'Bacillus anthracis and Anthrax': The past, present and future of anthrax research

'Bacillus anthracis and Anthrax': The past, present and future of anthrax research

Few strands of bacteria have achieved such a central place in public consciousness as Bacillus anthracis, the bacteria which causes Anthrax. While today it is a feared weapon of bioterrorism Bacillus anthracis has played a significant historical role, especially through the research of the celebrated 19th century scientists Robert Koch and Louis Pasteur, in shaping our understanding of infectious diseases and immunology.

In his new book Bacillus Anthracis and Anthrax Dr Nicholas Bergman brings both of these perspectives together to present a definitive 'state of the field' summary for anthrax research, providing a comprehensive guide to all aspects of the organism, ranging from basic biology to central public health issues.

"With all the attention and research that have been focused on Bacillus anthracis and anthrax in recent years, our understanding of both the organism and the disease has improved dramatically," said Bergman. "This book aims to provide a up-to-date reference that will be useful to scientists, medical and public health personnel, and those playing roles in shaping public policy."

Bacillus Anthracis and Anthrax covers all major aspects of Anthrax biology, from basic biology and pathogenesis to diagnosis, treatment, and prevention of anthrax bioterror-associated issues.

Throughout the book anthrax is considered historically as well biologically, with chapters ranging from the impact of anthrax on human history from 5000 BC by Peter Turnbull and Sean Shadomy, to an assessment of current anthrax vaccination research by Elke Saile and Conrad Quinn, as well as an analysis by Leonard Cole of anthrax as a weapon of war.

The biological analysis of B. anthracis, includes research on life cycle, differentiation, cellular structure, as well as the interaction of B. anthracis with the immune system.

Bacillus Anthracis and Anthrax also considers the clinical features, diagnosis and treatment of the different forms of human anthrax, as well as a review of animal models of anthrax and their use in research.

While Bacillus Anthracis and Anthrax will be a premier reference tool to B. Anthracis and anthrax for microbiologists, immunologists and physiologists it will also serve as an invaluable resource for medical professionals, bioterror experts and all those involved in the issues of public health.

Researchers ID molecular link key for cell growth

Researchers ID molecular link key for cell growth

Enlarge

Images of yeast cells expressing fluorescent proteins. Microtubules (part of the cytoskeleton) are shown in green and the lipid PI4P in red. The white arrows here denotes the direction of the cell growth.

(PhysOrg.com) -- When a cell is preparing to grow or replicate, it starts the way a monarch planning to expand his territory might: by identifying and marshaling the necessary resources, loading them onto the appropriate vehicles, and transporting them to the front line.

For cells, that means connecting key molecules with so-called motor proteins, which are neatly equipped to recognize them and carry them to their designated positions at the intended site of growth.

In research published Jan. 18 in Developmental Cell, Cornell scientists report on two molecules that work together to initiate that process in yeast cells. The research is a step toward understanding how cells orient themselves within their surroundings and, ultimately, toward finding new ways to fix the process when it goes awry.

In yeast and other organisms, membrane-bound organelles known as secretory vesicles and other compartments are responsible for providing membranes needed to build another cell. In the current research, Anthony Bretscher, professor of molecular biology and genetics, graduate student Felipe Santiago-Tirado and colleagues studied how the motor protein myosin-V, which transports secretory vesicles along filaments of the cytoskeleton, recognizes and binds with its appointed cargo.

They found that two key molecules -- the protein Sec4 and the lipid PI-4P -- work together to facilitate the selection and binding. By requiring two molecular indicators rather than just one, the researches say, cells can closely regulate the cascade of processes that lead to cell growth.

The findings also demonstrate that lipids, normally thought to be just simple barriers, can play a vital role in molecular processes.

An image from a movie of a yeast cell expressing a marker for the lipid PI4P. Multiple frames where superimposed to illustrate the directed movement of a PI4P rich compartment towards the growing bud. The compartment starts at the bottom and one second later is at the top of the cell.

To understand the process, the researchers used confocal microscopy to observe genetically modified yeast cells in which the key protein and lipid molecules were visualized with green fluorescent proteins.

They then tested the cells' ability to grow under a variety of conditions. "By looking at both the lipid and protein markers at the same time, we found that the lipid co-localized with specific proteins and not with others. And it turns out that the ones that co-localized with the lipids are the ones that are associated with secretory compartments," said Santiago-Tirado, who led the study.

If the lipid was present without the protein, or vice versa, the myosin-V protein still trundled along its path to the site of growth -- but with no secretory vesicle attached. "It recognizes these cargoes through both binding a protein, and indirectly through binding a lipid," said Bretscher. "So it's actually a coincidence detector -- it has to bind both."

By greatly boosting either the protein or the lipid when the other element wasn't present, the researchers were able to prompt some cell growth; but the new cells were far less robust.

The research is a step toward deciphering the way cells organize their structure and respond to changes, Bretscher said. The same type of mechanism is likely to apply in mammalian cells, he added.

An image of yeast cells expressing fluorescent proteins. The proteins localize to specific parts of the cell, specifically the areas undergoing active cell growth. The secretory vesicles are in green and the plasma membrane in red.

The work also suggests the need for further studying the role of lipids in cellular processes, Santiago-Tirado said.

"Proteins have been shown to play a role in many things in the cells, but lipids were thought to be just a physical component of the membrane and not involved in this process," he said. "But here we show that lipids also are important -- they do something by themselves."

Ultimately, the findings could lead to treatments for a variety of diseases, Santiago-Tirado said.

"Many diseases are related to the failure of moving these components; so if we understand how the myosin binds to the cargo, then maybe we can try to fix a condition where the myosin is unable to bind to the cargo."

The research was funded by the National Institutes of Health.

Human embryonic stem cells in culture created

Human embryonic stem cells in culture created

Enlarge

Human embryonic stem cells in culture created by UCLA researchers

(PhysOrg.com) -- In regenerative medicine, large supplies of safe and reliable human embryonic stem (hES) cells are needed for implantation into patients, but the field has faced challenges in developing cultures that can consistently grow and maintain clinical-grade stem cells.

Standard culture systems use mouse "feeder" cells and media containing bovine sera to cultivate and maintain hES cells, but such animal product–based media can contaminate the cells. And because of difficulties in precise quality control, each batch of the medium can introduce new and unwanted variations.

Now, a team of stem cell biologists and engineers from UCLA has identified an optimal combination and concentration of small-molecule inhibitors to support the long-term quality and maintenance of hES cells in feeder-free and serum-free conditions. The researchers used a feedback system control (FSC) scheme to innovatively and efficiently select the small-molecule inhibitors from a very large pool of possibilities.

The research findings, published today in the journal Nature Communications, represent a major advance in the quest to broadly transition regenerative medicine from the benchtop to the clinic.

"What is significant about this work is that we've been able to very rapidly develop a chemically defined culture medium to replace serum and feeders for cultivating clinical-grade hES cells, thereby removing a major roadblock in the area of regenerative medicine," said Chih-Ming Ho, the Ben Rich–Lockheed Martin Professor at the UCLA Henry Samueli School of Engineering and Applied Science and a member of the National Academy of Engineering.

Unlike current animal product–based media, the new medium is a "defined" culture medium — one in which every component is known and traceable. This is important for clinical applications and as drugs or cells enter the world of regulatory affairs, including good manufacturing practice compliance and Food and Drug Administration supervision.

Enlarge

Representation of the feedback system control scheme used by UCLA researchers to identify an optimal combination and concentration of small-molecule inhibitors to support the long-term quality and maintenance of hES cells in culture. (Image credit: UCLA)

"It is also the first defined medium to allow for long term single-cell passage," said the paper's senior author, Hong Wu, the David Geffen Professor of Molecular and Medical Pharmacology at the David Geffen School of Medicine at UCLA and a researcher with UCLA's Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research.

