Chaperone enzyme provides new target for cancer treatments

Chaperone enzyme provides new target for cancer treatments

UNC scientists who study how cells repair damage from environmental factors like sunlight and cigarette smoke have discovered how a "chaperone" enzyme plays a key role in cells' ability to tolerate the DNA damage that leads to cancer and other diseases.

The enzyme, known as Rad18, detects a protein called DNA polymerase eta (Pol eta) and accompanies it to the sites of sunlight-induced DNA damage, enabling accurate repair. When Pol eta is not present, alternative error-prone polymerases take its place – a process that leads to DNA mutations often found in cancer cells.

In one known example, faulty DNA repair due to Pol eta- deficiency is responsible for the genetic disease xeroderma pigmentosum-variant, which makes patients extremely susceptible to skin cancers caused by exposure to sunlight. However, scientists did not know how the cells selected the correct DNA Polymerase for error-free repair of each type of DNA damage.

"We found that the mechanism that promotes the 'chaperone' enzyme to recruit Pol eta to sites of DNA damage is managed by another signaling protein termed 'Cdc7' which we know is essential to normal regulation of the cellular lifecycle," said lead author Cyrus Vaziri, PhD, who is an associate professor of pathology and laboratory medicine and member of UNC Lineberger Comprehensive Cancer Center. Thus cells employ Cdc7 to ensure accurate DNA repair during the stage of their lifecycle that is most vulnerable to cancer-causing mutations.

The study was published in November in the Journal of Cell Biology.

According to Vaziri, the dual role that Cdc7 plays in the cell lifecycle and DNA repair offers a promising target for potential cancer therapies.

"We know that cancer cells have high levels of Cdc7 activity and can evade some DNA-damaging therapies such as cis-Platinum through Rad18 and Pol eta activity. We may be able to target this pathway in platinum-resistant tumors to prevent DNA repair and enhance cancer cell killing by platinating agents," he said.

Global view of blood cell development reveals new and complex circuitry

Global view of blood cell development reveals new and complex circuitry

A small pool of stem cells replenishes the human body with about 200 billion new blood cells daily. But the elaborate circuitry that determines if a cell will develop into a T cell, red blood cell, or one of the nine or more other blood cell types remains largely unknown. A research team led by scientists from the Broad Institute and Brigham and Women's Hospital has taken a systematic approach to help decipher this circuitry, compiling a comprehensive catalog of the factors that determine a blood cell's fate. Their work appears in the January 21 issue of Cell.

The researchers found that blood cells are directed by a multitude of transcription factors, proteins that turn on and off genes. While many previous studies have focused on individual transcription factors or types of blood cells, this study examined the expression and regulation of all transcription factors throughout blood development. The findings point to densely, interconnected circuits that control this process, suggesting that the wiring for blood cell fate is far more complex than previously thought.

"One assumption in the field had been that there are a small number of transcription factors that orchestrate this process," said Aviv Regev, a Broad Institute core member and co-senior corresponding author of the study. "Some people have always thought there would be a lot of factors and that it would just take time to find them. It turns out there are more masters than we would have thought."

The researchers looked globally at how the expression of all 20,000 or so genes in the genome change as blood stem cells become specialized cell types (a process known as differentiation). They discovered that while a small fraction of genes are uniquely expressed in a single type of cell, other genes are more broadly expressed — present in a variety of cell types but at varying levels. Some of these genes are turned on in the blood stem cells and switched off at certain points in development while others are reused in several parallel developmental branches. The researchers found about 80 of these patterns of variable genes, called modules. Each kind of specialized cell has a unique profile, or combination, of these modules.

Looking at the genes modulated in the course of healthy cell development could give researchers clues about what events lead to blood cancers, such as leukemia, a disease where differentiation has gone wrong.

"When you look at leukemia cells beneath a microscope, they have a lack of differentiation and they look abnormal," said Broad associate member Ben Ebert, an associate physician of hematology at Brigham and Women's Hospital and a senior corresponding author of the study. "They've ended up in a place that doesn't exist in normal development." Now that the researchers have a clearer picture of the modules that normal cells exhibit, they can apply this knowledge to help identify the similarities and critical changes in leukemia cells' profiles.

"Leukemia cells have the same set of building blocks as normal blood cells – some, they keep the right way so a piece of the profile is right, and a piece of the profile is wrong," said Regev, who is also an assistant professor in the department of biology at MIT and an Early Career Scientist at Howard Hughes Medical Institute.

The research team included co-first author Noa Novershtern from the School of Computer Science at the Hebrew University of Jerusalem, co-first author Aravind Subramanian in Todd Golub's laboratory at the Broad, and Lee Lawton and other collaborators in Richard Young's laboratory at the Whitehead Institute. All of their results will be made publicly available online through a database known as the Differentiation Map Portal (or D-Map). Ebert, Regev and their colleagues intend for D-Map to be a starting point for other researchers, empowering their investigations into the biology of blood cells as well as leukemia and other human diseases.

"Already, many people are asking for the data. Other groups can now combine their data with ours to ask new questions," said Novershtern. "What's also exciting is that people can see the power of computational models, tools that can be used to find new biological insights from the data."

Scientists find measles' natural nemesis

Scientists find measles' natural nemesis

Scientists at The Scripps Research Institute have found that a known enzyme in cells protects against measles virus, likely by altering the virus's genetic material, RNA. Cells lacking the enzyme become highly vulnerable to the virus's destructive effects. The enzyme also protects against several other respiratory viruses, including influenza A.

"We believe that host cells use this RNA-editing enzyme to slow these viruses' ability to replicate," said Michael B. A. Oldstone, the study's senior author and a professor at Scripps Research's La Jolla, California campus. The study's first authors are Simone V. Ward, a senior research associate in the Scripps Research Oldstone laboratory, and Cyril X. George of the University of California, Santa Barbara.

