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September 1, 2016

Research Notes

Testing 3-D printed partial dentures

The ADEAGies Foundation has awarded John Ference the 2016 Gies Award for Outstanding Innovation by an Academic Dental Institution.

The award includes a grant that will enable Ference, faculty member in the Department of Prosthodontics, to continue his research. Ference’s recent study compared the mechanical and physical properties of a 3-D-printed removable partial denture framework with traditionally manufactured frameworks.

This work is being performed in conjunction with Markus Chmielus, faculty member in the Swanson School of Engineering’s Department of Mechanical Engineering and Materials Science.

The research uses an Inconel 625 metal alloy. During the initial phase, biocompatible frameworks were made to test the mechanical and microstructural properties of the alloy according to the ASTM standards and compared to the values of the metal alloy. Ference and Chmielus recorded descriptive statistics (mean, median and standard deviations) and tested removable partial denture frameworks for specific gravity, tensile strength, elongation, compressive strength, flexural strength, impact and coefficient of linear thermal expansion.

The physical and mechanical properties of the completed partial denture were very similar to the stated manufacturer’s data sheet. The next phase of testing involves printing additional biocompatible metal alloy partial dentures and subjecting them to ASTM standards.


Website aids evolutionary biology researchers

DNA travels its own peculiar path over time to ensure we come out on the right side of the “adapt or die” equation. At the genetic level, we share our bodies with creatures great and small whose mutations in response to evolutionary pressures can bring new clarity to the cellular functions, proteins and other regulatory molecules linked with human disease.

This bioinformatics approach — known as evolutionary rate covariation (ERC) — developed by Nathan Clark, faculty member in computational and systems biology in the School of Medicine, and colleagues correlates branch-specific evolutionary rates of a chosen pair of genes. The fundamental concept behind ERC is that functionally related genes should respond similarly to evolutionary pressures and, therefore, be easier to track and characterize.

By parsing such genetic “guilt by association” in Drosophila and other species, Clark and his colleagues previously identified new protein networks that influence reproduction, amino acid transporters that affect signal transmission in the glutamatergic neuromuscular junction and key evolutionary signatures among a variety of disease genes that could help to untangle heretofore unknown relationships between clinically distinct disease processes.

Over the past year, the Clark team has built a publicly accessible internet portal to assist researchers with ERC-based investigations.

“Imagine you’re a kidney disease researcher interested in a pathway that leads to sodium permeability on the cell surface,” says Clark. “Take a gene that you know influences that pathway, put it into the portal, and it will serve you up genes that are highly correlated with your gene. We’ve shown now through a number of published studies that this works.”

The portal structure, described in Bioinformatics last year, contains data on three taxonomic groups — 33 mammal species, 12 Drosophila species and 18 kinds of yeasts. Website analyses are designed to provide statistically supported results that are easy to interpret, Clark explains, adding that as of the 12 months ending in July 2016, more than 3,000 unique users representing “all inhabited continents” had used the portal.

Clark’s lab most recently used a modified ERC method to correlate evolutionary patterns of genes with environmental influences in marine mammals compared to their terrestrial compatriots. These analyses traced molecular-level contributions to morphological and physiological adaptations necessary to support species transition from landlubber to air-breathing sea dweller.

Published in Molecular Biology and Evolution, the Clark team’s findings report accelerated changes in genes affecting muscle formation, lipid metabolism, sensory systems, skin, lung and connective tissue — broadly recognized functional adaptations for which underlying mechanisms are not well understood.


Cleft lip/palate project among first from NIH

A federal initiative to accelerate research into pediatric diseases and conditions will fund a Pitt-led effort to examine the entire genomes of nearly 1,300 people to learn more about the causes of cleft lip and palate, and to look for treatments.

In its first round of funding under the Gabriella Miller Kids First Research Act, the National Institutes of Health’s Office of the NIH Director selected a proposal from Pitt’s School of Dental Medicine and Graduate School of Public Health to sequence the whole genomes of 430 children with clefts, as well as their parents. According to the NIH, this is among the largest whole-genome sequencing efforts to examine an oral condition that it has ever initiated.

