Breathing in diesel exhaust leads to changes 'deep under the hood'

A student participates in the study while seated in a booth.
Credit: Image courtesy of University of British Columbia

Just two hours of exposure to diesel exhaust fumes can lead to fundamental health-related changes in biology by switching some genes on, while switching others off, according to researchers at the University of British Columbia and Vancouver Coastal Health.

The study involved putting volunteers in a polycarbonate-enclosed booth -- about the size of a standard bathroom -- while breathing in diluted and aged exhaust fumes that are about equal to the air quality along a Beijing highway, or a busy port in British Columbia.

The researchers examined how such exposure affected the chemical "coating" that attaches to many parts of a person's DNA. That carbon-hydrogen coating, called methylation, can silence or dampen a gene, preventing it from producing a protein -- sometimes to a person's benefit, sometimes not. Methylation is one of several mechanisms for controlling gene expression, which is the focus of a rapidly growing field of study called epigenetics.

The study, published this month in Particle and Fibre Toxicology, found that diesel exhaust caused changes in methylation at about 2,800 different points on people's DNA, affecting about 400 genes. In some places it led to more methylation; in more cases, it decreased methylation.

How these changes in gene expression translate to health is the next step for researchers. But this study shows how vulnerable our genetic machinery can be to air pollution, and that changes are taking place even if there are no obvious symptoms.

"Usually when we look at the effects of air pollution, we measure things that are clinically obvious -- air flow, blood pressure, heart rhythm," said senior author Dr. Chris Carlsten, an associate professor in the Division of Respiratory Medicine. "But asthma, higher blood pressure or arrhythmia might just be the gradual accumulation of epigenetic changes. So we've revealed a window into how these long-term problems arise. We're looking at changes 'deep under the hood.'"

The fact that DNA methylation was affected after only two hours of exposure has positive implications, said Carlsten, the AstraZeneca Chair in Occupational and Environmental Lung Disease.

"Any time you can show something happens that quickly, it means you can probably reverse it -- either through a therapy, a change in environment, or even diet," he said.

Carlsten's team, having catalogued the changes along the entire human genome, is now sharing its data with scientists who are further exploring the function of specific genes.

Source: University of British Columbia

ALS progression linked to increased protein instability

The new study provides evidence that proteins linked to more severe forms of ALS are less stable structurally and more prone to form clusters or aggregates. Mutants of the superoxide dismutase (SOD) protein formed long, rod-shaped aggregates (shown here as red lattice), compared to the compact folded structure of wild-type SOD (purple ribbons). Credit: Image courtesy of the Getzoff and Tainer labs, The Scripps Research Institute.

A new study by scientists from The Scripps Research Institute (TSRI), Lawrence Berkeley National Laboratory (Berkeley Lab) and other institutions suggests a cause of amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease.

"Our work supports a common theme whereby loss of protein stability leads to disease," said John A. Tainer, professor of structural biology at TSRI and senior scientist at Berkeley Lab, who shared senior authorship of the new research with TSRI Professor Elizabeth Getzoff.

Getzoff, Tainer and their colleagues, who focused on the effects of mutations to a gene coding for a protein called superoxide dismutase (SOD), report their findings this week in the online Early Edition of the Proceedings of the National Academy of Sciences. The study provides evidence that those proteins linked to more severe forms of the disease are less stable structurally and more prone to form clusters or aggregates.

"The suggestion here is that strategies for stabilizing SOD proteins could be useful in treating or preventing SOD-linked ALS," said Getzoff.

Striking in the Prime of Life

ALS is notorious for its ability to strike down people in the prime of life. It first leapt into public consciousness when it afflicted baseball star Lou Gehrig, who succumbed to the disease in 1941 at the age of only 38. Recently, the ALS Association's Ice Bucket Challenge has enhanced public awareness of the disease.

ALS kills by destroying muscle-controlling neurons, ultimately including those that control breathing. At any one time, about 10,000 Americans are living with the disease, according to new data from the Centers for Disease Control and Prevention, but it is almost always lethal within several years of the onset of symptoms.

