Poisonous cure: Toxic fungi may hold secrets to tackling deadly diseases

Take two poisonous mushrooms, and call me in the morning, said no doctor ever. Credit: Photo by G.L. Kohuth

Take two poisonous mushrooms, and call me in the morning. While no doctor would ever write this prescription, toxic fungi may hold the secrets to tackling deadly diseases.

A team of Michigan State University scientists has discovered an enzyme that is the key to the lethal potency of poisonous mushrooms. The results, published in the current issue of the journal Chemistry and Biology, reveal the enzyme's ability to create the mushroom's molecules that harbor missile-like proficiency in attacking and annihilating a single vulnerable target in the human liver.

The team revealed how the enzyme contributes to the manufacture of chemical compounds known as cyclic peptides, a favorite type of molecule that pharmaceutical companies use to create new drugs. These findings could lead to single-minded medicines with zero side effects, said Jonathan Walton, professor of plant biology and co-lead author.

"Mushrooms are prolific chemical factories, yet only a few of their peptides are poisonous," he said. "These toxins survive the high temperatures of cooking and the acids of digestion, and yet they're readily absorbed by the bloodstream and go directly to their intended target. These are the exact qualities needed for an effective medicine."

Walton published the paper with fellow MSU scientists Hong Luo, Sung-Yong Hong, R. Michael Sgambelluri and Evan Angelos. Working with the mushroom species Amanita, Walton and his teammates disassembled one of its poisonous peptides, which can be compared to a laser-guided missile with a nuclear warhead.

By removing the molecular equivalent of the deadly warhead, they now have a sturdy, precise delivery system that can supply medicine -- rather than poison -- to a single target. By taking a laser, rather than a shotgun approach, scientists could develop medicines capable of curing disease without the patient suffering any side effects.

The enzyme the team discovered is called POPB, and it converts toxins from their initial linear shape into cyclic peptides, fortress-like molecular circles comprising eight amino acids.

Harnessing the distinct properties of POPB will allow scientist to create billions of variant molecules, which can be tested against many different medical targets such as pathogenic bacteria and cancer.

"We've found some variables that are key," said Walton, an AgBioResearch scientist. "By making more variants, we can add or replace molecules that may or may not work. To date we've created a library of a hundred or so, and we eventually plan to create millions."

The challenge of the next stage of research, though, is testing the variants against diseases such as cancer. The bottleneck lies in the screening process. While Walton's team has discovered a missile capable of carrying a million different potential medicines, as of yet the payload that will be effective remains a mystery.

Source: Michigan State University

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Native fungus suggested as another tool for restoring ghostly whitebark pine forests

Siberian slippery jack is a native fungus that may help in the effort to restore whitebark pine forests.
Credit: Cathy Cripps

Cathy Cripps doesn't seem to worry about the grizzly bears and black bears that watch her work, but she is concerned about the ghosts and skeletons she encounters.

The ghosts are whitebark pine forests that have been devastated by mountain pine beetles and white pine blister rust, said the Montana State University scientist who studies fungi that grow in extreme environments. The skeletons are dead trees that no longer shade snow or produce pine cones. The round purple pine cones hold the seeds that feed bears, red squirrels and Clark's nutcracker birds. Shade at the top of watersheds keeps snow from melting too fast in the spring, preventing trout streams from drying up too early in the summer.

Fortunately, she has found hope in a native fungus called Siberian slippery jack, or Suillus sibiricus, said Cripps, a mycologist in MSU's Department of Plant Sciences and Plant Pathology.

Cripps conducted a three-year study in collaboration with Waterton Lakes National Park in Canada that showed a 10 to 15 percent increase in the survival rate of whitebark pine seedlings when Siberian slippery jack spores are injected into the soil around them. The injection takes place in nurseries before the seedlings are transplanted in the mountains.

That increase is significant and good news for those trying to reinstate whitebark pine trees to the north-central Rocky Mountains and Pacific Northwest, Cripps said. The whitebark pine is a keystone species that grows at high elevations where other trees cannot, but it has been declared an endangered species in Canada and awaits the designation in the United States.

"That (jump in survival rates) might not sound like a big difference, but a small amount is a big deal considering the labor-intensive process," Cripps said.

Cyndi Smith, scientist emeritus at Waterton, said "The positive results have encouraged Waterton Lakes National Park to continue inoculating both whitebark and limber pine seedlings, to give them the best opportunity we can to establish and survive to maturity."

Participants in the research project, in addition to Cripps' and Smith's teams, were the U.S. Forest Service, National Park Service and volunteers from the United States and Canada.

Explaining how the collaboration began, Smith said, "Cathy gave a presentation on some of her Yellowstone work at the annual science meeting of the Whitebark Pine Ecosystem Foundation in Hailey, Idaho, in 2006. I was really taken with the idea that the ecosystem may have lost the beneficial fungi because our forests have been dead and dying for so long and that perhaps there was a way to reverse that trend so I approached Cathy with the idea.

"I'm a big believer in collaboration, whether locally or internationally, but certainly working with someone of Cathy's academic stature has been very helpful when I have applied for funding for whitebark and limber pine projects," Smith said, adding that, "Cathy's enthusiasm is very infectious, and it is a delight to work with her."

To carry out the research project, the participants placed cages around whitebark pine trees to collect pine cones without interference from wildlife. Then they tested the cones to see if they were resistant to white pine blister rust, removed seeds by hand from the resistant cones, grew the seeds into seedlings and shipped them to the nursery in Glacier National Park.

Cripps and former graduate student Erin Lonergan drove to Waterton Lakes 

National Park and throughout the Greater Yellowstone area, hiked to the tops of mountains and collected the Siberian slippery jack and other fungi from whitebark pine forests. Then they returned to MSU where they used a coffee grinder to process the spongy outer layer of the fungi. They added water to create a spore slurry, stored the mixture at MSU and later injected about 3 million spores into the soil around each seedling temporarily housed at the Glacier National Park nursery.

A few months later, volunteers planted more than 1,000 seedlings into MSU's test plots. Most of those test plots were located in Waterton, while others were in the neighboring Glacier National Park. The two parks together comprise Waterton-Glacier International Peace Park.