Said project director and principal investigator Mary L. Marazita, faculty member and vice chair of the Department of Oral Biology in Dental Medicine and director of the Center for Craniofacial and Dental Genetics: “This sequencing will provide a wealth of data that will be made available to scientists everywhere, providing the basis for years of research into causes, prevention and treatment of cleft lip and palate.”

The Gabriella Miller Kids First Research Act — named for a 10-year-old girl who died of brain cancer in 2013, after working to raise support for research into childhood illnesses — allows NIH to direct the funding of $12 million in pediatric researchScanning the night skies: Pitt part of worldwide effort to discover new planetsprojects each year for the next 10 years.


New drug targets pulmonary hypertension

A study led by researchers from Pitt and UPMC has identified a new group of compounds that could have robust effects in treating pulmonary hypertension (PH), an enigmatic but sometimes fatal disease of the blood vessels of the lungs that currently has no cure. The findings, published in the Journal of Clinical Investigation, highlight the use of these drugs to alter vessel stiffness and its downstream control of metabolism, a link previously unknown for people suffering from the progressive disease.
Said Stephen Y. Chan, faculty member in the School of Medicine, director of the UPMC Center for Pulmonary Vascular Biology and Medicine at the Vascular Medicine Institute and senior author of the study: “If we aim to cure this disease, the next set of medications and treatments should be those that target the origin at the molecular level. As a community, we are struggling right now to understand those origins of PH, and this study aimed to address that untapped need.”

Affecting tens of millions of people worldwide, PH is high blood pressure in the arteries in the lungs, which makes it difficult for blood to flow from the heart to the lungs. Symptoms of the disease, which can lead to heart failure, include shortness of breath, fatigue and chest pain. In its early stages, it might not be noticeable for months or even years. Often a life-threatening condition, it becomes progressively worse, making early and accurate diagnosis important to allow treatments that extend and improve the quality of life for many patients.

To make these discoveries, Chan and his colleagues used a comprehensive array of tools derived from cells and tissues of animals and humans with PH. These findings also were relevant to PH caused by human immunodeficiency virus (HIV) infection, a particularly mysterious form of this disease for which the underlying molecular processes have remained unknown for decades.
Chan’s team found that stiffening or hardening of the vessels in the lung is an early event in PH that triggers the activation of two critical signaling molecules called YAP and TAZ. These molecules in turn activate a protein called GLS1, which controls how cells in the vessel produce and use energy.
“The link between vessel hardening and energy production is absolutely central to this disease,” said Chan. “That discovery offers us so many new ways to design drugs tailor-made to stop PH in its tracks.”

As proof-of-concept, Chan’s team tested both the YAP inhibitor verteporfin, a Food and Drug Administration-approved medication for macular degeneration, and a GLS1 inhibitor called CB-839, which is in clinical trials for treating cancer. They found that both of these compounds displayed robust effects in improving PH in a rodent model of disease.

“We are working to repurpose these drugs for treatment of human PH, which now can include long-neglected disease types such as HIV-related conditions and others,” said Chan. “We hope that we can do so without the delay of decades that often happens when developing new compounds from scratch.”
Given that vessel stiffness is prevalent in other diseases — including cancer progression — these results also may be important beyond PH, noted Chan.


Ultrasound, microbubbles may fight heart disease, cancer

Combining ultrasound energy and microbubbles to poke holes in cells may prove to be a new tool in the fight against cardiovascular disease and cancer, according to researchers from Pitt and UPMC. A study of this gene therapy approach, called sonoporation, appears in the Proceedings of the National Academy of Sciences.

Said Brandon Helfield, lead author of the study and a postdoctoral fellow at the Center for Ultrasound Molecular Imaging and Therapeutics at UPMC: “We can use ultrasound energy in combination with small, gas-filled bubbles to selectively open up cells to allow the delivery of therapeutic agents. With a focused ultrasound beam, this approach lets us tune this delivery to the precise location of disease while sparing healthy tissue. Our study looks at some of the biophysics at play and helps us get closer to refining this technique as a clinical tool.”

Current approaches to gene therapy often use viruses to gain access inside cells, which can cause severe side effects, including inflammatory immune system reactions. To address this, researchers have developed gene-loaded intravascular microbubbles that can be targeted to release their payloads by direct navigation of focused ultrasound energy.

