Rob Knight, a scientist at CU-Boulder’s BioFrontiers Institute, will be working with a research team led by Dr. Jeffrey I. Gordon at Washington University School of Medicine in St. Louis. The goal is to discover novel dietary and microbial therapeutics targeting infants and children living in countries with rampant malnutrition.

“Our hope is that by understanding the differences in individual gut microbial communities in both healthy and malnourished individuals, together with influences in diet including compounds and microbes transferred in breast milk, we will be able to better understand and develop new treatments for malnutrition,” said Knight, also an associate professor in CU-Boulder’s chemistry and biochemistry department. “Such treatments could include prebiotics, probiotics, antibiotics or nutrition which might help reverse severe malnutrition in different individuals.”

Severe malnutrition has long been thought to stem simply from a lack of adequate food, but now scientists understand the condition is far more complex and may involve a breakdown in the way gut microbial communities process various diet components.

“A complex relationship exists between diet, gut microbial communities and the immune system in severely malnourished children,” says Gordon, the Dr. Robert J. Glaser Distinguished University Professor and director of Washington University’s Center for Genome Sciences and Systems Biology. “We now have a way to tease apart these influences. Recreating the human gut ecosystem in mice gives us a way to control these variables. The lead compounds derived from these well-controlled, pre-clinical studies can be considered for future clinical trials in malnourished infants and children.”

Research led by Knight has shown that people carry “personalized” bacteria on many individual areas of the body, including the intestine. His CU-Boulder lab will host the Gates Foundation study database integrating various types of information, including whole-genome sequencing and microbial community analyses.

As part of the project, Knight and his CU-Boulder team will develop methods for high-throughput sequencing and analysis of bacterial genomes from “personalized culture collections,” in which hundreds of strains of bacteria will be isolated and characterized from the intestines of individual people. The personalized culture collections will be used to colonize lab mice in different combinations to test which strains are most important, said Knight.

The community of intestinal microbes and its vast collection of genes, known as the gut microbiome, are assembled right from birth and influenced by babies’ early environments and the first foods they consume, such as breast milk. As part of the Gates Foundation’s Breast Milk, Gut Microbiome and Immunity, or BMMI, Project, the scientists will evaluate the relationship among first foods, the developing community of microbes in the intestine and the developing immune system.

The new research builds on ongoing clinical studies in Africa, South Asia and South America of malnourished and healthy infants and children and their mothers, which also are funded by the Gates Foundation.

As part of the new project, scientists will evaluate the function of gut microbial communities in malnourished and healthy infants and children living in multiple countries where malnutrition is prevalent. They also will characterize the nutritional content and immune activity present in breast milk samples obtained from the children’s mothers during periods of exclusive and supplemental breastfeeding.

In parallel, the scientists will use a preclinical discovery pipeline recently developed in Gordon’s laboratory to identify next-generation probiotics and nutrient supplements or combinations of the two — known as synbiotics — that may promote healthy growth in infants and children.

The investigators will transplant communities of intestinal microbes obtained from stool samples from both malnourished and healthy children into germ-free mice raised under sterile conditions. These mice will essentially harbor collections of human gut microbes that mimic those found in the children, and they will be fed the same diets as the children.

Then using the mice, the scientists can carefully evaluate how various nutritional interventions influence the workings of the gut microbiomes obtained from the children. They will be able to determine which microbes respond, how they respond and how they affect the overall function of the gut microbiomes. The researchers also will evaluate certain aspects of childhood development.

CU-Boulder graduate students will be involved in the BMMI Project, including doctoral students in the Interdisciplinary Quantitative Biology, or IQ Biology, program recently launched by the BioFrontiers Institute directed by Nobel laureate Tom Cech. The students are involved in semester-long rotations that immerse them in mathematical biology, computational biology, biophysics and bio-imaging as they work toward doctoral degrees.

“IQ Biology students are being trained with the exact mixture of mathematical, computational and biological techniques essential for progress on complex, challenging projects like the Gates BMMI Project,” said Knight. Dan Knights, the first graduate of the IQ Biology program, helped to lay the groundwork for the CU portion of the BMMI effort with novel research on applying machine learning to studies of the human microbiome, said Knight, who also is a Howard Hughes Medical Institute Early Career Scientist.

Other scientists involved in the Gates BMMI project include Per Ashorn of the University of Tampere School of Medicine in Finland; Kathryn Dewey of the University of California, Davis; Michael Gottlieb of the Foundation for the National Institutes of Health; Kenneth Maleta of the University of Malawi College of Medicine; David Mills of the University of California, Davis; Jeremy Nicholson of Imperial College, London; and Linda Saif of Ohio State University.” -from University of Colorado – Boulder

PacBio RS and 454 DNA sequencing at engencore.sc.edu

The scientists tested their approach by creating a generator that produces enough current to operate a small liquid-crystal display. It works by tapping a finger on a postage stamp-sized electrode coated with specially engineered viruses. The viruses convert the force of the tap into an electric charge.