Two years into the study, Lonergan, Cripps and Smith reported success in the journal, American Forests. One year later, they announced their final results in the spring/summer 2014 issue of Nutcracker Notes, a small journal hosted by the Whitebark Pine Ecosystem Foundation.

"We wanted to get the word out that results after three years showed that inoculation with these native fungi significantly improved the survival of rust resistant seedlings, especially when inoculated seedlings were planted in burned areas near shelter objects such as stumps and logs," Cripps said.

High nitrogen fertilizations and fungicides prevent the inoculations from working, Cripps said. The age of the seedlings is important because they need to grow plenty of side roots before being inoculated.

When successful, the injected fungi slip like tiny socks over the ends of every root of the whitebark pine seedling and form a relationship that benefits both the tree and the fungi, Cripps said. The fungi help the seedling take in more nutrients and water from the soil. The tree produces the sugars that feed the fungi.

"Instead of being bad guys, these fungi are beneficial," Cripps said of the Siberian slippery jack. "They help plants take up nitrogen and phosphorus from the soil. That's a big deal."

Her study is one of many research projects involving whitebark pine forests, but it's unique because it focuses on beneficial native fungi, Cripps said. She added that land managers might want to incorporate MSU's findings into their overall strategy for restoring whitebark pine forests. She noted that large-scale inoculations are already planned for nurseries in Canada. She said inoculating beneficial fungi into nursery stock is common in Europe.

"As we work to save the vital whitebark pine from disappearing from the landscape, it is essential to use all available tools," Cripps said. "Ectomycorrhizal fungi are an integral part of forest integrity, ecology and health. Showing respect for these mighty microbes might just mean the difference between the restoration and death of a forest."

"Ectomycorrhizal fungi" refers to beneficial fungi that form a symbiotic relationship with the roots of trees. Siberian slippery jack is one of those fungi, and it only associates with five-needle pines. White pine blister rust is another type of fungus, one of the "bad guys."

Source: Montana State University

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Bacteria could be rich source for making terpenes

Odoriferous terpene metabolites: A phylogenetic tree of terpene synthases shows the synthases (bold face or underlined) found by researchers in Japan and at Brown University using bacterial sequences. Credit: Image courtesy of Brown University

If you've ever enjoyed the scent of a pine forest or sniffed a freshly cut basil leaf, then you're familiar with terpenes. The compounds are responsible for the essential oils of plants and the resins of trees. Since the discovery of terpenes more than 150 years ago, scientists have isolated some 50,000 different terpene compounds derived from plants and fungi. Bacteria and other microorganisms are known to make terpenes too, but they've received much less study.

New research at Brown University, published in the Proceedings of the National Academy of Sciences, shows that the genetic capacity of bacteria to make terpenes is widespread. 

Using a specialized technique to sift through genomic databases for a variety of bacteria, the researchers found 262 gene sequences that likely code for terpene synthases -- enzymes that catalyze the production terpenes. The researchers then used several of those enzymes to isolate 13 previously unidentified bacterial terpenes.

The findings suggest that bacteria "represent a fertile source for discovery of new natural products," the researchers write.

David Cane, a professor of chemistry at Brown and one of the authors on the new paper, began working about 15 years ago to understand how bacteria make terpenes.

"At that time, the first genomic sequences of certain classes of bacteria were just beginning to come out," he said. "We had this idea that maybe you could find the enzymes responsible for making terpenes by looking at the sequences of the genes that were being discovered."

To do that, Cane searched through the genome data gathered for a group of bacteria called Streptomyces, looking for sequences similar those known to produce terpene synthases in plants and fungi. Eventually, he found that Streptomyces did indeed have genes encoding terpene synthases and that those enzymes could be used to make terpenes.

The verified bacterial sequences found by Cane and others enabled researchers to refine subsequent searches for additional terpene synthase genes. "Instead of using plant sequences or fungal sequences as your search query, we can now use bacterial sequences, which should yield a greater degree of similarity," he said. "So now we're fishing in the right waters with the right kind of bait, and you can find more matches."

This latest paper made use of the third generation of iterative searches and a powerful search technique developed by Haruo Ikeda of Kitasato University in Japan. Previous work had identified 140 probable sequences for terpene synthases. This latest work expanded that to 262.

The next step was to verify that these sequences did indeed code for enzymes capable of making terpenes. Testing all 262 wasn't practical, so the team chose a few they thought might give them the best chance of finding terpene compounds that hadn't previously been identified. They looked for sequences that didn't seem to fit clearly into previously known categories of terpenes.

After they had selected a few, the team made use of a genetically engineered Streptomyces bacterium as a bio-refinery to generate the terpene products.

"What Professor Ikeda did, in collaboration with us, is develop a variant of a very well-studied Streptomyces system," Cane said. "He eliminated the genes that were responsible for making most of its native products, but he left behind all of the capacity to provide the starting materials and handle the accumulation of products."

By taking some of the gene sequences they found and splicing them into their test organism, the researchers could let the organisms generate the product using the instructions from the newly introduced gene. Using this method, they were able to make 13 previously unknown terpenes, their structures verified by mass spectrometry and nuclear magnetic resonance spectroscopy.

"It's a big step forward in the area in that it provides a paradigm for how one could go about discovering many new substances," Cane said. "It's a good example of how one can use sequence analysis to identify genes of interest and then apply molecular genetic and microbiological techniques to produce the chemical substances of interest."

The work also suggests that there may be many new terpene products as yet undiscovered hiding in the genomes of bacteria.

Source: Brown University

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Mother's diet affects the 'silencing' of her child's genes

An infant from the Gambia. Credit: Felicia Webb

A mother's diet before conception can permanently affect how her child's genes function, according to a study published in Nature Communications.

The first such evidence of the effect in humans opens up the possibility that a mother's diet before pregnancy could permanently affect many aspects of her children's lifelong health.

Researchers from the MRC International Nutrition Group, based at the London School of Hygiene & Tropical Medicine and MRC Unit, The Gambia, utilized a unique 'experiment of nature' in rural Gambia, where the population's dependence on own grown foods and a markedly seasonal climate impose a large difference in people's dietary patterns between rainy and dry seasons.

