Stanford bioengineers develop tool for reprogramming genetic code

Stanford bioengineers have developed a new tool that allows them to preferentially activate or deactivate genes in living cells. VITSTUDIO/SHUTTERSTOCK

Biology relies upon the precise activation of specific genes to work properly. If that sequence gets out of whack, or one gene turns on only partially, the outcome can often lead to a disease.

Now, bioengineers at Stanford and other universities have developed a sort of programmable genetic code that allows them to preferentially activate or deactivate genes in living cells. The work is published in the current issue of Cell, and could help usher in a new generation of gene therapies.

The technique is an adaptation of CRISPR, itself a relatively new genetic tool that makes use of a natural defense mechanism that bacteria evolved over millions of years to slice up infectious virus DNA.

Standard CRISPR consists of two components: a short RNA that matches a particular spot in the genome, and a protein called Cas9 that snips the DNA in that location. For the purposes of gene editing, scientists can control where the protein snips the genome, insert a new gene into the cut and patch it back together.

Inserting new genetic code, however, is just one way to influence how the genome is expressed. Another involves telling the cell how much or how little to activate a particular gene, thus controlling how much protein a cell produces from that gene and altering its behavior.

It's this action that Lei Stanley Qi, an assistant professor of bioengineering and of chemical and systems biology at Stanford, and his colleagues aim to manipulate.

Influencing the genome
In the new work, the researchers describe how they have designed the CRISPR molecule to include a second piece of information on the RNA, instructing the molecule to either increase (upregulate) or decrease (downregulate) a target gene's activity, or turn it on/off entirely.

Additionally, they designed it so that it could affect two different genes at once. In a cell, the order or degree in which multiple genes are activated can produce different metabolic products.

"It's like driving a car. You control the wheel to control direction, and the engine to control the speed, and how you balance the two determines how the car moves," Qi said. "We can do the same thing in the cell by up- or downregulating genes, and produce different outcomes."

As a proof of principle, the scientists used the technique to take control of a yeast metabolic pathway, turning genes on and off in various orders to produce four different end products. They then tested it on two mammalian genes that are important in cell mobility, and were able to control the cell's direction and how fast it moved.

Future therapies
The ability to control genes is an attractive approach in designing genetic therapies for complex diseases that involve multiple genes, Qi said, and the new system may overcome several of the challenges of existing experimental therapies.

"Our technique allows us to directly control multiple specific genes and pathways in the genome without expressing new transgenes or uncontrolled behaviors, such as producing too much of a protein, or doing so in the wrong cells," Qi said. "We could eventually synthesize tens of thousands of RNA molecules to control the genome over a whole organism."

Next, Qi plans to test the technique in mice and refine the delivery method. Currently the scientists use a virus to insert the molecule into a cell, but he would eventually like to simply inject the molecules into an organism's blood.

"That is what is so exciting about working at Stanford, because the School of Medicine's immunology group is just around the corner, and working with them will help us address how to do this without triggering an immune response," said Qi, who is a member of the interdisciplinary Stanford ChEM-H institute. "I'm optimistic because everything about this system comes naturally from cells, and should be compatible with any organism."

Source: Stanford university

An Advanced Method of DNA Nanostructure Formation Developed

Figure 1: Uni-molecular magnetic tweezers orchestrating the DNA nanostructure formation

Professor Tae-Young Yoon’s research team from the Department of Physics at KAIST has developed a new method to form DNA nanostructures by using magnetic tweezers to observe and to induce the formation of the structure in real time.

Unlike traditional designs of "DNA origami" which relies on thermal or chemical annealing methods, the new technology utilizes a completely different dynamic in DNA folding. This allows the folding to be done within only ten minutes.

Developed in 2006, the "DNA origami" allows a long skeleton of DNA to be folded into an arbitrary structure by using small stapler DNA pieces. This has been a prominent method in DNA nanotechnology.
 

Figure 2: The evolution of DNA nanostructure formation using magnetic tweezers. The DNA nanostructure with a 21-nanometer size was formed in about eight minutes.

However, the traditional technology which adopts thermal processes could not control the DNA formation during the folding because every interaction among DNAs occurs simultaneously. Thus, the thermal processes, which take dozens of hours to complete, had to be repeated multiple times in order to find the optimal condition.

The research team designed a DNA folding using uni-molecular magnetic tweezers that applied force to a single DNA molecule while measuring the state of the DNA. Through this technology, they were able to induce the formation of DNA nanostructure and observe it at the same time.