Their generator is the first to produce electricity by harnessing the piezoelectric properties of a biological material. Piezoelectricity is the accumulation of a charge in a solid in response to mechanical stress.

The milestone could lead to tiny devices that harvest electrical energy from the vibrations of everyday tasks such as shutting a door or climbing stairs.

It also points to a simpler way to make microelectronic devices. That’s because the viruses arrange themselves into an orderly film that enables the generator to work. Self-assembly is a much sought after goal in the finicky world of nanotechnology.

The scientists describe their work in a May 13 advance online publication of the journal Nature Nanotechnology.

“More research is needed, but our work is a promising first step toward the development of personal power generators, actuators for use in nano-devices, and other devices based on viral electronics,” says Seung-Wuk Lee, a faculty scientist in Berkeley Lab’s Physical Biosciences Division and a UC Berkeley associate professor of bioengineering.

He conducted the research with a team that includes Ramamoorthy Ramesh, a scientist in Berkeley Lab’s Materials Sciences Division and a professor of materials sciences, engineering, and physics at UC Berkeley; and Byung Yang Lee of Berkeley Lab’s Physical Biosciences Division.

The piezoelectric effect was discovered in 1880 and has since been found in crystals, ceramics, bone, proteins, and DNA. It’s also been put to use. Electric cigarette lighters and scanning probe microscopes couldn’t work without it, to name a few applications.

But the materials used to make piezoelectric devices are toxic and very difficult to work with, which limits the widespread use of the technology.

Lee and colleagues wondered if a virus studied in labs worldwide offered a better way. The M13 bacteriophage only attacks bacteria and is benign to people. Being a virus, it replicates itself by the millions within hours, so there’s always a steady supply. It’s easy to genetically engineer. And large numbers of the rod-shaped viruses naturally orient themselves into well-ordered films, much the way that chopsticks align themselves in a box.

These are the traits that scientists look for in a nano building block. But the Berkeley Lab researchers first had to determine if the M13 virus is piezoelectric. Lee turned to Ramesh, an expert in studying the electrical properties of thin films at the nanoscale. They applied an electrical field to a film of M13 viruses and watched what happened using a special microscope. Helical proteins that coat the viruses twisted and turned in response—a sure sign of the piezoelectric effect at work.

Next, the scientists increased the virus’s piezoelectric strength. They used genetic engineering to add four negatively charged amino acid residues to one end of the helical proteins that coat the virus. These residues increase the charge difference between the proteins’ positive and negative ends, which boosts the voltage of the virus.

The scientists further enhanced the system by stacking films composed of single layers of the virus on top of each other. They found that a stack about 20 layers thick exhibited the strongest piezoelectric effect.

The only thing remaining to do was a demonstration test, so the scientists fabricated a virus-based piezoelectric energy generator. They created the conditions for genetically engineered viruses to spontaneously organize into a multilayered film that measures about one square centimeter. This film was then sandwiched between two gold-plated electrodes, which were connected by wires to a liquid-crystal display.

When pressure is applied to the generator, it produces up to six nanoamperes of current and 400 millivolts of potential. That’s enough current to flash the number “1” on the display, and about a quarter the voltage of a triple A battery.

“We’re now working on ways to improve on this proof-of-principle demonstration,” says Lee. “Because the tools of biotechnology enable large-scale production of genetically modified viruses, piezoelectric materials based on viruses could offer a simple route to novel microelectronics in the future.”

Berkeley Lab’s Laboratory Directed Research and Development fund and the National Science Foundation supported this work.” – article from the Lawrence Berkeley National Laboratory

PacBio RS and 454 DNA sequencing at engencore.sc.edu

“My programs would have been substantially weaker had I not had the challenge of my fellow competitors. The mix of competition and open discussion really produced amazing results,” James said. “But, perhaps the most exciting thought is where this work will go next. Several entrants shared ideas with each other and I suspect that we can produce an even better solution by combining the best parts from each of our entries.”

This competition was created by the Pistoia Alliance – a precompetitive alliance of research groups, pharmaceutical companies and scientific societies seeking to improve worldwide genetic research by solving the problems that all researchers in the field face. The aim was to drive the creation of solutions to one of the most pressing problems in genetic research today: the storage and sharing of the vast volumes of genetic data that researchers need to find disease-causing gene variants.