Through a selection process involving over 2,000 women, the researchers enrolled pregnant women who conceived at the peak of the rainy season (84 women) and the peak of the dry season (83 women). By measuring the concentrations of nutrients in their blood, and later analysing blood and hair follicle samples from their 2-8 month old infants, they found that a mother's diet before conception had a significant effect on the properties of her child's DNA.

While a child's genes are inherited directly from their parents, how these genes are expressed is controlled through 'epigenetic' modifications to the DNA. One such modification involves tagging gene regions with chemical compounds called methyl groups and results in silencing the genes. The addition of these compounds requires key nutrients including folate, vitamins B2, B6 and B12, choline and methionine.

Experiments in animals have already shown that environmental influences before conception can lead to epigenetic changes that affect the offspring. A 2003 study found that a female mouse's diet can change her offspring's coat colour by permanently modifying DNA methylation.1 But until this latest research, funded by the Wellcome Trust and the MRC, it was unknown whether such effects also occur in humans.

Senior author Dr Branwen Hennig, Senior Investigator Scientist at the MRC Gambia Unit and the London School of Hygiene & Tropical Medicine, said: "Our results represent the first demonstration in humans that a mother's nutritional well-being at the time of conception can change how her child's genes will be interpreted, with a life-long impact."

The researchers found that infants from rainy season conceptions had consistently higher rates of methyl groups present in all six genes they studied, and that these were linked to various nutrient levels in the mother's blood. Strong associations were found with two compounds in particular (homocysteine and cysteine), and the mothers' body mass index (BMI) had an additional influence. However, although these epigenetic effects were observed, their functional consequences remain unknown.

Professor Andrew Prentice, Professor of International Nutrition at the London School of Hygiene & Tropical Medicine, and head of the Nutrition Theme at the MRC Unit, The Gambia, said: "Our on-going research is yielding strong indications that the methylation machinery can be disrupted by nutrient deficiencies and that this can lead to disease. Our ultimate goal is to define an optimal diet for mothers-to-be that would prevent defects in the methylation process. Pre-conceptional folic acid is already used to prevent defects in embryos. Now our research is pointing towards the need for a cocktail of nutrients, which could come from the diet or from supplements."

Dr Rob Waterland of Baylor College of Medicine in Houston, who conducted the epigenetic analyses said: "We selected these gene regions because our earlier studies in mice had shown that establishment of DNA methylation at metastable epialleles is particularly sensitive to maternal nutrition in early pregnancy."

The authors note that their study was limited by including only one blood sampling point during early pregnancy, but estimates of pre-conception nutrient concentrations were calculated using results from non-pregnant women sampled throughout a whole calendar year. The authors also plan to increase the sample size in further studies.

Source: London School of Hygiene & Tropical Medicine

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Identifying gene-enhancers: New technique

Diane Dickel is the lead author of Nature Methods paper describing a new technique for identifying gene enhancers in the genomes of humans and other mammals. Credit: Roy Kaltschmidt

An international team led by researchers with the Lawrence Berkeley National Laboratory (Berkeley Lab) has developed a new technique for identifying gene enhancers -- sequences of DNA that act to amplify the expression of a specific gene -- in the genomes of humans and other mammals. Called SIF-seq, for site-specific integration fluorescence-activated cell sorting followed by sequencing, this new technique complements existing genomic tools, such as ChIP-seq (chromatin immunoprecipitation followed by sequencing), and offers some additional benefits.

"While ChIP-seq is very powerful in that it can query an entire genome for characteristics associated with enhancer activity in a single experiment, it can fail to identify some enhancers and identify some sites as being enhancers when they really aren't," says Diane Dickel, a geneticist with Berkeley Lab's Genomics Division and member of the SIF-seq development team. "SIF-seq is currently capable of testing only hundreds to a few thousand sites for enhancer activity in a single experiment, but can determine enhancer activity more accurately than ChIP-seq and is therefore a very good validation assay for assessing ChIP-seq results."

Dickel is the lead author of a paper in Nature Methods describing this new technique. The paper is titled "Function-based identification of mammalian enhancers using site-specific integration." The corresponding authors are Axel Visel and Len Pennacchio, also geneticists with Berkeley Lab's Genomics Division.

With the increasing awareness of the important role that gene enhancers play in normal cell development as well as in disease, there is strong scientific interest in identifying and characterizing these enhancers. This is a challenging task because an enhancer does not have to be located directly adjacent to the gene whose expression it regulates, but can instead be located hundreds of thousands of DNA base pairs away. The challenge is made even more difficult because the activity of many enhancers is restricted to specific tissues or cell types.

"For example, brain enhancers will not typically work in heart cells, which means that you must test your enhancer sequence in the correct cell type," Dickel says.

Currently, enhancers can be identified through chroma­tin-based assays, such as ChIP-seq, which predict enhancer elements indirectly based on the enhancer's association with specific epigenomic marks, such as transcription factors or molecular tags on DNA-associated histone proteins. Visel, Pennacchio, Dickel and their colleagues developed SIF-seq in response to the need for a higher-throughput functional enhancer assay that can be used in a wide variety of cell types and devel­opmental contexts.

"We've shown that SIF-seq can be used to identify enhancers active in cardiomyocytes, neural progenitor cells, and embryonic stem cells, and we think that it has the potential to be expanded for use in a much wider variety of cell types," Dickel says. "This means that many more types of enhancers could potentially be tested in vitro in cell culture."

In SIF-seq, hundreds or thousands of DNA fragments to be tested for enhancer activity are coupled to a reporter gene and targeted into a single, reproducible site in embryonic cell genomes. Every embryonic cell will have exactly one potential enhancer-reporter. 

Fluorescence-activated sorting is then used to identify and retrieve from this mix only those cells that display strong reporter gene expression, which represent the cells with the most active enhancers.

"Unlike previous enhancer assays for mammals, SIF-seq includes the integration of putative enhancers into a single genomic locus," says Visel. "Therefore, the activity of enhancers is assessed in a reproducible chromosomal context rather than from a transiently expressed plasmid. Furthermore, by making use of embryonic stem cells and in vitro differentia­tion, SIF-seq can be used to assess enhancer activity in a wide variety of disease-relevant cell types."