The finding represents a significant improvement in the understanding of measles infections, which still kill about 150,000 children and adults around the world every year. The paper, which was published recently in Proceedings of the National Academy of Sciences, has prompted commentaries in the journals Nature Reviews Microbiology and Viruses.

The focus of the study was the enzyme ADAR1 ("adenosine deaminase acting on RNA, 1"), which is known to be produced in high amounts in measles-infected cells. ADAR1 has been suspected as a "restriction factor" that inhibits viral replication.

ADAR1's role against measles has been difficult to nail down, however. In mice genetically engineered to be infectable by measles – a virus that normally infects only humans – ADAR1 is required for embryonic development, as in all mice. Thus the standard "gene knockout" technique, which would enable scientists to see how measles infections proceed without ADAR1, hasn't been feasible.

In this study, Ward, George, and Oldstone, and their colleagues knocked out only one of the two forms of ADAR1 produced in cells. This form, p150, is the one produced in response to infections. For reasons that still aren't clear, mouse embryos cannot grow for long without p150, so the researchers used a standard technique to "immortalize" these p150-knockout embryonic cells—ensuring their continuous supply—and in this way created a useful cell model.

When infected by measles virus, the p150-knockout cells succumbed quickly compared to immortalized control cells that produced p150 normally. "When I looked at the cells only 21 hours after infection, the p150-knockout cells already showed the signs of cell damage typical of measles infection," said Ward. "But the control cells looked exactly like uninfected cells."

In further tests, Ward found p150 also provided significant protection for cells against Newcastle disease virus, Sendai virus, canine distemper virus, and influenza A virus – which are all respiratory viruses like measles, and all members of the paramyxovirus or orthomyxovirus families.

Nine years ago, it was reported that a cellular enzyme known as APOBEC3G protects cells from DNA-based viruses such as HIV, by mutating viral genes. "We're now showing that an analogous gene-editing enzyme also seems to exist for RNA viruses," said Oldstone.

With the new cell model, and advanced "conditional knockout" techniques that allow genes to be disrupted in specific organs in adult mice, Ward, Oldstone, and their colleagues now hope to study ADAR1-p150's role in more detail.

One key issue to be resolved is the enzyme's role during brain infections. Measles virus usually results in a relatively mild illness lasting only a week or two, but in rare cases it spreads to the brain and becomes a persistent, always fatal infection known as subacute sclerosing panencephalitis (SSPE). In such cases, the virus doesn't have to spread via cell-to-cell contact, thus exposing itself to the immune system; it can spread more stealthily along the axons and dendrites that connect neurons.

"What we hope to show with our ongoing work is that host neurons are using ADAR1 to slow down this process, turning it into a gradual neurological disease," said Oldstone. ADAR1 might also be exacerbating the neurological symptoms of SSPE, he adds, because its enzymatic activity is known to affect the production of the important neurotransmitter receptors for serotonin and glutamate: "It's an enzyme that has multiple roles," Oldstone concluded.

How bacteria keep us healthy

How bacteria keep us healthy

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Joerg Graf, associate professor of molecular and cell biology, with medicinal leeches at his lab. His research on bacteria in the gut uses the medicinal leech as a model system. Photo by Peter Morenus

(PhysOrg.com) -- Joerg Graf is studying medicinal leeches for clues about how changes in diet affect microorganisms in the digestive tract.

Many people think of bacteria as disease-causing agents that should be avoided for fear of getting sick. But molecular and cell biologist Joerg Graf points out that we can’t live without them.

He says there are actually more bacteria on and in humans than there are human cells. “If you took a person and removed all the human cells, you would still see the outline of a human body,” made up of bacterial cells, says Graf, an associate professor of molecular and cell biology in the College of Liberal Arts and Sciences.

Bacteria are on our skin, in our mouth and throat, in our intestines, nose, and virtually every nook and cranny of our bodies that is connected with the outside – and almost of all them are not bad for us.

Graf recently received a $1.6 million grant from the National Institutes of Health (NIH) to study bacteria that live in digestive tracts. The project is a collaboration with Pieter Visscher, a professor of marine sciences, and Hillary Morrison, a researcher at the Marine Biological Laboratory at Woods Hole. Their work will help scientists understand the vital role that bacteria play in our everyday health.

Many bacteria are essential to the normal functioning of physiological processes, including digestion and immune responses. The gut microbiome consists of all of the bacteria in the human gut and, for example, digests food that humans otherwise can’t, such as some plant material, as well as providing nutrients in forms that humans can use. Humans acquire these bacteria from their mothers as they pass through the birth canal and from the outside world through the course of their childhood. This is why, explains Graf, people in different parts of the world have different combinations of gut bacteria.

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Joerg Graf displays a leech. Photo by Peter Morenus

Graf and his colleagues are interested in how relationships between bacteria and their hosts have evolved, and how they change over an organism’s lifetime.

“We don’t understand what drives the changes in the gut microbiome,” he says, adding that it’s difficult to study the human gut because there are hundreds of different kinds of bacteria present.

Instead Graf and his colleagues work on a model system. “By having a simple system, we can understand it better using molecular tools.”

The model system they use is the medicinal leech, because the microbiome of its digestive tract is normally dominated by only two types of bacteria. These species help to degrade blood that the leech consumes, providing nutrients to the leech. With only two species to observe, Graf can study how this bacterial community changes with dietary shifts in the organism.

Like humans, leeches undergo a shift in their diet at a young age: while humans spend the first stages of their life drinking only milk, leeches also spend their first few days living only on protein provided by their mother. After feeding the leech its first actual blood meal, the scientists will observe the changes in the leeches’ gut bacteria by looking at the RNA they make, which will in turn produce different proteins that allow the bacteria to degrade the food and provide nutrients to the leech. To perform this work, they will use a gas chromatography-mass spectrometer that was purchased by Kenneth Noll, a professor of molecular and cell biology, using funds from the Provost’s Intermediate Research Equipment Competition.