“The latest high-speed sequencing machines are opening up the genetic study of disease and biological pathways in incredible depth because they allow hundreds or thousands of genes or genomes to be read and compared,” said Tony Cox, Head of Operation Production Software and Informatics at the Sanger Institute. “However, this major leap forward is creating mountains of data that need to be stored and distributed around the world. Current storage solutions and internet transfer methods are struggling to cope, which is why James’ work is so vital. It literally reduces the size of the problem.”

The competition itself was a demonstration of a novel way to drive forward innovation through its open and interactive set up. The Alliance encouraged continual innovation by posting an interactive leaderboard that showed, day by day, which entrant had produced the most efficient approach. In addition, the collaborative nature of the competition saw entrants sharing their problems and ideas on a variety of discussion forums.

“Seeing my entry being beaten by others spurred me on to improve my code again and again,” James said. “Forums, such as encode.ru, had numerous and surprisingly open discussions on ideas, particularly from respected programmer Matt Mahoney, who went as far as to post code snippets. The views on that thread gave me ideas for improving my own program, so the final outcome was better than if I had worked purely in isolation.”

Out of the more than 100 entries, James’ solutions were judged to be the best overall for compressing the avalanche of information produced by the latest high-speed sequencing machines into forms that can be easily stored and transferred across the internet. The judging panel evaluated the approaches on their ability to:

• Squeeze the data into the smallest possible space (have the highest compression ratio)
• Achieve this in the shortest possible time (fastest compression and time)
• Allow others to unpack the compressed data as quickly as possible for use (fastest decompression time)
• Use the least amount of computing memory to compress and decompress the data

James’ algorithms scored highly in the top three criteria and ensured that alignment data was preserved to allow genetic sequences to be put together quickly and efficiently.

James will be giving half of his prize money to the British Heart Foundation.

“We are delighted for James that his work has been recognised in this way,” said Emma Millican, Head of DNA Pipelines and responsible for sequencing at the Sanger Institute. “We hope that his efforts will benefit the Institute and genetic researchers around the world for years to come.” ” – article from Wellcome Trust Sanger Institute

PacBio RS and 454 DNA sequencing at engencore.sc.edu

“The cells that slough off from a cancerous tumor into the bloodstream are a genetically diverse bunch, Stanford University School of Medicine researchers have found. Some have genes turned on that give them the potential to lodge themselves in new places, helping a cancer spread between organs. Others have completely different patterns of gene expression and might be more benign, or less likely to survive in a new tissue. Some cells may even express genes that could predict their response to a specific therapy. Even within one patient, the tumor cells that make it into circulating blood vary drastically.

The finding underscores how multiple types of treatment may be required to cure what appears outwardly as a single type of cancer, the researchers say. And it hints that the current cell-line models of human cancers, which showed patterns that differed from the tumor cells shed from human patients, need to be improved upon.

The new study, published May 7 in PLoS ONE, is the first to look at so-called circulating tumor cells one by one, rather than taking the average of many of the cells. And it’s the first to show the extent of the genetic differences between such cells.

“Within a single blood draw from a single patient, we’re seeing heterogeneous populations of circulating tumor cells,” said senior study author Stefanie Jeffrey, MD, professor of surgery and chief of surgical oncology research.

For over a century, scientists have known that circulating tumor cells, or CTCs, are shed from tumors and move through the bloodstreams of cancer patients. And over the past five years, there’s been a growing sense among many cancer researchers that these cells — accessible by a quick blood draw — could be the key to tracking tumors non-invasively. But separating CTCs from blood cells is hard; there can be as few as one or two CTCs in every milliliter of a person’s blood, mixed among billions of other blood cells.

To make their latest discovery, Jeffrey, along with an interdisciplinary team of engineers, quantitative biologists, genome scientists and clinicians, relied on a technology they developed in 2008. Called the MagSweeper, it’s a device that lets them isolate live CTCs with very high purity from patient blood samples, based on the presence of a particular protein — EpCAM — that’s on the surface of cancer cells but not healthy blood cells.

With the goal of studying CTCs from breast cancer patients, the team first tested whether they could accurately detect the expression levels of 95 different genes in single cells from seven different cell-line models of breast cancer — a proof of principle since they already knew the genetics of these tumors. These included four cell lines generally used by breast cancer researchers and pharmaceutical scientists worldwide and three cell lines specially generated from patients’ primary tumors.

“Most researchers look at just a few genes or proteins at a time in CTCs, usually by adding fluorescent antibodies to their samples consisting of many cells,” said Jeffrey. “We wanted to measure the expression of 95 genes at once and didn’t want to pool our cells together, so that we could detect differences between individual tumor cells.”