During high temperature heat treatment, the first stage of conventional thermal processes, the internal structure of the long skeleton DNA untangles. To induce such state, after attaching one side of the skeleton DNA to the surface of glass and the other side to a magnetic material, the team unfolded the internal structure of the DNA by pulling the two sides apart with magnetic force.

Unlike the conventional thermal processes, this method lets the stapler DNA swiftly adhere to the skeleton DNA within a minute because the sites are revealed at room temperature.

After the stapler pieces connected to the skeleton, the team removed the magnetic force. Next, the structure folded through self-assembly as the stapler DNAs stuck to different sites on the skeleton DNA.

Professor Yoon said, “With the existing thermal methods, we could not differentiate the reactions of the DNA because the response of each DNA pieces mutually interacted with each other.” He added that “Using the magnetic tweezers, we were able to sort the process of DNA nanostructure formation into a series of reactions of DNA molecules that are well known, and shorten the time taken for formation in only ten minutes.”

He commented, “This nanostructure formation method will enable us to create more intricate and desirable DNA nanostructures by programming the folding of DNA origami structures.”

Conducted by Dr. Woori Bae under the guidance of Professor Yoon, the research findings were published online in the December 4th issue of Nature Communications.


Source: KAIST

Skull sheds light on human-Neanderthal relationship

Retrieved from a cave in northern Israel, the partial skull provides the first evidence that Homo sapiens inhabited that region at the same time as Neanderthals. (Reuters: Nikola Solic)

A partial skull, found in a cave in Israel, is shedding light on the pivotal moment in early human history when our species left Africa and encountered our close cousins the Neanderthals.

Anthropologist Israel Hershkovitz, from Tel Aviv University, called the skull "an important piece of the puzzle of the big story of human evolution."

The findings of the research, led by Hershkovitz, are published today in the journal Nature.

The upper part of the skull - the domed portion without the face or jaws - was unearthed in Manot Cave in Israel's Western Galilee.

Scientific dating techniques determined the skull was about 55,000 years old, a time period when members of our species were thought to have been marching out of Africa,

The researchers say characteristics of the skull suggest the individual was closely related to the first Homo sapiens populations that later colonized Europe.

They also say the skull provides the first evidence that Homo sapiens inhabited that region at the same time as Neanderthals, our closest extinct human relative.

Previous genetic evidence suggests our species and Neanderthals interbred around the time the skull is dated to, with all people of Eurasian ancestry still retaining a small amount of Neanderthal DNA as a result.

"It is the first direct fossil evidence that modern humans and Neanderthals inhabited the same area at the same time," says palaeontologist Bruce Latimer of Case Western Reserve University in Cleveland, another of the researchers.

"The co-existence of these two populations in a confined geographic region at the same time that genetic models predict interbreeding promotes the notion that interbreeding may have occurred in the Levant region," Hershkovitz says.

The robust, large-browed Neanderthals prospered across Europe and Asia from about 350,000 to 40,000 years ago, going extinct sometime after Homo sapiens arrived.

Scientists say our species first appeared about 200,000 years ago in Africa and later migrated outwards. The cave is located along the sole land route for ancient humans to take from Africa into the Middle East, Asia and Europe.

Latimer says he suspects the skull belonged to a woman, although the researchers could not say definitively.

The cave, sealed off for 30,000 years, was discovered in 2008 during sewage line construction work. Hunting tools, perforated seashells perhaps used ornamentally and animal bones have been excavated from the cave, along with further human remains.

Source: ABC

Source of most cases of invasive bladder cancer identified

Philip Beachy and his team found a single type of cell in mice that gives rise to invasive bladder cancers. Credit: Steve Fisch

A single type of cell in the lining of the bladder is responsible for most cases of invasive bladder cancer, according to researchers at the Stanford University School of Medicine.

Their study, conducted in mice, is the first to pinpoint the normal cell type that can give rise to invasive bladder cancers. It's also the first to show that most bladder cancers and their associated precancerous lesions arise from just one cell, and explains why many human bladder cancers recur after therapy.

"We've learned that, at an intermediate stage during cancer progression, a single cancer stem cell and its progeny can quickly and completely replace the entire bladder lining," said Philip Beachy, PhD, professor of biochemistry and of developmental biology. "All of these cells have already taken several steps along the path to becoming an aggressive tumor. Thus, even when invasive carcinomas are successfully removed through surgery, this corrupted lining remains in place and has a high probability of progression."

Although the cancer stem cells, and the precancerous lesions they form in the bladder lining, universally express an important signaling protein called sonic hedgehog, the cells of subsequent invasive cancers invariably do not -- a critical switch that appears vital for invasion and metastasis. This switch may explain certain confusing aspects of previous studies on the cellular origins of bladder cancer in humans. It also pinpoints a possible weak link in cancer progression that could be targeted by therapies.