Single-cell passaging — a process in which hES cells are dissociated into single cells and subcultured through single-cell–derived colonies — is important in overcoming the massive cell death associated with hES cell dissociation during routine passage, and it allows for genetic manipulation at the clonal level.

"Although other studies have demonstrated growth of hES cells under defined media formulations and/or on defined surfaces, to the best of our knowledge, this is the first study that combines defined cultures with routine single-cell passaging, which plays an important role in supplying a large mass of clinically applicable cells," said Hideaki Tsutsui, a UCLA postdoctoral scholar and lead author of the study. "Thus, our hES cell culture system, guided by the FSC technique, will bring hES cells one step closer to clinical therapies."

Initially, the very large number of small molecules in the culture medium and their unknown synergistic effects made it difficult for researchers to assess the proper concentration of each for achieving long-term expansion of hES cells. The major challenge was to find the best way  to sort out those molecules and rapidly determine the best combinatorial concentrations.

The breakthrough, ultimately, was the product of a close interdisciplinary collaboration.

Tsutsui, then a UCLA Engineering graduate student, and Bahram Valamehr, then a graduate student at the Geffen School of Medicine, started working on the project two years ago. Armed with biological readouts and analyses of stem cells mastered in Hong Wu's laboratory through the lab's extensive accomplishments in stem cell research, Tsutsui and Valamehr used the FSC scheme — developed previously by Ho's group to search for optimal drug combinations for viral infection inhibition and cancer eradication — to facilitate the rapid screening of a very large number of possibilities.

Working together, the team was able to discover a unique combination of three small-molecule inhibitors that supports long-term maintenance of hES cell cultures through routine single-cell passaging.

"There are certain research projects biologists can dream about, and we know we can eventually get there, but we don't have the capacity to achieve them in a timely manner, especially in a study like this," Wu said. "It would have taken 10 graduate students another 10 years to test all the possible combinations of molecules. Having an opportunity to collaborate with the engineering school has been invaluable in making this dream a reality."

"This is the best example of demonstrating the strength and potential of interdisciplinary collaborations," said Ho, who is also director of the Center for Cell Control at UCLA Engineering and a senior author of the paper. "Engineers and biologists working side by side can accomplish a mission impossible."

Other authors of the study included Antreas Hindoyan, Rong Qiao, Xianting Ding, Shuling Guo, Owen N. Witte and Xin Liu.

Shedding light on the elegant mechanisms that control the push and shove of cells in living organisms

Shedding light on the elegant mechanisms that control the push and shove of cells in living organisms

Enlarge

Figure 1. a-Catenin and myosin in epithelial cells. a-Catenin (blue) is a protein that forms part of an ?adhesion apparatus? for joining adjacent cells and is uniformly distributed at cell boundaries. Myosin (green) is another protein that produces a force that deforms the inner structure. The areas undergoing the resultant structural change are shown in pink.

The many amazing photomicrographs on display at the entrance to the Electron Microscope Laboratory in the RIKEN Center for Developmental Biology (CDB) were all taken by laboratory leader Shigenobu Yonemura and his colleagues. In addition to its own research program, the laboratory also offers technical assistance with electron microscopic analysis to researchers at other laboratories in the CDB. “Moving cells seem to have a life of their own. I have long been fascinated by the motion force generated by cells,” says Yonemura. His team’s recent clarification of the mechanism by which adjacent cells pull and pull against each other has attracted considerable attention.

Yonemura clearly remembers his days as a graduate student examining the development of the sea urchin. “The fertilized egg of a sea urchin cleaves and replicates, doubling the number of cells in sequence to build up a spherical body. At one point, part of the sphere suddenly sinks, taking surface cells into the body itself. This process is known as archenteron invagination. I was fascinated by the change, in which it looks as if the surface cells move under their own volition. Since then, I have been interested in the force generated by cells.”

That force is produced by two proteins: actin and myosin. Individual actins occur in spherical form, with many actins joining together to form actin filaments. As myosins move on the filaments, a force that pulls the filaments is produced, which in turn allows the cells to move or change their form. “It is not necessary for the cells to be in the process of morphogenesis, such as during archenteron invagination, for them to generate a force. For example, adjacent epithelial cells are constantly pulling at each other.”

Epithelial cells play an important role in separating the internals of a living body from the outer world by arranging themselves in a sheet to form the surface of the skin and the gastrointestinal tract. Adjacent epithelial cells are linked together by adhesion apparatus (Figs 1, 2). The key component of this apparatus is cadherin, a protein discovered in 1982 by Masatoshi Takeichi, director of the CDB. Cadherin penetrates the cell membrane, with one end exposed to the cell surface, and the other end protruding into the cell. Bound to the end of the cell are a-catenin and ß-catenin. As the actin filaments bind to the a-catenin and the myosin acts on the filaments, a pulling force is produced.

Enlarge

Figure 2. Mechanism producing tensile forces among adjacent cells. Cadherin binds to actin filaments via a-catenin and ß-catenin, forming an adhesion apparatus. When not bound to a nearby cell, myosin does not act on the actin filament, and the a-catenin assumes a bent structure. When cadherin is bound to that in an adjoining cell, it is pulled strongly, inducing myosin to act on the actin filament. As both ends of the a-catenin are pulled in opposite directions, the bent portion of the a-catenin stretches. Vinculin then binds to the a-catenin, causing the actin filament to bind even more strongly to the a-catenin. The adhesion apparatus is thus enhanced, producing a pull-back force. enlarge image

“As cadherin adheres to the same protein in an adjoining cell, the adjoining cell is pulled by tensile force. If that pulling force continues, however, the cell is deformed. A good balance cannot be achieved unless the adjoining cell pulls back. When pulled strongly, the cell pulls back strongly, and when pulled weakly, it pulls back weakly. Believing in the existence of a mechanism behind this, I have been trying to find what it is, and recently succeeded in clarifying it. The key player proved to be a-catenin."

The mechanism is remarkable. Each cell contains a-catenin, which assumes a bent structure (Fig. 2). When pulled by an adjoining cell, the bent portion stretches. The vinculin molecule then binds to the a-catenin, and as the actin filaments continue to bind to the vinculin, the adhesion apparatus is enhanced and a pulling-back force is produced.

“This can be compared to a tug-of-war in which one team is about to lose the game as it is pulled with a strong force by its opponent, then many helpers come to join and pull back strongly,” says Yonemura. When the opponent's pulling force weakens, the helpers are no longer necessary. The a-catenin then bends again, and the vinculin and actin filaments leave the adhesion apparatus, restoring the original state. “From this system, I realized that a-catenin is responsible for two tasks: sensing the forces and enhancing the adhesion apparatus.”

When a cell is pulled by an adjacent cell, it must immediately pull back at the same location. However, transducing information about such a response by the release of a signal transmitter, as occurs in many biological processes, would not provide the responsiveness required to balance rapid changes in pulling location and strength. “It is reasonable that one molecule plays the dual roles of sensing the forces and enhancing the adhesion apparatus. We have arrived at a definite answer to the inveterate problem of how cells perceive forces and respond to them.”

Yonemura is planning to make observations of how the a-catenin structure changes to allow binding to the vinculin, and to quantify the pulling force required to cause morphological changes in the cells.

“Pulling at each other is very important for cells. I think that mutual pulling is utilized in, for example, sensing whether an adjoining cell is alive or dead,” says Yonemura.

Dead epithelial cells are quickly eliminated from the sheet structure. “The mechanism by which cells sense the death of adjoining cells remains unclear. Although it is said that dead cell detection may be achieved by molecular exchanges on the cell surfaces, I do not think that’s the whole story.”

To investigate the mechanism behind the sensing of dead cells, Yonemura artificially damaged epithelial cells and examined them in the process of repair. Similar experiments undertaken before had not yielded good results because the experimentally inflicted damage affected a larger range than intended due to cell scratching. Yonemura succeeded in selectively killing a target cell by laser irradiation. His technique has made it possible to make extensive observations of the behavior of cells adjacent to the dead cell (Fig. 3).

