Landmark Study Unlocks Stem Cell, DNA Secrets to Speed Therapies

In a groundbreaking study led by an eminent molecular biologist at Florida State University, researchers have discovered that as embryonic stem cells turn into different cell types, there are dramatic corresponding changes to the order in which DNA is replicated and reorganized.

The findings bridge a critical knowledge gap for stem cell biologists, enabling them to better understand the enormously complex process by which DNA is repackaged during differentiation -- when embryonic stem cells, jacks of all cellular trades, lose their anything-goes attitude and become masters of specialized functions.

As a result, scientists now are one significant step closer to the central goal of stem cell therapy, which is to successfully convert adult tissue back to an embryo-like state so that it can be used to regenerate or replace damaged tissue. Such therapies hold out hope of treatments or cures for cancer, Parkinson’s disease, multiple sclerosis, spinal cord injuries and a host of other devastating disorders.

Using mouse and human embryonic stem cells, FSU researchers employed advanced imaging techniques and state-of-the-art genomics technology to demonstrate, with unprecedented resolution along long stretches of chromosomes, which sequences are replicated first, and which occur later in the process of differentiation.

“Understanding how replication works during embryonic stem cell differentiation gives us a molecular handle on how information is packaged in different types of cells in manners characteristic to each cell type,” said David M. Gilbert, the study’s principal investigator. “That handle will help us reverse the process in order to engineer different types of cells for use in disease therapies.” Internationally renowned for his body of cutting-edge research on chromosomal structure and reproduction that he began as a doctoral student at Stanford University in the 1980’s, Gilbert joined the FSU faculty and was appointed as the first J. Herbert Taylor Distinguished Professor of Molecular Biology in 2006.

Results from the FSU study, which includes contributions from researchers at three other institutions, are described in a paper published in the October 7, 2008, edition of PLoS (Public Library of Science) Biology, a peer-reviewed journal that showcases biological science research of exceptional significance. So prodigious were the findings that the current paper -- “Global Reorganization of Replication Domains During Embryonic Stem Cell Differentiation” -- is focused solely on results observed in the mouse embryonic stems cells; data on the human cells will be detailed in a future report.

“We know that all the information (DNA) required to take on the identity of any tissue type is present in every cell, because we already can, albeit very inefficiently, create whole animals from adult tissue through cloning,” Gilbert said. “We also can make a kind of artificial embryonic stem cells, called induced pluripotent stem cells, out of many adult cell types, but there are two major hurdles remaining. First, the methods currently used rely on the unnatural retroviral insertion of genes into patients’ cells, and these genes are capable of forming tumors. Second, this method is very inefficient as well because only one in 1,000 cells into which the genes are inserted becomes pluripotent. We must learn how cells lose pluripotency in the first place so we can do a better job of reversing the process without risks to patients.

“The challenge is, adult cells are highly specialized and over the course of their family history over many generations they’ve made decisions to be certain cell types rather than others,” he said. “In doing so, they have tucked away the information they no longer need on how to become other cell types. Hence, all cells contain the same genetic information in their DNA, but during differentiation they package it with proteins into ‘chromatin’ in characteristic ways that define each cell type. The rules that determine how cells package DNA are complicated and have been difficult for scientists to decipher.”

But, Gilbert noted, one time that the cell “shows its cards” is during DNA replication.

“During this process, which was the focus of our FSU research, it’s not just the DNA that replicates,” he said. “All the packaging must be replicated as well in each cell division cycle.”

He explained that embryonic stem cells have many more, smaller “domains” of organization than differentiated cells, and it is during differentiation that they consolidate information.

“In fact, ‘domain consolidation’ is what we call the novel concept we discovered,” he said.

Gilbert likened the concept of domain consolidation to the undeclared or “undifferentiated” college student who then consolidates her literature resources during the course of declaring a major and specialization. “From a student with books on all subjects on all of her bookshelves comes a student who has placed all texts pertaining to her major on the eye-level shelf and moved the distantly-related, potentially distracting texts to the hard-to-reach bottom or top shelves,” he said.

“Now, our challenge as scientists,” said Gilbert, “is to build on what we’ve learned about domain consolidation so that we can efficiently and safely create patient-specific induced pluripotent stem cells or even coax the body’s cells to change their specialization in response to medications.”

“Black Gold Agriculture” May Revolutionize Farming, Curb Global Warming


Fifteen hundred years ago, tribes people from the central Amazon basin mixed their soil with charcoal derived from animal bone and tree bark. Today, at the site of this charcoal deposit, scientists have found some of the richest, most fertile soil in the world. Now this ancient, remarkably simple farming technique seems far ahead of the curve, holding promise as a carbon-negative strategy to rein in world hunger as well as greenhouse gases.