"This could be a game changer in terms of therapeutic and diagnostic approaches," said Michael Hsieh, MD, PhD, assistant professor of urology and a co-author of the study. "Until now, it's not been clear whether bladder cancers arise as the result of cancerous mutations in many cells in the bladder lining as the result of ongoing exposure to toxins excreted in the urine, or if it's due instead to a defect in one cell or cell type. If we can better understand how bladder cancers begin and progress, we may be able to target the cancer stem cell, or to find molecular markers to enable earlier diagnosis and disease monitoring."

Beachy is the senior author of the study, which will be published online April 20 in Nature Cell Biology. He is the Ernest and Amelia Gallo Professor in the School of Medicine and a member of the Stanford Cancer Institute and the Stanford Institute for Stem Cell Biology and Regenerative Medicine. He is also a Howard Hughes Medical Institute investigator. Kunyoo Shin, PhD, an instructor at the institute, is the lead author.

Bladder cancer is the fourth most common cancer in men and the ninth most common in women. Smoking is a significant risk factor. There are two main types of the disease: one that invades the muscle around the bladder and metastasizes to other organs, and another that remains confined to the bladder lining. Unlike the more-treatable, noninvasive cancer -- which comprises about 70 percent of bladder cancers -- the invasive form is largely incurable. It is expensive and difficult to treat, and the high likelihood of recurrence requires ongoing monitoring after treatment.

In 2011, Shin and Beachy and their colleagues identified a cell type in the bladder that is capable of completely replacing the lining of the organ after infection or damage. The fact that it could give rise to multiple cell types (even forming small, multilayered, bladder-like spheres when cultured in vitro), and also self-renew, showed that it was a bladder stem cell. 

They found that the cell, which came from the basal layer of the bladder epithelium, used a protein called sonic hedgehog to "talk" to other cells in the bladder and stimulate proliferation and specialization into other cell types. (Beachy identified the first hedgehog protein in fruit flies in 1992; the hedgehog signaling pathway has since been shown to play a vital role in embryonic development and in many types of cancers.)

Many animal models of cancer rely on prior knowledge or hunches as to what genes or cell types are involved. Researchers may genetically alter an animal, or a certain cell type, to induce the overexpression of a gene known to be involved in tumorigenesis, for example, or block the expression of a gene that inhibits cancer development.

Although prior work suggested that basal cells may play a role in bladder cancer, the researchers chose an unbiased approach when developing their mouse model that more closely mimicked what happens in humans: They put a chemical compound called N-butyl-N-4-hydroxybutyl nitrosamine, or BBN, in the mice's drinking water and watched the animals over a period of months.

Nitrosamines are carcinogens found in cigarette smoke; BBN is a form of the chemical that is specifically activated in the bladder. After four months, many of the animals had developed precancerous lesions, or carcinomas in situ, in their bladders that very closely resemble those seen in human patients. By six months, all of the animals had developed invasive bladder cancers.

With their model in place, the researchers then conducted two main experiments in the mice: In the first experiment, they looked to see what would happen in animals exposed to BBN when the sonic-hedgehog-expressing cells were marked with a distinctive fluorescent color. In the second, they used genetic techniques to selectively kill those same cells in animals prior to exposure with BBN.

In the first case, they saw something startling: After just a few months of BBN exposure, nearly the entire lining of the bladder was labeled with the fluorescent green marker that indicated the cells had arisen from the sonic-hedgehog-expressing basal stem cells. When transplanted into other mice, those labeled cells were able to give rise to bladder cancers, but cells not expressing sonic hedgehog did not.

In the second case, no tumors grew in the animals in which the stem cells had been selectively killed -- although the bladder architecture became severely compromised in the absence of stem cells to regenerate cells lost during the normal course of bladder function.

"So now we have two lines of evidence indicating that the bladder stem cells are solely responsible for tumorigenesis," Shin said. "When we mark the bladder stem cells, the tumors are also marked. When we remove, or ablate, the stem cells, no tumors arise after BBN treatment."

Next the researchers tackled the question of whether bladder cancers arise as the result of genetic changes to one or more of these bladder stem cells. To do so, they used a genetically engineered mouse with cells that fluoresce green, but which can be triggered to randomly fluoresce one of three other colors: red, blue or yellow. Known as a "rainbow mouse," the animal allows researchers to more precisely determine the origin of groups of cells. If all cells in a tumor are red, for example, it is much more likely that they originated from a single cell.