“When a cell dies, a change begins to occur in the cells adjacent to the dead cell. Actin filaments and myosins gather on the surfaces in contact with the dead cell as if they were joining hands to form a ring surrounding the dead cell. The ring gradually shrinks, and eventually the dead cell is ejected from the sheet.”

This accumulation of actin filaments and myosins begins as soon as a cell dies. “The repair of epithelial cells cannot begin immediately as it does unless actin filaments and myosins are involved in sensing cellular death,” says Yonemura. “Cells are constantly pulling at each other. When an adjacent cell has died, however, the living cell in question continues to pull the dead cell, but is no longer pulled back. It is through this process that living cells may sense the death of an adjacent cell. Such a process would ensure that death signals are detected earlier and more accurately, allowing the elimination of the dead cell to start more quickly than if cellular death were detected by molecular exchange on cell surfaces.”

Enlarge

Figure 3. Elimination of dead epithelial cells. Epithelial cells visualized by transmission microscopy (upper) and fluorescence microscopy (lower) showing intracellular myosin. When an epithelial cell is damaged (center of each plate) by laser irradiation, actin filaments and myosins in adjoining cells gather to form a ring around the dead cell. The ring then gradually shrinks to expel the dead cell out of the sheet. enlarge image

However, that may not be the full story. “When actin filaments and myosins have formed a ring surrounding the dead cell, the forces remain balanced. It is natural that the process ceases in that state. In actuality, however, the ring continues to shrink further to eliminate the dead cell. There must be another, unknown mechanism that breaks the balance of forces. Clarification of the mechanism behind the series of processes from the sensing of cellular death to its elimination is an issue that remains to be addressed.”

Of course, gene expression and molecular signal transduction are vitally important to the functioning of cells. “Although this is true, there is also a wide variety of mechanisms that act like machinery in cells. I am much more interested in those types of machinery. I want to discover these exquisite mechanisms, which are remarkably simple and controlled by feedback,” says Yonemura.

In addition to its own research program, the Electron Microscope Laboratory provides technical support for electron microscopic analysis to researchers at the CDB. Yonemura also provided such support while conducting his own research at the Laboratory for Cellular Morphogenesis in the CDB, for which he served as team leader between 2001 and 2006. Even after the team was renamed the Electron Microscope Laboratory in April 2007, its tasks have continued basically unchanged.

“Only a few researchers are good at using electron microscopes,” says Yonemura. Electron microscopes have a resolution of as fine as 0.1 nanometers, compared with the much coarser resolution of 200 nanometers achievable using optical microscopes, making it possible to observe much smaller features. “It is likely that users realize the performance of the electron microscope but tend to view it as something that is difficult to operate, possibly because of the large amount of instrumentation, so they are reluctant to use it. Myself and two technical staff members provide technical assistance on their use.”

At the Electron Microscope Laboratory, members manage two transmission electron microscopes and one scanning electron microscope, as well as peripheral equipment including two microtomes for preparing sample sections. “We want researchers to be able to examine as many samples as possible, so we chose to install more pieces of standard equipment, rather than one or two highly specialized, high-performance units.”

Currently, the laboratory provides assistance to about 20 research projects annually. In some cases, support is provided for all steps from the preparation of resin-fixed sectional samples to examination and photographic recording. In other cases, it is only necessary to provide instructions on how to use the instruments. Many different types of materials are brought into the laboratory, including mice, drosophila fruit flies, nematodes, zebrafish and chickens. The types of tissue examined are also diverse, and include embryos, nerves and kidneys. “New types of samples can be quite difficult to handle the first time, so it can take a lot of time to prepare them properly. However, since all trial-and-error experimentation represents the accumulation of knowhow, we always endeavor to work steadily without rushing. State-of-the-art techniques are not the only key to success in this field. We must have a broad range of knowledge about the wide variety of tissues of various organisms, and be able to hold scientific discussions with the users.”

When providing support, Yonemura discusses the project and desired goals with the requestor. “We do this to determine whether the target is in fact suitable for analysis using an electron microscope. Electron microscopy only allows a relatively narrow range of examination compared with optical microscopy. The sample must also be fixed in resin, and this is a time-consuming procedure. Our discussions sometime lead us to recommend use powerful optical microscopes. To ensure the best outcomes, we make it a rule to first discuss things in detail with the requestor.”

Yonemura’s support role also has its own benefits. “The support work allows me to get involved in many kinds of research that I would otherwise never do myself, and it provides me with opportunities to see the very latest achievements before they are published. For me, the greatest fascination is in meeting many different people. It makes me very happy to be able to continue to do my own research and at the same time balance that with our support work.”

Skin provides Australia's first adult stem cells for rare genetic disease

Skin provides Australia's first adult stem cells for rare genetic disease

(PhysOrg.com) -- Scientists have developed Australia’s first adult induced pluripotent stem cell lines using skin biopsies from patients with the rare genetic disease Friedreich Ataxia (FA).

The study was conducted by the University of Melbourne and Monash Institute of Medical Research and is published in the current online edition of the international journal Stem Cell Reviews and Reports. It is the first time adult pluripotent stem cells, known as iPS cells have been developed for a specific disease in Australia, allowing for the development of new treatments for FA and related conditions such as diabetes and heart disease.

Induced pluripotent stem (iPS) cells result from the reprogramming of adult cells, such as skin cells, and are similar to embryonic stem cells in that they have the potential to generate any cell type of the body.

Dr Alice Pébay and Dr Mirella Dottori, co leaders of the study from University of Melbourne, characterized and directed the Friedreich Ataxia iPS cells to become specific cell types, including heart cells and nerves, which are normally not functioning well in the disease.
“By focusing on the heart and nerve cell types, we hope to be able to develop treatments to improve heart function and the loss of movement experienced by patients with FA,” Dr Pébay said.

Friedreich Ataxia affects one in 30,000 people globally, and Dr Paul Verma of the Monash Institute of Medical Research said this research could be applied to other diseases.

“Due to the number of symptoms experienced by people with FA, including diabetes and heart disease, this resource could be applied to developing treatment for those conditions and helping even more people, “ he said.

Dr Dottori said the research could not have been achieved without a significant network of experts and support from the Friedreich Ataxia Research Association (Australasia) (FARA-A) and the Friedreich Ataxia Research Alliance (FARA) in the United States.

“It is the collective effort of clinicians, scientists, patients and FARA that has made this discovery possible,” she said.

Ms Varlli Beetham, Executive Director of FARA said the finding provided real hope for people suffering the debilitating condition. “We are proud to have supported this research effort and look forward to the next stage of research, the development of new trial treatments,” she said.

A pesky bacterial slime reveals its survival secrets

A pesky bacterial slime reveals its survival secrets

January 7, 2011

Enlarge

Slimy bacterial coatings known as biofilms (Bacillus subtilis colony superimposed at center) exhibit an unmatched ability to repel a wide range of liquids and even vapors. An electron microscope image shows the biofilm surface's resilient meshwork made from proteins and polysaccharides and assembled into a multiscale, hierarchical structure. Credit: Courtesy of the laboratory of Joanna Aizenberg, Harvard School of Engineering and Applied Sciences.

By rethinking what happens on the surface of things, engineers at Harvard University have discovered that Bacillus subtilis biofilm colonies exhibit an unmatched ability to repel a wide range of liquids -- and even vapors.

Centimeters across yet only hundreds of microns thick, such slimy bacterial coatings cling to the surfaces of everything from pipes to teeth and are notoriously resistant to antimicrobial agents. The researchers now suspect they know the secret to a biofilm's resiliency.

Published in the January 5th early edition of the Proceedings of the National Academy of Sciences (PNAS), the study holds promise for both creating bio-inspired non-wetting materials and developing better ways to eliminate harmful biofilms that can clog pipes, contaminate food production and water supply systems, and lead to infections.