So once Jeffrey and her collaborators isolated CTCs using the MagSweeper, they turned to a different kind of technology: real-time PCR microfluidic chips, invented by a Stanford collaborator, Stephen Quake, PhD, professor of bioengineering. They purified genetic material from each CTC and used the high-throughput technology to measure the levels of all 95 genes at once. The results on the cell-line-derived cells were a success; the genes in the CTCs reflected the known properties of the cell-line models. So the team moved on to testing the 95 genes in CTCs from 50 human breast cancer patients — 30 with cancer that had spread to other organs, 20 with only primary breast tumors.

“In the patients, we ended up with a subset of 31 genes that were most dominantly expressed,” said Jeffrey. “And by looking at levels of those genes, we could see at least two distinct groups of circulating tumors cells.” Depending on which genes they used to divide the CTCs into groups, there were as many as five groups, she said, each with different combinations of genes turned on and off. And if they’d chosen genes other than the 95 they’d picked, they likely would have seen different patterns of grouping. However, because the same individual CTCs tended to group together in multiple different analyses, these cells likely represent different types of spreading cancer cells.

The diversity, Jeffrey said, means that tumors may contain multiple types of cancer cells that may get into the bloodstream, and a single biopsy from a patient’s tumor doesn’t necessarily reflect all the molecular changes that are driving a cancer forward and helping it spread. Moreover, different cells may require different therapies. One breast cancer patient studied, for example, had some CTCs positive for the marker HER2 and others lacked the marker. When the patient was treated with a drug designed to target HER2-positive cancers, the CTCs lacking the molecule remained in her bloodstream.

When the team went on to compare the diverse genetic profiles of the breast cancer patients’ CTCs with the cells they’d studied from the cell lines, they were in for another surprise: None of the human CTCs had the same gene patterns as any of the cell-line models.

“These models are what people are using for drug discovery and initial drug testing,” said Jeffrey, “but our finding suggests that perhaps they’re not that helpful as models of spreading cancers.” While the human cell-line cells did show diversity between each of the seven cell lines, they didn’t fall into any of the same genetic profiles as the CTCs from human blood samples.

These results don’t have immediate impacts for cancer patients in the clinic because more work is needed to discover whether different types of CTCs respond to different therapies and whether that will be clinically useful for guiding treatment decisions. But the finding is a step forward in understanding the basic science behind the bits of tumors that circulate in the blood. It’s the first time that scientists have used high-throughput gene analysis to study individual CTCs, and opens the door for future experiments that delve even more into the cell diversity. The Stanford team is now working on different methods of using CTCs for drug testing as well as studying the relationship between CTC genetic profiles and cancer treatment outcomes. They’ve also expanded their work to include primary lung and pancreatic cancers as well as breast tumors.

The first authors of the study are former postdoctoral scholars Ashley Powell, PhD, and AmirAli Talasaz, PhD, and research scientist Haiyu Zhang, PhD. The other corresponding authors are Ronald Davis, PhD, professor of biochemistry, and Shanaz Dairkee, PhD, visiting professor. Other Stanford co-authors include Quake; Marc Coram, PhD, assistant professor of health research and policy; former research scientist Glenn Deng, PhD; Fabian Pease, PhD, emeritus professor of electrical engineering; Michael Mindrinos, PhD, senior research scientist; Melinda Telli, MD, assistant professor of medicine; Ranjana Advani, MD, professor of medicine; Robert Carlson, MD, professor of medicine; Joseph Mollick, MD, PhD, clinical instructor of medicine; Shruti Sheth, MD, clinical instructor of medicine; Allison Kurian, MD, assistant professor of medicine; James Ford, MD, associate professor of medicine and of genetics; and Frank Stockdale, MD, PhD, professor emeritus of medicine. The team also collaborated with researchers at Rutgers University, the Cancer Institute of New Jersey and the Simons Center for Systems Biology in New Jersey.

The MagSweeper is licensed by Stanford to the sequencing company Illumina. Jeffrey, Powell, Talasaz, Mindrinos, Pease and Davis receive royalties for their contributions to the technology; Jeffrey donated her royalties to a nonprofit.

The work was supported by the National Institutes of Health, the California Breast Cancer Research Grants Program Office of the University of California, the John and Marva Warnock Cancer Research Fund and donations from Andrew and Debra Rachleff and Vladimir and Natalie Ermakoff.

