold that of the vehicle control (P < 0.05).
Finally, we attempted to examine whether CEL had
an anti-atrophy effect on muscle cells even under the
influence of DEX. As shown in Figure 3, the diameter
of DEX-treated cells decreased 0.27-fold that of
DMSO (P < 0.05), whereas the diameter of CELtreated cells did not change significantly. More
importantly, the atrophy-inducing effect of DEX was
Figure 2. DEX treatment significantly reduced the
diameter of the C2C12 cells at concentrations > 50 μM.
Data: mean ± SEM (n = 3). a versus b, P < 0.05.
73
Gwag et al. -– Attenuation of Muscle Atrophy
Conclusively, our data demonstrate that CEL was a
potent HSP72 inducer and diminished the atrophic
effect of DEX in the muscle cell. Such efficacy
seemed to be due to overexpression of HSP72 that
might suppress the proteolytic signals involving the
ubiquitin-proteasomal system (Walcott and Heikkila,
2010). Further studies on anabolic and catabolic
signaling pathways will reveal a precise mechanism in
which celastrol works for the anti-atrophy effect on
the muscle cell.
REFERENCES
Adams, G.R., Caiozzo, V.J., and Baldwin, K.M. 2003.
Skeletal muscle unweighting: spaceflight and groundbased models. J Appl Physiol. 95: 2185–2201.
Hoffman, E.P. and Nader, G.A. 2004. Balancing muscle
hypertrophy and atrophy. Nat Med. 10(6):584-5.
Langley, R.A. 1971. Practical statistics simply explained.
New York: Dover Publications.
Naito, H, Powers, S.K., Demirel, H.A., Suqiura, T., Dodd,
S.L., and Aoki, J. 2000. Heat stress attenuates skeletal
muscle atrophy in hindlimb-unweighted rats. J Appl
Physiol 88:359-363.
Srikakulam, R. and Winkelmann, D.A. 2004. Chaperone
mediated folding and assembly of myosin in striated
muscle. J Cell Sci. 117: 641–652.
Westerheide, S.D., Bosman, J.D., Mbadugha, B.N.,
Kawahara, T.L., Matsumoto, G., Kim, S., Gu, W.,
Devlin, J.P., Silverman, R.B., and Morimoto, R.I. 2004.
Celastrols as inducers of the heat shock response and
cytoprotection. J Biol Chem 279:56053-56060.
Walcott, S.E. and Heikkila, J.J. 2010. Celastrol can inhibit
proteasome activity and upregulate the expression of
heat shock protein genes, hsp30 and hsp70, in Xenopus
laevis A6 cells. Comp Biochem Physiol A Mol Integr
Physiol 156:285-293.
Figure 3. The atrophy-inducing effect of DEX was
abolished when the cells were treated simultaneously
with CEL. Cell counts: 230 ~ 264. *, P < 0.05.
Zheng, B., Ohkawa, S., Li, H., Roberts-Wilson, T.K., and
Price, S.R. 2010. FOXO3a mediates signaling crosstalk
that coordinates ubiquitin and atrogin-1 expression
during glucocorticoid- induced skeletal muscle atrophy.
FASEB J. 24: 2660-2669.
ACKNOWLEDGEMENTS
This study was supported by National Space
Lab program funded by the Ministry of
Education, Science and Technology (grant No.
2011-0018654).
Short Communication
Rapid Phenotypic Response of LSMMG-“Primed” Candida albicans
1
2
1,3
Stephen C. Searles , Linda E. Hyman , and Sheila M. Nielsen-Preiss
1
2
Immunology and Infectious Diseases, Montana State University, Bozeman, MT; Boston University School of
3
Medicine, Boston, MA; Division of Health Sciences, Montana State University, Bozeman, MT
Candida albicans is an opportunistic fungal
pathogen responsible for a wide range of illnesses,
including oral thrush, onychomycoses, vaginitis, and
disseminated candidiasis.