Adds Pennacchio, "The range of biologically or disease-relevant enhancers that SIF-seq can be used to identify is limited only by currently available stem cell differentiation methods. Although we did not explicitly test the activity of species-specific enhancers, such as those derived from certain classes of repetitive elements, our results strongly suggest that SIF-seq can be used to identify enhancers from other mammalian genomes where desired cell types are difficult or impossible to obtain."

The ability of SIF-seq to use reporter assays in mouse embryonic stem cells to identify human embryonic stem cell enhancers that are not present in the mouse genome opens the door to intriguing research possibilities as Dickel explains.

"Human and chimpanzee genes differ very little, so one hypothesis in evolutionary genomics holds that humans and chimpanzees are so phenotypically different because of differences in the way they regulate gene expression. It is very difficult to carry out enhancer identification through ChIP-seq that would be useful in studying this hypothesis," she says. 

"However, because SIF-seq only requires DNA sequence from a mammal and can be used in a variety of cell types, it should be possible to compare the neuronal enhancers present in a large genomic region from human to the neuronal enhancers present in the orthologous chimpanzee region. This could potentially tell us interesting things about the genetic differences that differentiate human brain development from that of other primates."

Source DOE/Lawrence Berkeley National Laboratory

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Gene silencing instructions acquired through 'molecular memory' tags on chromatin

Epigenetic inheritance is a two-step process, with a heritable molecular memory first forming to maintain a chromatin state required later for actual silencing of a genetic locus. Credit: Craig Pikaard and Todd Blevins

Scientists at Indiana University have unlocked one of the mysteries of modern genetics: how acquired traits can be passed between generations in a process called epigenetic inheritance. The new work finds that cells don't know to silence some genes based on information hardwired into their DNA sequences, but recognize heritable chemical marks that are added to the genes. These chemical tags serve as a form of molecular memory, allowing cells to recognize the genes and remember to silence them again in each new generation.

The discovery made by a 12-member all-Indiana University team of scientists led by IU biologist and biochemist Craig Pikaard provides important new insight into how plant cells know to silence a genetic locus -- that specific place on a chromosome where a gene is located -- in every successive generation.

Rather than rely on intrinsic, DNA sequence-based information, the cells instead must recall the need to silence specific loci by relying on chemical marks displayed on the complex of DNA and proteins called chromatin. Addition, or removal, of one-carbon (methyl) or two-carbon (acetyl) chemical tags are ways of modifying chromatin that can impart additional, epigenetic (literally, "above genetic") information to a locus beyond the genetic information encoded in the DNA.

The ability to perpetuate chromatin marks serves as a form of epigenetic memory that confers what Pikaard calls silent locus identity, a pre-established state that is needed for the cell to deliver to the loci the machinery that actually accomplishes silencing in a multi-step process known as RNA-directed DNA methylation (RdDM). RdDM involves short-interfering RNAs (siRNA), tiny RNA molecules that are 24 nucleotides long and that guide the addition of methyl groups to matching DNA strands, ultimately rendering the genes inactive.

"Importantly, this work shows that silent locus identity is required for, but separable from, actual gene silencing," Pikaard said. "We've found that epigenetic inheritance is a two-step process, with the heritable specification of silent locus identity occurring before actual silencing of the locus can occur."

Scientists are interested in epigenetic inheritance because it's a process by which heritable modifications occur in gene function without changes in the base sequence of an organism's DNA being required. Disease states such as cancer, which occur sporadically during an individual's lifetime, are increasingly recognized as having an epigenetic basis. Pikaard said the new work not only sheds important new light on the mechanisms responsible for epigenetic inheritance, a topic of broad interest in the fields of genetics and chromosome biology, but it also helps explain the basis for the recruitment of two plant-specific gene silencing enzymes -- the RNA polymerases Pol IV and Pol V -- first identified by Pikaard in 1999.

Specifically, the researchers tested and identified the relationship between histone deacetylase 6 (HDA6), an enzyme that removes acetyl groups from histones, and the CG DNA sequence maintenance methyltransferase, MET1, and discovered that their partnership in maintenance methylation can explain the perpetuation of epigenetic memory that accounts for silent locus identity.

"Collectively, our results show that silent locus identity is perpetuated from generation to generation through the actions of HDA6 and MET1," Pikaard said. "These activities are not sufficient to silence the loci but maintain a chromatin state that is required for Pol IV recruitment, siRNA biogenesis and RdDM, which is what ultimately silences the loci." When the team removed the RdDM pathway in Pol IV and Pol V mutant strains of the model plant Arabidopsis thaliana (rockcress), all gene silencing was lost, but silent locus identity remained. They then removed the HDA6 and MET1-dependent process that specifies silent locus identity and, importantly, the epigenetic memory required for silent locus identity was lost and unable to be regained.

Source:Indiana University

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Mysteries of 'molecular machines' revealed: Phenix software uses X-ray diffraction spots to produce 3-D image

This is a membrane protein called cysZ, imaged in 3 dimensions with Phenix software using data that could not previously be analyzed. Credit: Los Alamos National Laboratory

Scientists are making it easier for pharmaceutical companies and researchers to see the detailed inner workings of molecular machines.

'Inside each cell in our bodies and inside every bacterium and virus are tiny but complex protein molecules that synthesize chemicals, replicate genetic material, turn each other on and off, and transport chemicals across cell membranes,' said Tom Terwilliger, a Los Alamos National Laboratory scientist.

'Understanding how all these machines work is the key to developing new therapeutics, for treating genetic disorders, and for developing new ways to make useful materials.'

To understand how a machine works you have to be able to see how it is put together and how all its parts fit together. This is where the Los Alamos scientists come in. These molecular machines are very small: a million of them placed side by side would take up less than an inch of space. Researchers can see them however, using x-rays, crystals and computers. Researchers produce billions of copies of a protein machine, dissolve them in water, and grow crystals of the protein, like growing sugar crystals except that the machines are larger than a sugar molecule.