Information about Stanford’s departments of Surgery and of Medicine, which also supported the work, is available at http://surgery.stanford.edu/ and http://medicine.stanford.edu.” – article from Stanford University School of Medicine

PacBio RS and 454 DNA sequencing at engencore.sc.edu

“We found that patients were able to tolerate the chemotherapy better and without negative side effects after transplantation of the gene-modified stem cells than patients in previous studies who received the same type of chemotherapy without a transplant of gene-modified stem cells,” said Hans-Peter Kiem, M.D., senior and corresponding author of the study published in the May 9 issue of Science Translational Medicine.

Kiem, a member of the Clinical Research Division at the Hutchinson Center, said that a major barrier to effective use of chemotherapy to treat cancers like glioblastoma has been the toxicity of chemotherapy drugs to other organs, primarily bone marrow. This results in decreased blood cell counts, increased susceptibility to infections and other side effects. Discontinuing or delaying treatment or reducing the chemotherapy dose is generally required, but that often results in less effective treatment.

In the current study, Kiem and colleagues focused on patients with glioblastoma, an invariably fatal cancer. Many of these patients have a gene called MGMT (O6-methylguanine-DNA-methyltransferase) that is turned on because the promoter for this gene is unmethylated. MGMT is a DNA repair enzyme that counteracts the toxic effect of some chemotherapy agents like temozolomide. Patients with such an unmethylated promoter status have a particularly poor prognosis.

A drug called benzylguanine can block the MGMT gene and make tumor cells sensitive to chemotherapy again, but when given with chemotherapy, the toxic effects of this combination are too much for bone marrow cells, which results in marrow suppression.

By giving bone marrow stem cells P140K, which is a modified version of MGMT, those cells are protected from the toxic effects of benzylguanine and chemotherapy, while the tumor cells are still sensitive to chemotherapy. “P140K can repair the damage caused by chemotherapy and is impervious to the effects of benzylguanine,” Kiem said.

“This therapy is analogous to firing at both tumor cells and bone marrow cells, but giving the bone marrow cells protective shields while the tumor cells are unshielded,” said Jennifer Adair, Ph.D., who shares first authorship of the study with Brian Beard, Ph.D., both members of Kiem’s lab.

The three patients in this study survived an average of 22 months after receiving transplants of their own circulating blood stem cells. One, an Alaskan man, remains alive 34 months after treatment. Median survival for patients with this type of high-risk glioblastoma without a transplant is just over a year.

“Glioblastoma remains one of the most devastating cancers with a median survival of only 12 to 15 months for patients with unmethylated MGMT,” said Maciej Mrugala, M.D., the lead neuro oncologist for this study.

As many as 50 percent to 60 percent of glioblastoma patients harbor such chemotherapy-resistant tumors, which makes gene-modified stem cell transplant therapy applicable to a large number of these patients. In addition, there are also other brain tumors such as neuroblastoma or other solid tumors with MGMT-mediated chemo resistance that might benefit from this approach.

The researchers also found that chemotherapy increased the number of gene-modified blood and bone marrow cells in these patients. Kiem said this finding will have implications for other stem cell gene therapy applications where defective bone marrow stem cells can be corrected by gene therapy but their numbers need to be increased to produce a therapeutic benefit, or for patients with HIV/AIDS to increase the number of HIV-resistant stem and T cells.

The clinical trial is open and is recruiting more patients. For more information go to: http://clinicaltrials.gov/ct2/show/NCT00669669.

Researchers from Washington State University, the University of Washington, Dana Farber/Children’s Hospital Cancer Center and the Harvard Medical School contributed to the study. The research was funded by grants from the National Institutes of Health and the Heath Foundation.

Note to media: Please contact Dean Forbes to arrange an interview or to obtain a copy of the Science Translational Medicine paper, “Extended Survival of Glioblastoma Patients After Chemoprotective HSC Gene Therapy.” ” – from Fred Hutchinson Cancer Research Center

PacBio RS and 454 DNA sequencing at engencore.sc.edu

The carnivorous plant Nepenthes bicalcarata grows in the nutrient-poor peatswamp forests of Borneo but bears insect-trapping pitchers with poor retentive and digestive abilities. However it has a symbiotic relationship with the ant species Camponotus schmitzi, shown in the current study to act as the “gizzard” of its carnivorous host by recycling nitrogen from insects it preys upon in the trap.Vincent Bazile and researchers from University Montpellier 2, CNRS, INRA (UMR AMAP in France) and from the Universities of Brunei and Royal Roads (Canada), also found that plants inhabited by the ants produced more, larger leaves, and that the ants provided a striking increase in the nitrogen available to the plants. On the other hand, plants without ants were determined to be nutrient stressed.”