“The function of these proteins is related to the bacterial species and can determine how diverse the functions are within one species,” and how the function changes as the microbial community changes, says Graf.

One of Graf’s hypotheses is that one of the bacteria uses a “molecular syringe” to inject a toxin that prevents the leech’s natural immune cells from attacking it. This molecular syringe is also a virulence factor that is required to cause disease in other animals, but not to the leech. In the leech, it simply signals that the bacteria are not enemies, and should be left alone.

Additionally, Graf is interested to find out whether these bacteria rely on each other for nutrients. Like the leech itself, one of the bacteria might rely on the other to converting food into a form that it can use, allowing it to grow faster.

All of these questions will help Graf and his colleagues determine how physiology changes when the gut microbiome changes, which could lead to insights about our dependence on the bacteria themselves. For example, people with inflammatory bowel disease have a different microbial community than those without, says Graf. Learning about the changes in bacterial gut communities and its contribution to the host animal could give clues about other diseases.

Graf is also involved with the Human Microbiome Project, which aims to characterize human microbial communities, including that of the gut, and to analyze their role in health and disease. Hundreds of scientists across the U.S. are working on this NIH-funded initiative.

Graf says that for him, being involved with these projects is like working to solve a puzzle.

“There are many different ways to do science,” he says. “We’re at a very exciting time in biology, with new techniques to sequence millions of DNA and RNA molecules. These techniques are proving very powerful.”

Cell death pathway linked to mitochondrial fusion

Cell death pathway linked to mitochondrial fusion

New research led by UC Davis scientists provides insight into why some body organs are more susceptible to cell death than others and could eventually lead to advances in treating or preventing heart attack or stroke.

In a paper published Jan. 21 in the journal Molecular Cell, the UC Davis team and their collaborators at the National Institutes of Health and Johns Hopkins University report that Bax, a factor known to promote cell death, is also involved in regulating the behavior of mitochondria, the structures that provide energy inside living cells.

Mitochondria constantly split and fuse. The proteins that control the splitting of mitochondria also promote a process called apoptosis, or programmed cell death. In contrast, the proteins that control mitochondrial fusion help protect against cell death. Cell death can happen when cells are starved of oxygen, for example during a heart attack or stroke.

Yeast have a single protein that controls outer membrane fusion, but both human and mouse cells have two proteins, called MFN1 and MFN2, which control outer membrane fusion. Using mitochondria from cells derived from genetically modified "knockout" mice, Suzanne Hoppins, a postdoctoral researcher at UC Davis, and Jodi Nunnari, a professor of molecular cell biology, studied how these two proteins work together and the role specific genes play in that process.

The research team discovered that these proteins combine with themselves or each other to form a tether between two mitochondria, leading to fusion. All three combinations -- MFN1/MFN1, MFN1/MFN2 and MFN2/MFN2 -- can promote membrane fusion, but the combination of MFN1/MFN2 is by far the most efficient, Hoppins said.

Hoppins also found that a soluble form of Bax, a protein that triggers apoptosis, can also stimulate mitochondria to fuse. It acts only through the MFN2/MFN2 combination, she found.

The form of Bax that promotes mitochondrial fusion is different from the type that leads to cell death, Nunnari said. Bax leads to cell death when it inserts itself in the mitochondrial membrane. In its soluble, free-floating form, it causes mitochondria to fuse instead.

MFN1 and MFN2 are found in different amounts in different body organs. MFN2 is more abundant in the brain and heart -- tissues where cell death can have disastrous consequences.

The paper shows how MFN2 could act to protect the brain or heart from cell death, by using Bax in a different form, Nunnari said.

"This shows that the fusion machine is both positively and negatively regulated in cells and opens doors to finding the regulatory mechanisms and discovering ways to increase or decrease the sensitivity of cells to apoptosis," Hoppins said. That could lead to new drugs that save cells, for heart disease and stroke, or that kill cells, for cancer.

The genius of bacteria: Scientists develop IQ test to assess and outsmart bacteria's 'social intelligence'

The genius of bacteria: Scientists develop IQ test to assess and outsmart bacteria's 'social intelligence'

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This is a "smart community" of Paenibacillus vortex bacteria. Credit: Prof. Eshel Ben-Jacob, Tel Aviv University

IQ scores are used to assess the intelligence of human beings. Now Tel Aviv University has developed a "Social-IQ score" for bacteria ? and it may lead to new antibiotics and powerful bacteria-based "green" pesticides for the agricultural industry.

An international team led by Prof. Eshel Ben-Jacob of Tel Aviv University's Department of Physics and Astronomy and his research student Alexandra Sirota-Madi says that their results deepen science's knowledge of the social capabilities of bacteria, one of the most prolific and important organisms on earth. "Bacteria are our worst enemies but they can also be our best friends. To better exploit their capabilities and to outsmart pathogenic bacteria, we must realize their social intelligence," says Prof. Ben-Jacob.

The international team was first to sequence the genome of pattern-forming bacteria, the Paenibacillus vortex (Vortex) discovered two decades ago by Prof. Ben-Jacob and his collaborators. While sequencing the genome, the team developed the first "Bacteria Social-IQ Score" and found that Vortex and two other Paenibacillus strains have the world's highest Social-IQ scores among all 500 sequenced bacteria. The research was recently published in the journal BMC Genomics.

Highly evolved communities

The impact of the team's research is three-fold. First, it shows just how "smart" bacteria can really be –– a new paradigm that has just begun to be recognised by the science community today. Second, it demonstrates bacteria's high level of social intelligence –– how bacteria work together to communicate and grow. And finally, the work points out some potentially significant applications in medicine and agriculture.