"After four months of BBN treatment," Beachy said, "we'd most often see just one color dominating the entire epithelium. This clearly indicates that a single cell has taken over the lining of the entire bladder, elbowing out its neighbors in a way that's not been seen in other organs."

Further studies showed that, surprisingly, none of the cells in the most advanced, invasive carcinomas in the BNN-treated animals expressed sonic hedgehog -- despite the fact that only sonic-hedgehog-expressing cells are able to give rise to the earlier stages of bladder cancer. One obvious implication of the lack of sonic hedgehog expression in these cells is that the hedgehog pathway somehow inhibits steps required for tissue invasion or metastasis.

"We know that the hedgehog pathway is widely used throughout the animal kingdom to tightly regulate cellular and tissue differentiation," Hsieh said. "So its loss could make sense in this context because cancer is essentially a loss of normal regulation."

"One really important lesson from this study," Beachy said, "is the idea that, by the time you get to a full-blown tumor, the properties of the cells in that tumor may have changed quite significantly from the cell type that gives rise to the tumors. This can complicate understanding how human tumors arise, because even if you identify the tumor-propagating cells within a mature tumor, conclusions about the origins of a cancer based on properties of these cells may be inaccurate."

Source: Stanford University Medical Center

Charged graphene gives DNA a stage to perform molecular gymnastics

DNA interacts with charged graphene and contorts into sequence-specific shapes when the charge is changed. Credit: Photo courtesy Alek Aksimentiev

When Illinois researchers set out to investigate a method to control how DNA moves through a tiny sequencing device, they did not know they were about to witness a display of molecular gymnastics.

Fast, accurate and affordable DNA sequencing is the first step toward personalized medicine. Threading a DNA molecule through a tiny hole, called a nanopore, in a sheet of graphene allows researchers to read the DNA sequence; however, they have limited control over how fast the DNA moves through the pore. In a new study published in the journal Nature Communications, University of Illinois physics professor Aleksei Aksimentiev and graduate student Manish Shankla applied an electric charge to the graphene sheet, hoping that the DNA would react to the charge in a way that would let them control its movement down to each individual link, or nucleotide, in the DNA chain.

"Ideally, you would want to step the DNA through the nanopore one nucleotide at a time," said Aksimentiev. "Take a measurement and then have another nucleotide in the sensing hole. That's the goal, and it hasn't been realized yet. We show that, to some degree, we can control the process by charging the graphene."

The researchers found that a positive charge in the graphene speeds up DNA movement through the nanopore, while a negative charge stops the DNA in its tracks. However, as they watched, the DNA seemed to dance across the graphene surface, pirouetting into shapes they had never seen, specific to the sequence of the DNA nucleotides.

"It reminds me of Swan Lake," Aksimentiev said. "It's very acrobatic. We were very surprised by the variety of DNA conformations that we can observe at the surface of graphene when we charge it. There is one sequence that starts out laying down on the surface, and when we change the charge, they all tilt on the side like they are doing a one-armed push-up. Then we also have nucleotides that would lay back, or go up like a ballerina en pointe."

Aksimentiev hypothesizes that the conformations are so different and so specific to the sequence because each nucleotide has a slightly different distribution of electrons, the negatively charged parts of the atoms. There is even a visible difference when a nucleotide is methylated, a tiny chemical change that can turn a gene on or off.

By switching the charge in the graphene, the researchers can control not only the DNA's motion through the pore, but also the shape the DNA contorts into.

"Because it's reversible, we can force it to adopt one conformation and then force it to go back. That's why we call it gymnastics," Aksimentiev said.

The researchers extensively used the Blue Waters supercomputer at the National Center for Supercomputing Applications, housed at the University of Illinois. They mapped each individual atom in the complex DNA molecule and ran numerous simulations of many different DNA sequences. Supercomputing power was essential to carrying out the work, Aksimentiev said.

"This is a really computationally intensive project," he said. "Having access to Blue Waters was essential because with the sheer number of simulations, we would not have been able to finish them. It would have taken too long."

The next step is to combine a charged nanopore setup with a sensor to build a DNA sequencing device that would incorporate both motion control and nucleotide recognition. The researchers also hope to explore the unexpected conformational changes for insights into epigenetics, the field that studies how genes are expressed and moderated.

"DNA is much more complicated than just a double helix. It's a complex molecule that has many properties, and we are still uncovering them," Aksimentiev said.

Video animation of DNA dancing as the graphene charge changes:


Source: University of Illinois at Urbana-Champaign

Were Neanderthals a sub-species of modern humans? New research says no

Depiction of Neanderthal (stock image). Credit: © procy_ab / Fotolia

In an extensive, multi-institution study led by SUNY Downstate Medical Center, researchers have identified new evidence supporting the growing belief that Neanderthals were a distinct species separate from modern humans (Homo sapiens), and not a subspecies of modern humans.