At the 235th national meeting of the American Chemical Society, scientists report that charcoal derived from heated biomass has an unprecedented ability to improve the fertility of soil — one that surpasses compost, animal manure, and other well-known soil conditioners.

They also suggest that this so-called “biochar” profoundly enhances the natural carbon seizing ability of soil. Dubbed “black gold agriculture,” scientists say this “revolutionary” farming technique can provide a cheap, straight-forward strategy to reduce greenhouse gases by trapping them in charcoal-laced soil.

“Charcoal fertilization can permanently increase soil organic matter content and improve soil quality, persisting in soil for hundreds to thousands of years,” Mingxin Guo, Ph.D., and colleagues report. In what they describe as a “new and pioneering” ACS report — the first systematic investigation of soil improvement by charcoal fertilization — Guo found that soils receiving charcoal produced from organic wastes were much looser, absorbed significantly more water and nutrients and produced higher crop biomass. The authors, with Delaware State University, say “the results demonstrate that charcoal amendment is a revolutionary approach for long-term soil quality improvement.”

Soil deterioration from depletion of organic matter is an increasingly serious global problem that contributes to hunger and malnutrition. Often a result of unsustainable farming, overuse of chemical fertilizers and drought, the main weapons to combat the problem —compost, animal manure and crop debris — decompose rapidly.

“Earth’s soil is the largest terrestrial pool of carbon,” Guo said. “In other words, most of the earth’s carbon is fixed in soil.” But if this soil is intensively cultivated by tillage and chemical fertilization, organic matter in soil will be quickly decomposed into carbon dioxide by soil microbes and released into the atmosphere, leaving the soil compacted and nutrient-poor.

Applying raw organic materials to soil only provides a temporary solution, since the applied organic matter decomposes quickly. Converting this unutilized raw material into biochar, a non-toxic and stable fertilizer, could keep carbon in the soil and out of the atmosphere, says Guo.

“Speaking in terms of fertility and productivity, the soil quality will be improved. It is a long-term effect. After you apply it once, it will be there for hundreds of years,” according to Guo. With its porous structure and high nutrient- and water-holding capabilities, biochar could become an extremely attractive option for commercial farmers and home gardeners looking for long-term soil improvement.

The researchers planted winter wheat in pots of soil in a greenhouse. Some pots were amended with two percent biochar, generated from readily available ingredients like tree leaves, corn stalk and wood chips. The other pots contained ordinary soil.

The biochar-infused soil showed vastly improved germination and growing rates compared to regular soil. Guo says that even a one-percent charcoal treatment would lead to improved crop yield.

Guo is “positive” that this ground-breaking farming technique can help feed countries with poor soil quality. “We hope this technology will be extended worldwide,” says Guo.

“The production of current arable land could be significantly improved to provide more food and fiber for the growing populations. We want to call it the second agricultural revolution, or black gold revolution!”

He suggests that charcoal production has been practiced for at least 3000 years. But until now, nobody realized that this charcoal could improve soil fertility until archaeologists stumbled on the aforementioned Amazonian soil several years ago.

Biochar production is straightforward, involving a heating process known as pyrolysis. First, organic residue such as tree leaves and wood chips is packed into a metal container and sealed. Then, through a small hole on top, the container is heated and the material burns. The raw organic matter is transformed into black charcoal. Smokes generated during pyrolysis can also be collected and cooled down to form bio-oil, a renewable energy source, says Guo.

In lieu of patenting biochar, Guo says he is most interested in extending the technology into practice as soon as possible. To that end, his colleagues at Delaware State University are investigating a standardized production procedure for biochar. They also foresee long-term field studies are needed to validate and demonstrate the technology. Guo noted that downsides of biochar include transportation costs resulting from its bulk mass and a need to develop new tools to spread the granular fertilizer over large tracts of farmland.

The researchers are about to embark on a five-year study on the effect of “black gold” on spinach, green peppers, tomatoes and other crops. They seek the long-term effects of biochar fertilization on soil carbon changes, crop productivity and its effect of the soil microorganism community.

“Through this long-term work, we will show to people that biochar fertilization will significantly change our current conventional farming concepts,” says Guo.

The American Chemical Society — the world’s largest scientific society — is a nonprofit organization chartered by the U.S. Congress and a global leader in providing access to chemistry-related research through its multiple databases, peer-reviewed journals and scientific conferences. Its main offices are in Washington, D.C., and Columbus, Ohio.

Wrapping up a cell biology riddle


New research has revealed how protein filaments drive a key cellular process by physically wrapping around and constricting bits of cell membrane

The process of endocytosis, by which a cell internalizes molecules bound to outward-facing receptors, is essential for a wide variety of cellular functions. During the first steps of endocytosis, the cell membrane invaginates, puckering inward to form a pocket that is ultimately pinched off to become a bubble-like vesicle, which can act as a vehicle for delivering encapsulated molecules to various locations within the cell.