The researchers looked at genes which allow the bacteria to communicate and process information about their environment, making decisions and synthesizing agents for defensive and offensive purposes. This research shows that bacteria are not simple solitary organisms, or "low level" entities, as earlier believed ? they are highly social and evolved creatures. They consistently foil the medical community as they constantly develop strategies against the latest antibiotics. In the West, bacteria are one of the top three killers in hospitals today.

The recent study shows that everyday pathogenic bacteria are not so smart: their S-IQ score is just at the average level. But the social intelligence of the Vortex bacteria is at the "genius range": if compared to human IQ scores it is about 60 points higher than the average IQ at 100. Armed with this kind of information on the social intelligence of bacteria, researchers will be better able to outsmart them, says Prof. Ben-Jacob.

This information can also be directly applied in "green" agriculture or biological control, where bacteria's advanced offense strategies and toxic agents can be used to fight harmful bacteria, fungi and even higher organisms.

Tiny biotechnology factories

Bacteria are often found in soil, and live in symbiotic harmony with a plant's roots. They help the roots access nutrients, and in exchange the bacteria eat sugar from the roots.

For that reason, bacteria are now applied in agriculture to increase the productivity of plants and make them stronger against pests and disease. They can be used instead of fertilizer, and also against insects and fungi themselves. Knowing the Social-IQ score could help developers determine which bacteria are the most efficient.

"Thanks to the special capabilities of our bacteria strain, it can be used by researchers globally to further investigate the social intelligence of bacteria," says co-author Sirota-Madi. "When we can determine how smart they really are, we can use them as biotechnology factories and apply them optimally in agriculture."

Research shows when stem cell descendants lose their versatility

Research shows when stem cell descendants lose their versatility

Becoming hair. To create a new hair follicle (blue), the body taps stem cells from a reservoir called the niche (green). Researchers have determined at what point stem cells irreversibly become hair follicle cells, and have found that these descendent cells send signals back to the niche that regulate the stem cells' activity.

(PhysOrg.com) -- Stem cells are the incomparably versatile progenitors of every cell in our body. Some maintain this remarkable plasticity throughout the life of an animal, prepared to respond as needed to repair an injury, for instance. Others differentiate into specialized cells, regenerating tissue or facilitating some other process before dying. Now new research from Rockefeller University defines the point at which hair follicle stem cells abandon their trademark versatility, or “stemness,” having left their niche to make new hairs. It also shows how these fated stem cell descendants then regulate the activity of their forebears.

“We and others have been focusing on what mobilizes stem cells to make tissue,” says Elaine Fuchs, Rebecca C. Lancefield Professor and head of the Laboratory of Mammalian Cell Biology and Development. “However, it is just as important for stem cells to know when to stop the process, which is what we’ve found here.”

The researchers, led by Ya-Chieh Hsu, a postdoctoral associate in Fuchs’ lab, focused on mouse hair follicles, which undergo cyclical bouts of growth, destruction and rest, a process requiring the activation of stem cells. Stem cells are usually inactive, at rest in their niche, but when activated, they proliferate and leave that niche to make new hairs. In the new research, published last week by Cell, the researchers drilled down on this cycle, defining the point at which activated stem cells become irreversibly committed to becoming the specialized cells needed to grow hair.

Through gene expression analysis and experiments designed to test the cells’ function at different stages in the cycle, the researchers show that early stem cell descendents can retain their stemness and return back to their niche when hair growth stops. In fact, even after their proliferating descendants irreversibly lose their stemness, some can still find their way back to the niche, where they continue to serve two primary purposes: they hold the hairs tightly in place to prevent hair loss, and they release inhibitory signals that prevent the stem cells from activating too early.

“This study shows that committed stem cell descendents transmit inhibitory signals back to the stem cells and return them to a dormant state,” says Fuchs, who is also a Howard Hughes Medical Insititute investigator. “The finding gives us new insights into why our spurts of hair growth are followed by a resting period. For many tissues of the body, such negative feedback loops could provide the necessary signals to prevent tissue overgrowth.”

These findings is work represents a new concept in stem cell biology — that an irreversibly committed cell that is downstream in a stem cell lineage can become an essential regulator of stem cells, the researchers say. In other words, the children tell the parents how to behave. “In many systems, stem cells and their differentiated progeny coexist in close proximity,” Hsu says. “The ability of the progeny to regulate stem cell activity could be a general but previously unrecognized phenomenon which enables stem cells to know when to stop making tissue.”

Antibiotics improve with visualization techniques

Antibiotics improve with visualization techniques

Researchers from the Universities of Dundee and Oxford have made a significant breakthrough in understanding how resistance to antibiotics might be overcome, by producing the first ever 3D molecular image of a key drug target and showing how drugs bind to it.

Professor William Hunter, from the College of Life Sciences at Dundee, coordinates the EU-funded Aeropath project, which is designed to advance early stage drug discovery against Pseudomonas aeruginosa (P. aeruginosa), a common bacterium that can cause serious diseases in animals and humans.

In collaboration with colleagues from Oxford’s Division of Structural Biology, and the Oxford Protein Production Facility (OPPF) using state of the art facilities at the Diamond Light Source synchrotron in Oxfordshire, the team were able to determine the accurate chemical structure of the penicillin binding protein PBP3 from Pseudomonas aeruginosa.

This bacterium is particularly troublesome since it has developed resistance to many common antibiotics and is an important pathogen of burns victims, immuno-compromised patients for example due to chemotherapy or other conditions, and also to cystic fibrosis patients.

The research was made possible by using a machine called a synchrotron. It houses a large ring-shaped vacuum tube similar in size to Hampden Park, in which electrons are accelerated at close to the speed of light and manipulated by special magnets to give off very intense X-rays. These X-rays were then used to probe tiny PBP3 crystals through diffraction methods, enabling the researchers to determine the 3D structure of the protein.