Then they shine a beam of X-rays at a crystal and measure the brightness of each of the thousands of diffracted X-ray spots that are produced. Then researchers use the powerful Phenix software, developed by scientists at Los Alamos, Lawrence Berkeley National Laboratory, Duke and Cambridge universities, to analyze the diffraction spots and produce a three-dimensional picture of a single protein machine. This picture tells the researchers exactly how the protein machine is put together.

The 3-D Advance

Recently Los Alamos scientists worked with their colleagues at LBNL and Cambridge University to make it even easier to visualize a molecular machine. In a report in the journal Nature Methods this month, Los Alamos scientists and their team show that they can obtain three-dimensional pictures of molecular machines using X-ray diffraction spots that could not previously be analyzed.

Some molecular machines contain a few metal atoms or other atoms that diffract X-rays differently than the carbon, oxygen, nitrogen, and hydrogen atoms that make up most of the atoms in a protein. The Phenix software finds those metal atoms first, and then uses their locations to find all the other atoms. For most molecular machines, however, metal atoms have to be incorporated into the machine artificially to make this all work.

The major new development to which Los Alamos scientists have contributed was showing that powerful statistical methods could be applied to find metal atoms even if they do not scatter X-rays very differently than all the other atoms. Even metal atoms such as sulfur that are naturally part of almost all proteins can be found and used to generate a three-dimensional picture of a protein. Now that it will often be possible to see a three-dimensional picture of a protein without artificially incorporating metal atoms into them, many more molecular machines can be studied.

Cracking the Cascade

Molecular machines that have recently been seen in three-dimensional detail include a 'huge' molecular machine called Cascade that was reported in the journal Science this summer. The Cascade machine is present in bacteria and can recognize DNA that comes from viruses that infect the bacteria. The Cascade machine is made up of 11 proteins and an RNA molecule and looks like a seahorse, with the RNA molecule winding through the whole 'body' of the seahorse. If a foreign piece of DNA in the bacterial cell is complementary to part of the RNA molecule then another specialized machine can come by and chop up the foreign DNA, saving the bacterium from infection.

Los Alamos and Cambridge University scientists who were developing the Phenix software were part of the team that visualized this protein machine for the first time. The Phenix software has been used to determine the three-dimensional shapes of over 15,000 different protein machines and has been cited by over 5000 scientific publications.

Source: DOE/Los Alamos National Laboratory

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In search of the origin of our brain

Nervous system in Nematostella vectensis embryos with different nerve cell populations, where the different neurons (here in green, blue and magenta) evidence asymmetry. Credit: Hiroshi Watanabe, Thomas Holstein / Nature Communication 5:5536, Macmillan Publishers Limited

While searching for the origin of our brain, biologists at Heidelberg University have gained new insights into the evolution of the central nervous system (CNS) and its highly developed biological structures. The researchers analysed neurogenesis at the molecular level in the model organism Nematostella vectensis. Using certain genes and signal factors, the team led by Prof. Dr. Thomas Holstein of the Centre for Organismal Studies demonstrated how the origin of nerve cell centralization can be traced back to the diffuse nerve net of simple and original lower animals like the sea anemone. The results of their research will be published in the journal "Nature Communications."

Like corals and jellyfish, the sea anemone -- Nematostella vectensis -- is a member of the Cnidaria family, which is over 700 million years old. It has a simple sack-like body, with no skeleton and just one body orifice. The nervous system of this original multicellular animal is organised in an elementary nerve net that is already capable of simple behaviour patterns. Researchers previously assumed that this net did not evidence centralization, that is, no local concentration of nerve cells. In the course of their research, however, the scientists discovered that the nerve net of the embryonic sea anemone is formed by a set of neuronal genes and signal factors that are also found in vertebrates.

According to Prof. Holstein, the origin of the first nerve cells depends on the Wnt signal pathway, named for its signal protein, Wnt. It plays a pivotal role in the orderly evolution of different types of animal cells. The Heidelberg researchers also uncovered an initial indication that another signal path is active in the neurogenesis of sea anemones -- the BMP pathway, which is instrumental for the centralization of nerve cells in vertebrates.

Named after the BMP signal protein, this pathway controls the evolution of various cell types depending on the protein concentration, similar to the Wnt pathway, but in a different direction. The BMP pathway runs at a right angle to the Wnt pathway, thereby creating an asymmetrical pattern of neuronal cell types in the widely diffuse neuronal net of the sea anemone. "This can be considered as the birth of centralization of the neuronal network on the path to the complex brains of vertebrates," underscores Prof. Holstein.

While the Wnt signal path triggers the formation of the primary body axis of all animals, from sponges to vertebrates, the BMP signal pathway is also involved in the formation of the secondary body axis (back and abdomen) in advanced vertebrates. "Our research results indicate that the origin of a central nervous system is closely linked to the evolution of the body axes," explains Prof. Holstein.

Source: Heidelberg, Universität

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Mechanics of cells' long-range communication modeled by researchers

As fibrosis progresses, "bridges" of extracellular matrix appear between cells. Credit: Image courtesy of University of Pennsylvania

Interdisciplinary research at the University of Pennsylvania is showing how cells interact over long distances within fibrous tissue, like that associated with many diseases of the liver, lungs and other organs.

By developing mathematical models of how the collagen matrix that connects cells in tissue stiffens, the researchers are providing insights into the pathology of fibrosis, cirrhosis of the liver and certain cancers.

Tissue stiffness has long been know to be clinically relevant in these diseases, but the underlying changes that alter the mechanics of tissues are poorly understood. Consisting of a complex network of fibers, tissues have proven difficult to simulate and model beyond local, neighbor-to-neighbor interactions.

Developing a better understanding of the large-scale mechanical changes that occur over longer distances, specifically the process by which the extracellular matrix is pulled into compact, highly-aligned "bridges," could eventually form the basis of treatments for related diseases.

Vivek Shenoy, professor in the Department of Materials Science and Engineering in Penn's School of Engineering and Applied Science, has led an interdisciplinary research team to tackle this problem, authoring a pair of papers that were published in Biophysical Journal.