"By looking at biofilms from a materials perspective rather than a cellular or biochemical one, we discovered that they have a remarkable ability to resist wetting to an extent never seen before in nature," says lead author Alex Epstein, a graduate student at the Harvard School of Engineering and Applied Sciences (SEAS). "In fact the biofilm literally resisted our initial efforts to study it."

The finding came about serendipitously, as the original intention of the researchers was to study the structure of the biofilm. To image the interior of the biofilm, the team had to soak it with liquids such as ethanol and acetone, which normally spread and seep easily into a surface.

Enlarge

Four drops of ethanol solution placed on a Bacillus subtilis biofilm colony, the light-colored film on top of the sectioned agar gel, bead up instead of readily wetting and seeping in. This macroscopic phenomenon reveals a critical clue to what may be responsible for biofilms' broad antimicrobial resistance. Credit: Courtesy of the laboratory of Joanna Aizenberg, Harvard School of Engineering and Applied Sciences

"But to our surprise, it was impossible. The liquids kept beading up on the surface and wouldn't infiltrate the colonies," says Epstein, who is a member of the laboratory of Joanna Aizenberg, Amy Smith Berylson Professor of Materials Science at SEAS; Susan S. and Kenneth L. Wallach Professor at the Radcliffe Institute; and a core member of the Wyss Institute for Biologically Inspired Engineering at Harvard.

As the Aizenberg lab studies materials and wetting, the engineers immediately recognized the significance of what they were observing. It turns out that biofilm has an unprecedented liquid-repellent surface, thereby revealing a critical clue to what may be responsible for its broad antimicrobial resistance.

Nature offers numerous examples of water-resistant surfaces, such as the lotus leaf, a longstanding inspiration for creating synthetic materials. Until now, however, no model natural systems have been found for broadly repellent materials.

While such surfaces can be manufactured, the top-down process is costly, labor intensive, and reliant on toxic chemicals and brittle structures. A biofilm, however, is living proof that only the simplest and most natural of components are required—namely, a resilient meshwork made from proteins and polysaccharides assembled into a multi-scale, hierarchical structure.

At the same time, the finding offers a completely new perspective on how biofilms are immune to so many different types of biocides. Even the most sophisticated biochemical strategy will be ineffective if a biocide cannot enter the slime to reach the bacteria. In short, the antimicrobial activity of alcohols and other solvents becomes compromised by the strongly non-wetting behavior at clinically relevant concentrations.

The team expects that their newfound knowledge will help alert researchers to the need to consider this requirement when designing ways to destroy harmful biofilms.

"Their notorious resistance to a broad range of biocide chemistries has remained a mysterious and pressing problem despite two decades of biofilm research," says Aizenberg, a pioneer in the field of biomimicry. "By looking at it as a macroscopic problem, we found an explanation that was just slightly out of view: antimicrobials can be ineffective simply by being a non-wetting liquid that cannot penetrate into the biofilm and access subsurface cells."

Aizenberg and her colleagues speculate that such strong liquid repellence may have evolved in response to the bacteria's natural soil environment where water can leach heavy metals and other toxins.

Enlarge

A Bacillus subtilis biofilm, the light-colored, slimy, bacterial coating on top, displays unprecedented liquid repellency as it beads up a drop of ethanol solution that has been placed on it. This remarkable ability offers both a completely new perspective on how biofilms can protect themselves from a wide range of biocides and introduces the first model natural system for broadly non-wetting materials. Credit: Courtesy of the laboratory of Joanna Aizenberg, Harvard School of Engineering and Applied Sciences.

Moreover, the property may underlie the recent success of the use of biofilm as an eco-friendly form of biocontrol for agriculture, protecting plant roots from water-borne pathogens.

Looking ahead, the Harvard team plans to investigate precisely how the biochemical components of biofilms give rise to their exceptional resistance and to test the properties of other bacterial species.

"The applications are exciting, but we are equally thrilled that our findings have revealed a previously undocumented phenomenon about biofilms," says Aizenberg. "The research should be an inspiring reminder that we have only scratched the surface of how things really work."

Just as with biofilm, she adds, "It has been a challenge to get deep into the core of the problem."

Discovering how microbes cooperate

Discovering how microbes cooperate

Ever wonder what microorganisms do on a Saturday night? In professor Derek Lovley's lab at the University of Massachusetts, Amherst, doctoral candidate Zarath Summers and her colleagues made a point to find out. In the process, Summers discovered a new cooperative behavior in bacteria.

"Interspecies electron transfer" entails one microorganism forming a direct electrical connection to another.

Scientists have known since the 1960s that microorganisms can indirectly exchange electrons through a process called hydrogen transfer, in which one microbe produces hydrogen and then another microbe consumes it. But this discovery takes hydrogen transfer and goes a step further. Rather than a baton pass of sorts, it is two species directly plugging into each other.

The microbes Summers and her colleagues are studying - Geobacter - are of particular interest because of their role in environmental restoration. For example, the organisms can destroy petroleum contaminants and remove radioactive metal from polluted groundwater.

Summers recently talked with the Los Angeles Times about her lab work:

Question: What were you originally testing in the lab?

Answer: In the natural environment, there are all sorts of species everywhere, so we wanted to get a handle on what microbes are doing with their friends in the dirt.

We know that the microorganisms living in soil and water are essential for a healthy environment, but we have very little information on how different microbes live together. We wanted to learn more about how they cooperate. What we discovered is that different types of microbes can make electrical connections and pass electricity from one microbe to the other. This allows them to share energy in a highly efficient way that no one has ever seen before.

Q: You essentially placed the microbes in conditions that forced them to work together to survive, using alcohol as an energy source. What is their process?

A: We took two different microbes - Geobacter sulfurreducens and Geobacter metallireducens. They are different species, and only one of them - G. metallireducens - can consume ethanol for energy. They grow in clumps. So the second microbe attaches to the first and, rather than transfer substrates between each other, they are "wired" together and transfer electrons directly. In order to live, the G. sulfurreducens are sucking electrons off of the G. metallireducens.

We essentially put them in a position where they either adapt to work together or they both die. How they are working together has never been seen before in microbiology.

Q: What do they look like?

A: In order to grow and transfer energy, they have to be in these huge clumps together. They are the size of Nerds candy and they're pink, so they look like strawberry Nerds. The clumps are a millimeter or two, which is surprisingly large. You no longer need a microscope to see them. It's microbiology that turned into macrobiology.

Q: You've called these two "the ultimate drinking buddies." Why?

A: Because they have to work together or there's no ethanol consumed.

Q: What are the implications for renewable energy sources?

A: This is a new way to look at how microbe species can live together. Until now people have assumed that when microbes are working together they are transferring substrates between them. What we're saying is direct transfer through "wired" connections is also a possibility. This way of transfer is a much more streamlined way to transfer electrons between microbes. It's like taking an extension cord and plugging it into a neighbor.

Say you want to make a better wastewater treatment reactor. With this new information in hand, now we can possibly help to optimize these systems for faster waste degradation, and more effectively produce valuable biofuels from that waste, thanks to our microbe friends.

Q: What will you test next?

A: There are lots of cases all over the world where there are naturally occurring communities of bacteria that are in close contact, where multiple microbial species are touching - the touching is key for this type of electron transfer. For example, in microbial mats that are as thick as a doormat, or thicker, which grow in temperate climates along coastal regions (such as along the coasts of L.A., San Diego and Mexico), this type of transfer could also be happening.

With the mats, every millimeter you go down within it, there is something different going on - one of the microbes might consume something else that another does not like. There is probably some direct electron transfer going on there.

In wastewater treatment plants, we can take what we know and test different reactors processing all types of waste around the world, to see if there is a direct form of electron transfer happening.