Knowing the 3D structure of an antibiotic bound to its target protein elucidates the molecular mechanism - revealing how the drug works and how it could be modified, for example, to overcome resistance. The structures identified suggest that there could be scope to develop new drugs that work in combination with existing PBP inhibitors to make them more effective and able to overcome resistance.

The research has been published in the most recent edition of the Journal of Molecular Biology. Now that the Dundee/Oxford team have established the exact chemical structure of the protein, researchers at these and other institutions will be able to try and develop new inhibitors and therapies.

Using the Diamond synchrotron the team were able to efficiently determine the 3D structure of the penicillin binding protein PBP3, and this may have significant impact in the future development of antibiotics, according to Professor Hunter.

'Having this accurate 3D picture of the enzyme and knowing where these molecules called inhibitors bind is critical,' he said. 'It gives us a clear understanding of the molecular interactions that are involved in inhibiting this drug target. If we didn’t have that information we would be looking to characterise interactions by other less accurate methods.

'In those circumstances, you’re essentially working blind. You have to do many more experiments to get the understanding we have been able to get by doing this crystal structure.

'The objective of our work is to help advance understanding of potentially new targets for the development of therapies against Gram negative infections, which are a real problem because of the increase in drug resistance and in some cases, these are just tough beasts to kill.

'Pseudomonas aeruginosa is something we’re particularly interested in as this causes a lot of problems for people whose immune system is compromised either due to transplant surgery, chemotherapy, people who have HIV/AIDS, people who are recovering from burns and also young people with cystic fibrosis whose lungs struggle to cope with dangerous Gram negative bacteria.

'Because these organisms are so tough, we need new ideas for drugs and the way to do that is to find new targets or exploit old targets and come out with new compounds that will hit the old targets.

'We interact with a number of colleagues to look at these targets. In order to make sure we were casting our net as wide as possible I set up a collaboration with this group in Oxford to get them to take on additional targets and, using the excellent facilities at the Diamond synchrotron, they were fantastic and did a very good job of getting the structure of the penicillin binding protein PBP3.

'For many years, this has been a key therapeutic target but the organisms can change, mutate, and develop drug resistance.

'Now that we've got this 3D picture, we know where all the atoms are, we know how this molecule works, and the challenge is to use this information to come up with new small molecules that will stop PBP3 from working. This is a medical problem that deserves a solution and we are looking at several possible pathways of modifying existing drugs to supplement the arsenal of antibiotics.'

The team used Diamond Light Source's protein crystallography facilities to solve three structures: the protein in native form; and the protein linked to two important antibiotics, carbenicillin and ceftazidime.

A common feature observed in the crystal structures of PBPs is the flexibility of the active sites. This property plays a key role in the bacteria's ability to develop a resistance to drugs; the resistance mechanism works by mutating the protein. For binding of the drug to occur, the protein bends towards the inhibitor. The conformational change introduced by the inhibitor opens up a small pocket within the active site.

From their 3D structures of the mechanism, the group are able to characterise these pockets with a view to targeting them with a new type of inhibitor that restricts the active site 'breathing' i.e. restrict the flexibility of the protein. This new inhibitor could be used in conjunction with existing antibiotics to combat drug resistance.

Overall, the group's findings will allow discussions of different approaches to inhibit PBPs and potentially lead to new ways of combating P. aeruginosa and related bacteria which cause respiratory diseases and skin infections, gastro-intestinal and sexually transmitted diseases.

Single cell studies identify coactivator role in fat cell maturation

Single cell studies identify coactivator role in fat cell maturation

All fat cells are not the same – a fact that has implications in the understanding and treatment of type 2 diabetes and obesity, said researchers from Baylor College of Medicine in a report that appears in the current issue of the Journal of Cell Biology.

The amount of fat in each cell and the central transcription factor, PPAR gamma (peroxisome proliferator activated receptor gamma), can vary widely, but the fat cells (adipocytes) can maintain stable levels of master switches known as steroid receptor coactivators (SRC)-2 and -3, said Dr. Sean M. Hartig, a postdoctoral fellow, and Dr. Michael Mancini, an associate professor of molecular and cellular biology at BCM and the director of the Integrated Microscopy Core at BCM. Hartig is first author and Mancini senior author of the report.

"The difference was the SRCs," said Hartig. "They control the transcriptional switch for PPAR gamma to maximize fat accumulation."

PPAR gamma is known to regulate the production of adipocytes or fat cells. It regulates transcription – making an RNA copy of DNA, which is the first step in gene expression.

"Our research shows that there isn't always a linear connection between this transcriptional regulator PPAR gamma and the lipid in a cell," said Mancini. "It's dogma that one equals the other, but as you dive into the population of cells using high throughput microscopy and with custom-built software 'pipelines,' you find lots of exceptions. Then Sean (Hartig) connected it to the coregulators."

New drug-screening technology that automates both microscopy and image analysis allows experts like Mancini and Hartig to collect pictures and quantify thousands of cells in a short period of time. In this case, it allowed them to analyze the composition of different populations of human fat cells.

"Sean measured the amount of lipid in every cell," said Mancini. "This new technology uses fluorescent dyes and antibodies and enabled him to quantify both the amount of fat in each cell, but also how much of the transcriptional regulator PPAR gamma was expressed."

"There was a continuum," said Hartig. "There were cells that did not have any PPAR gamma but still had somehow become adipocytes. There were cells that had increased levels of PPAR gamma but had never developed the characteristics of adipocytes."

The finding supports the theory that these cells represent a continuum of factors with modulated levels of PPAR gamma and lipids. "PPAR-equals-fat simply didn't hold up to this level of scrutiny," Mancini said.