“Citation: Bazile V, Moran JA, Le Mogue´dec G, Marshall DJ, Gaume L (2012) A Carnivorous Plant Fed by Its Ant Symbiont: A Unique Multi-Faceted Nutritional Mutualism. PLoS ONE 7(5): e36179. doi:10.1371/journal.pone.0036179

Disclaimer: This press release refers to upcoming articles in PLoS ONE. The releases have been provided by the article authors and/or journal staff. Any opinions expressed in these are the personal views of the contributors, and do not necessarily represent the views or policies of PLoS. PLoS expressly disclaims any and all warranties and liability in connection with the information found in the release and article and your use of such information.

Financial Disclosure: The work was supported by Centre National de la Recherche Scientifique (CNRS) through the PEPS/INEE-2010 grant devoted to the ”CarniBiop” project supervised by LG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interest: The authors have declared that no competing interests exist.

PLEASE LINK TO THE SCIENTIFIC ARTICLE IN ONLINE VERSIONS OF YOUR REPORT (URL goes live after the embargo ends): http://dx.plos.org/10.1371/journal.pone.0036179

Disclaimer: This press release refers to upcoming articles in PLoS ONE. The releases have been provided by the article authors and/or journal staff. Any opinions expressed in these are the personal views of the contributors, and do not necessarily represent the views or policies of PLoS. PLoS expressly disclaims any and all warranties and liability in connection with the information found in the release and article and your use of such information.” – from PLoS ONE

PacBio RS and 454 DNA sequencing at engencore.sc.edu

“Imagine tracking a deer through a forest by clipping a radio transmitter to its ear and monitoring the deer’s location remotely. Now imagine that transmitter is the size of a house, and you understand the problem researchers may encounter when they try to use nanoparticles to track proteins in live cells.

Understanding how a protein moves around a cell helps researchers understand the protein’s function and the cellular mechanisms for making and processing proteins. This information also helps researchers study disease, which at a cellular level may mean that a protein is malfunctioning, stops being made, or is sent to the wrong part of the cell. But nanoparticle probes that are too big can disrupt a protein’s normal activities.

Now a team of scientists led by Bruce Cohen of Lawrence Berkeley National Laboratory’s Molecular Foundry, a U.S. Department of Energy (DOE) nanoscience center, has figured out how to grow light-emitting nanocrystals small enough to not disrupt cell activity but bright enough to be imaged one at a time. Cohen is corresponding author of a paper in the February 16, 2012 issue of ACS Nano describing this work titled, “Controlled Synthesis and Single-Particle Imaging of Bright, Sub-10 nm Lanthanide-Doped Upconverting Nanocrystals.” Coauthors are Alexis Ostrowski, Emory Chan, Daniel Gargas, Elan Katz, Gang Han, James Schuck, and Delia Milliron.

“Scientists have been trying for years to study protein behavior by tagging them with light-emitting probes,” said Cohen. “But the problem is finding the right kind of probe. Our approach is to make upconverting-nanoparticle probes small enough that they shouldn’t disrupt protein behavior.”

Making a better probe

In the past, researchers used fluorescent molecules or quantum dots as probes. Using state-of-the art optics and microscopes, researchers can resolve light coming from single molecules attached to proteins, which tells them where the protein is in a cell. The probe molecules in these experiments tend to degrade or “photobleach” rapidly, limiting researchers to just a few seconds of continuous imaging or a series of images taken seconds apart. The alternative probes, quantum dots, suffer less from photobleaching but instead they flicker on and off, similarly limiting their usefulness as probes.

The Foundry team wanted to avoid both blinking and bleaching, so they turned to nanocrystals of sodium yttrium fluoride (NaYF4) with trace amounts of lanthanide elements ytterbium and erbium, which, they discovered, emit bright, steady light ideal for bioimaging. More importantly, these nanocrystals “upconvert” light, absorbing low energy photons and re-emitting them at higher energies.

“Typically when something fluorescent absorbs light it then emits light at a slightly lower energy. Upconversion goes the other way, actually increasing the energy of the light being emitted,” Cohen said. “In our case we’re exciting with fairly low energy light, near infrared (beyond red in the visible spectrum), and then the nanocrystals emit light in the visible range, like green or red, which is actually higher in energy.”

The advantage of upconverting nanocrystals is that cells don’t upconvert light themselves. Normally when scientists image a cell using molecular probes, they use visible-wavelength light to both excite and image. Unfortunately, lots of things in the cell also reemit absorbed light at these wavelengths, which creates background noise in the image and forces scientists to use more probes and brighter light sources. With upconverting nanocrystals, researchers can gently stimulate with infrared light and look at visible light from single probes that stand clearly against a dark background.