One, "Remodeling of Fibrous Extracellular Matrices by Contractile Cells: Predictions from Discrete Fiber Network Simulations" involved developing simulations that extrapolated the overall remodeling of the extracellular matrix based on the behavior of neighboring pairs of cells. The other, "Long Range Force Transmission in Fibrous Matrices Enabled by Tension-Driven Alignment of Fibers," took a more mathematical approach, producing a coarse-grained model of this remodeling that could be more broadly applied to fibrotic tissue.

"We're trying to understand how force is transmitted in tissues," Shenoy said. "Cells are the ones that generate force, and it has to be transmitted through what surrounds the cell, the extracellular matrix, or ECM. But imagine trying to model the ECM by trying to keep track of each collagen fibril in your liver; there are tens of millions of those. So we're taking what we learn from simulating those networks to turn it into a model that captures the main features with only a few parameters.

"The key here is the mechanics," he said. "In particular, how does ECM, as a fibrous material, differ from solids, gels and other materials that are better studied."

Rebecca Wells, an associate professor in Penn's Perelman School of Medicine and a co-author on the latter paper, provided insight into the clinical relevance of the mechanics that characterize ECM-related disorders.

"Fibrosis occurs when you have an injury and the tissue responds by depositing ECM, forming scar tissue," Wells said. "In liver fibrosis, the liver can stiffen by up to an order of magnitude, so measuring stiffness is a common diagnostic test for the disease. Increased stiffness also occurs in cancer, where tumors are typically stiffer than the surrounding tissue."

Existing experimental evidence showed that mechanical forces were at play in the changes in both fibrosis and cancer and that these forces were important to their development and progression but could not explain the long-ranging changes cells were able to produce to change their environments. When put in tissue-simulating gels, cells can deform their immediate surroundings but are unable to pull on more distant cells. In real, ECM-linked tissue, however, cells' range of influence can be up to 20 times their own diameter.

"If you look at a normal tissue," Shenoy said, "you see the cells are more rounded, and the network of ECM fibers is more random. But as cancer progresses, you see more elliptical cells, more ECM, and you see that the ECM fibers are more aligned. The cells are the ones generating force, so they're contracting and pulling the fibers, stretching them out into bridges."

"That's also the pathology of cirrhosis," Wells said. "My group had been looking at the early mechanical changes associated with liver fibrosis, which progresses to cirrhosis, but then, by collaborating with Vivek, we started to wonder if these large scale changes in the architecture of the liver could have a mechanical basis and if something similar to what is seen in gels might be occurring in the liver. This is a new way of approaching the problem, which has largely been thought of as biochemical in origin. And there are other tissues where it is probably the same thing, the lung, for example."

The researchers found that the critical difference between the existing models and ECM's long-range behavior was rooted in its elastic properties. Materials with linear elasticity cannot transmit force over the distances observed, but the team's simulations showed that nonlinear elasticity could arise from the ECM's fibrous structure.

"In our model, every component is linearly elastic," Shenoy said, "but the collective behavior is nonlinear; it emerges because of the connectivity. When you deform the network, it's easy to bend the 'sticks' that represent collagen fibers but hard to stretch them. When you deform it to a small extent, it's all the bending of the fibers, but, as you deform further, it can't accommodate bending any more and moves over to stretching, forming the bridges we see in the tissue."

Such simulations can't predict which fibers will end up in which bridge, necessitating the coarser-grained model the researchers described in their second paper. By showing the point at which linear elasticity gives way to its nonlinear counterpart, the team produced a more complete picture of how the alignment of collagen bridges under tension transmit force between distant cells.

Further studies are needed to elucidate the feedback loops between ECM stiffening and cell contraction strength. The team is conducting physical experiments to confirm and refine their in silico findings.

"Right now," Wells said," we're hypothesizing that the mechanical interactions modeled by the Shenoy lab explain aspects of cancer and fibrosis, and we're developing the experimental systems to confirm it with real cells."

Source: University of Pennsylvania

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Revolutionizing genome engineering

Streptococcus pyogenes is one of the bacteria in which the HZI scientists have studied the CRISPR-Cas system. Credit: © HZI / M. Rohde

Genome engineering with the RNA-guided CRISPR-Cas9 system in animals and plants is changing biology. It is easier to use and more efficient than other genetic engineering tools, thus it is already being applied in laboratories all over the world just a few years after its discovery. This rapid adoption and the history of the system are the core topics of a review published in the journal Science. The review was written by the discoverers of the system Prof. Emmanuelle Charpentier, who works at the Helmholtz Centre for Infection Research (HZI) and is also affiliated to the Hannover Medical School and Umeå University, and Prof. Jennifer Doudna from the University of California, Berkeley, USA.

Many diseases result from a change of an individual's DNA -- the letter code that genes consist of. The defined order of the letters within a gene usually codes for a protein. Proteins are the workforce of our body and responsible for almost all processes needed to keep us running. When a gene is altered, its protein product may lose its normal function and disorders can result. "Making site-specific changes to the genome therefore is an interesting approach to preventing or treating those diseases," says Prof Emmanuelle Charpentier, head of the HZI research department "Regulation in Infection Biology." Due to this, ever since the discovery of the DNA structure, researchers have been looking for a way to alternate the genetic code.

First techniques like zinc finger nucleases and synthetic nucleases called TALENs were a starting point but turned out to be expensive and difficult to handle for a beginner. "The existing technologies are dependent on proteins as address labels and customizing new proteins for any new change to introduce in the DNA is a cumbersome process," says Charpentier. In 2012, while working at Umeå University, she described what is now revolutionising genetic engineering: the CRISPR-Cas9 system.

It is based on the immune system of bacteria and archaea but is also of value in the laboratory. CRISPR is short for Clustered Regularly Interspaced Palindromic Repeats, whereas Cas simply stands for the CRISPR-associated protein. "Initially we identified a novel RNA, namely tracrRNA, associated to the CRISPR-Cas9 system, which we published in 2011 in Nature. We were excited when Krzysztof Chylinski from my laboratory subsequently confirmed a long term thinking: Cas9 is an enzyme that functions with two RNAs," says Charpentier.