Mancini pointed to a population of cells with high lipid levels and low levels of PPAR gamma. There were cells with the opposite situation.

Hartig said reduced levels of SRC-2 and 3 resulted in more cells with low levels of lipid and increased PPAR gamma.

This is important because some drugs used to treat type 2 diabetes increase the activity of PPAR gamma. These include the thiazolidines such as Actos and Avandia, which increase the levels of genes associated with sensitivity to insulin. However, because PPAR gamma stimulates fat cell production, these drugs can also lead to increased abdominal fat and, more recently, cardiovascular complications.

"If you could find a way to increase the proportion of cells that have PPAR gamma but don't accumulate lipids, you might have a positive outcome. That would probably require a drug with a different structure," said Mancini.

The automated microscopy makes it possible to monitor the effects of drugs on different populations of a large number of cells, said Mancini.

"Had we not been able to analyze the cell-to-cell differences, we would not necessarily have understood how this favorable switch controlling PPAR gamma transcriptional activity might manifest itself. Identification of compounds that target the SRC and PPAR gamma interface might be alternatives to current therapeutic strategies for type 2 diabetes," said Hartig.

Biologists' favorite worm gets viruses

Biologists' favorite worm gets viruses

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Scientists have discovered that C. elegans, a microscopic worm biologists have used in the lab to identify important biological phenomena, suffers from natural viral infections. This may mean that C. elegans can help scientists learn more about how hosts and viruses interact. Credit: Marie-Anne Felix, the Monod Institute

A workhorse of modern biology is sick, and scientists couldn't be happier.

Researchers at Washington University School of Medicine in St. Louis, the Jacques Monod Institute in France and Cambridge University have found that the nematode C. elegans, a millimeter-long worm used extensively for decades to study many aspects of biology, gets naturally occurring viral infections.

The discovery means C. elegans is likely to help scientists study the way viruses and their hosts interact.

"We can easily disable any of C. elegans' genes, confront the worm with a virus and watch to see if this makes the infection worse, better or has no effect," says David Wang, PhD. "If it changes the worm's response to infection, we will look to see if similar genes are present in humans and other mammals."

Wang notes that several fundamental aspects of human biology, including the ability of cells to self-destruct to prevent cancer, and RNA interference, an important process for regulating how genes are used to make proteins, were first identified in C. elegans and later affirmed to be present in humans.

The findings appear online in PLoS Biology.

Marie-Anne Felix, PhD, a researcher who studies nematodes at the Monod Institute, began the study by gathering C. elegans from rotting fruit in French orchards. Felix noted that some of her sample worms appeared to be sick. Treatment with antibiotics failed to cure them.

Felix then repeated a classic biology experiment that led to the discovery of viruses.

"She ground up the sick worms, passed them through a filter fine enough to remove any bacterial or parasitic infectious agents, and exposed a new batch of worms to the ground-up remains of the first batch," Wang says. "When the new batch got sick, she knew that a viral infection was likely to be present."

Wang, associate professor of pathology and immunology and of molecular microbiology, specializes in the identification of novel viruses. He found the worms had been suffering infections from two viruses related to nodaviruses, a class of viruses previously found to infect insects and fish. Nodaviruses are not currently known to infect humans. Tests showed one of the new viruses can infect the strain of C. elegans most commonly used in research.

"Model organisms are essential to important steps forward in biology, and we're eager to see what C. elegans can teach us about the way hosts and viruses interact," Wang says.

Researchers work towards pharmacological targets for cholera

Researchers work towards pharmacological targets for cholera

Just over a year after the earthquake in Haiti killed 222,000 people there's a new problem that is killing Haitians. A cholera outbreak has doctors in the area scrambling and the water-borne illness has already claimed 3600 lives according to officials with Médicin Sans Frontières (Doctors without Borders).

Stefan Pukatzki, a bacteriologist in the Faculty of Medicine & Dentistry at the University of Alberta, is hoping that down the road he can help prevent deadly cholera outbreaks.

His lab studies Vibrio cholerae, the bacteria that makes up the disease, and he has discovered how it infects and kills other bacteria and host cells. This discovery, published in November's Proceedings of the National Academy of Sciences, could explain how this organism survives between epidemics. Pukatzki says Vibrio cholerae lives in fresh water mix between epidemics and no one has known how it competed and survived with other bacteria in the water.

A better understanding of how this organism infects cells means Pukatzki may be able to devise novel strategies to block the function.

Pukatzki discovered that Vibrio cholerae uses molecular nano-syringes to puncture host cells and secrete toxins straight in to the other organism; this is called the type six secretion system.

"Vibrio cholerae uses these syringes so when it comes in contact with another bacteria, like E. coli, which is a gut bacterium, it kills it," said Pukatzki. "That's a novel phenomenon. We knew it [Vibrio cholerae] competed with cells of the immune system but we didn't know it was able to kill other bacteria.

"Keep in mind these syringes are sitting on the outside of the bacterium so they make good vaccine targets," said Pukatzki. "That's actually better because you could either inhibit the type six function or you could induce an immune response with these components that are sitting on the outside."

Pukatzki is excited because the type six secretion system isn't unique to Vibrio cholera; it is found in most major pathogens.

"We really think that even though this pathway is important for cholera, I think that whatever we find we can apply to other diseases as well because they're so highly conserved," said Pukatzki.

Pukatzki's lab is now looking to get a better understanding of this mechanism and continue working towards a potential pharmaceutical target.

Newly discovered group of algae live in both fresh water and ocean

Newly discovered group of algae live in both fresh water and ocean

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A collection of rappemonad cells photographed by a high-powered microscope. Each cell contains at least two chloroplasts (green dots) and a nucleus (blue dots). Credit: Image from Kim, Harrison, Sudek et al. PNAS 2010.