SOD1 mutations, the most studied factors in ALS, are found in about a quarter of hereditary ALS cases and seven percent of ordinary "sporadic" ALS cases. SOD-linked ALS has nearly 200 variants, each associated with a distinct SOD1 mutation. Scientists still don't agree, though, on just how the dozens of different SOD1 mutations all lead to the same disease.

One feature that SOD1-linked forms of ALS do have in common is the appearance of SOD clusters or aggregates in affected motor neurons and their support cells. Aggregates of SOD with other proteins are also found in affected cells, even in ALS cases that are not linked to SOD1 mutations.

In 2003, based on their and others' studies of mutant SOD proteins, Tainer, Getzoff and their colleagues proposed the "framework destabilization" hypothesis. In this view, ALS-linked mutant SOD1 genes all code for structurally unstable forms of the SOD protein. 
Inevitably some of these unstable SOD proteins lose their normal folding enough to expose sticky elements that are normally kept hidden, and they begin to aggregate with one another, faster than neuronal cleanup systems can keep up -- and that accumulating SOD aggregation somehow triggers disease.

Faster Clumping, Worse Disease

In the new study, the Tainer and Getzoff laboratories and their collaborators used advanced biophysical methods to probe how different SOD1 gene mutations in a particular genetic ALS "hotspot" affect SOD protein stability.

To start, they examined how the aggregation dynamics of the best-studied mutant form of SOD, known as SOD G93A, differed from that of non-mutant, "wild-type" SOD. To do this, they developed a method for gradually inducing SOD aggregation, which was measured with an innovative structural imaging system called SAXS (small-angle X-ray scattering) at Berkeley Lab's SIBYLS beamline.

"We could detect differences between the two proteins even before we accelerated the aggregation process," said David S. Shin, a research scientist in Tainer's laboratories at Berkeley Lab and TSRI who continues structural work on SOD at Berkeley.

The G93A SOD aggregated more quickly than wild-type SOD, but more slowly than an SOD mutant called A4V that is associated with a more rapidly progressing form of ALS.

Subsequent experiments with G93A and five other G93 mutants (in which the amino acid glycine at position 93 on the protein is replaced with a different amino acid) revealed that the mutants formed long, rod-shaped aggregates, compared to the compact folded structure of wild-type SOD. The mutant SOD proteins that more quickly formed longer aggregates were again those that corresponded to more rapidly progressing forms of ALS.

What could explain these SOD mutants' diminished stability? Further tests focused on the role of a copper ion that is normally incorporated within the SOD structure and helps stabilize the protein. Using two other techniques, electron-spin resonance (ESR) spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS), the researchers found that the G93-mutant SODs seemed normal in their ability to take up copper ions, but had a reduced ability to retain copper under mildly stressing conditions -- and this ability was lower for the SOD mutants associated with more severe ALS.

"There were indications that the mutant SODs are more flexible than wild-type SOD, and we think that explains their relative inability to retain the copper ions," said Ashley J. Pratt, the first author of the study, who was a student in the Getzoff laboratory and postdoctoral fellow with Tainer at Berkeley Lab.

Toward New Therapies

In short, the G93-mutant SODs appear to have looser, floppier structures that are more likely to drop their copper ions -- and thus are more likely to misfold and stick together in aggregates.

Along with other researchers in the field, Getzoff and Tainer suspect that deviant interactions of mutant SOD trigger inflammation and disrupt ordinary protein trafficking and disposal systems, stressing and ultimately killing affected neurons.

"Because mutant SODs get bent out of shape more easily," said Getzoff, "they don't hold and release their protein partners properly. By defining these defective partnerships, we can provide new targets for the development of drugs to treat ALS."

The researchers also plan to confirm the relationship between structural stability and ALS severity in other SOD mutants.

"If our hypothesis is correct," said Shin, "future therapies to treat SOD-linked ALS need not be tailored to each individual mutation -- they should be applicable to all of them."

Source: The Scripps Research Institute

Sharing that crowded holiday flight with countless hitchhiking dust mites

Predicted structure of the group 1 allergen protein from an American house dust mite. Arrow points to the location of a novel mutation discovered by the University of Michigan-led team.
Credit: Rubaba Hamid

As if holiday travel isn't stressful enough. Now University of Michigan researchers say we're likely sharing that already overcrowded airline cabin with countless tiny creatures including house dust mites.