As an opportunistic
pathogen, C. albicans is capable of sensing and
responding to a variety of environmental stresses,
perhaps even microgravity. Research has suggested
that some, but not all, microbial pathogens respond to
microgravity with increased virulence and virulenceassociated phenotypes (Nickerson, et al., 2004;
Rosenzweig, et al., 2010). This is troubling from the
perspective of astronaut health, particularly as
spaceflight missions become potentially longer and
exposure to the extreme environment of microgravity
increases. Furthermore, immunological research has
indicated that spaceflight adversely affects the human
immune system, possibly leaving astronauts more
susceptible to infections that would otherwise be
limiting in a healthy host (Kaur, et al., 2005). Fungal
infections are difficult to treat, resulting in a high
mortality rate from disseminated disease. Therefore it
is important to better understand the response of C.
albicans to microgravity from the perspective of
maintaining astronaut health.
At the 2010 American Society for Gravitational
and Space Biology Annual Conference, our laboratory
described a variety of phenotypic changes observed in
a eukaryotic pathogen (C. albicans) following
exposure to low-shear modeled microgravity
(LSMMG) (Searles, et al., submitted).
The
environmentally
induced
phenotypes
and
morphological alterations included filamentation,
antifungal resistance, altered colony morphology, and
biofilm formation (Altenburg, et al., 2008; Woolley,
et al., 2010; Searles, et al., 2011, submitted). Notably,
the morphologic changes occurred following
relatively long-term LSMMG exposure (5-12 days).
The physiological alterations are consistent with
phenotypes associated with increased virulence and
may be very useful in predicting C. albicans response
to microgravity, but ultimately, flight studies are
necessary for confirmation (Mitchell, 1998). Some
genetic and phenotypic alterations have been detected
following short-term spaceflight (25 hrs, unpublished
data), but our research also indicates that long-term
microgravity exposure elicits a more complex set of
adaptations that are of particular importance,
especially in consideration of the potential duration
of future spaceflight missions to the moon, asteroids,
or Mars.
Currently, spaceflight experiments without
repetitive astronaut involvement are restricted to the
time it takes for cells to reach stationary phase
(approximately 1 day); therefore, additional exposure
is required in order to detect long-term phenotypic
and genotypic alterations. This report explores a
possible method to circumvent inherent limitations
associated with short-term spaceflight experiments
without requiring additional astronaut assistance. We
propose that cells can be pre-exposed (or “primed”) in
LSMMG prior to being subjected to spaceflight,
where they will continue on the adaptation trajectory
initiated during preliminary LSMMG exposure. To
test this hypothesis, cells were primed in a groundbased LSMMG bioreactor and stored in water to
mimic conditions that may exist prior to launch.
However, for priming to ‘extend’ spaceflight
exposure, the LSMMG-induced phenotype must be
maintained during water storage. The studies outlined
in this report aim to determine the feasibility of
utilizing this priming principle in spaceflight
experiments in order to elicit a more robust response
during limited zero-gravity exposure.
Two separate eight-day ground-based LSMMG
exposure experiments were performed in HARV
bioreactors. One experiment was inoculated with a
standard overnight culture (standard), whereas the
other was inoculated with cells that had been primed
by a five-day LSMMG exposure and stored in water
for seven days (primed). These two experiments were
compared using previously characterized phenotypic
outcome measures to evaluate the effect of priming on
the environmental adaptive response.
75
Searles et al. -– Candida albicans Phenotype Response
Figure 1. Hwp1 expression analysis of C. albicans
exposed to LSMMG for one or eight days.
Expression of Hwp1, a hyphal-specific gene, was
evaluated following exposure of C. albicans to
LSMMG for either one or eight days (Figure 1). In
cells cultured in standard conditions, Hwp1
expression is increased nearly 1.6-fold by 8 days of
LSMMG exposure, consistent with prior findings
from our laboratory (Altenburg, et al., 2008). Hwp1
levels also increase following priming (1.75-fold by 8
days), but with considerably less variability than in
standard LSMMG conditions. This data suggest that
priming results in a more uniform transcriptional
response to subsequent LSMMG exposure.
In
addition, when comparing the Hwp1 response
between cells grown in primed relative to standard
conditions, there is a modest trend towards a more
rapid response at both 1 and 8 days. These data
indicate that some genetic alterations underlying
filamentation may be synchronized and stabilized
following the priming process and water storage.