A team of biologists has discovered an entirely new group of algae living in a variety of marine and freshwater environments. This group of algae, which the researchers dubbed "rappemonads," have DNA that is distinctly different from that of other known algae. In fact, humans and mushrooms are more closely related to each other than rappemonads are to some other common algae (such as green algae). Based on their DNA analysis, the researchers believe that they have discovered not just a new species or genus, but a potentially large and novel group of microorganisms.

The rappemonads were found in a wide range of habitats, in both fresh and salt water, and at temperatures ranging from 52 degrees to 79 degrees Fahrenheit. According to MBARI Senior Research Technician Sebastian Sudek, co-first author of the paper reporting the discovery of these algae, "Based on the evidence so far, I think it's fair to say that rappemonads are likely to be found throughout many of the world's oceans. We don't know how common they are in fresh water, but our samples were not from unusual sources—they were from small lakes and reservoirs."

Researchers Sebastian Sudek, Heather Wilcox, and Alexandra Worden of the Monterey Bay Aquarium Research Institute (MBARI), along with collaborators at Dalhousie University and the Natural History Museum (NHM), London, discovered these microscopic algae by following up on an unexpected DNA sequence listed in a research paper from the late 1990s. They named the newly identified group of algae "rappemonads" after Michael Rappé a professor at the University of Hawaii, who was first author of that paper.

Following up on their initial lead, the research team developed two different DNA "probes" that were designed to detect the unusual DNA sequences reported by Rappé. Using these new probes, the researchers analyzed samples collected by Worden's group from the Northeast Pacific Ocean, the North Atlantic, the Sargasso Sea, and the Florida Straits, as well as samples collected from several freshwater sites by co-author Thomas Richards' group at NHM. To the teams' surprise, they discovered evidence of microscopic organisms containing the unusual DNA sequence at all five locations.

Although the rappemonads were fairly sparse in many of the samples, they appear to become quite abundant under certain conditions. For example, water samples taken from the Sargasso Sea near Bermuda in late winter appeared to have relatively high concentrations of rappemonads.

When asked why these apparently widespread algae had not been detected sooner, Sudek speculates that it may in part be due to their size. "They are too small to be noticed by people who study bigger algae such as diatoms, yet they may be filtered out by researchers who study the really small algae, known as picoplankton."

Sudek says, "The rappemonads are just one of many microbes that we know nothing about—this makes it an exciting field in which to work." Worden, in whose lab the research was conducted, and who first noticed the unique sequence in the 1990 paper then initiated research to "chase down" the story behind that sequence, continues, "Right now we treat all algae as being very similar. It is as if we combined everything from mice up to humans and considered them all to have the same behaviors and influence on ecosystems. Clearly mice and humans have different behaviors and different impacts!"

Even though DNA analysis demonstrated that rappemonads were present in their water samples, the researchers were still unable to visualize the tiny organisms because they didn't know what physical characteristics to look for. However, by attaching fluorescent compounds to the newly developed DNA probes, and then applying these probes to intact algae cells, Eunsoo Kim at Dalhousie was able to make parts of the rappemonads glow with a greenish light. This allowed the researchers to see individual rappemonads under a microscope.

The greenish glow highlighted the rappemonad's "chloroplasts," which contain the unique DNA sequence tagged by the new probes. Chloroplasts are used by plants and algae to harvest energy from sunlight in a process called photosynthesis. Because all of the rappemonads contain chloroplasts, the researchers believe they "make a living" through photosynthesis. However, Worden points out that it still needs to be shown that the chloroplasts are functional.

One of the primary goals of Worden's research is to study marine algae in the context of their environment. Worden feels that such an approach is imperative to understanding how rappemonads and other microorganisms affect large-scale processes in the ocean and in the atmosphere. In coming years her lab will be building upon their recent insights, including the discovery of the rappemonads, to study the roles that different algal groups play in the cycling of carbon dioxide between the atmosphere and the ocean.

Worden says, "There is a tremendous urgency in gaining an understanding of biogeochemical cycles. Marine algae are key players in these cycles, taking up carbon dioxide from the atmosphere and releasing oxygen, which we breathe. Until we have a true census of marine algae and understanding of how each group thrives, it will be very difficult to model global biogeochemical cycles. Such modeling is essential for predicting how climate change will impact life on earth."

Scientists sequence gut microbes of premature infant

Scientists sequence gut microbes of premature infant

Scientists have for the first time sequenced and reconstructed the genomes of most of the microbes in the gut of a premature newborn and documented how the microbe populations changed over time.

Further studies involving more infants could eventually help researchers understand the causes of various intestinal problems that afflict preemies, in particular the sometimes fatal necrotizing enterocolitis, according to researchers at the University of California, Berkeley, the University of Pittsburgh School of Medicine and Stanford University. One unresolved question is whether these illnesses are caused by pathogenic strains of bacteria or just an imbalance in the microbe populations in the gut.

The study was posted online Dec. 29 in advance of print publication in the journal Proceedings of the National Academy of Sciences.

While this is not the first time that microbes in the human intestinal tract have been sequenced as a community, this is the first comprehensive look at a time series documenting colonization of the gut of a premature newborn, and one of few completely assembled community genomic datasets, said Jill Banfield, a UC Berkeley professor of earth and planetary science and of environmental science, policy and management.

"Sequencing of microbial communities has become exceedingly common, but many researchers work with essentially unassembled data and often analyze very short contiguous DNA sequences – genome fragments," she said. "We actually go in and work out where the assemblies failed and fix them – what's called curating the data – so we can build very complete genomes for most of the microbes."