"What people might not realize when they board a plane is that they can share the flight with a myriad of microscopic passengers-- including house dust mites--that take advantage of humanity's technological progress for their own benefit," said U-M biologist Pavel Klimov.

"House dust mites can easily travel on an airline passenger's clothes, skin, food and baggage," said Klimov, an assistant research scientist in the U-M Department of Ecology and Evolutionary Biology. "Like humans, they use air travel to visit new places, where they establish new populations, expand their ranges and interact with other organisms through various means."

Air travel likely explains some of the findings of a new genetic study conducted by Klimov and U-M visiting scholar Rubaba Hamid that looked at the connections between house dust mite populations in the United States and South Asia.

They found genetic mutations shared by mites in the U.S. and Pakistan that demonstrate the eight-legged creatures' propensity for intercontinental dispersal, according to a research paper scheduled for online publication Dec. 10 in the journal PLOS ONE.

"What we found suggests that mite populations are indeed linked through migration across continents, though geographic differences still can be detected," Hamid said. "Every time a mite successfully migrates to a new place, it brings its own genetic signature that can be detected in the resident population a long time after the migration event."

The study focused on two medically important mite species, the American and European house dust mite. Both species have global distributions, though the former is more abundant in the U.S.

Ancestors of the two species probably separated from each other nearly 81 million years ago--long before the origin of humans--when they inhabited bird nests. Today, house dust mites are blamed for causing allergic reactions in more than 65 million people worldwide and thrive in the mattresses, sofas and carpets of even the cleanest homes.

Hamid, Klimov and their colleagues examined genetic variation in the group 1 allergen gene from samples of the two mite species collected in the U.S. and Pakistan. The group 1 allergen gene encodes for the most important allergy-causing protein in house dust mites.

An inactive form of this protein is used in clinics worldwide as part of the standard skin-prick test for allergies. Though the test can be inaccurate if it does not include local genetic variants of the allergy-causing protein, geographical variation in group 1 allergen proteins has not been extensively studied in the U.S., Klimov said.

"We need to have a better idea about the diversity of allergenic proteins around the world, and particularly in the United States," he said.

In genetic sequences from American house dust mites (Dermatophagoides farinae), the 
researchers observed mutations at 14 positions along the length of the group 1 allergen gene.

All but one of the mutations are "silent," meaning they occur at the DNA level without changing the amino acid structure of the protein. Only mutations at the protein level have medical significance because they can change allergenic properties.

"The most unexpected result was the finding that a previously unknown mutation occurred at the active site of the protein at position 197," Klimov said. "This was a rare mutation, found in only a single population of house dust mite in South Asia.

"Our analysis indicates that this mutation might alter the enzyme activity of the protein. But allergenic properties, immune response and cross-reactivity of the protein are unknown at this time," he said. "Follow-up experiments to elucidate these issues are underway in our lab."

Source: University of Michigan

Genes tell story of birdsong and human speech

The activity of genes related to singing shows a unique pattern in the brain of an Australian Budgerigar.
Credit: Duke University

His office is filled with all sorts of bird books, but Duke neuroscientist Erich Jarvis didn't become an expert on the avian family tree because of any particular interest in our feathered friends. Rather, it was his fascination with how the human brain understands and reproduces speech that brought him to the birds.

"We've known for many years that the singing behavior of birds is similar to speech in humans -- not identical, but similar -- and that the brain circuitry is similar, too," said Jarvis, an associate professor of neurobiology at the Duke University Medical School and an investigator at the Howard Hughes Medical Institute. "But we didn't know whether or not those features were the same because the genes were also the same."

Now scientists do know, and the answer is yes -- birds and humans use essentially the same genes to speak.

After a massive international effort to sequence and compare the entire genomes of 48 species of birds representing every major order of the bird family tree, Jarvis and his colleagues found that vocal learning evolved twice or maybe three times among songbirds, parrots and hummingbirds.

Even more striking is that the set of genes involved in each of those song innovations is remarkably similar to the genes involved in human speaking ability.