“The other advantage to upconverting nanocrystals is that near-infrared light is a lot less damaging to cells than, say, visible or ultraviolet light,” said Cohen. “That means when we do these very long imaging experiments using intense powers of light to see single molecules, we’re using wavelengths that are pretty benign to cells.”

A combinatorial solution

Nanocrystals of NaYF₄ can form in two different geometries called alpha and beta. The beta-phase nanocrystals are more efficient at upconversion and thus better for bioimaging, but they’re also harder to grow. In order to nail down the growth parameters to get reproducible beta-NaYF4 nanocrystals, the team used the Molecular Foundry’s WANDA robot – the Workstation for Automated Nanomaterial Discovery and Analysis – developed by Berkeley Lab’s Emory Chan and Delia Milliron.

“None of this would be possible without being able to do what we at the Foundry call combinatorial nanoscience. Basically that means running lots and lots of different reactions in WANDA to learn how to control the size or the color of the nanoparticles,” said Cohen. “We’ve run thousands of different reactions to learn how to grow these things.”

Smaller nanoparticles means less light, so the team had to find the sweet spot:

How small could they make them and still be able to image individual nanocrystals in a live system? “That’s one of the nice things about having this control is that we can not only make them down to, say, 5 nanometers, but we also know the conditions for making them bigger if we need to make them brighter,” Cohen said.

To help understand the geometry of their nanocrystals, coauthor James Schuck asked a summer intern to make a computer model of the crystal structure. Andrew Mueller, a high school student from Vistamar School in Los Angeles, went well beyond a simple crystal structure though.

“I started out just putting shapes together based on what was in the literature for the crystal,” said Mueller. “Then I wanted to show how it looked in a nanocrystal so I moved the camera around in the structure and panned out to show how atoms come together in a nanocrystal.” Mueller later added animation of two photons being absorbed and upconverted to a single emitted photon.

“The video is a good answer to the question, what is a nanocrystal?” said Cohen. “You can see that this is really just a few hundred or maybe a couple of thousand atoms in a nanocrystal, arranged in small, regular patterns.”

Next, the team wants to put the upconverting nanocrystals into action and actually map single proteins moving through a cell. “One of the things we’d like to study is how two neurons come together, how two brain cells come together to form a synapse — the spaces between neurons responsible for all brain activity,” Cohen said. “It’s known that there are certain pairs of proteins that come together from two neurons and they find each other and form a synapse but the question is, how many of those do you need? How many pairs of proteins? Is just one interaction enough to cause a synapse to form, do they reverse themselves, and so forth? Now that we know how to make exactly the nanoparticles we want, the next step is to test them in a cell.” -article from Lawrence Berkeley National Laboratory

PacBio RS and 454 DNA sequencing at engencore.sc.edu

Looking closely at one large deletion — a so-called copy-number alteration or CNA on 8p, the short arm of chromosome 8 — in mouse models of human liver cancer, the team validated the presence of a number of genes which normally serve to suppress the formation of tumors, and demonstrated that they act together, and not singly, to suppress tumors. The 8p deletion is commonly seen in human liver cancer and in other epithelial cancers including those of the breast, colon and lung.

The research team, which was co-led by now-adjunct CSHL Professor Scott W. Lowe of Memorial Sloan-Kettering Cancer Center and CSHL Professor Michael Wigler, publishes their results online today in Proceedings of the National Academy of Sciences.

PacBio RS and 454 DNA sequencing at engencore.sc.edu

“This is one of the most beautiful examples to date of the mapping of a simple genetic trait in humans,” said David Reich, PhD, a professor of genetics at Harvard University, who was not involved in the study.

The study identifying the gene responsible for blond hair in the Solomon Islands, a nation in the South Pacific, represents a rare case of simple genetics determining human appearance, and shows the importance of including understudied populations in gene mapping studies, said co-senior author Carlos D. Bustamante, PhD, professor of genetics at Stanford. The findings were published May 4 in Science.

“Since most studies in human genetics only include participants of European descent, we may be getting a very biased view of which genes and mutations influence the traits we investigate. Here, we sought to test whether one of the most striking human traits, blond hair, had the same — or different — genetic underpinning in different human populations,” Bustamante said.

Globally, blond hair is rare, occurring with substantial frequency only in northern Europe and in Oceania, which includes the Solomon Islands and its neighbors. “Its frequency is between 5 and 10 percent across the Solomon Islands, which is about the same as where I’m from,” said co-first author Eimear Kenny, PhD, who was born in Ireland.

Many assumed the blond hair of Melanesia was the result of gene flow — a trait passed on by European explorers, traders and others who visited in the preceding centuries. The islanders themselves give several possible explanations for its presence, said co-senior author Sean Myles, PhD, a former Stanford postdoctoral scholar who is now an assistant professor at the Nova Scotia Agricultural College. They generally chalked it up to sun exposure, or a diet rich in fish, he said.

