LOOKING TO CURE ANTIBIOTIC RESISTANCE

FROM:  NATIONAL SCIENCE FOUNDATION
Researcher studies how to prevent antibiotic resistance
Solution could be in bacterial protein called UmuD

The widespread and indiscriminate use of antibiotics has prompted many bacteria to mutate, an adaptation that often renders the drugs useless. The increasing threat of resistance worries infectious disease experts who fear that the era of public health successes brought by the introduction of antibiotics in the 1940s is seriously eroding, or soon even may be at an end.

But what if science could improve existing antibiotics in such a way as to not only destroy bacteria, but prevent them from mutating?

At least one research team, in seeking to better understand bacterial mutation, may provide scientific answers that ultimately could lead to thwarting the organisms' ability to mutate, thus blunting the increasing threat of antibiotic resistance.

"The idea would be a one-two punch," says Penny Beuning, an associate professor of chemistry and chemical biology at Northeastern University's college of science. "We need a good therapeutic target that will both kill the bacteria and prevent mutagenesis."

To be sure, the approach almost certainly is years away. Still, the National Science Foundation (NSF)-funded scientist thinks it may be possible. She and her colleagues are studying an important bacterial protein known as UmuD that regulates mutagenesis and may provide important clues about how to stop the process that eventually results in antimicrobial resistance.

Using the bacterium E. coli as a model, she has learned that UmuD interacts with the machinery that replicates DNA, and, when altered, may provide the switch that triggers mutation. UmuD exists in two forms, a full length version when first expressed, and later, if DNA is damaged, a much shorter form. It is this shorter version that allows bacteria to mutate.

Once there is DNA damage, "there is an SOS response, and the levels of some specific proteins go up," she says. "There is a massive stress response, and UmuD responds by cutting its arms off."

In cells where only the full-length version of the protein is present, the bacteria cannot mutate. "But when it forms its shorter self, the cells are mutable," she says.

The fact that UmuD is not present outside bacteria makes it a viable antibiotic target.

"The hope would be to find something that targets UmuD together with an existing antibiotic to prevent bacteria from mutating and developing a resistance to that particular drug," she says. "Among the things we have been looking at: how does UmuD work, and what controls the cleavage of the arms?"

Beuning is conducting her research under an NSF Faculty Early Career Development (CAREER) grant awarded in 2009 under the American Recovery and Reinvestment Act. The award supports junior faculty who exemplify the role of teacher-scholars through outstanding research, excellent education and the integration of education and research within the context of the mission of their organization. NSF is funding her work with $994,655 over five years.

Beuning specifically is looking at the cleavage process of UmuD using gel electrophoresis, which separates proteins according to size.

"UmuD is a small protein--139 amino acids--which loses 24 amino acids from the arm. So it goes from 139 to 115," she says. "We can observe this difference with electrophoresis, allowing us to determine how different conditions or other proteins might affect UmuD cleavage."

The team is studying different UmuD protein interactions in the lab, using biochemistry to see when and how different proteins bind to one another. Essentially "we light up the proteins and measure how they change when other proteins bind, using a method called FRET, which stands for fluorescence resonance energy transfer," she says.

"This measures energy transfer between two proteins using light emission," she adds. "The proteins have to be close to each other for energy transfer to occur, so it's a way of detecting whether two things bind to each other. People often call the technique a molecular ruler, because it can be used to measure precise distances, but we use it simply to measure proximity."

Using FRET, they discovered that UmuD prevents specific protein interactions in the replication process. That is, it stops or slows down replication by keeping two proteins that need to interact for replication from binding to each other. "Protein-protein interactions are generally hard to target with drugs, but the approach has some potential," she says.

They also use another technique that measures how floppy or flexible proteins are by putting them in heavy water and measuring how much heavier the protein gets as it trades its regular hydrogens with heavy hydrogens from the heavy water. "The floppier parts swap out the hydrogens faster than the less floppy parts," she says.

As part of the grant's education component, she has up to ten undergraduates--as well as local high school students and teachers--working in her research lab. Several students have worked in her lab as part of Northeastern's signature co-op program, in which students work full-time for six months in positions related to their career goals.