The study looked at the entire nasal complex of Neanderthals and involved researchers with diverse academic backgrounds. Supported by funding from the National Science Foundation and the National Institutes of Health, the research also indicates that the Neanderthal nasal complex was not adaptively inferior to that of modern humans, and that the Neanderthals' extinction was likely due to competition from modern humans and not an inability of the Neanderthal nose to process a colder and drier climate.

Samuel Márquez, PhD, associate professor and co-discipline director of gross anatomy in SUNY Downstate's Department of Cell Biology, and his team of specialists published their findings on the Neanderthal nasal complex in the November issue of The Anatomical Record, which is part of a special issue on The Vertebrate Nose: Evolution, Structure, and Function (now online).

They argue that studies of the Neanderthal nose, which have spanned over a century and a half, have been approaching this anatomical enigma from the wrong perspective. Previous work has compared Neanderthal nasal dimensions to modern human populations such as the Inuit and modern Europeans, whose nasal complexes are adapted to cold and temperate climates.

However, the current study joins a growing body of evidence that the upper respiratory tracts of this extinct group functioned via a different set of rules as a result of a separate evolutionary history and overall cranial bauplan (bodyplan), resulting in a mosaic of features not found among any population of Homo sapiens. Thus Dr. Márquez and his team of paleoanthropologists, comparative anatomists, and an otolaryngologist have contributed to the understanding of two of the most controversial topics in paleoanthropology -- were Neanderthals a different species from modern humans and which aspects of their cranial morphology evolved as adaptations to cold stress.

"The strategy was to have a comprehensive examination of the nasal region of diverse modern human population groups and then compare the data with the fossil evidence. We used traditional morphometrics, geometric morphometric methodology based on 3D coordinate data, and CT imaging," Dr. Márquez explained.

Anthony S. Pagano, PhD, anatomy instructor at NYU Langone Medical Center, a co-author, traveled to many European museums carrying a microscribe digitizer, the instrument used to collect 3D coordinate data from the fossils studied in this work, as spatial information may be missed using traditional morphometric methods. "We interpreted our findings using the different strengths of the team members," Dr. Márquez said, "so that we can have a 'feel' for where these Neanderthals may lie along the modern human spectrum."

Co-author William Lawson, MD, DDS, vice-chair and the Eugen Grabscheid research professor of otolaryngology and director of the Paleorhinology Laboratory of the Icahn School of Medicine at Mount Sinai, notes that the external nasal aperture of the Neanderthals approximates some modern human populations but that their midfacial prognathism (protrusion of the midface) is startlingly different. That difference is one of a number of Neanderthal nasal traits suggesting an evolutionary development distinct from that of modern humans. Dr. Lawson's conclusion is predicated upon nearly four decades of clinical practice, in which he has seen over 7,000 patients representing a rich diversity of human nasal anatomy.

Distinguished Professor Jeffrey T. Laitman, PhD, also of the Icahn School of Medicine and director of the Center for Anatomy and Functional Morphology, and Eric Delson, PhD, director of the New York Consortium in Evolutionary Primatology or NYCEP, are also co-authors and are seasoned paleoanthropologists, each approaching their fifth decade of studying Neanderthals. Dr. Delson has published on various aspects of human evolution since the early 1970's.

Dr. Laitman states that this article is a significant contribution to the question of Neanderthal cold adaptation in the nasal region, especially in its identification of a different mosaic of features than those of cold-adapted modern humans. Dr. Laitman's body of work has shown that there are clear differences in the vocal tract proportions of these fossil humans when compared to modern humans. This current contribution has now identified potentially species-level differences in nasal structure and function.

Dr. Laitman said, "The strength of this new research lies in its taking the totality of the Neanderthal nasal complex into account, rather than looking at a single feature. By looking at the complete morphological pattern, we can conclude that Neanderthals are our close relatives, but they are not us."

Ian Tattersall, PhD, emeritus curator of the Division of Anthropology at the American Museum of Natural History, an expert on Neanderthal anatomy and functional morphology who did not participate in this study, stated, "Márquez and colleagues have carried out a most provocative and intriguing investigation of a very significant complex in the Neanderthal skull that has all too frequently been overlooked." Dr. Tattersall hopes that "with luck, this research will stimulate future research demonstrating once and for all that Homo neanderthalensis deserves a distinctive identity of its own."