The Pitt researchers developed an ultrafast imaging camera capable of reaching speeds up to 25 million frames per second — the only one of its kind in North America. Using the camera, these researchers examined the biophysics of sonoporation. They determined that the oscillating bubbles need to generate a minimum amount of localized shear stress, beyond which cell membranes perforate and allow entry of a targeted therapeutic.

Said Xucai Chen, medicine faculty member in the Division of Cardiology and the Pittsburgh Heart, Lung and Blood Vascular Medicine Institute, who co-developed the camera system: “By allowing us to actually see the microbubbles vibrating at millions of times per second, our unique camera enabled us to determine that microbubble-induced shear stress is the critical factor for sonoporation. This new information, in turn, will facilitate the intelligent design of treatment protocols and microbubble fabrication to preferentially cause the desired effect of opening nearby cells. It also gives us a starting point to investigate how cells cope with this treatment.”

Researchers believe the findings will help them understand how the process of sonoporation works, as well as how experts can tailor the approach, including ultrasound amplitude levels and microbubble designs, toward its eventual clinical use.

Said Flordeliza Villanueva, medicine faculty member, director of the Center for Ultrasound Molecular Imaging and Therapeutics and the senior author of the investigation: “It’s critical for us to understand the biophysical mechanisms of sonoporation in order to translate this approach into an effective gene or drug delivery tool for patients. Building on the PNAS study, we are continuing to investigate how sonoporation affects the function of treated cells and to develop strategies to maximize its therapeutic effects.”

This work was partly funded by NIH, the Fonds de Recherche Natures et Technologies in Quebec, Canada, and the Center for Ultrasound Molecular Imaging and Therapeutics.


Insights into how mind influences body

Neuroscientists have identified the neural networks that connect the cerebral cortex to the adrenal medulla, which is responsible for the body’s rapid response in stressful situations. These findings, reported in Proceedings of the National Academy of Sciences, provide evidence for the neural basis of a mind-body connection.

The findings shed new light on how stress, depression and other mental states can alter organ function, and show that there is a real anatomical basis for psychosomatic illness. The research also provides a concrete neural substrate that may help explain why meditation and certain exercises such as yoga and Pilates can be so helpful in modulating the body’s responses to physical, mental and emotional stress.

In their experiments, the scientists traced the neural circuitry that links areas of the cerebral cortex to the adrenal medulla (the inner part of the adrenal gland, which is located above each kidney). The scientific team included senior author Peter L. Strick, Thomas Detre Chair of the Department of Neurobiology in the School of Medicine and scientific director of the University of Pittsburgh Brain Institute; lead author Richard P. Dum, neurobiology faculty member; and David J. Levinthal, Department of Medicine faculty member.

The scientists were surprised by the sheer number of neural networks they uncovered. Other investigators had suspected that one or two cortical areas might be responsible for the control of the adrenal medulla. The actual number and location of the cortical areas were uncertain. The Strick laboratory used a tracing method involving the rabies virus, which reveals long chains of interconnected neurons. Strick and his colleagues demonstrated that the control of the adrenal medulla originates from multiple cortical areas. According to their findings, the biggest influences arise from motor areas of the cerebral cortex and from other cortical areas involved in cognition and affect.

Why does it matter which cortical areas influence the adrenal medulla? Acute responses to stress include a wide variety of changes such as a pounding heart, sweating and dilated pupils. These responses help prepare the body for action and often are characterized as fight-or-flight responses. Many situations in modern life call for a more thought-out reaction than simply fight or flight, and it is clear that we have some cognitive control (or what neuroscientists call “top-down” control) over our responses to stress.

Said Strick: “Because we have a cortex, we have options. If someone insults you, you don’t have to punch them or flee. You might have a more nuanced response and ignore the insult or make a witty comeback. These options are part of what the cerebral cortex provides.”

Another surprising result was that motor areas in the cerebral cortex involved in the planning and performance of movement provide a substantial input to the adrenal medulla. One of these areas is a portion of the primary motor cortex that is concerned with the control of axial body movement and posture. This input to the adrenal medulla may explain why core body exercises are so helpful in modulating responses to stress. Calming practices such as Pilates, yoga, tai chi and even dancing in a small space all require proper skeletal alignment, coordination and flexibility.