The findings are part of a package of eight scientific papers in a Dec. 12 special issue of Science and 21 additional papers appearing nearly simultaneously in Genome Biology, GigaScience and other journals. Jarvis' name appears on 20 papers and he is a corresponding author for 8 of them.

Jarvis co-led the Avian Phylogenomics Consortium with Guojie Zhang of the National Genebank at BGI in China and the University of Copenhagen and M. Thomas P. Gilbert of the Natural History Museum of Denmark. His Duke lab contributed to preparing samples, sequencing and annotating the genomes, performing the analyses and coordinating the overall project.

The Jarvis lab in the Bryan Research Building prepared DNA of many of the species, pulling it from little chunks of frozen, pink bird flesh collected over the past 30 years by museums and other institutions around the world. To ensure the DNA being sequenced really belonged to the Golden-collared manakin and not an undergraduate lab assistant, the lab has been kept spotlessly clean and many of its tools are used only once, to avoid the possibility of subsequent contamination.

"We change gloves a lot," said Carole Parent, the lab research analyst who set up a DNA isolation pipeline for the next stage of the project to sequence still more birds and supervised sample prep with a team of Duke undergrads and a student from East Chapel Hill High School.

All of this meticulous and somewhat tedious work has given Jarvis and hundreds of colleagues around the world a crack at an unprecedented amount of genomic data generated by BGI in China. The whole-genome comparison of the 48 bird species required new algorithms written at the University of Illinois and University of Texas that ran for 400 years of CPU time on three supercomputers in the U.S.

Of the 29 papers covering everything from penguin evolution to color vision, eight are devoted to bird song.

One of the Dec. 12 papers in Science found there is a consistent set of just over 50 genes that show higher or lower activity in the brains of vocal learning birds and humans. These changes were not found in the brains of birds that do not have vocal learning and of non-human primates that do not speak, according to this Duke team, which was led by Jarvis; Andreas Pfenning, a graduate of the Ph.D. program in computational biology and bioinformatics (CBB); and Alexander Hartemink, professor of computer science, statistical science and biology.

"This means that vocal learning birds and humans are more similar to each other for these genes in song and speech brain areas than other birds and primates are to them," Jarvis said.

These genes are involved in forming new connections between neurons of the motor cortex and neurons that control the muscles that produce sound.

A companion study by another CBB doctorate, Rui Wang, looked at the specialized activity of a pair of genes involved in the regions of the brain that control song and speech. This study, appearing in the Journal of Comparative Neurology, found that these genes are down- and up-regulated in one brain region of song-learning birds during the juvenile period of their vocal learning , changes that last into adulthood. This study, and that of Pfenning, hypothesize that changes in these genes could be critical for the evolution of song in birds and speech in humans.

"You can find those same genes in the genomes of all species, but they're active at much higher or lower levels in the specialized song or speech brain regions of vocal learning birds and humans," Jarvis said. "What this suggests to me is that when vocal learning evolves, there may be a limited way in which the brain circuits can evolve."

Another paper in Science from Duke, led by post-doc Osceola Whitney, Pfenning, Hartemink and Anne West, an associate professor of neurobiology, looked at gene activation in different areas of the brain during singing. This team found activation of 10 percent of the expressed genome during singing, with diverse activation patterns in different song-learning regions of the brain. The diverse gene patterns are best explained by epigenetic differences in the genomes of the different brain regions, meaning that individual cells in different brain regions can regulate genes at a moment's notice when the birds sing.

Among the three main groups of vocal learning birds, parrots are clearly different in their ability to mimic human speech. Mukta Chakraborty, a postdoc in the Jarvis lab, led a project that used the activity of some of the specialized genes to discover that the parrot's speech center is organized somewhat differently. It has what the researchers call a "song-system-within-a-song-system" in which the area of the brain with different gene activity for producing song has an outer ring of still more differences in gene expression.

Parrots are very social animals, Chakraborty said, and having the ability to quickly pick up "dialects" of parrot speech may account for their super-charged speech center. The "shell" or outer regions were found to be proportionally larger in the parrot species, which are believed to have the highest vocal, cognitive and social abilities. These species include Amazon parrots, the African Grey and the Blue and Gold Macaw.