Also, she teaches an upper level chemical biology class to undergraduates, and created a lab research project for the students that takes place during half of the semester that actually involves them directly in her mutagenesis research.

"A lot of these students had not yet conducted any research, so they were really motivated by the idea of doing something that someone would use as part of a bigger project," she says. "Particularly at Northeastern, where co-op is such a large part of the culture, it is fun to take advantage of the laboratory as the ultimate in experiential education.”

-- Marlene Cimons, National Science Foundation
Investigators
Penny Beuning
Related Institutions/Organizations

GENE EDITITING AND REGULATION TO IMPROVE IMMUNE SYSTEM

FROM:  NATIONAL SCIENCE FOUNDATION
Rewriting genetic information to prevent disease

Breakthrough Prize winner harnesses CRISPR to improve immune system
For the last few years, scientists have been studying an ancient but only recently understood mechanism of bacterial immunity that has the potential to provide immeasurable benefits to plant and animal health.

The phenomenon known as CRISPR (for Clustered Regularly Interspaced Short Palindromic Repeats) is a natural immune system found in many bacteria with the ability to identify and destroy the genomes of invading viruses and plasmids.

Researchers are trying to harness this system for gene editing and regulation, a process that could transform "the genome of plants or animals in ways that will improve their health, or introduce genetic changes that will resist disease of climate change," says Jennifer Doudna, a Howard Hughes Medical Institute investigator and professor of biochemistry, biophysics and structural biology at the University of California, Berkeley. "The explosion of research using this technique has been amazing."

Doudna, collaborating with Emmanuelle Charpentier of Sweden's Helmholtz Center for Infection Research and Umeå University, identified how the system works and engineered it in new ways that broadened its scope. The two researchers, who described their work in a 2012 paper in the journal Science, developed a technique that enables the rewriting of genetic information and the correction of mutations that otherwise can cause disease, and also can knock out the cell's ability to make harmful proteins, she says.

"Many labs have shown in principle that this can be used to correct such mutations as those that occur in cystic fibrosis, or sickle cell disease," she says. "They are showing it in cell lines and lab animals. We're still some period of time away from using this in humans, but the pace in the field has been truly remarkable, and really exciting to see."

Many bacteria have this CRISPR-based immune system capable of identifying and destroying hostile invaders. Doudna and Charpentier showed that, in doing so, CRISPR produces the protein Cas9, a DNA-cutting enzyme guided by RNA, which relies on two short RNA guide sequences to find foreign DNA, then cleaves, or cuts, the target sequences, thereby muting the genes of the invaders.

Cas9 has evolved to provide protection against viruses that could infect the bacterium, and uses pieces of RNA derived from CRISPRS to direct its activity. The system is specific and efficient enough to stave off viral infections in bacteria.

Doudna and her colleagues programmed the process so that it can be directed by a single short RNA molecule; researchers who use it to edit genomes can customize the RNA so that it sends Cas9 to cleave, like "scissors," at their chosen location in the genome.

"When we figured out how it worked, we realized we could alter the design of RNA and program Cas9 to recognize any DNA sequence," she says. "One can therefore target Cas9 to any region of a genome simply by providing a short guide RNA that can pair with the region of interest. Once targeted, different versions of Cas9 can be used to activate or inhibit genes, as well as make target cuts within the genome. Depending on the experimental design, research can use these latter cuts to either disrupt genes or replace them with newly engineered versions."

Recently Douda and Charpentier and four other scientists received the Breakthrough Prize in life sciences, which honors transformative advances toward understanding living systems and extending human life. The prizes recognize pioneering work in physics, genetics, cosmology, neurology and mathematics, and carry a $3 million award for each researcher. The Breakthrough committee specifically cited Doudna and Charpentier for their advances in understanding the CRISPR mechanism.

Doudna has been the recipient of several National Science Foundation (NSF) grants to support her research in recent years totaling more than $1.5 million. In 2000, she received NSF's prestigious $500,000 Alan T. Waterman Award, which recognizes an outstanding young researcher in any field of science or engineering supported by NSF.

She also was a founder of the Innovative Genomics Initiative, established in 2014 at the Li Ka Shing Center for Genomic Engineering at UC Berkeley. Its goal is to promote and support genome editing research and technology in both academic and commercial research communities.