The study also revealed that the areas of the cortex that are active when we sense conflict, or are aware that we have made an error, are a source of influence over the adrenal medulla. “This observation raises the possibility that activity in these cortical areas when you re-imagine an error, or beat yourself up over a mistake, or think about a traumatic event, results in descending signals that influence the adrenal medulla in just the same way as the actual event,” said Strick. These anatomical findings have relevance for therapies that deal with post-traumatic stress.

Additional links with the adrenal medulla were discovered in cortical areas that are active during mindful meditation and areas that show changes in bipolar familial depression. “One way of summarizing our results is that we may have uncovered the stress and depression connectome,” said Strick.

Overall, these results indicate that circuits exist to link movement, cognition and affect to the function of the adrenal medulla and the control of stress. This circuitry may mediate the effects of internal states like chronic stress and depression on organ function and thus provide a concrete neural substrate for some psychosomatic illness.

This research was funded by NIH and the Pennsylvania Department of Health.


New prevention, treatment possible for organ rejection

An international team led by researchers from the School of Medicine found that targeting certain donor cells lowered the risk of organ rejection in mice that underwent kidney and heart transplants. The study results, published in Nature Communications, could lead to new ways of preventing or treating organ transplant rejection in humans.

Said Fadi Lakkis, Frank & Athena Sarris Chair in Transplantation Biology, faculty member in surgery and immunology, scientific director of the Thomas E. Starzl Transplantation Institute and co-author of the study: “The success of organ transplantation has reached a plateau over the past 10 or 20 years, with a significant proportion of patients still losing their grafts to rejection despite immunosuppressive treatment. New methods to tackle rejection are needed, and this discovery is another step toward finding a solution.”

Without immunosuppressive treatment, transplanted organs are quickly rejected by the recipient’s immune system — in particular, by T cells. Successful engraftment traditionally has relied upon preventing the activation of T cells in the lymph nodes and spleen or in the graft by administering anti-rejection drugs. If T cell activation does occur, stopping rejection becomes increasingly difficult.

To become fully activated, T cells need to make physical contact and receive help from a highly specialized type of cell called dendritic cells. Post-transplant, the main function of the dendritic cells is to present donor-derived antigens to donor-reactive T cells in lymphoid tissues, causing an immune response in the body.

In the study, researchers found that dendritic cells play a key role in driving rejection of transplanted organs by activated T cells that already have entered the transplanted organ. The donor dendritic cells that accompanied heart or kidney grafts in mice were rapidly replaced by the recipient’s dendritic cells, which propagated T lymphocyte activation within the graft and increased the risk of rejection.

Said Adrian E. Morelli, surgery and immunology faculty member at the Thomas E. Starzl Transplantation Institute and study co-author: “We demonstrated that dendritic cells not only exert a key role as antigen-presenting cells in graft-draining lymphoid organs, but also play a critical function within the transplanted organs. Our study indicates that eliminating transplant-infiltrating dendritic cells reduces proliferation and survival of T cells within the graft with the consequent prolongation of transplant survival.”

Added Lakkis: “The next step would be to devise methods to specifically target dendritic cells within transplanted organs. Such methods carry the promise of preventing or interrupting rejection without compromising the patient’s overall immune defenses.”

The study was supported by NIH, the American Society of Nephrology, the American Heart Association and the National Natural Science Foundation of China.


Federal grants will aid early cancer detection

The University of Pittsburgh Cancer Institute (UPCI), partner with UPMC CancerCenter, is sharing in $15.5 million over five years from the National Cancer Institute (NCI) to accelerate research into the early detection of cancer.

The NCI Early Detection Research Network (EDRN) projects seek to discover, develop and validate tests that use blood or other bodily fluids to detect cancer biomarkers — the biological signatures left by cancerous cells — to detect cancer at an earlier stage or to better monitor cancer progression. The projects typically involve dozens of scientists at multiple institutions.