Jarvis was also part of a team with Claudio Mello and his Ph.D. student Morgan Wirthlin at Oregon Health & Science University that found ten more genes that are unique to song-control regions of songbirds. This paper appears in BMC Genomics.

A paper in Science led by Zhang, Gilbert and Jarvis found the genomes of vocal learners are more rapidly evolving and have more chromosomal rearrangements compared to other bird species. This genomic comparison also found similar changes occurred independently in in the song-learning area of different birds' brains.

Jarvis said knowing more of this history of how speech evolved in birds makes vocal learning birds even more valuable model organisms for helping to answer the questions he and other researchers are addressing about human speech.

"Speech is difficult to study in human brains," he said. "Whales and elephants learn speech and songs, but they're too big to house in the lab. Now that we have a deeper understanding of how similar birdsong brain regions are to human speech regions at the genetic level, I think they'll be a better model than ever."

Jarvis' general exploration of the bird brain over his 16 years at Duke has also led to several unexpected discoveries unrelated to song.

In 2005, he and colleagues found a center of the brain in migratory birds that apparently enables sensing of magnetic fields through "night vision." That year he also led a revision of the understanding of bird brain organization and vertebrate brain evolution. Last year, he led a re-drawing of the geography of the bird brain based on analysis of 52 genes that are active in 23 areas of the brains of eight species of birds. This new map shows neuron groupings in the birds' brains to be organized in columns like the brains of humans and other mammals.

He also branched out a bit and learned about the brain structures that enable mice to "sing" in ultrasonic ranges beyond human hearing.

Jarvis said this first wave of findings from the Avian Phylogenomics Consortium is just the beginning of an exciting new era of genomic analysis. The international group is already sequencing more birds at the whole-genome level.

"This is an exciting moment," said Jarvis, who is also a member of the Duke Institute for Brain Sciences. "Lots of fundamental questions now can be resolved with more genomic data from a broader sampling. I got into this project because of my interest in birds as a model for vocal learning and speech production in humans, and it has opened up some amazing new vistas on brain evolution."

Source: Duke University

Ancient human genome from southern Africa throws light on our origins

Professor Vanessa Hayes in the field.

The skeleton of a man who lived 2,330 years ago in the southernmost tip of Africa tells us about ourselves as humans, and throws some light on our earliest common genetic ancestry.

What can DNA from the skeleton of a man who lived 2,330 years ago in the southernmost tip of Africa tell us about ourselves as humans? A great deal when his DNA profile is one of the 'earliest diverged' -- oldest in genetic terms -- found to-date in a region where modern humans are believed to have originated roughly 200,000 years ago.

The man's maternal DNA, or 'mitochondrial DNA', was sequenced to provide clues to early modern human prehistory and evolution. Mitochondrial DNA provided the first evidence that we all come from Africa, and helps us map a figurative genetic tree, all branches deriving from a common 'Mitochondrial Eve'.
When archaeologist Professor Andrew Smith from the University of Cape Town discovered the skeleton at St. Helena Bay in 2010, very close to the site where 117,000 year old human footprints had been found -- dubbed "Eve's footprints" -- he contacted Professor Vanessa Hayes, an expert in African genomes.

At the time, Hayes was Professor of Genomic Medicine at the J. Craig Venter Institute in San Diego, California. She now heads the Laboratory for Human Comparative and Prostate Cancer Genomics at Sydney's Garvan Institute of Medical Research.

The complete 1.5 metre tall skeleton was examined by Professor Alan Morris, from the University of Cape Town. A biological anthropologist, Morris showed that the man was a 'marine forager'. A bony growth in his ear canal, known as 'surfer's ear', suggested that he spent some time diving for food in the cold coastal waters, while shells carbon-dated to the same period, and found near his grave, confirmed his seafood diet. Osteoarthritis and tooth wear placed him in his fifties.

Due to the acidity of the soil within the region, acquiring DNA from skeletons has proven problematic. The Hayes team therefore worked with the world's leading laboratory in ancient DNA research, namely that of paleogeneticist Professor Svante Pääbo at the Max Planck Institute for Evolutionary Anthropolgy in Leipzig, Germany, who successfully sequenced a Neanderthal.