Pediatric surgeon Michael J. Morowitz, until recently at The University of Chicago Medical Center but now with Children's Hospital of Pittsburgh of the University of Pittsburgh Medical Center and an assistant professor of surgery at the University of Pittsburgh School of Medicine, first approached Banfield because of her pioneering work over the past decade sequencing microbial communities in extreme environments, such as the acid drainage from underground mines. He suggested that she tackle a unique human environment, the newborn intestinal tract. Unlike the adult gut, which may contain a couple of thousand microbial species, the newborn's intestinal tract may be colonized by only a handful, making it feasible to sequence the entire community.

His interest stemmed from work with premature infants, most of whom spend anywhere from two weeks to six months in the intensive care unit before they're deemed healthy enough to go home. Between 5 and 10 percent of these preemies develop symptoms of necrotizing enterocolitis (NEC), which requires rounds of antibiotics to halt, and perhaps a third of these babies eventually require surgery to remove parts of their intestines that have died.

"The actual impact of necrotizing enterocolitis in the ICU is even larger, because feeding routines and other care are conducted around a fear of NEC developing," Morowitz said.

Previous studies, however, have produced conflicting results about NEC's cause. Some have found pathogenic bacteria associated with NEC, while others have found no difference between the bacteria in babies with and without NEC. Banfield, Morowitz and their collaborators suspect that these results reflect the fact that researchers have looked broadly at species or families of bacteria in the gut, rather than at variants or strains. Although coexisting strains may have genes that are 99 percent identical, their genomes could be sufficiently distinct to make one bad and the other good.

"We already know that just a few genes can make one strain a pathogen and one beneficial or commensal," meaning that the microbes live amicably with their host, Banfield said. "We expect that a lot of the issues with the colonization process in the gut that leads to disease may be tracked to subtle differences in strains," she said. "So one question on the table is, 'Are these very closely related strains physiologically distinct, and in what ways'?"

The only way to get at these differences, she said, is to sequence the entire genomes of the intestinal microbiota – not merely DNA fragments or short DNA tags, which can be used to identify the genus or even species of a microbe, but not the specific strain.

"Although a primary target of our research is NEC, it's become very apparent that there are some fundamental unanswered questions just about the colonization process under normal circumstances," Morowitz added. "It's really important to get a handle on what the normal process is first, and then, eventually, we can look closely at babies with NEC and see if they deviate from what appears to be the normal colonization process."

Other human diseases, including asthma, diabetes and obesity, have been linked to problems with microbial colonization of the gut, and several papers have reported symptomatic improvement after "transplanting" fecal material from healthy individuals to patients with a range of intestinal disorders.

Banfield, Morowitz and their colleagues followed a single premature infant that had been delivered by cesarean and identified three distinct communities of intestinal microbes present at different times during the first month of the infant's life. The microbe populations in these communities seemed to change after alterations in medication and feeding, Morowitz said. Although it was presumably sterile at birth, the infant's gut was quickly colonized by a set of known intestinal microbes – bacteria and Archaea, primarily, but also viruses, bacterial viruses (phage) and the naked lengths of DNA called plasmids. When the baby went off antibiotics and switched from breast feeding to intravenous feeding, the microbe populations completely changed, with minor microbial members suddenly dominating and dominant members declining.

Vincent Denef, a post-doctoral researcher in Banfield's lab who contributed to the study, noted that such real-time studies are powerful because "very rarely do we have the opportunity to observe the dynamics of a naturally occurring system, such as the infant GI tract, as it is transformed from sterile to functionally diverse."

The populations again shifted when intravenous feeding was replaced by formula. Morowitz stopped collecting feces from dirty diapers after 21 days, and the infant was sent home healthy after 9 weeks in the ICU.

Though fecal samples were taken nearly every day, a complete genome analysis was performed only for samples collected on days 10, 16, 18 and 21. For the other days, the microbial community was estimated based on DNA tags (16S rRNA) that identify microbe families and species, but not specific strains.

What surprised the researchers is that the microbial population was comprised of members of at least 20 groups, many of which include harmful as well as benign organisms. These included Staphylococcus, a frequent cause of hospital infections; Pseudomonas, "the cause of an enormous amount of morbidity in ICU patients, both children and adults," Morowitz said; Serratia, a common cause of sepsis in general; and Citrobacter, which can cause meningitis in babies. Yet, the baby in this study appeared healthy throughout.

"The gut populations are highly dynamic, with large shifts through three stages over time, but we saw an overabundance of gram-negative organisms that we often associate with disease," he said. "Particularly striking was the dominance of Pseudomonas for several days, though the infant was clinically stable."

The seeming contradiction of a healthy infant with disease-causing bacteria in her gut could be explained if the strains in the infant's gut were benign, or if the balance of other microbes prevented pathogenic microbes from causing problems.

Citrobacter, for example, is one type of bacteria that is reportedly associated with NEC: one study found Citrobacter in three of four infants with NEC, but in no control infants. Yet, in the current study, sequencing of the gut microbiome on days 16, 18 and 21 revealed the presence of two strains of Citrobacter, which fluctuated significantly in proportions on the three days. "Those big shifts could potentially have been very important for the medical state of that baby," Banfield said. "Fortunately, the baby was fine."

The researchers found that those two strains were 99 percent similar over areas of the genome that could be compared.

"Of particular interest were hot spots of rapid DNA evolution within and between genes. Those potentially could be very important and interesting," she said. "Though the two Citrobacter genotypes are very, very similar over most of the genome, the results suggest that they could be functioning in different ways because their genomes are regulated differently."

Banfield noted that the intestinal community of the infant no doubt would continue to shift repeatedly for a year or more after birth, as the child encounters new microbes – courtesy of family, friends and pets. These populations shift with the influx of new strains and species and potentially because the resident microbes themselves evolve by picking up new traits from the plasmids and phages living alongside them.

"This is an ecological study," she emphasized. "One of the things we are trying to do is bring into the field of medicine a high resolution, ecological approach."

'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

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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

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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.

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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

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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.

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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.”

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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

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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.

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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.

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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."