Source: SUNY Downstate Medical Center

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

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

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

Ancient Europeans intolerant to lactose for 5,000 years after they adopted agriculture

Milk

By analysing DNA extracted from the petrous bones of skulls of ancient Europeans, scientists have identified that these peoples remained intolerant to lactose (natural sugar in the milk of mammals) for 5,000 years after they adopted agricultural practices and 4,000 years after the onset of cheese-making among Central European Neolithic farmers.

The findings published online in the scientific journal Nature Communications (21 Oct) also suggest that major technological transitions in Central Europe between the Neolithic, Bronze Age and Iron Age were also associated with major changes in the genetics of these populations.

For the study, the international team of scientists examined nuclear ancient DNA extracted from thirteen individuals from burials from archaeological sites located in the Great Hungarian Plain, an area known to have been at the crossroads of major cultural transformations that shaped European prehistory. The skeletons sampled date from 5,700 BC (Early Neolithic) to 800 BC (Iron Age).

It took several years of experimentation with different bones of varying density and DNA preservation for the scientists to discover that the inner ear region of the petrous bone in the skull, which is the hardest bone and well protected from damage, is ideal for ancient DNA analysis in humans and any other mammals.

According to Professor Ron Pinhasi from the UCD Earth Institute and UCD School of Archaeology, University College Dublin, the joint senior author on the paper, "the high percentage DNA yield from the petrous bones exceeded those from other bones by up to 183-fold. This gave us anywhere between 12% and almost 90% human DNA in our samples compared to somewhere between 0% and 20% obtained from teeth, fingers and rib bones."

For the first time, these exceptionally high percentage DNA yields from ancient remains made it possible for scientists to systematically analyse a series of skeletons from the same region and check for known genetic markers including lactose intolerance.

"Our findings show progression towards lighter skin pigmentation as hunter and gatherers and non-local farmers intermarried, but surprisingly no presence of increased lactose persistence or tolerance to lactose" adds Professor Pinhasi.

"This means that these ancient Europeans would have had domesticated animals like cows, goats and sheep, but they would not yet have genetically developed a tolerance for drinking large quantities of milk from mammals," he says.

According to Professor Dan Bradley from the Smurfit Institute of Genetics, Trinity College Dublin, co-senior author on the paper, "our results also imply that the great changes in prehistoric technology including the adoption of farming, followed by the first use of the hard metals, bronze and then iron, were each associated with the substantial influx of new people. We can no longer believe these fundamental innovations were simply absorbed by existing populations in a sort of cultural osmosis."

Source: University College Dublin

Visualizing DNA double-strand break process for the first time

The enzyme I-DmoI (purple) is specifically associated to the double strand of DNA (yellow and green). Credit: CNIO

Scientists from the Spanish National Cancer Research Centre (CNIO), led by Guillermo Montoya, have developed a method for producing biological crystals that has allowed scientists to observe --for the first time-- DNA double chain breaks. They have also developed a computer simulation that makes this process, which lasts in the order of millionths of a second, visible to the human eye. The study is published today by the journal Nature Structural & Molecular Biology.

"We knew that enzymes, or proteins, endonucleases, are responsible for these double strand breaks, but we didn't know exactly how it worked until now," said Montoya. "In our study, we describe in detail the dynamics of this basic biological reaction mediated by the enzyme I-Dmol. Our observations can be extrapolated to many other families of endonucleases that behave identically."

DNA breaks occur in several natural processes that are vital for life: mutagenesis, synthesis, recombination and repair. In the molecular biology field, they can also be generated synthetically. Once the exact mechanism that produces these breaks has been uncovered, this knowledge can be used in multiple biotechnological applications: from the correction of mutations to treat rare and genetic diseases, to the development of genetically modified organisms.

Slow-motion reaction

Enzymes are highly specialised dynamic systems. Their nicking function could be compared, said Montoya, to a specially designed fabric-cutting machine that "it would only make a cut when a piece of clothing with a specific combination of colours passed under the blade."

In this case, researchers concentrated on observing the conformational changes that occurred in the I-Dmol active site; the area that contains the amino acids that act as a blade and produces DNA breaks.

By altering the temperature and pH balance, the CNIO team has managed to delay a chemical reaction that typically occurs in microseconds by up to ten days. Under those conditions, they have created a slow-motion film of the whole process.

"By introducing a magnesium cation we were able to trigger the enzyme reaction and subsequently to produce biological crystals and freeze them at -200ºC," said Montoya. "In that way, we were able to collect up to 185 crystal structures that represent all of the conformational changes taking place at each step of the reaction."