Intelligent microscopy: New software runs experiments on its own (w/ Video)

Intelligent microscopy: New software runs experiments on its own (w/ Video)

Enlarge

Micropilot detected cells at particular stages of cell division (each row shows one cell), and then instructed the microscope to remove fluorescent tags from proteins in half the cell?s nucleus (left), and record what happened next (middle and right). Credit: EMBL

The sight of a researcher sitting at a microscope for hours, painstakingly searching for the right cells, may soon be a thing of the past, thanks to new software created by scientists at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany. Presented today in Nature Methods, the novel computer programme can rapidly learn what the scientist is looking for and then takes over this laborious and time-consuming task, automatically performing complex microscopy experiments when it detects cells with interesting features.

Called Micropilot, the software brings machine learning to microscopy. It analyses low-resolution images taken by a microscope and, once it has identified a cell or structure the scientists are interested in, it automatically instructs the microscope to start the experiment. This can be as simple as recording high-resolution time-lapse videos or as complex as using lasers to interfere with fluorescently tagged proteins and recording the results.

You need Flash installed to watch this video

Micropilot detected these cells and then instructed the microscope to remove fluorescent tags from proteins in half of each cell?s nucleus, and record whether fluorescently-tagged proteins moved in from elsewhere. Credit: EMBL

The software is a boon to systems biology studies, as it generates more data, faster. In a mere four nights of unattended microscope operation, Micropilot detected 232 cells in two particular stages of cell division and performed a complex imaging experiment on them, whereas an experienced microscopist would have to work full-time for at least a month just to find those cells among the many thousands in the sample. With such high throughput, Micropilot can easily and quickly generate enough data to obtain statistically reliable results, allowing scientists to probe the role of hundreds of different proteins in a particular biological process.

You need Flash installed to watch this video

Dividing cells were automatically recorded by Micropilot. Credit: EMBL

Jan Ellenberg and Rainer Pepperkok, whose teams at EMBL designed Micropilot, have used the software to deploy several different microscopy experiments, investigating various aspects of cell division. They determined when structures known as endoplasmic reticulum exit sites form, and uncovered the roles of two proteins, CBX1 and CENP-E, in condensing genetic material into tightly-wound chromosomes and in forming the spindle which helps align those chromosomes. This software will be a key tool for the European systems biology projects Mitosys and SystemsMicroscopy, for which Ellenberg and Pepperkok are developing technology.

Taking mating cues from many sources, pathogen adapts to thrive and infect

Taking mating cues from many sources, pathogen adapts to thrive and infect

The success of a fungal pathogen in becoming a persistent and opportunistic source of infection in human beings may be due to a mating strategy that can best be described as "don't be too choosy." A new Brown University study finds that Candida albicans will respond to the pheromones of several different species, not just its own, and if an opposite-sex partner isn't around, it can switch over to same-sex mating. In affairs of DNA exchange — for the yeast has no heart — Candida is exquisitely pragmatic.

When the yeast is not in a sexually active state, the same wide variety of pheromones can inspire it to clump together in tough-to-treat biofilms, said Richard Bennett, professor of biology and co-author of the paper published online in the Proceedings of the National Academy of Sciences.

The surprising finding that Candida albicans is so indiscriminate about pheromones could help in the fight against infections, which can sometimes be deadly for patients with compromised immune systems, Bennett said. The study illuminates both how the yeast may genetically adapt and how it can be induced to form biofilms.

"Sex can potentially generate recombinant forms of the species that may have increased drug resistance or altered pathogenic properties, a theory that we are currently testing," Bennett said. "Also, there are now direct links between mating and pathogenesis, as pheromone signaling can increase biofilm formation, an important first step in the establishment of many clinical infections."

In lab experiments led by first author and graduate student Kevin Alby, the researchers synthesized pheromones from several other Candida species. To varying degrees, many of the pheromones they used inspired mating in sexually active "opaque" C. albicans cells, including same-sex mating, while causing biofilm formation in sexually inactive "white" cells.

Bennett said the experiments were a "first step" to understanding mating signals and conditions in infectious Candida species. The next step is to determine whether other infectious fungi are similarly promiscuous and whether their previously underappreciated propensity for mating is perhaps based on a form of pathogenic peer pressure.

"We speculate that this could be a general advantage for fungi," he said. "If they sense other species are trying to mate, perhaps they decide they should too."

Another question, Bennett says, is whether the fungi could even be similarly receptive to chemical signals emitted by their human hosts.

Like humans, amoebae pack a lunch before they travel

Like humans, amoebae pack a lunch before they travel

"Fruiting bodies" discovered in amoebae collected in Virginia and Minnesota. Credit: Scott Solomon

(PhysOrg.com) -- Some amoebae do what many people do. Before they travel, they pack a lunch. In results of a study reported today in the journal Nature, evolutionary biologists Joan Strassmann and David Queller of Rice University show that long-studied social amoebae Dictyostellum discoideum (commonly known as slime molds) increase their odds of survival through a rudimentary form of agriculture.

Research by lead author Debra Brock, a graduate student at Rice, found that some amoebae sequester their food--particular strains of bacteria--for later use.

"We now know that primitively social slime molds have genetic variation in their ability to farm beneficial bacteria as a food source," says George Gilchrist, program director in the National Science Foundation's Division of Environmental Biology, which funded the research. "But the catch is that with the benefits of a portable food source, comes the cost of harboring harmful bacteria."

After these "farmer" amoebae aggregate into a slug, they migrate in search of nourishment--and form a fruiting body, or a stalk of dead amoebae topped by a sorus, a structure containing fertile spores. Then they release the bacteria-containing spores to the environment as feedstock for continued growth.

The findings run counter to the presumption that all "Dicty" eat everything in sight before they enter the social spore-forming stage.

Non-farmer amoebae do eat everything, but farmers were found to leave food uneaten, and their slugs don't travel as far.

Perhaps because they don't have to.

The advantages of going hungry now to ensure a good food supply later are clear, as farmers are able to thrive in environments in which non-farmers find little food.

The researchers found that about a third of wild-collected Dicty are farmers.

Petri plate containing bacteria harvested from amoebae. Credit: Scott Solomon

Instead of consuming all the bacteria they encounter, these amoebae eat less and incorporate bacteria into their migratory systems.

Brock showed that carrying bacteria is a genetic trait by eliminating all living bacteria from four farmers and four non-farmers--the control group--by treating them with antibiotics.

All amoebae were grown on dead bacteria; tests confirmed that they were free of live bacteria.

When the eight clones were then fed live bacteria, the farmers all regained their abilities to seed bacteria colonies, while the non-farmers did not.

Dicty farmers are always farmers; non-farmers never learn.

Rice graduate student Tracy Douglas co-authored the paper with Brock, Queller and Strassmann. She confirmed that farmers and non-farmers belong to the same species and do not form a distinct evolved group.

Still, mysteries remain.

The researchers want to know what genetic differences separate farmers from non-farmers. They also wonder why farmer clones don't migrate as far as their counterparts.

It might be a consequence of bacterial interference, they say, or an evolved response, since farmers carry the seeds of their own food supply and don't need to go as far.

An alternate view of amoebae fruiting bodies, with spores and bacteria. Credit: Owen Gilbert

Also, some seemingly useless or even harmful bacteria are not consumed as food, but may serve an as-yet-undetermined function, Brock says.

That has implications for treating disease as it may, for instance, provide clues to the way tuberculosis bacteria invade cells, says Strassmann, infecting the host while resisting attempts to break them down.

The results demonstrate the importance of working in natural environments with wild organisms whose complex ties to their living environment have not been broken.

Nailing down a crucial plant signaling system

Nailing down a crucial plant signaling system

Plant biologists have discovered the last major element of the series of chemical signals that one class of plant hormones, called brassinosteroids, send from a protein on the surface of a plant cell to the cell's nucleus. Although many steps of the pathway were already known, new research from a team including Carnegie's Ying Sun and Zhiyong Wang fills in a missing gap about the mechanism through which brassinosteroids cause plant genes to be expressed. Their research, which will be published online by Nature Cell Biology on January 23, has implications for agricultural science and, potentially, evolutionary research.