To further evaluate phenotypic stability,
filamentation was quantified by microscopic analysis
(Figure 2). Primed cells showed a more robust
filamentation response to LSMMG as compared to
cells prepared using standard procedures. These data
suggest that the rate of filamentation was amplified by
priming.
Figure 2. Filamentation analysis of C. albicans
exposed to LSMMG for eight days.
Colony morphology phenotypic switching is
another alteration induced by LSMMG. Over time,
cells exposed to LSMMG convert from smooth to
‘hyper’ irregular wrinkled (HIW) colony morphology
(Figure 3A and B, respectively). To evaluate colony
morphology, cells cultured in LSMMG following
standard or priming conditions were diluted and
plated daily.
The percentage of colonies
demonstrating the HIW morphology was determined
based on visual inspection of spread plates (Figure
3C). Under standard conditions, about six days of
exposure to LSMMG are required before the
appearance of HIW colonies. By eight days of
standard LSMMG exposure, nearly 25% of colonies
are HIW. Conversely, primed cells demonstrate
altered colony morphology after as little as three days
following LSMMG exposure. Within eight days of
exposure to LSMMG, over 65% of colonies are HIW.
Therefore, primed cells can be induced to undergo
phenotypic switching following a relatively shortterm exposure to LSMMG. These data suggest that
the precursor adaptations necessary to trigger the
HIW phenotype are stable during water storage.
Figure 3. (A, B) Representative stereoscopic
images of colonies following LSMMG exposure:
(A) smooth and (B) ‘hyper’ irregular wrinkle
(HIW).
(C) Emergence of HIW colony
morphology during LSMMG exposure.
Taken together these data suggest the priming
concept outlined in this report may be useful to better
exploit opportunities associated with spaceflight and
may be effective at shortening the amount of
microgravity exposure required to elicit comparable
longer-term phenotypic alterations. A precedent for
consistency in prokaryotic cellular physiology in
LSMMG and true microgravity has been established
Searles et al. -– Candida albicans Phenotype Response
(Wilson, et al., 2002); therefore, combining these
methodologies to provide an extended analysis of
yeast response to microgravity is logical. To better
understand the changes induced by long-term
exposure to microgravity, cells can be primed by
exposure to LSMMG prior to launch. The ability of
primed cells to respond more quickly to LSMMG
than standard cells implicates a certain degree of
phenotypic stability incurred by priming.
The
stability of the phenotype induced by LSMMG would
permit primed cells to be stored in water prior to
spaceflight and accommodate the timeline often
associated with flight opportunities.
Continued
research on the effects of LSMMG and true
microgravity on C. albicans and other human
pathogens is needed to prepare for eventual long-term
space missions.
ACKNOWLEDGEMENTS
Work was supported by NASA
#NNX09AM79G to SMNP and LEH.
grant
REFERENCES
Altenburg, S.D., Nielsen-Preiss, S.M., and Hyman, L.E.
2008.
Increased filamentous growth of Candida
albicans in simulated microgravity.
Genomics
Proteomics Bioinformatics. 6: 42-50
Kaur, I., Simons, E.R., Castro, V.A., Ott, C.M., and Pierson,
D.L. 2005. Changes in monocyte functions of
astronauts. Brain, Behavior, and Immunity. 19: 547554.
Mitchell, A.P. 1998. Dimorphism and virulence in Candida
albicans. Current Opinion in Microbiology. 1: 687692.
Nickerson, C.A., Ott, C.M., Wilson, J.W., Ramamurthy, R.,
and Pierson, D.L. 2004. Microbial responses to
microgravity and other low-shear environments.
Microbiology and Molecular Biology Reviews. 68: 345361.
Rosenzweig, J.A., Abogunde, O., Thomas, K., Lawal, A.,
Nguyen, Y., Sodipe, A., and Jejelowo, O. 2010.
Spaceflight and modeled microgravity effects on
microbial growth and virulence. Applied Microbiology
and Biotechnology. 85: 885-891.
Searles, S.C., Woolley, C.M., Petersen, R.A., Hyman, L.E.,
and Nielsen-Preiss, S.M.
(submitted).