Finally, using computational analysis, the researchers illustrated the seven intermediate stages of the DNA chain separation process. "It is very exciting, because the elucidation of this mechanism will give us the information we need to redesign these enzymes and provide precise molecular scissors, which are essential tools for modifying the genome," he concluded.

Source: Centro Nacional de Investigaciones Oncologicas (CNIO)

Controlling genes with your thoughts

Thoughts control a near-infrared LED, which starts the production of a molecule in a reaction chamber. Credit: Martin Fussenegger et al., Copyright ETH Zurich

It sounds like something from the scene in Star Wars where Master Yoda instructs the young Luke Skywalker to use the force to release his stricken X-Wing from the swamp: Marc Folcher and other researchers from the group led by Martin Fussenegger, Professor of Biotechnology and Bioengineering at the Department of Biosystems (D-BSSE) in Basel, have developed a novel gene regulation method that enables thought-specific brainwaves to control the conversion of genes into proteins -- called gene expression in technical terms.

"For the first time, we have been able to tap into human brainwaves, transfer them wirelessly to a gene network and regulate the expression of a gene depending on the type of thought. Being able to control gene expression via the power of thought is a dream that we've been chasing for over a decade," says Fussenegger.

A source of inspiration for the new thought-controlled gene regulation system was the game Mindflex, where the player wears a special headset with a sensor on the forehead that records brainwaves. The registered electroencephalogram (EEG) is then transferred into the playing environment. The EEG controls a fan that enables a small ball to be thought-guided through an obstacle course.

Wireless Transmission to Implant

The system, which the Basel-based bioengineers recently presented in the journal Nature Communications, also makes use of an EEG headset. The recorded brainwaves are analysed and wirelessly transmitted via Bluetooth to a controller, which in turn controls a field generator that generates an electromagnetic field; this supplies an implant with an induction current.

A light then literally goes on in the implant: an integrated LED lamp that emits light in the near-infrared range turns on and illuminates a culture chamber containing genetically modified cells. When the near-infrared light illuminates the cells, they start to produce the desired protein.

Thoughts Control Protein Quantity

The implant was initially tested in cell cultures and mice, and controlled by the thoughts of various test subjects. The researchers used SEAP for the tests, an easy-to-detect human model protein which diffuses from the culture chamber of the implant into the mouse's bloodstream.

To regulate the quantity of released protein, the test subjects were categorised according to three states of mind: bio-feedback, meditation and concentration. Test subjects who played Minecraft on the computer, i.e. who were concentrating, induced average SEAP values in the bloodstream of the mice. When completely relaxed (meditation), the researchers recorded very high SEAP values in the test animals. For bio-feedback, the test subjects observed the LED light of the implant in the body of the mouse and were able to consciously switch the LED light on or off via the visual feedback. This in turn was reflected by the varying amounts of SEAP in the bloodstream of the mice.

New Light-sensitive Gene Construct

"Controlling genes in this way is completely new and is unique in its simplicity," explains Fussenegger. The light-sensitive optogenetic module that reacts to near-infrared light is a particular advancement. The light shines on a modified light-sensitive protein within the gene-modified cells and triggers an artificial signal cascade, resulting in the production of SEAP. Near-infrared light was used because it is generally not harmful to human cells, can penetrate deep into the tissue and enables the function of the implant to be visually tracked.

The system functions efficiently and effectively in the human-cell culture and human-mouse system. Fussenegger hopes that a thought-controlled implant could one day help to combat neurological diseases, such as chronic headaches, back pain and epilepsy, by detecting specific brainwaves at an early stage and triggering and controlling the creation of certain agents in the implant at exactly the right time.

Source: ETH Zurich

Making lab-grown tissues stronger

Connective tissues like cartilage are made of cross-linked bundles of collagen fibers. UC Davis biomedical engineers have discovered that reducing oxygen or adding an enzyme called LOX can make these bundles stronger. The technique can be used to strengthen both natural cartilage kept in the lab for transplant, and artificial cartilage grown in culture. Credit: Eleftherios Makris and Kyriacos Athanasiou, UC Davis

Lab-grown tissues could one day provide new treatments for injuries and damage to the joints, including articular cartilage, tendons and ligaments.

Cartilage, for example, is a hard material that caps the ends of bones and allows joints to work smoothly. UC Davis biomedical engineers, exploring ways to toughen up engineered cartilage and keep natural tissues strong outside the body, report new developments this week in the journal Proceedings of the National Academy of Sciences.

"The problem with engineered tissue is that the mechanical properties are far from those of native tissue," said Eleftherios Makris, a postdoctoral researcher at the UC Davis Department of Biomedical Engineering and first author on the paper. Makris is working under the supervision of Professor Kyriacos A. Athanasiou, a distinguished professor of biomedical engineering and orthopedic surgery, and chair of the Department of Biomedical Engineering.