"We have a team of scientists working with various collaborative partners," she says. "We want to ensure that the technology gets into as many hands as possible, and explore ways to make it even better. We are trying to bring about fundamental change in biological and biomedical research by enabling scientists to read and write in genomes with equal ease. It's a bold new effort that embraces a new era in genomic engineering."

-- Marlene Cimons, National Science Foundation
Investigators
Jennifer Doudna
Related Institutions/Organizations
University of California-Berkeley

THE GENETICS OF ALZHEIMER'S

FROM:  THE NATIONAL SCIENCE FOUNDATION 
Uncovering Alzheimer's complex genetic networks
Researchers from the Mayo Clinic use NSF-supported Blue Waters supercomputer to understand gene expression in the brain
February 3, 2015

The release of the film, "Still Alice," in September 2014 shone a much-needed light on Alzheimer's disease, a debilitating neurological disease that affects a growing number of Americans each year.

More than 5.2 million people in the U.S. are currently living with Alzheimer's. One out of nine Americans over 65 has Alzheimer's, and one out of three over 85 has the disease. For those over 65, it is the fifth leading cause of death.

There are several drugs on the market that can provide relief from Alzheimer's symptoms, but none stop the development of disease, in part because the root causes of Alzheimer's are still unclear.

"We re interested in studying the genetics of Alzheimer's disease," said Mariet Allen, a post-doctoral fellow at the Mayo Clinic in Florida. "Can we identify genetic risk factors and improve our understanding of the biological pathways and cellular mechanisms that can play a role in the disease process?"

Allen is part of a team of researchers from the Mayo Clinic who are using Blue Waters, one of the most powerful supercomputers in the world, to decode the complicated language of genetic pathways in the brain. In doing so, they hope to provide insights into what genes and proteins are malfunctioning in the brain, causing amyloid beta plaques, tau protein tangles and brain atrophy due to neuronal cell loss--the telltale signs of the disease--and how these genes can be detected and addressed.

In the case of late onset Alzheimer's disease (LOAD), it is estimated that as much as 80 percent of risk is due to genetic factors. In recent years, researchers discovered 20 common genetic loci, in addition to the well-known APOE gene, that are found to increase or decrease risk for the disease. (Loci are specific locations of a gene, DNA sequence, or position on a chromosome.) These loci do not necessarily have a causal connection to the disease, but they provide useful information about high-risk patients.

Despite all that doctors have learned in recent years about the genetic basis of Alzheimer's, according to Allen, a substantial knowledge gap still exists. It has been estimated that likely less than 40 percent of genetic risk for LOAD can be explained by known loci. Furthermore, it is not always clear which are the affected genes at these known loci.

In other words, scientists have a long way to go to get a full picture of which genes are involved in processes related to the disease and how they interact.

The Mayo team and their colleagues had been very successful in the past in finding genetic risk factors using a method that matched individual differences in the DNA code--single-nucleotide polymorphisms or SNPs, to phenotypes--the outward appearances of the disease. In particular, the Mayo team focused on identifying SNPs that influence expression of genes in the brain. However, they now hypothesize that the single SNP method may be too simplistic to find all genetic factors, and is likely not an accurate reflection of the complex biological interactions that take place in an organism.

For that reason, the Mayo researchers have recently turned their attention to investigating the brain using genetic interaction (epistasis) studies. Such studies allow researchers to understand the effects of pairs of gene changes on a given phenotype and can uncover additional genetic variants that influence gene expression and disease.

The process involves the analysis of billions of DNA base pairs (the familiar C, G, A and T) to find statistically significant correlations. Importantly, the search is not to discover simple one-to-one connections, since these have largely been found, but to study the interaction effects of pairs of DNA sequence variations.

Solving a problem of this size and complexity requires a huge amount of computational processing time, so the researchers turned to the Blue Waters supercomputer at the National Center for Supercomputing Applications (NCSA).

Supported by the National Science Foundation and the University of Illinois at Urbana-Champaign, Blue Waters allows scientists and engineers across the country to tackle a wide range of challenging problems using massive computing and data processing power. From predicting the behavior of complex biological systems to simulating the evolution of the cosmos, Blue Waters assists researchers whose computing problems are at a scale or complexity that cannot be reasonably approached using any other method.