Randall Brand, medicine faculty member and director of UPCI’s gastrointestinal malignancy early detection, diagnosis and prevention program, is principal investigator with a colleague from the University of Nebraska, on the first EDRN Clinical Validation Center for pancreatic cancer. These centers manage large repositories of blood and tissue samples against which promising biomarkers can be tested. This is the last phase of the NCI-funded early detection process. Once a promising biomarker is validated, typically it will be developed further for clinical use by a biotech or pharmaceutical company.

Brand also is a co-investigator on an EDRN grant for a Biomarker Development Laboratory headed by the University of Texas MD Anderson Cancer Center that is geared toward discovering biomarkers for pancreatic cancer.

Said Brand: “Pancreatic cancer is one of the deadliest cancers because it is rarely found early. That is why teamwork is so essential to saving lives. Our infrastructure will make it possible to hit the ground running in coordinating with other institutions toward the goal of an accurate, effective test for the early detection of pancreatic cancer.”

Robert Schoen, faculty member in medicine and epidemiology and interim chief of the Division of Gastroenterology, is principal investigator with colleagues from Johns Hopkins University on another EDRN Biomarker Development Laboratory focused on colon cancer.

Schoen’s group plans to evaluate a blood screening test that will compare people with colon cancer to those without it to see if their biomarker test can accurately detect subjects with colon cancer. They also will evaluate a blood-monitoring test for people with newly diagnosed stage III colorectal cancer to see if their monitoring test is better than the standard monitoring test at detecting subjects with recurrent disease.

Said Schoen: “Of course, a blood test to screen for cancer is highly desirable compared to colonoscopy or stool-based tests that currently are in use. A test that can tell you things after you’ve been diagnosed also would be valuable. For example, you could determine if the cancer treatment is working or if it needs to be changed, and you could potentially identify patients with recurrence earlier.”

Anna Lokshin, faculty member in medicine and pathology with UPCI, is a principal investigator on a subcontract of the EDRN Clinical Validation Center led by a colleague from the Fred Hutchinson Cancer Research Center to evaluate potential biomarkers for breast, colorectal and ovarian cancers.
Said Lokshin: “We believe we’ve selected some very strong biomarker test candidates for validation and that these tests will not only help detect cancer earlier, but also lessen the difficulty some patients find with preventative screenings, such as mammography and colonoscopy. We anticipate that the work we do with this project will yield a clinical impact relatively soon.”


Metabolic improvements may help depression symptoms

Identifying and treating metabolic deficiencies in patients with treatment-resistant depression can improve symptoms and in some cases even lead to remission, according to research from the School of Medicine published in the American Journal of Psychiatry.

This research is funded through a 2014 Pitt Innovation Challenge Award from Pitt’s Clinical and Translational Science Institute.

Said David Lewis, Thomas Detre Professor and chair of the Department of Psychiatry: “What’s really promising about these new findings is that they indicate that there may be physiological mechanisms underlying depression that we can use to improve the quality of life in patients with this disabling illness.”

Major depressive disorder, also referred to simply as depression, affects nearly 15 million American adults and is one of the most common mental disorders. Unfortunately, at least 15 percent of patients don’t find relief from conventional treatments such as antidepressant medications and psychotherapy, according to lead study investigator Lisa Pan, faculty member in psychiatry and clinical and translational science in the School of Medicine. Depression also is the cause of more than two-thirds of suicides.

The groundwork for the current study was laid five years ago when Pan and David Brent, Endowed Chair in Suicide Studies and faculty member in psychiatry, pediatrics, epidemiology and clinical and translational science, treated a teen with a history of suicide attempts and long-standing depression. “Over a period of years, we tried every treatment available to help this patient, and yet he still found no relief from his depression symptoms,” Pan said.

Searching for answers, Pan contacted Jerry Vockley, pediatrics faculty member and chair of genetics at Children’s Hospital, and David Finegold, human genetics faculty member in public health. Through a series of biochemical tests, the three discovered that the patient had a cerebrospinal fluid deficiency in biopterin, a protein involved in the synthesis of several brain signaling chemicals called neurotransmitters.

After receiving an analogue of biopterin to correct the deficiency, the patient’s depression symptoms largely disappeared and today he is a thriving college student.

The success prompted the researchers to examine other young adults with depression who were not responding to treatment, said Pan.