Together the system has the ability to detect specific sequences of letters within the genetic code and to cut DNA at a specific point. In this process the Cas9 protein functions as the scissors and an RNA snippet as the address label ensuring that the cut happens in the right place. In collaboration with Martin Jinek and Jennifer Doudna, the system could be simplified to use it as a universal technology. Now the user would just have to replace the sequence of this RNA to target virtually any sequence in the genome.

After describing the general abilities of CRISPR-Cas9 in 2012 it was shown in early 2013 that it works as efficiently in human cells as it does in bacteria. Ever since, there has been a real hype around the topic and researchers from all over the world have suggested new areas in which the new tool can be used. The possible applications extend from developing new therapies for genetic disorders caused by gene mutations to changing the pace and course of agricultural research in the future all the way to a possible new method for fighting the AIDS virus HIV.

"The CRISPR-Cas9 system has already breached boundaries and made genetic engineering much more versatile, efficient and easy," Charpentier says. "There really does not seem to be a limit in the applications."

Source: Helmholtz Centre for Infection Research

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Squid supplies blueprint for printable thermoplastics

This is a whimsical image of a squid creating 3-D printed devices. Credit: Adriás Bago

Squid, what is it good for? You can eat it and you can make ink or dye from it, and now a Penn State team of researchers is using it to make a thermoplastic that can be used in 3-D printing.

"Most of the companies looking into this type of material have focused on synthetic plastics," said Melik C. Demirel, professor of engineering science and mechanics. "Synthetic plastics are not rapidly deployable for field applications, and more importantly, they are not eco-friendly."

Demirel and his team looked at the protein complex that exists in the squid ring teeth (SRT). The naturally made material is a thermoplastic, but obtaining it requires a large amount of effort and many squid.

"We have the genetic sequence for six squid collected around the world, but we started with the European common squid," said Demirel, who with his team collected the cephalopods.

The researchers looked at the genetic sequence for the protein complex molecule and tried synthesizing a variety of proteins from the complex. Some were not thermoplastics, but others show stable thermal response, for example, the smallest known molecular weight SRT protein was a thermoplastic. The results of their work were published in today's (Dec. 17) issue of Advanced Functional Materials and illustrates the cover.

Most plastics are currently manufactured from fossil fuel sources like crude oil. Some high-end plastics are made from synthetic oils. Thermoplastics are polymer materials that can melt, be formed and then solidify as the same material without degrading materials properties.

This particular thermoplastic can be fabricated either as a thermoplastic, heated and extruded or molded, or the plastic can be dissolved in a simple solvent like acetic acid and used in film casting. The material can also be used in 3D printing machines as the source material to create complicated geometric structures.

To manufacture this small, synthetic SRT molecule, the researchers used recombinant techniques. They inserted SRT protein genes into E. coli, so that this common, harmless bacteria could produce the plastic molecules as part of their normal activity and the thermoplastic was then removed from the media where the E. coli lived. Wayne Curtis, professor of chemical engineering and Demirel collaborating on this project together with their students worked on this aspect of the project.

"The next generation of materials will be governed by molecular composition -- sequence, structure and properties," said Demirel.

The thermoplastic the researchers created is semi-crystalline and can be rigid or soft. It has a very high tensile strength and is a wet adhesive; it will stick to things even if it is wet.

This thermoplastic protein has a variety of tunable properties, which can be adjusted to individual requirements of manufacturing. Because it is a protein, it can be used for medical or cosmetic applications.

"Direct extraction or recombinant expression of protein based thermoplastics opens up new avenues for materials fabrication and synthesis, which will eventually be competitive with the high-end synthetic oil based plastics," the researchers report.

Source: Penn State

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Scrapie could breach the species barrier

Scrapie is a neurodegenerative disease that has been known for centuries and which affects sheep and goats. Credit: INRA/Florent Giffard

INRA scientists have shown for the first time that the pathogens responsible for scrapie in small ruminants (prions) have the potential to convert the human prion protein from a healthy state to a pathological state. In mice models reproducing the human species barrier, this prion induces a disease similar to Creutzfeldt-Jakob disease. These primary results published in Nature Communications on 16 December 2014, stress the necessity to reassess the transmission of this disease to humans.

Scrapie is a neurodegenerative disease that has been known for centuries and which affects sheep and goats. Similar to Bovine Spongiform Encephalopathy (BSE) or mad cow disease, scrapie is caused by a transmissible pathogen protein called prion.

However, and contrary to BSE[1], epidemiological studies have never been able to establish a link between this disease and the occurrence of prion diseases in humans. "Risks of transmitting scrapie to humans (zoonose) were hitherto considered negligible because of the species barrier that naturally prevents prion propagation between species," said Olivier 

Andreoletti, INRA scientist who led the present study.

Researchers at INRA studied the permeability of the human transmission barrier to pathogens responsible for scrapie, using animal models specifically developed for this purpose. This approach previously allowed the confirmation of the zoonotic nature of prions responsible for BSE in cows and of the variant of Creutzfeldt-Jakob disease in humans (vCJD).

Unexpectedly, in these rodent models, certain pathogens responsible for scrapie were able to cross the transmission barrier. Moreover, the pathogens that propagated through this barrier were undistinguishable from the prions causing the sporadic form of Creutzfeldt-Jakob disease (sCJD). This data suggest a potential link between the occurrence of certain sCJD and these animal prions.

"Since CJD is scarce, about 1 case per million and per year, and incubation periods are usually long -several decades- it is extremely difficult for epidemiological studies to try and make this link," explains Olivier Andreoletti.

In their conclusions, the authors stress the fact that CJD cases are rare though scrapie has been circulating for centuries in small ruminants for which we eat the meat. Even if in future studies scrapie is finally confirmed to have a zoonotic potential, the authors consider that this disease does not constitute a new major risk for public health.

Source: INRA-France

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The business-minded veterinarian

Veterinarians are needed not only to treat our pets and livestock, but in a broader context, to help with zoonotic disease maintenance. Credit: Image courtesy of Kansas State University Research and Extension

Animals can teach us more about the human body than we might realize. Crack open New York Times bestseller "Zoobiquity," and you'll learn about a human cardiologist's experiences at the Los Angeles Zoo that allowed her to more closely connect human and animal medicine. Even in the first chapter -- Dr. House, Meet Doctor Dolittle -- author Barbara Natterson-Horowitz reveals how doctors and veterinarians could learn from each other to effectively diagnose and treat all species.