While engineered cartilage has yet to be tested or approved for use in humans, a current method for treating serious joint problems is with transplants of native cartilage. But it is well known that this method is not sufficient as a long-term clinical solution, Makris said.
The major component of cartilage is a protein called collagen, which also provides strength and flexibility to the majority of our tissues, including ligaments, tendons, skin and bones. Collagen is produced by the cells and made up of long fibers that can be cross-linked together.

Engineering new cartilage

Researchers in the Athanasiou group have been maintaining native cartilage in the lab and culturing cartilage cells, or chondrocytes, to produce engineered cartilage.

"In engineered tissues the cells produce initially an immature matrix, and the maturation process makes it tougher," Makris said.

Knee joints are normally low in oxygen, so the researchers looked at the effect of depriving native or engineered cartilage of oxygen. In both cases, low oxygen led to more cross-linking and stronger material. They also found that an enzyme called lysyl oxidase, which is triggered by low oxygen levels, promoted cross-linking and made the material stronger.

"The ramifications of the work presented in the PNAS paper are tremendous with respect to tissue grafts used in surgery, as well as new tissues fabricated using the principles of tissue engineering," Athanasiou said. Grafts such as cadaveric cartilage, tendons or ligaments -- notorious for losing their mechanical characteristics in storage -- can now be treated with the processes developed at UC Davis to make them stronger and fully functional, he said.
Athanasiou also envisions that many tissue engineering methods will now be altered to take advantage of this strengthening technique.

Source: University of California - Davis

PHS gene prevents wheat from sprouting: Fewer crop losses anticipated

Preharvest sprouting can cause significant losses in wheat crops, particularly in white wheat crops. Credit: Kansas State University Photo Services

A new study about the common problem of preharvest sprouting, or PHS, in wheat is nipping the crop-killing issue in the bud.

Researchers at Kansas State University and the U.S. Department of Agriculture-Agricultural Research Service, or USDA-ARS, found and cloned a gene in wheat named PHS that prevents the plant from preharvest sprouting. Preharvest sprouting happens when significant rain causes the wheat grain to germinate before harvest and results in significant crop losses.

"This is great news because preharvest sprouting is a very difficult trait for wheat breeders to handle through breeding alone," said Bikram Gill, university distinguished professor of plant pathology and director of the Wheat Genetics Resource Center. "With this study, they will have a gene marker to expedite the breeding of wheat that will not have this problem."

Gill conducted the study with Guihau Bai, a researcher with the Hard Winter Wheat Genetics Research Unit of the USDA-ARS, adjunct professor of agronomy at Kansas State University and the study's lead author. Also involved were Harold Trick, professor of plant pathology; Shubing Liu, research associate in agronomy; Sunish Sehgal, senior scientist in plant pathology; Jiarui Li, research assistant professor; and Meng Lin, doctoral student in agronomy, all from Kansas State University; and Jianming Yu, Iowa State University.

Their study, "Cloning and Characterization of a Critical Regulator for Pre-Harvest Sprouting in Wheat," appears in a recent issue of the scientific journal Genetics.

The finding will to be most beneficial to white wheat production, which loses $1 billion annually to preharvest sprouting, according to Gill.

He said consumers prefer white wheat to the predominant red wheat because white wheat lacks the more bitter flavor associated with red wheat. Millers also prefer white wheat to red because it produces more flour when ground. The problem is that white wheat is very susceptible to preharvest sprouting.

"There has been demand for white wheat in Kansas for more than 30 years," Gill said. "The very first year white wheat was grown in the state, though, there was rain in June and then there was preharvest sprouting and a significant loss. The white wheat industry has not recovered since and has been hesitant to try again. I think that this gene is a big step toward establishing a white wheat industry in Kansas."

Gill said identifying the PHS gene creates a greater assurance before planting a crop that it will be resistant to preharvest sprouting once it grows a year later. Wheat breeders can now bring a small tissue sample of a wheat plant into a lab and test whether it has the preharvest sprouting resistance gene rather than finding out once the crop grows.

Much of the work to isolate the PHS gene came from Gill and his colleagues' efforts to fully sequence the genome -- think genetic blueprint -- of common wheat. Wheat is the only major food plant not to have its genome sequenced. The genome of wheat is nearly three times the size of the human genome.

Researchers were able to study sequenced segments of the common wheat genome and look for a naturally occurring resistance gene. Gill said without the sequenced segments, finding the PHS gene would have been impossible.

Source: Kansas State University