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

Enlarge

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."