In the published trial, the researchers looked for metabolic abnormalities in 33 adolescents and young adults with treatment-resistant depression and 16 controls. Although the specific metabolites affected differed among patients, the researchers found that 64 percent of the patients had a deficiency in neurotransmitter metabolism, compared with none of the controls.

In almost all of these patients, treating the underlying deficiency improved their depression symptoms, and some patients even experienced complete remission. In addition, the further along the patients progress in the treatment, the better they are getting, Pan added.

Additional Pitt School of Medicine collaborators on the study are David Peters, Petra Martin, Thomas Zimmer, Anna Maria Segreti, Sivan Kassiff, Brian McKain, Cynthia Baca, Manivel Rengasamy, Nicolette Walano, Marion Hughes, Steven Dobrowolski, Michele Pasquino, Rasim Diler and James Perel. Colleagues from the University of California-San Diego, MNG Laboratories in Atlanta and the University Medical Center Gottingen in Germany also contributed.

This research also was supported by the American Foundation for Suicide Prevention, a Brain and Behavior Research Foundation NARSAD Young Investigator Award and the Beck and Lohman families through the Children’s Hospital of Pittsburgh Foundation.


Pitt continues flu vaccine evaluation

The Vaccination Research Group (PittVax) has earned a $5.5 million, five-year renewal from the U.S. Centers for Disease Control and Prevention (CDC) to continue evaluation of the annual influenza vaccine and, once licensed, the respiratory syncytial virus (RSV) vaccine. The PittVax team collaborates with investigators in medicine and public health and across UPMC, including Children’s Hospital.

Starting in 2011, PittVax became one of five U.S. Influenza Vaccine Effectiveness Network sites that provide data and analysis needed for public health officials to make or adjust recommendations for vaccination, antiviral and other treatments. PittVax is directed by Richard K. Zimmerman and Tricia Nowalk, faculty members in the School of Medicine’s Department of Family Medicine.

Said Zimmerman, also a faculty member in public health’s Department of Behavioral and Community Health Sciences: “The information we collect and share is one of the primary data sources that the CDC uses in setting its vaccination policies and recommendations for clinicians treating patients in any given flu season. In past years, we’ve given the evidence needed to prompt earlier and more widespread use of flu antiviral drugs. This season, our work has led to the recommendation to discontinue nasal spray flu vaccine because it has not been effective at preventing type A flu.”

The Pittsburgh site collects data from hundreds of patients seen at UPMC outpatient facilities with symptoms of an acute respiratory infection who consent to participate. They are tested to determine if they have flu or another illness, such as RSV, or simply a cold. The researchers also confirm whether or not the participants were immunized against flu earlier in the season and conduct a follow-up survey on participants’ recovery.

In the past five years, Pitt-Vax participants had the highest follow-up survey completion rate of any of the sites and higher enrollment rates than required, with well over the 1,100 necessary participants annually. The program is supported by ongoing outbreak surveillance at six UPMC sites, with more than 14,000 respiratory virus tests performed each flu season.

These data allow PittVax and the other sites to determine if the flu vaccine — which is designed to work against the three or four strains of flu that the World Health Organization predicts most likely to be circulating when the flu vaccine is manufactured, months before flu season actually starts — is effective against whatever strains of flu ultimately circulate.

Last season, the flu vaccine was nearly 60 percent effective, which means that the chances someone vaccinated against flu would get sick with the virus were less than half the chances of illness in someone who didn’t get the flu vaccine. In the 2014-15 season, the vaccine was only 23 percent effective, the lowest in nearly a decade, but it still offered more protection against flu than not being vaccinated. On average, the vaccine is about 50-60 percent effective.

Based on 2015-16 data collected by PittVax and other sites, the nasal spray flu vaccine often offered to children was found to be only 3 percent effective, and the CDC’s advisory committee on immunization practices recommended it not be offered this season, instead recommending the traditional influenza vaccination with a needle for everyone six months and older.

In the event of a flu pandemic — when a new strain of flu to which people have little existing immunity emerges and spreads globally — PittVax will be prepared to collect respiratory samples and conduct studies estimating how many people are affected and how well antivirals or existing flu vaccines work against it.
New with this grant, PittVax also will be prepared to collect and provide data and analysis on the effectiveness of the RSV vaccine, which is expected to be offered to older adults within the next five years, if clinical trials go well.