"Brassinosteroids are found throughout the plant kingdom and regulate many aspects of growth and development, as well as resistance from external stresses," said Wang. "Mutant plants that are deficient in brassinosteroids show defects at many phases of the plant life cycle, including reduced seed germination, irregular growth in the absence of light, dwarfism, and sterility."

Previous research had identified a pathway of chemical signals that starts when a brassinosteroid binds to a receptor on the surface of a plant cell and activates a cascade of activity that consists of adding and removing phosphates from a series of proteins.

When brassinosteroids are not present, a protein in this pathway called BIN2 acts to add phosphates to two other proteins called BZR1 and BZR2, which are part of a special class of proteins called transcription factors. The phosphates inhibit the transcription factors. But when a brassinosteroid binds to the cell-surface receptor, BIN2 is deactivated, and as a result phosphates are removed from the two transcription factors. As a result, BZR1 and BZR2 can enter the cell's nucleus, where they bind directly to DNA molecules and promote a wide variety of gene activity.

Before this new research, the protein that detaches the phosphates and allows BZR1 and BZR2 to work was unknown. Using an extensive array of research techniques, the team was able to prove that a protein called protein phosphatase 2A (PP2A) is responsible.

"We discovered that PP2A is a key component of the brassinosteroid signaling pathway," Wang said. "This discovery completes the core signaling module that relays extracellular brassinosteroids to cue activity in the nucleus."

Further research is needed to determine whether brassinosteroid binding activates PP2A, or just deactivates BIN2, thus allowing PP2A to do this job. Additionally, PP2A is involved in a plant's response to gravity and light, among other things.

This aspect of the brassinosteroid signaling pathway bears some surprising resemblances to signaling pathways found in many members of the animal kingdom. More research could demonstrate details of the evolutionary split between non-protozoan animals and plants.

Unlocking the secret(ase) of building neural circuits

Unlocking the secret(ase) of building neural circuits

Enlarge

Presenilin, better known for its role in Alzheimer's disease, aids with the correct wiring of the embryonic nervous system. Credit: Image: Courtesy of Dr. Sam Pfaff, Salk Institute for Biological Studies

Mutant presenilin is infamous for its role in the most aggressive form of Alzheimer's disease -- early-onset familial Alzheimer's -- which can strike people as early as their 30s. In their latest study, researchers at the Salk Institute uncovered presenilin's productive side: It helps embryonic motor neurons navigate the maze of chemical cues that pull, push and hem them in on their way to their proper targets. Without it, budding motor neurons misread their guidance signals and get stuck in the spinal cord.

By putting genes associated with Alzheimer's disease in a new light, their findings, published in the Jan. 7, 2011, issue of the journal Cell, reveal an important link between the formation of neural circuits and neurodegenerative disorders. "It was a bit of a surprise since we always thought about presenilin in the context of severing neuronal connections rather than wiring the nervous system during embryonic development," says Howard Hughes Medical Institute investigator Samuel Pfaff, Ph.D., a professor in the Gene Expression Laboratory, who led the study.

Presenilin is a component of the enzyme gamma secretase, which cleaves the amyloid precursor protein, resulting in accumulation of beta amyloid fragments. In Alzheimer's, these fragments form hard, insoluble plaques, one of the hallmarks of the disease.

Many embryonic guidance molecules persist in the adult central nervous system, where they participate in maintenance, repair and plasticity of neural circuits. "This could explain how a deregulation of guidance signaling by abnormal presenilin may play a role in the pathogenesis of Alzheimer's disease," proposes Pfaff.

The Salk study also adds an important new piece to the clockwork mechanism that guides growing nerve cells through the embryo and that depends as much on timing as on spatial accuracy. Understanding how axons find their destinations may help restore movement in people following spinal cord injury, or in those with motor neuron diseases such as Lou Gehrig's disease, spinal muscle atrophy and post-polio syndrome.

During normal development, trillions of neurons reach out for others with long, slender extensions to touch, connect and wire the budding nervous system. As the hair-like protrusions, called axons, grope around in the developing embryo, trying to find their proper targets, molecular ushers stationed along their path steer them in the right direction.

"Because of the vast number of neurons in the nervous system, ensuring that every single cell is on target creates more biological complexity than we can account for with the genetic information encoded in our genome," says Pfaff. "There are an estimated 100 trillion connections in our brain and only about 20,000 genes."

To find their course, growing neurons, especially motor neurons, which need to travel very long distances to reach their targets, navigate their path one small segment at a time, guided at each intersection by intermediate guideposts—chemical cues that attract or repel approaching axons. What's more, in a tightly regulated choreography, axons often switch allegiances when they reach a critical junction.

"It provides a way of creating some of these intermediate temporal steps," explains postdoctoral researcher and first author Ge Bai. "It allows the use of a small number of genes to regulate axonal growth by regulating the signals' effects in a very precise temporal and spatial ways."

He and his team found presenilin's unexpected role in controlling the activity of axon guidance signals during a search for genes involved in the fetal development of the nervous system. They had developed a method of engineering mice so that all of their motor neurons glow green. This fluorescence allowed them to visually identify mutant mice that have errors in motor neuron development and function.

One mouse, whose specific defect the researchers had mapped to the gene coding for presenilin, stood out. Failing to exit the spinal cord, its motor neurons got stuck at the midline, a row of cells that lie, moat-like, in the middle of the developing embryo. Bai discovered that in presenilin mutant mice, they were irresistibly attracted to Netrin, which is expressed by the midline.

In normal mice, motor neurons turn a deaf ear to Netrin's siren call and head out to the periphery. They are able to ignore Netrin because the receptor for Netrin is blocked by the so-called Slit/Robo tag team. Without presenilin, however, Netrin receptor fragments that are resistant to Slit/Robo silencing accumulate in the cell, and the motor neurons are now attracted to Netrin.

"The most satisfying thing we have learned about presenilin is that this is a component that is not directly involved in the detection of signals either as a ligand or a receptor but functions as a very important regulator of their spatiotemporal activity," says Bai.

Unearthing the mechanisms controlling plant size

Unearthing the mechanisms controlling plant size

Enlarge

Mechanism underlying increases in plant size. Photograph showing a cross-section of Arabidopsis roots. Plant cells divide mainly in the meristems at the tips of roots and stems. After dividing several times, cells begin to increase their size due to endoreduplication. When the cell reaches a certain size, endoreduplication and cell growth cease. The HPY2 gene controls the transition from cell division to endoreduplication, whereas the GTL1 gene stops cell growth. The green fluorescence indicates the HPY2 expression.

Plants have been cultivated and studied from the earliest days of human civilization, yet much remains unknown about them. A good example is the mechanism by which the size of plant cells is determined. Keiko Sugimoto, leader of the Cell Function Research Unit in RIKEN's Plant Science Center, in working to elucidate this mechanism has discovered a series of genes that control cell division or cell growth, attracting the attention of researchers and companies worldwide. "Our research focuses on the cellular aspects of plants," says Sugimoto.

A garden of lilies

While a high-school student, Sugimoto noticed that a single lily that had blossomed in her garden the year before had become three lilies a year later, followed by ten the next year and as many as 100 the year after. “But what impressed me most was that all of those flowers were the same size, and had the same color and same shape every year,” she says. “Although I knew that this was a manifestation of heredity, which I had learned at school, I was fascinated. I wanted to understand the mystery of plants, and this led me into research.”

After completing her master’s course in Japan, Sugimoto gained her PhD in plant science at the Australian National University. She then went to work at the John Innes Center in the UK—a Mecca for researchers studying plant biology—and in 2007 she set up the Cell Function Research Unit in RIKEN’s Plant Science Center. “How is plant cell size controlled? We are now working to solve this difficult problem.”