The team generated a complete mitochondrial genome, using DNA extracted from a tooth and a rib. The findings provided genomic evidence that this man, from a lineage now presumed extinct, as well as other indigenous coastal dwellers like him, were the most closely related to 'Mitochondrial Eve'.

The study underlines the significance of southern African archaeological remains in defining human origins, and is published in the journal Genome Biology and Evolution, now online.

"We were thrilled that archaeologist Andrew Smith understood the importance of not touching the skeleton when he found it, and so did not contaminate its DNA with modern human DNA," said Professor Hayes.

"I approached Svante Pääbo because his lab is the best in the world at DNA extraction from ancient bones. This skeleton was very precious and we needed 
to make sure the sample was in safe hands."

"Alan Morris undertook some incredible detective work. He used his skills in forensics and murder cases to assemble a profile of the man behind the St Helena skeleton."

"Alan helped establish that this man was a marine hunter-gatherer -- in contrast to the contemporary inland hunter-gatherers from the Kalahari dessert. We were very curious to know how this man related to them."

"We also know that this man pre-dates migration into the region, which took place around 2,000 years ago when pastoralists made their way down the coast from Angola, bringing herds of sheep. We could demonstrate that our marine hunter-gatherer carried a different maternal lineage to these early migrants -- containing a DNA variant that we have never seen before."

"Because of this, the study gives a baseline against which historic herders at the Cape can now be compared."

While interested in African lineages, and how they interact with each other, Professor Hayes is especially keen for Africa to inform genomic research and medicine worldwide.

"One of the biggest issues at present is that no-one is assembling genomes from scratch -- in other words, when someone is sequenced, their genome is not pieced together as is," she said.

"Instead, sections of the sequenced genome are mapped to a reference genome. Largely biased by European contribution, the current reference is poorly representative of indigenous peoples globally."

"If we want a good reference, we have to go back to our early human origins."
"None of us that walk on this planet now are pure anything -- we are all mixtures. For example 1-4% of Eurasians even carry Neanderthal DNA"

"We need more genomes that don't have extensive admixture. In other words, we need to reduce the noise."

"In this study, I believe we may have found an individual from a lineage that broke off early in modern human evolution and remained geographically isolated. That would contribute significantly to refining the human reference genome."

Source: Garvan Institute of Medical Research

Uncovering one of humankind’s most ancient lineages

A Khoisan hunter/gatherer with his bow and arrows. Credit: Image courtesy of Nanyang Technological University

Scientists at Nanyang Technological University (NTU Singapore) and Penn State University in the United States have successfully discovered one of modern humans' ancient lineages through the sequencing of genes.

A geneticist from NTU, Professor Stephan Christoph Schuster, who led an international research team from Singapore, United States and Brazil, said this is the first time that the history of humankind populations has been analysed and matched to Earth's climatic conditions over the last 200,000 years.
Their breakthrough findings are published today (4 Dec) in Nature Communications.

The team has sequenced the genome of five living individuals from a hunter/gatherer tribe in Southern Africa, and compared them with 420,000 genetic variants across 1,462 genomes from 48 ethnic groups of the global population.

Through advanced computation analysis, the team found that these Southern African Khoisan tribespeople are genetically distinct not only from Europeans and Asians, but also from all other Africans.

The team also found that there are individuals of the Khoisan population whose ancestors did not interbreed with any of the other ethnic groups for the last 150,000 years and that Khoisan was the majority group of living humans for most of that time until about 20,000 years ago.

Their findings mean it is now possible to use genetic sequencing to reveal the ancestral lineage of any ethnic group even up to 200,000 years ago, if non-admixed individuals are found, like in the case of the Khoisan. This will show when in history there have been important genetic changes to an ancestral lineage due to intermarriages or geographical migrations that may have occurred over the centuries.

"Khoisan hunter/gatherers in Southern Africa have always perceived themselves as the oldest people," said Prof Schuster, an NTU scientist at the Singapore Centre on Environmental Life Sciences Engineering (SCELSE) and a former Penn State University professor.

"Our study proves that they truly belong to one of mankind's most ancient lineages, and these high quality genome sequences obtained from the tribesmen will help us better understand human population history, especially the understudied branch of mankind such as the Khoisan.