Allen and her colleagues used Blue Waters to rapidly advance their Alzheimer's epistasis study through NCSA's Private Sector Program, which lets teams outside of academia access the system.

Instead of requiring as much as a year or more of processing on a single workstation or university cluster, the research team was able to do each analysis on Blue Waters in less than two days.

The researchers conducted three sets of analysis to investigate brain gene expression levels in a group of individuals without Alzheimer's, a group of individuals with Alzheimer's and then a combined analysis of both groups together. To date, these analyses have been completed for the almost 14,000 genes expressed in the majority of the brain samples studied.

Through their work with collaborators at NCSA and the University of Illinois at Urbana-Champaign (including Victor Jongeneel and Liudmila Mainzer), the Mayo team overcame many of the challenges that a project of this scope presented.

"The analysis of epistatic effects in large studies, such as ours, requires powerful computational resources and would not be possible without the unique computing capabilities of Blue Waters," wrote project lead Nilufer Ertekin-Taner from the Mayo Clinic.

"The Mayo Clinic project is emblematic of the type of problem that is beginning to emerge in computational medicine," said Irene Qualters, division director of Advanced Computing Infrastructure at NSF. "Through engagement with the Blue Waters project, researchers at Mayo have demonstrated the potential of new analytic approaches in addressing the challenges of a daunting medical frontier."

The team reported on their progress at the Blue Waters Symposium in May 2014. Allen and her colleagues are currently processing and filtering the results so they can be analyzed.

"Recent studies by our collaborators and others have shown that both the risk for late onset Alzheimer's disease and gene expression are likely influenced by epistasis. However little is known about the effect of genetic interactions on brain gene expression specifically and how this might influence risk for neurological diseases such as LOAD," said Allen. "The goal of our study is to address this knowledge gap; something we have been uniquely positioned to do using our existing data and the resources available on Blue Waters."

-- Aaron Dubrow, NSF

STAMPEDE SUPERCOMPUTER AND DRIVING DNA THROUGH THE NANOPORE

FROM:  NATIONAL SCIENCE FOUNDATION 

Blueprint for the affordable genome

Stampede supercomputer powers innovations in DNA sequencing technologies
Aleksei Aksimentiev, a professor of physics at the University of Illinois-Urbana Champaign, used the National Science Foundation-supported Stampede supercomputer to explore a cutting-edge method of DNA sequencing. The method uses an electric field to drive a strand of DNA through a small hole, or "nanopore," either in silicon or a biological membrane.

By controlling this process precisely and measuring the change in ionic current as the DNA strands move through the pore of the membrane, the sequencer can read each base pair in order.

"Stampede is by far the best computer system my group has used over the past 10 years," Aksimentiev said. "Being able to routinely obtain 40-80 nanoseconds of molecular dynamic simulations in 24 hours, regardless of the systems' size, has been essential for us to make progress with rapidly evolving projects."

Aksimentiev and his group showed that localized heating can be used to stretch DNA, which significantly increases the accuracy of nanopore DNA sequencing. In addition, he and his team used an all-atom molecular dynamics method to accurately describe DNA origami objects, making it possible to engineer materials for future applications in biosensing, drug delivery and nano-electronics. These results were published in ACS Nano and the Proceedings of the National Academy of Sciences.

-- Aaron Dubrow, NSF
Investigators
Aleksei Aksimentiev
Related Institutions/Organizations
University of Texas at Austin
University of Illinois at Urbana-Champaign

LOS ALAMOS SCIENTIST TO DISCUSS IF BEHAVIOR IS PRODUCT OF DNA OR ENVIRONMENT

Photo Caption: Cells in the human body contains strands of DNA nearly 10 feet long that look like this and are packed into cellular sacks less than a millionth of an inch in diameter.  Credit:  LANL

FROM:  LOS ALAMOS NATIONAL LABORATORY

Lab’s Frontiers in Science lectures focus on epigenetics

Is behavior hardwired by DNA or a product of environment?

LOS ALAMOS, N.M., August 7, 2013—Los Alamos National Laboratory scientist Karissa Sanbonmatsu, will discuss epigenetics in a series of Frontiers in Science lectures beginning Tuesday, Aug. 13, at the New Mexico Museum of Natural History and Science in Albuquerque.