Indeed, veterinarians are needed not only to treat our pets and livestock, but in a broader context, to help with zoonotic disease maintenance. The interaction between animals and humans secures the continuous demand for the profession, and the fewer veterinarians we have, the larger potential for catastrophic disease, according to Michael Dicks, director of the economics division for the American Veterinary Medical Association (AVMA).

Although the profession is needed, Dicks said financial struggles do exist, especially for those beginning to practice. Many veterinarians who are just starting out find that they need to make enough money to pay off their high educational debt while trying to make a living, which can pose a major challenge.

According to Kansas State University's College of Veterinary Medicine, the average debt reported by its 2014 graduates was $170,380, and graduates in 2013 had similar debt at $170,919.

The average practice salary reported by 2014 K-State graduates was $64,678 and for 2013 graduates, $63,294. For those practicing outside of Kansas, the average starting salaries were a bit higher at $66,057 for 2014 graduates and $66,939 for those who graduated in 2013.

"The downturn of the economy impacted veterinary medicine and what graduates could earn in their first year," said Roger Fingland, executive associate dean for K-State's College of Veterinary Medicine and director of the Veterinary Health Center. "It is important to educate people who want to be veterinarians about the financial realities. But, I think the value of being a veterinarian has to always be in the discussion."

Seeking opportunities

Dicks, a veteran agricultural economist, said the objective of the AVMA's economics division is to find ways to enhance the lifelong value of a veterinary degree. Understanding the market for veterinary services and how individual veterinary practices make money are important components that add value.

The biggest area of demand in veterinary medicine is working with companion animals, or pets, which accounts for about six out of 10 practicing veterinarians, he said. Food animal veterinarians, those who work with cattle, sheep and pigs as examples, account for one out of 10. Other veterinarians might choose mixed animal practice or work in zoos, animal hospitals, the education field or other industries.

Scholarship opportunities are available for students in many interest areas, particularly for those who want to work in rural areas and seek mixed animal practice or large animal practice, including food animals and other livestock. Fingland said at K-State, rural scholarship recipients receive $25,000 a year if they intend to practice in a Kansas county that is declared rural. Most counties in Kansas have a rural designation.

If the students take the scholarship and don't practice in a rural area, however, they have to pay that money back, he said. To his knowledge, no students have had to pay the money back, but some graduates will find that some rural areas don't have enough animals to support a full-time veterinarian. Or, perhaps the environment will not allow the veterinarian to charge the going rate for various services.

"I believe there is a need for large animal and mixed animal practitioners in some rural areas," Fingland said. "Need means there are consumers in that area who perceive that they need veterinary services. Needing veterinary services and having an environment that financially supports veterinary services are two different things. Some communities can't financially support the service."

Certain aspects of veterinary medicine are different than others, and veterinarians can choose certain avenues of practice to potentially increase their salaries. Industry veterinarians typically have higher earning potential than mixed animal practitioners, Fingland said.

Like human doctors, veterinarians can specialize as cardiologists, surgeons, internists and radiologists, as examples. Veterinarian specialists tend to make higher salaries, Fingland said, but they also have to go through much more training than general practitioners. Practice ownership also lends itself to higher pay.

Combining business with a calling

Most people go into business, because that's what they want to do. Veterinarians, no matter what area of practice they prefer, are no different, Dicks said. He encourages veterinarians, like any other business-minded professionals, to plan ahead to maximize their opportunities.

"We know just like farmers and ranchers, we all weigh life in some ways with the amount of money we're making," Dicks said. "People must give up a little return to have the style of life they want. For veterinarians, that huge (college) debt can be a restraint. If I have debt when I get out of school, that means I may be driving a 10-year-old car, living with my roommate for another five years, and not going out or buying anything. I may be paying my debt and trying to make a life."

Fingland, who teaches veterinary business courses, said on the first day of orientation at K-State, he presents new students and their parents with numbers showing what the education will cost for the next four years of veterinary school to make them aware.

"There is no question that there is a financial problem at work, and I worry about it like other people in my position worry about it," Fingland said. "But, there is value in doing what is your calling. I understand as a veterinarian that I'm not going to make as much money as someone in another profession. I don't want to be in that other profession, so what difference does it make if that person makes more money than I do?"

"We can't tell young people who aspire to be veterinarians, 'You shouldn't do this, because you won't make as much money as you could doing something else,'" he continued. "Is that what we're going to tell people who want to teach? Imagine if somebody would have turned away the wonderful teachers that we had in grade school, high school and college. That would have been very unfortunate."

Fingland said preparing students to be business-minded veterinarians involves work in and out of the classroom. In addition to one required business and finance course, veterinary students at K-State are allowed to take elective business courses and join organizations such as the Veterinary Business Management Association. They also learn about planning and budgeting through K-State's Powercat Financial Counseling, available as a free resource for all students.

Involving the veterinarian

As a livestock producer, Dicks said he believes it is essential to have a veterinarian as part of your health team if you own animals. Sometimes the veterinarian might be considered a provider of last resort or someone whose job can be handled by salesmen or technicians for artificial insemination, embryo transfer, ultrasounding for carcass characteristics or pregnancy, hoof trimming and nutritional planning, as examples.

But, he said in the last 10 years the cattle industry has experienced diseases such as trichomoniasis and curly calf syndrome, among others, that may have surprised some producers. Having a close relationship with a veterinarian could help protect animals from diseases and producers from major financial losses.

"Maybe because a veterinarian wasn't part of our herd health program, we only found out about these diseases once they happened to us," Dicks said. "Some of those things cause 20 to 30 percent losses."

"What we focus on is teaching veterinary students to thrive in a competitive environment, not in an unrealistic environment where there's no competition," Fingland said. "There are many things veterinarians can do that others can't do who don't have the level of training. No one will ever replace the veterinarians' intellect, when they go to a farm to analyze the nutrition that the rancher or farmer is providing, and the environment and how that environment might lead to disease."

Source: Kansas State University Research and Extension

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