"The new data gathered will also enable scientists to better understand how the human genome has evolved and hopefully lead to more effective treatment options for certain genetic diseases and illnesses."

Of the five tribesmen who were the oldest members of the Ju/'hoansi tribe and other tribes living in protected areas of northwest Namibia, two individuals were found to have a genome which had not admixed with other ethnic groups.

The Ju/'hoansi tribe was made famous in the 80s and 90s by the box-office hit movie series "The Gods Must Be Crazy." The main character of the series was a hunter/gatherer tribesman, played by Nǃxau, a bushman.

The research paper's first author, Dr Hie Lim Kim, a SCELSE senior research fellow, said "it was very surprising that this group apparently did not intermarry with non-Khoisan neighbours for thousands of years." This is because the Khoisan peoples and the rest of modern humanity shared their most recent common ancestor around 150,000 years ago.

The current Khoisan culture and tradition, where marriage occurs either among Khoisan groups or results in female members leaving their tribes after marrying non-Khoisan men, appears to be long-standing.

"A key finding from this study is that even today after 150,000 years, single non-admixed individuals or descendants of those who did not interbreed with separate populations can be identified within the Ju/'hoansi population, which means there might be more of such unique individuals in other parts of the world," added Dr Kim.

The Khoisan tribespeople participating in this study had parts of their genomes sequenced in an earlier study by the same team in 2010. The new study generated complete genome sequences at high quality, which enabled the analysis of admixture and population history. The availability of such high quality Southern African genomes will allow further investigation of the population history of this largely understudied branch of humankind at high resolution.

This research project involving six investigators was led by NTU and Penn State University. Other institutions participating in the study include the Ohio State University and Sao Paulo State University, Brazil.

Moving forward, Prof Schuster added that they will be looking to find more non-admixed individuals who are in the other parts of the world, such as in South Asia and South America, where uncontacted tribes still exist. The team will also be seeking more funding to further their research which will have large impact on the study of life sciences.

Source: Nanyang Technological University

The shape of infectious prions

Structural changes were located in the prion protein N-terminus, where a novel reorganization of the beta sheet (in yellow) was observed. In the background, the X-ray diffraction pattern of the crystal composed by the complex prion protein-Nanoboy.

Prions are unique infective agents -- unlike viruses, bacteria, fungi and other parasites, prions do not contain either DNA or RNA. Despite their seemingly simple structure, they can propagate their pathological effects like wildfire, by "infecting" normal proteins. PrPSc (the pathological form of the prion protein) can induce normal prion proteins (PrPC) to acquire the wrong conformation and convert into further disease-causing agents.

"When they are healthy, they look like tiny spheres; when they are malignant, they appear as cubes" stated Giuseppe Legname, principal investigator of the Prion Biology Laboratory at the Scuola Internazionale Superiore di Studi Avanzati (SISSA) in Trieste, when describing prion proteins. Prions are "misfolded" proteins that cause a group of incurable neurodegenerative diseases, including spongiform encephalopathies (for example, mad cow diseases) and Creutzfeldt-Jakob disease. Legname and coworkers have recently published a detailed analysis of the early mechanisms of misfolding. Their research has just been published in the Journal of the American Chemical Society, the most authoritative scientific journal in the field.

"For the first time, our experimental study has investigated the structural elements leading to the disease-causing conversion" explains Legname. "With the help of X-rays, we observed some synthetic prion proteins engineered in our lab by applying a new approach -- we used nanobodies, i.e. small proteins that act as a scaffolding and induce prions to stabilize their structure." Legname and colleagues reported that misfolding originates in a specific part of the protein named "N-terminal." "The prion protein consists of two subunits. The C-terminal has a clearly defined and well-known structure, whereas the unstructured N-terminal is disordered, and still largely unknown. This is the very area where the early prion pathological misfolding occurs" adds Legname. "The looser conformation of the N-terminal likely determines a dynamic structure, which can thus change the protein shape."

"Works like ours are the first, important steps to understand the mechanisms underlying the pathogenic effect of prions" concludes Legname. "Elucidating the misfolding process is essential to the future development of drugs and therapeutic strategies against incurable neurodegenerative diseases."

Source: Sissa Medialab

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

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

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

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

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