The 7 p.m. talk, titled “Nature, Nurture or Neither: The New Science of Epigenetics,” focuses on the age-old question of “nature versus nurture,” and also looks at how social interactions and environmental factors play a role in programming your DNA.

“Over the past decade, epigenetics research has and continues to unveil a whole new kind of biological circuitry,” Sanbonmatsu said. “The act of a mother nurturing or not nurturing her baby programs DNA; so literally, nurture directly affects nature in a way that nature and nurture are fused together.”

The new science of epigenetics studies how DNA is reprogrammed at the molecular level. DNA is often considered the blueprint of life, however, environmental factors and social interactions during formative years can affect genes for more than three generations. This heritable switching is called “epigenetics” and has been associated with diet, exercise, mate preference, depression, autism, eating disorders and response to abuse.

Sanbonmatsu, of Los Alamos’ Theoretical Biology and Biophysics Group, will discuss the new science of epigenetics and how it is related to a wide range of biological phenomena. Her research involves how DNA can be reprogrammed throughout life and how the missing link could be RNA molecules.

“We have been lucky enough to land on the cutting edge of this field, in the area of long non-coding RNAs, which has absolutely exploded in the last three years,” Sanbonmatsu said. “With many suggesting that the number of long non-coding RNAs may rival the number of proteins, the landscape of molecular biology may look entirely different ten years from now."

These Frontiers in Science lectures all begin at 7 p.m., at the following locations:

Tuesday, Aug. 13, New Mexico Museum of Natural History and Science, 1801 Mountain Road NW, Albuquerque

Thursday, Aug. 15, Nick L. Salazar Center for the Arts, Northern New Mexico College, 921 Paseo de Oñate, Española

Tuesday, Aug. 20, Duane W. Smith Auditorium, Los Alamos High School, Los Alamos

Thursday, Aug. 22, James A. Little Theater, New Mexico School for the Deaf, 1060 Cerrillos Road, Santa Fe.

Sponsored by the Fellows of Los Alamos National Laboratory, the Frontiers in Science lecture series is intended to increase local public awareness of the diversity of science and engineering research at the Laboratory.

THE MUTANT PIGEON GENE

Victoria Crown Pigeon.  Credit:  Wikimedia Commons.

FROM: NATIONAL SCIENCE FOUNDATION
Mutant Gene Responsible for Pigeons' Head Crests
Decoded genome reveals secrets of pigeon traits and origins
January 31, 2013

Scientists have decoded the genetic blueprint of the rock pigeon, unlocking secrets about pigeons' Middle East origins, feral pigeons' kinship with escaped racing birds and how mutations give pigeons traits like feather head crests.

"Birds are a huge part of life on Earth, but we know surprisingly little about their genetics," says Michael Shapiro, one of the study's two principal authors and a biologist at the University of Utah.

In the new study, "we've shown a way forward to find the genetic basis of traits--the molecular mechanisms controlling animal diversity in pigeons," he says. "Using this approach, we expect to be able to do this for other traits in pigeons, and it can be applied to other birds and many other animals as well."

The findings appear in a paper published this week in the online journal Science Express.

Shapiro conducted the research with Jun Wang of China's BGI-Shenzhen (formerly Beijing Genomics Institute) and other scientists from BGI, the University of Utah, Denmark's University of Copenhagen and the University of Texas M.D. Anderson Cancer Center in Houston.

"The research identified the genes contributing to variation in the avian head crest, using the domesticated pigeons that so fascinated and inspired Charles Darwin in developing his theory of natural selection," says George Gilchrist, program director in the National Science Foundation's (NSF) Division of Environmental Biology, which funded the research. "This finding illustrates the power of comparative genomics."

Pigeons were domesticated some 5,000 years ago in the Mediterranean region. Key results of this study include sequencing of the genome of the rock pigeon Columba livia, which is among the most common bird species.

There are some 350 breeds of rock pigeons--all with different sizes, shapes, colors, color patterns, beaks, bone structure, vocalizations and arrangements of feathers on the feet and head--including head crests in shapes known as hoods, manes, shells and peaks.