This invagination requires the assembly of various proteins into complexes that associate with the membrane and induce deformation and the subsequent formation of membrane ‘tubules’. Tadaomi Takenawa's group at the Kobe University Graduate School of Medicine has focused much of their work on these proteins, and recently identified a protein domain known as EFC/F-BAR that plays a direct role in membrane tubulation1. At the same time, Shigeyuki Yokoyama's research team at the RIKEN Genomic Sciences Center in Yokohama was studying the Cdc42-interacting protein (CIP4), which happens to contain a functional EFC domain. Yokoyama and Takenawa decided to collaborate on an in-depth structural analysis of EFC in an effort to clarify its function.

They found that EFC domains pair off to form crescent-shaped dimers—much like BAR, another known membrane-binding domain, although the curve is far subtler for EFC domains2. “This explains why the EFC domain generates tubular membranes with diameters that are several times larger than those induced by the BAR domain,” says Yokoyama. Surprisingly, the structural data also suggested that these EFC dimers can further assemble into lengthy filaments, which can tightly wrap around—and thereby extend—tubulations in the cell membrane (Fig. 1). Subsequent microscopic analysis of endocytotic cells would demonstrate that this model was accurate. “The physiological function of the EFC filament was predicted by the structure-function analysis of the EFC domain,” says Yokoyama.

Based on these findings, Yokoyama, Takenawa and colleagues were able to develop a more sophisticated model for endocytosis, where EFC proteins like CIP4 drive early stages of invagination through filament formation, then gradually recruit additional proteins like dynamin, which further constrict the tubules before pinching them off to form mature vesicles.

Yokoyama doesn’t think this is the end of the story, however, and his group is continuing to investigate EFC proteins with Takenawa’s team. “We would like to investigate the function of the full-length protein and its interaction partners,” he says, “because we believe that yet unknown, interesting regulatory mechanisms are hidden in this molecule.”

1. Tsujita, K., Suetsugu, S., Sasaki, N., Furutani, M., Oikawa, T. & Takenawa, T. Coordination between actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis. Journal of Cell Biology 172, 269–279 (2006).

2. Shimada, A., Niwa, H., Tsujita, K., Suetsugu, S., Nitta, K., Hanawa-Suetsugu, K., Akasaka, R., Nishino, Y., Toyama, M., Chen, L. et al. Curved EFC/F-BAR-domain dimers are joined end to end into a filament for membrane invagination in endocytosis. Cell 129, 761–772 (2007).

Chicken cathelicidin-B1, an antimicrobial guardian
at the mucosal M cell gateway

Ryo Goitsuka*†, Chen-lo H. Chen‡§, Lesley Benyon‡, Yusuke Asano*, Daisuke Kitamura*, and Max D. Cooper‡§¶
**††
*Research Institute for Biological Sciences, Tokyo University of Science, Noda, Chiba 278-0022, Japan; ‡Division of Developmental and Clinical Immunology,
University of Alabama at Birmingham, Birmingham, AL 35294-2812 ; and Departments of ¶Medicine,
Pediatrics, §Microbiology, and **Pathology, University
of Alabama at Birmingham, Birmingham, AL 35294-3300
Contributed by Max D. Cooper, July 26, 2007 (sent for review May 25, 2007)