Modeled
microgravity
increases
filamentation,
biofilm
complexity, phenotypic switching, and antimicrobial
resistance in Candida albicans.
Wilson, J.M., Ramamurthy, R., Porwollik, S., McClelland,
M., Hammond, T., Allen, P., Ott, C.M., Pierson, D.L.,
and Nickerson, C.A. 2002. Microarray analysis
identifies Salmonella genes belonging to the low-shear
modeled microgravity regulon. PNAS. 99 (21): 1380713812.
Woolley, C.M., Nielsen-Preiss, S.M., and Hyman, L.E.
2010. The response of Candida albicans to simulated
microgravity. Gravitational and Space Biology. 23(2):
93-94.
77
Index of Authors
Ahrendt, Steven R. ...............................
Bateman, Ted A. ...................................
Berkowitz, Dan E. ................................
Bushart, Thomas J. ...............................
Cai, Shiwei ...........................................
Chang, Polly Y. ....................................
Choi, Takeshi .......................................
Claudio, Pier Paolo ..............................
Cohen, Luchino Y. ...............................
Conlon, Colleen ...................................
Delfos, René .........................................
Dhumal, Kondiram N. ..........................
Diep, Quynh .........................................
Dubeau-Laramee, Genevieve ...............
Duda, Jessica E. ....................................
Duke, P. Jackie ....................................
English, Jerry .......................................
Ferl, Robert J. .......................................
Fortin, Michel ......................................
Foster, Jamie S. ....................................
Fuller, Charles A. .................................
Galicia, Eugene ....................................
Gomez, David ......................................
Griko, Yuri ...........................................
Grindeland, Richard .............................
Gupta, Kiran .........................................
Gwag, Taesik .......................................
Hyman, Linda E. ..................................
Jatap, Sagar S. ......................................
Kabarowski, Janusz H. .........................
Kerney, Krystal R. ...............................
Khaled, Saman .....................................
Khodadad, Christina L.M. ...................
Krooneman, Janneke ............................
Kucik, Dennis F. ..................................
LeBoeuf, William ................................
Leclair, Sebastien .................................
Leguy, Carole A. D. . ............................
Lloyd, Shane A.J. .................................
Luong, Heather ....................................
Martin-Brennan, Cynthia .....................
44
14
51
6
33
57
72
63
60
63
36
48
33
60
66
33
33
40
60
44
66
54
54
54
54
57
72
75
48
57
44
57
44
36
57
33
60
36
14
33
66
McKay, Christopher P. .........................
Mermut, Ozzy ......................................
Montgomery, Beronda L. .....................
Nelson, Gregory A. ..............................
Nielson-Preiss, Sheila M. .....................
Nikawa, Takeshi ...................................
Nyhan, Daniel ......................................
Oh, Sookyung .......................................
Palma, Ervin .........................................
Parks, Brian W. ....................................
Parrish, Mirina L. .................................
Parsons-Wingerter, Patricia ..................
Patel, Mihir ...........................................
Paul, Anna-Lisa ....................................
Platts, Steven H. ...................................
Poelma, Christian .................................
Pourquie, Mathieu J.B.M. ......................
Provencal, Daniel .................................
Roux, Stanley J. ....................................
Salmi, Mari L. ......................................
Searles, Stephen C. ...............................
Selch, Florian .......................................
Shin, Haksub ........................................
Shoukas, Artin A. .................................
Souza, Kenneth A. .................................
Srivastava, Roshni ................................
Tuday, Eric C. ......................................
Valluri, Jagan V. ..................................
Valluri, Vishnu S. .................................
van Loon, Jack J.W.A. .........................
Vernikos, Joan ......................................
Vickerman, Mary B. .............................
Vidyasagar, Pandit B. ...........................
Wagner, Erika B. ..................................
Wang, Aileen Y. ...................................
Westerweel, Jerry . ................................
Willey, Jeffrey S. ..................................
Wu, Xing ..............................................
Young, Laurence R. .............................
Yu, Shaohua .........................................
Yu, Tao .................................................
66
60
22
14
75
72
51
22
54
56
44
69
33
40
51
36
36
60
6
6
75
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72
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51
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63
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