The pigeon's genetic blueprint is among the few bird genomes sequenced so far, along with those of the chicken, turkey, zebra finch and a common parakeet known as a budgerigar or budgie. "This will give us new insights into bird evolution," Shapiro says.

Using software developed by paper co-author Mark Yandell, a geneticist at the University of Utah, the scientists revealed that a single mutation in a gene named EphB2 causes head and neck feathers to grow upward instead of downward, creating head crests.

"This same gene in humans has been implicated as a contributor to Alzheimer's disease, as well as prostate cancer and possibly other cancers," Shapiro says, noting that more than 80 of the 350 pigeon breeds have head crests, which play a role in attracting mates in many bird species.

The researchers compared the pigeon genome to those of chickens, turkeys and zebra finches. "Despite 100 million years of evolution since these bird species diverged, their genomes are very similar," Shapiro says.

A genome for the birds, a gene for head crests

The biologists assembled 1.1 billion base pairs of DNA in the rock pigeon genome; the researchers believe there are about 1.3 billion total, compared with 3 billion base pairs in the human genome. The rock pigeon's 17,300 genes compare in number with the approximately 21,000 genes in humans.

The researchers first constructed a "reference genome"--a full genetic blueprint--from a male of the pigeon breed named the Danish tumbler.

Shapiro says the study is the first to pinpoint a gene mutation responsible for a pigeon trait, in this case, head crests.

"A head crest is a series of feathers on the back of the head and neck," Shapiro says. "Some are small and pointed. Others look like a shell behind the head; some people think they look like mullets. They can be as extreme as an Elizabethan collar."

The researchers found strong evidence that the EphB2 (Ephrin receptor B2) gene acts as an on-off switch to create a head crest when mutant, and no head crest when normal.

They also showed that the mutation and related changes in nearby DNA are shared by all crested pigeons, so the trait evolved just once and was spread to numerous pigeon breeds by breeders.

Full or partial genetic sequences were analyzed for 69 crested birds from 22 breeds, and 95 uncrested birds from 57 breeds. The biologists found a perfect association between the mutant gene and the presence of head crests.

They also showed that while the head crest trait becomes apparent in juvenile pigeons, the mutant gene affects pigeon embryos by reversing the direction of feather buds--from which feathers later grow--at a molecular level.

Other genetic factors determine what kind of head crest each pigeon develops: shell, peak, mane or hood.

Tracking the origins of pigeons

A 2012 study by Shapiro provided limited evidence of pigeons' origins in the Middle East and some breeds' origins in India and indicated kinship between common feral or free-living, city pigeons and escaped racing pigeons.

In the new study, "we included some different breeds that we didn't include in the last analysis," Shapiro says. "Some of those breeds only left the Middle East in the last few decades. They've probably been there for hundreds if not thousands of years. If we find that other breeds are closely related to them, then we can infer those other breeds probably also came from the Middle East."

The scientists found that the owl breeds--pigeon breeds with very short beaks that are popular with breeders--likely came from the Middle East. They're closely related to breeds from Syria, Lebanon and Egypt.

The research also uncovered a shared genetic heritage between breeds from Iran and breeds likely from India, consistent with historical records of trade routes between those regions. People were not only sharing goods along those routes, but probably also interbreeding their pigeons.

As for the idea that free-living pigeons descended from escaped racing pigeons, Shapiro says his 2012 study was based on "relatively few genetic markers scattered throughout the genome. We now have stronger evidence based on 1.5 million markers, confirming the previous result with much better data."

The scientists analyzed partial genomes of two feral pigeons: one from a U.S. Interstate-15 overpass in Utah's Salt Lake Valley, the other from Lake Anna in Virginia.

"Despite being separated by 1,000 miles, they are genetically very similar to each other and to the racing homer breed," Shapiro says.

"Darwin used this striking example to communicate how natural selection works," he says. "Now we can get to the DNA-level changes that are responsible for some of the diversity that intrigued Darwin 150 years ago."

The study's co-authors from the University of Utah include Yandell, Eric Domyan, Zev Kronenberg, Michael Campbell, Anna Vickery and Sydney Stringham; Chad Huff is a co-author from the University of Texas.

The study was also funded by the Burroughs Wellcome Fund, the University of Utah Research Foundation, the National Institutes of Health and the Danish National Research Foundation.

-NSF-