The aspect that had impressed Sugimoto most as a high-school student was that the sizes of flowers, leaves, seeds and other plant organs depend roughly on the species of plant. Each organ grows as its cells self-divide and increase in number, and each cell expands. However, plant organs do not continue to grow infinitely. “Flowers and leaves stop growing when they reach a certain size. Research has shown that plant hormones such as auxin and cytokinin are involved in plant growth, but we still don’t know how plant hormones control cell division and cell expansion to determine ultimate organ size,” says Sugimoto. “Plant size cannot be understood without knowing what is happening in cells. We are conducting research focusing on the cellular aspects of plants, which is a unique approach.”

Determinants of plant growth

“Every time I cut radishes or carrots into long, thin strips for cooking, I cannot help admiring the slices for a moment,” says Sugimoto with a smile. “If you look closely at a slice, you can see a finely textured portion near the tip. This is called the meristem. It is dividing tissue where the cells self-divide. Try taking a look next time you’re preparing a meal.”

Enlarge

Endoreduplication and cell size in Arabidopsis A scanning electron microscopy image of the surface of an Arabidopsis leaf showing a trichome. Most cells have 2C nuclear DNA content, but some cells have increased nuclear DNA contents of 4C, 8C and 32C due to endoreduplication. The four insets show the correlation between cell size and the amount of nuclear DNA. The trichome has a nuclear DNA content of 32C.

In plants, meristems are found only in the tips of roots and stems. Plant growth is the result of cell division and proliferation at the meristem, which is followed by cell expansion. “There are two key time points in plant growth. One is a turning point when cell division switches to cell expansion. Once a cell begins expanding, it cannot return to the stage of proliferation by division. The other is the point when cell growth stops and cells no longer expand. Plants cannot grow normally unless these two points are strictly controlled.” Sugimoto and her colleagues have attracted global attention for their discovery of the genes that control these two growth points.

Genes control cell division and endoreduplication

Sugimoto used a new technique to discover genes that control the point at which cells stop dividing and begin increasing in size. “Many researchers have tried to look for genes that control cell size, but most of them were trying to find mutants with altered cell size. Mutants have been identified merely based on the appearance of cells. We developed a method to accurately measure nuclear DNA content and isolate mutants with altered DNA levels.”

The cells of Arabidopsis, a commonly used experimental material in plant science, just like human cells, have two sets of chromosomes, one from the mother and the other from the father. The DNA of a cell having two sets of chromosomes is denoted 2C. When a 2C cell divides, its DNA is first replicated to produce 4C, which is then equally distributed into the next two dividing cells, resulting in two 2C daughter cells. In Arabidopsis, however, 2C and 4C cells are not the only cell types to be found. Gametes (pollen, ovules) that have undergone meiosis, a special process of cell division that results in half the number of chromosomes as found in somatic cells, are 1C cells, but there are also 8C, 16C and 32C cells. “In plant cells, DNA replication is sometimes followed by doubling in DNA without mitosis,” says Sugimoto. “This phenomenon is called endoreduplication, which results in 8C, 16C and 32C cells. The nuclear DNA and cell size are correlated; cells expand as their nuclear DNA increases.”

Together with Takashi Ishida, a postdoc in her lab, Sugimoto examined the nuclear DNA content of Arabidopsis cells and discovered a mutant having fewer 2C and 4C cells and more 32C, 64C and 128C cells. “Usually in plants, 2C and 4C cells in meristems continue to divide at a constant rate. We assume that in the mutant we discovered, these meristematic cells have undergone endoreduplication and switched into cell expansion prematurely. A more detailed investigation revealed that this mutant had lost the function of the HPY2 gene. Hence, HPY2 plays a role in controlling the point of switching to endoreduplication, where cells stop dividing and instead grow in size.”

This achievement was announced in August 2009, drawing attention not only from plant biologists, but also from researchers studying a wide variety of other organisms. “This is because HPY2 is involved in the function of a small peptide known as SUMO, a small ubiquitin-like modifier. SUMO is found in a broad range of species, from humans to plants and yeasts. It binds to other proteins to enhance or weaken their functions, and to regulate the diverse functions of cells. The reason why my result attracted so much attention is that researchers studying diverse ranges of organisms have been interested in SUMO.”

Enlarge

Regulation of cell division by the HPY2 gene. (Upper) Nuclear DNA content of a cell population. The hpy2 mutant has lower ratios of 2C and 4C and higher ratios of 32C, 64C and 128C compared to the wild-type control. (Lower) Photographs of wild-type (left) and hpy2-mutant (right) plants ten days after germination. The mutant has very small roots and leaves. The Blue staining indicates defective cell division in the mutant.

Sugimoto’s group demonstrated that the protein produced by HPY2 mediates the binding of SUMO to other proteins, resulting in the regulation of cell division. This was the first report of SUMO being associated with the regulation of cell division in multicellular organisms. “I never thought that my studies on the mechanism of plant cell size control would lead to SUMO. Research is fascinating because it can lead to unexpected results.”

Sugimoto has also discovered three other genes that control the switch into endoreduplication like HPY2. Her next task is to clarify the differences in their functions.

A gene terminating cell growth

In September 2009, following the discovery of HPY2, Sugimoto’s group discovered a gene involved in the second point—when plant cells stop growing in size. “It began with the discovery of a mutant having very large trichomes by Christian Breuer, a posdoc in my lab, who was searching for mutants with abnormal cell size.”

Trichomes are hair-like outgrowths that cover the surfaces of Arabidopsis leaves to protect them from insects, pathogens and even ultraviolet radiation. “Each trichome comprises a single epidermal cell in Arabidopsis. It is large enough to be seen macroscopically.” While even a normal-sized trichome is 500 times larger than an ordinary cell, the mutant discovered by Breuer has trichomes that are more than twice this size.

The mutant was found to have the GTL1 gene partially modified and expressed in excess. When the function of GTL1 was artificially suppressed, the mutant’s trichomes became more than twice the size of wild-type trichomes. Based on these experiments, Sugimoto’s group hypothesized that GTL1 functions to terminate cell growth. To test the hypothesis, they examined when and where GTL1 is expressed. It was found not to be expressed in smaller trichomes in the early stage of growth or trichomes that had stopped growing, but to be expressed only in trichomes that have just expanded to maximum size.

Previously, it had been thought that cell growth ceases when the supply of cellulose and other components of the cell wall is stopped, or when water absorption in vacuoles ceases. However, the discovery of GTL1 shows that plants have an intrinsic mechanism for actively stopping cell growth. The discovery is groundbreaking, overturning the traditional concept of plant growth.

Trichomes are cells undergoing endoreduplication, which is known to cease at 32C. Sugimoto’s group is conducting research on the hypothesis that GTL1 may control endoreduplication. It is already known that the function of the gene necessary for endoreduplication is activated in mutants lacking the function of GTL1. “GTL1 produces a protein known as a transcription factor, which binds to the DNA of a certain gene to promote or suppress its transcription to RNA. In the future, I want to clarify how GTL1 controls transcription and of which genes, and to discover the mechanism of endoreduplication.”

Giant prospects

Since the announcement of the discovery of GTL1, Sugimoto has received a flood of offers for joint research, including many inquiries from industry, who have great expectations for creating larger fruits and vegetables by suppressing the function of GTL1.

Some cultivars are already available with increased yields thanks to artificial duplication of nuclear DNA with chemical agents. However, this chemical treatment unavoidably duplicates the nuclear DNA in all cells constituting the plant body, which in turn makes the plant unable to produce seeds. “Advanced research on GTL1 may allow us to promote endoreduplication at desirable portions of plants, such as fruits, flowers and leaves, or whenever needed, to change their sizes without preventing seed production,” says Sugimoto, who is keen to conduct joint research with industry.