Mucosal epithelial M cells provide an efficient portal of entry for
microorganisms. Initially defined by their irregular microvilli and
abundant transcytotic channels in the avian bursa of Fabricius,Mcells
also are found in the lymphoid follicle-associated epithelium of the
mammalian appendix, Peyer’s patches, and other mucosal surfacelymphoid
interfaces. We describe here a previously unrecognized
cathelicidin gene in chickens, chCATH-B1, that is expressed exclusively
in the epithelium of the bursa of Fabricius. Like the mature peptides
of previously identified cathelicidins, the carboxyl-terminal peptide of
chCATH-B1 has broad antimicrobial activity against Gram-positive
and Gram-negative bacteria. chCATH-B1 expression is restricted to
the secretory epithelial cell neighbors of the M cells, whereas its
mature peptide is transported to become concentrated on the fibrillar
network surrounding basolateral surfaces of the M cells that overlie
the bursal lymphoid follicles. We conclude that chCATH-B1 is well
placed to serve a protective antimicrobial role at the M cell gateway.
antimicrobial peptides  follicle-associated epithelium  innate immunity 
bursa of Fabricius
The survival of all multicellular organisms depends on an
effective immune response to microbial pathogens. The first
hurdle to microbial entry is provided by our epithelial surfaces.
Microbial pathogens that mount this barrier encounter innate
immunity elements as a first line of defense. Innate immune
responses also facilitate the ensuing adaptive immune responses
that vertebrates use to clear infectious agents. The mucosal M
cells are an important microbial portal of entry because of their
highly efficient pinocytotic channels. Initially identified as specialized
epithelial cells overlying the lymphoid follicles of the
avian bursa of Fabricius that have irregular microvilli and
efficient transcytotic capability (1),Mcells were also found to be
conserved in the lymphoid follicle-associated epithelium of
mammalian appendix and intestinal Peyer’s patches (1, 2). The
Mcells have since been found in other mucosal lymphoid tissues,
including those of the upper and lower airways, oropharynx,
salivary glands, stomach, colon, and eye (3, 4), where they
provide an efficient conduit for transporting microorganisms
and other antigenic substances into the underlying lymphoid
structures to initiate immune responses (5, 6). Despite the
physiological importance of this entry portal, there is limited
information about the differentiation of the M cells, their
transport mechanism(s), and how the microbes that constantly
enter the body via the M cells are rendered noninvasive.
Antimicrobial peptides are well known as front-line participants
in microbial defense (7–10). Two evolutionary groups of
antimicrobial peptides, the cathelicidins and the defensins, provide
endogenous peptide-based defense against microbial invasion
(11–13). Cathelicidins and defensins are produced by many
cell types and have broad spectrum antimicrobial activity against
bacteria, fungi, and viruses. As one example, the human cathelicidin
LL-37 (also called hCAP-18, FALL-39, and CAMP) is
produced by neutrophils, B cells,
 T cells, natural killer cells,
monocytes, and macrophages (14–16); it is also found in the
squamous epithelium of the mouth, tongue, and esophagus, as
well as in the colonic and bronchial mucosal epithelium (17).
LL-37 expression is negligible in normal skin, but epidermal cells
are induced to express high levels of LL-37 in inflammatory
conditions, such as psoriasis and contact dermatitis (18). Conversely,
deficiencies in the LL-37 cathelicidin and the HBD-2
defensin may underlie the Staphylococcus aureus skin infections
that plague patients with atopic dermatitis (19). Recent studies
have also indicated the importance of epithelial cathelicidin in
the maintenance of the sterility of the human urinary tract (20).
Remarkably, neither cathelicidins nor defensins have been identified
at the M cell interface, where one might anticipate their
need. We report here an avian cathelicidin that appears to fulfill
this expectation. This peptide was identified during a search for
genes expressed preferentially in the bursa of Fabricius.
Results
Identification of a Bursa-Specific Cathelicidin, chCATH-B1. Our search
for bursa-specific genes began with the cloning of bursal cDNA
subtracted by splenic cDNA and yielded cDNA clones, some of
which have been reported (21). Among these, BFG7 is expressed
exclusively in the bursa of Fabricius as shown by Northern blot
analysis (Fig. 1). Sequence analysis of a full-length BFG7 cDNA did
not yield a match with then-reported genes. However, a BLAST
search of the National Center for Biotechnology Information
protein database revealed aBFG7cathelin domain sequence, which
is a conserved hallmark of the cathelicidin gene family (Fig. 2).
Although conserved cathelin regions of mammalian cathelicidins
share 50% or greater amino acid identity (12), the BFG7 cathelin
region has only 20–30% homology with mammalian cathelicidins.
This is in the ‘‘twilight’’ zone of sequence similarity but is within the
range of identity shared by many avian and mammalian orthologs
(22). Provisionally, we have named this cathelicidin relative
‘‘chicken cathelicidin-B1,’’ or chCATH-B1, in view of its selective
expression in the bursa of Fabricius.
Proteolytic cleavage of mammalian cathelicidin proproteins
yields mature C-terminal peptides with antimicrobial activity. In
this context, a comparative alignment of chCATH-B1 with
mammalian cathelin region sequences predicted a cationic peptide
of 40 C-terminal amino acid residues with a high pI value
(pI
12.2) (Fig. 2).

This peptide sequence appears to belong to
Author contributions: R.G., C.-l.H.C., and M.D.C. designed research; R.G., C.-l.H.C., L.B., and
Y.A. performed research; C.-l.H.C. contributed new reagents/analytic tools; R.G., C.-l.H.C.,
D.K., and M.D.C. analyzed data; and R.G., C.-l.H.C., and M.D.C. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The sequences reported in this paper have been deposited in the DNA
Data Bank of Japan database, www.ddbj.nig.ac.jp (accession nos. AB307733 and AB308318
for chCATH-B1).
†To whom correspondence may be addressed at: Division of Development and Aging, 2669
Yamazaki, Noda, Chiba 278-0022, Japan. E-mail: ryogoi@rs.noda.tus.ac.jp.
††To whom correspondence may be addressed at: University of Alabama at Birmingham,
401 Shelby Research Building, 1825 University Boulevard, Birmingham, AL 35294-2812.
E-mail: max.cooper@ccc.uab.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0707037104/DC1.
© 2007 by The National Academy of Sciences of the USA