After researchers at UCSF generated genetic data from the samples, Kenny, a postdoctoral scholar in Bustamante’s lab, began the analysis in September 2010, the week she started at Stanford. “Within a week we had our initial result. It was such a striking signal pointing to a single gene — a result you could hang your hat on. That rarely happens in science,” she said. “It was one of the best experiences of my career.”

In terms of genetic studies, the analysis was straightforward, said Kenny. But gathering the data, accomplished in 2009 by Myles and co-first author Nicholas Timpson, PhD, was more difficult. Much of the Solomon Islands is undeveloped, without roads, electricity or telephones. It’s also one of the most linguistically diverse nations in the world, with dozens of languages spoken.

It was a return trip for Myles who had been there in 2004 as a graduate student with Max Planck Institute molecular anthropologist Mark Stoneking, PhD, (also a co-author of the study) to investigate whether the language variations correlated with genetic variations. While there, Myles was fascinated by the ubiquity of blond hair, which was especially common among children.

“They have this very dark skin and bright blond hair. It was mind-blowing,” said Myles. “As a geneticist on the beach watching the kids playing, you count up the frequency of kids with blond hair, and say, ‘Wow, it’s 5 to 10 percent.’”

A grant from the Wenner-Gren Foundation for Anthropological Research gave Myles, who at that time was doing a stint as a postdoctoral researcher at Cornell University, his chance to study the genetics of the Solomon Islanders’ hair color. Myles worked with Bustamante, who was also at Cornell, to design the study. Then back in the islands, Myles and Timpson went village to village explaining what they wanted to do and asking for permission to gather data, Myles speaking in Solomon Islands pidgin, the most widely understood language.

When the local chief gave the OK, the researchers recruited participants and assessed hair and skin color using a light reflectance meter, took blood pressure readings and measured heights and weights. They asked the villagers to spit into small tubes to provide saliva to be used for DNA extraction. In the span of a month they collected more than 1,000 samples.

While the islands fit many people’s notion of a tropical paradise, they lack amenities Westerners take for granted. For instance, simply finding a level spot for the scale to weigh study participants was a challenge.

Then in 2010 Bustamante joined Stanford’s faculty and, with funding from the Department of Genetics, the team looked for genes underlying this striking phenotype. Soon after, Kenny joined the lab and started the analysis, selecting 43 blond- and 42 dark-haired Solomon Islanders from the opposite 10 percent extremes of the hair pigmentation range. She used these in a genome-wide association study, a method to reveal differences in the frequency of genetic variants between two groups, that usually requires thousands of samples.

Because the vast majority of human physical characteristics analyzed to date have many genetic and environmental factors, Kenny expected an inconclusive result that would require much further study. Instead, she immediately saw a single strong signal on chromosome 9, which accounted for 50 percent of the variance in the Solomon Islanders’ hair color.

The team went on to identify the gene responsible, TYRP1, which encodes tyrosinase-related protein 1, an enzyme previously recognized as influencing pigmentation in mice and humans. Further research revealed that the particular variant responsible for blond hair in the Solomon Islands is absent in the genomes of Europeans.

“So the human characteristic of blond hair arose independently in equatorial Oceania. That’s quite unexpected and fascinating,” Kenny said.

The finding underscores the importance of genetic studies on isolated populations, said Bustamante. “If we’re going to be designing the next generation of medical treatments using genetic information and we don’t have a really broad spectrum of populations included, you could disproportionately benefit some populations and harm others.”

Bustamante is seeking funding to analyze the rest of the data gathered. “For instance, the genetics of skin pigmentation might be different there too — not the same as in Europe or Africa or India. We just don’t know.”

Additional co-authors were Stanford postdoctoral scholars Martin Sikora, PhD, and Andres Moreno Estrada, PhD; Stanford research assistant Muh-Ching Yee, PhD; and researchers from UCSF including professor of bioengineering & therapeutic sciences and medicine, Esteban González Burchard, MD. Nicholas Timpson is currently a lecturer at the University of Bristol, U.K.

In addition to the Wenner-Gren Foundation, the research was funded by the MRC Centre for Causal Analyses in Translational Epidemiology, the National Human Genome Research Institute, the National Heart, Lung, and Blood Institute and the Max Planck Society.

Information about Stanford’s Department of Genetics, which also supported the work, is available at http://genetics.stanford.edu/.” – article from Stanford University School of Medicine

PacBio RS and 454 DNA sequencing at engencore.sc.edu