“Now is the most enjoyable time in my academic career,” declares Sugimoto. However, she is not satisfied with just discovering the genes that control plant growth. Further extensive investigation of the functions of individual genes is needed. It is also necessary to identify the targets of HPY2 and GTL1 to determine on which genes and proteins they act. She is also interested in the relationship between HPY2 and GTL1, and their association with plant hormones. “Much remains to be done, and I have not found the answer to my question about lilies when I was a high school student. In the plant kingdom, there are so many unanswered questions. This is why I am fascinated by plant research.”

Study sheds new light on river blindness parasite

Study sheds new light on river blindness parasite

The team found that a bacterium inside the worm acts as a 'disguise' for the parasite, resulting in the immune system reacting to it in an ineffective way. The bacteria protect the worm from the body's natural defences, but once the bacteria are removed with antibiotics, the immune system responds appropriately, releasing cells, called eosinophils, that kill the worm.

Antibiotics are successful against the parasite, but the long treatment regime means that it has limited use across whole communities. These new findings suggest that if medics could prime the immune system to recognise the worm, a shorter duration of antibiotic treatment may be sufficient to overcome its bacterial defences.

River Blindness is caused by black flies that breed in rivers and deposit the larvae of a worm into the person they bite. The infection leads to severe itching of the skin and lesions of the eye which can result in blindness. It affects millions of people in developing countries, particularly in West and Central Africa. A closely related parasite also infects cattle, which causes lumps to appear on the animal's skin but does not cause blindness or other illness.

Dr Ben Makepeace, from the University's Institute of Infection and Global Health, explains: "Our team has already shown that removing the bacteria with antibiotics results in the death of the worm, but until now we were unaware of how the bacteria protected the parasite in the first instance. Antibiotics can rid the parasite of the bacteria, allowing the immune system to respond properly, but it is a long treatment process, lasting up to six weeks.

"Now we can begin to look for a way to 'prime' the body into reacting to the parasite more efficiently. Currently there is no vaccine for River Blindness, but if a candidate could be identified this may help boost the immune system ahead of antibiotic treatment and reduce the length of time patients have to take the drug. It is essential that whole communities are cured of the infection and the more we know about the mechanisms the parasite uses to survive in the body, the further we can progress with finding a practical treatment that kills adults worms and not just the larval stages."

High-pressure division

High-pressure division

Enlarge

One way to recognise dividing human cells (red) on a Petri dish is by the DNA stained green in their cell nuclei. The tip of the silicon spring (black) is positioned above a cell that has already rounded up to prepare for division. Its condensed DNA is visible as a green bar. Credit: zVg D. Müller/ETH Zurich

To be able to divide, a cell must first of all create enough space for itself in the tissue. How cells do that was a mystery, until now. Researchers from the Department of Biosystems Science and Engineering at ETH Zurich have now discovered that cells "inflate" themselves generating hydrostatic pressure during division. By doing this cells develop an enormous force, which they use to push other cells aside.

Tissues, e.g. skin or muscles, consist of a dense network of cells in close contact with one another. If an individual cell wants to divide into two daughter cells, it must first detach itself from its neighbouring cells and round up into a sphere. At the same time it increases its volume, for which it needs additional space. How the cell can achieve this dramatic change amongst the densely congregated cells was unknown until now.

Researchers led by Daniel Müller, Professor of Biophysics at ETH Zurich, have now solved this problem, in collaboration with researchers from Dresden. Their results, which they have published in the scientific journal Nature, show that animal cells absorb water during division. As a result they build up hydrostatic pressure, which inflates them like a balloon. This generates a force that can push aside the surrounding tissue. Daniel Müller is convinced that “This means cells have developed an active process to create space for themselves.”

Miniature muscleman

To measure the force cells develop during division, Martin Stewart a research team member used a scanning force microscope on which there is a silicon spring only a few hundredths of a millimetre long. The researchers positioned the spring above a cell that had grown on the bottom of a Petri dish and had just started to divide. At first the spring did not touch the cell, but as division progressed the cell expanded more and more until it pressed the spring upwards. The force that is measurable in this process is only about 0.0000001 Newton. Although that sounds small, it is very large on the scale of a single cell. It corresponds approximately to the force a person would need to hold back an elephant.

In further experiments the researchers tried to find out how the cell succeeds in generating so much force. They did this by searching for proteins that become particularly active during cell division. Eventually they discovered a transport protein in the cell envelope that transports sodium ions into the cell interior, thus regulating the salt balance. They observed that a particularly large amount of sodium is transported when a cell divides. This generates an osmotic effect that causes water to flow into the cell’s interior. The more water flows in, the more the cell expands.

Soft shell, hard core

However, that on its own would not be enough to enable the cell to exert a force on its surroundings, because its envelope would be too soft and unstable. Daniel Müller compares it to an inflated bicycle inner tube that gives way when pressed. The tube only becomes taut due to the tyre, which keeps it in shape. In cells this function is carried out by the cell skeleton, consisting of a network of actin molecules which are also proteins. The skeleton is located immediately beneath the cell envelope and offers it stability.

When the researchers used a chemical substance to destroy the actin network of a dividing cell, the cell expanded even further. Conversely, it shrank when the actin was left intact but the inflow of water was blocked. From this the scientists concluded that two forces interact in opposite directions: one directed outwards, which inflates the cell, and an opposing force directed inwards, which causes the pressure to increase.

So far Daniel Müller’s team has only studied individual growing cells, but not those inside tissue. “Our measurement method is not yet suitable for that”, he explains. However, studies by microscopy have yielded evidence that the same processes also take place in tissues. Moreover, division only succeeds if sufficient space is available. The researchers infer this from an experiment in which they used the silicon spring to exert so much pressure on the cell that it was unable to expand. More than seventy percent of the cells treated in this way did not complete the division and died.

However, sooner or later the cell must also release the pressure that it has built up during the division process. The researchers still cannot say how it knows when to do this. Müller suspects that “There must be a signal for it”. He and his team are already tracking this down in a new project.

The good, the bad and the 'green' -- harnessing the potential of bacteria

The good, the bad and the 'green' -- harnessing the potential of bacteria

Enlarge

(PhysOrg.com) -- A diverse family of bacteria that can cause a potentially fatal illness in humans but could offer a greener alternative to petrol to power our cars will be the subject of a talk by a University of Nottingham academic at an international conference.

Professor Nigel Minton, one of the world's leading experts on the Clostridium bacteria, will be presenting at the Society for Applied Microbiology (SfAM) annual Winter Meeting, being held at the Royal Society in London on January 12.

In his presentation Professor Minton will discuss the potential exploitation of the anaerobic, Gram-positive Clostridium bacteria — a few strains of which have given the genus a bad name.

Clostridium difficile infection is the most significant cause of hospital-acquired diarrhoea and is seven times more deadly than MRSA. The bacterium is present in the gut of up to three per cent of healthy adults and 66 per cent of infants. Usually it is kept in check by the healthy balance of bacteria in the gut but when this is disturbed by certain antibiotics, C.difficile can multiply rapidly and produce toxins that cause illness and death. The disease is spread through spores, usually from poor hygiene. The emergence of highly virulent clones means that cases and fatalities from the illness are on the increase.

In addition, there has been heightened public concern in recent years about the potential use by bioterrorists of the food-borne pathogen Clostridium botulinum, which causes the rare but serious paralytic illness botulism.

However, Professor Minton will argue, there is an urgent need to understand the basic biology of these important bacteria to enable scientists to both prevent and treat the diseases they cause and to harness the potentially beneficial properties which they also offer.

In particular, high oil prices and the need to move towards a more sustainable energy economy has sparked an increased interest in the use of clostridial fermentations for biofuel production. Strains of Clostridium can offer a highly effective method of producing renewable ethanol and butanol.

Latest research which has demonstrated that some Clostridia spores have a unique ability to selectively germinate in tumours indicates that the bacteria could even potentially play a part in treating cancer.

The meeting organised by SfAM, the UK's oldest microbiology society, will bring together more than 100 international scientists to learn about two of the hottest topics in microbiology — probiotics and anaerobic microbes.