NEH grants go to 2 faculty members

The National Endowment for the Humanities’ Office of Digital Humanities has awarded one of four Institutes for Advanced Topics in the Digital Humanities grants to David J. Birnbaum. Birnbaum is faculty member and chair of the Department of Slavic Languages and Literatures in the Dietrich School of Arts and Sciences.

The $156,251 grant will support “Make Your Edition: Models and Methods for Digital Textual Scholarship,” a three-week program in which participants, ranging from graduate students to professors, will learn how to develop systems to analyze manuscript material for research. Similar training is common in Europe but not in the United States. Birnbaum aims to host the program in summer 2017.

Said Birnbaum: “Digital scholarly editing (that is, the creation of digital critical editions) is an area of rapid change and development. Editions are made for many different reasons, and the appropriate way to make an edition is therefore not a one-size-fits-all matter. What we propose to do is to teach the attendees to ‘fish,’ as it were, so that they’ll be able to conceptualize their editions according to their own research questions and venture forth without fear to build what they need to realize it.”

Birnbaum is the principal investigator of the project. Other team members are from the University of Bern, Switzerland; Huygens Institute for the History of the Netherlands, Royal Netherlands Academy of Arts and Sciences; the University of Gothenburg, Sweden; and the University of Antwerp, Belgium.
English faculty member Michael Meyer received a grant in support of his book project through NEH’s public scholar program.

The $50,400 award for “Benjamin Franklin’s Last Bet: How a Founding Father’s Daring Philanthropy Reshaped the American Will,” will support Meyer’s research and writing toward publication of a book on the subject.


New cancer immunotherapy-boosting strategies

The microenvironment that supports a cancerous tumor also starves the immune cells that the body sends in to destroy the cancer, UPCI scientists revealed in a discovery that holds the potential to significantly boost the performance of breakthrough immunotherapy drugs.

The UPCI team showed that when immune T cells enter the tumor microenvironment, their mitochondria — which act as mini-factories inside cells, making energy and crucial reagents a cell needs to survive — begin to shrink and disappear, indicating that the T cell is out of fuel and can’t do its tumor-destroying job. The finding, reported in Immunity, opens the door to several potential clinical approaches that could help keep T cells functioning and boost the body’s ability to fight cancer.

Said senior author Greg M. Delgoffe, faculty member in immunology and member of the Tumor Microenvironment Center at UPCI, partner with UPMC CancerCenter: “Immunotherapy to stimulate the body’s immune system has increasingly become the way we treat people with aggressive cancers. It’s effective for a subset of patients, but the truth is that only about 20-40 percent of patients will respond to the treatment, and it is still unclear why. It’s a huge question in the cancer immunotherapy field, and we think we’ve found a big part of the answer.”

As tumors grow, they build a microenvironment, which develops its own blood supply and keeps the tumor thriving, protected and voraciously consuming all available nutrients.

When T cells enter the microenvironment, it’s as if they’re “automobiles that suddenly had the emergency brake applied; they can’t keep driving,” explained Delgoffe. Immunotherapies, like those that target negative regulators on the T cell surface, take these brakes off. “However, what we’re discovering in many cases is that even though the brakes have been taken off, there isn’t any fuel in the tank.” Or — in scientific terms — the lack of mitochondria in the tumor-infiltrating T cells keeps them from functioning. “This is an exciting discovery because we already have various strategies to fill the fuel tank and support T cell function in the tumor microenvironment.”

In laboratory experiments and tests with mice, Delgoffe and his team found that when they boosted the mitochondria in the T cells, they were better able to clear the tumor.

Delgoffe is partnering with other scientists to test various mitochondria-boosting strategies, including using drugs that already have proven safe in humans, such as those for type 2 diabetes, to stimulate T cell metabolism. He’s also working with existing immunotherapy studies to further modify the T cells so that their metabolism functions better in the tumor microenvironment.

Additional Pitt authors on this research were Nicole E. Scharping, Ashley V. Menk, Rebecca S. Moreci, Ryan D. Whetstone, Rebekah E. Dadey, Simon C. Watkins and Robert L. Ferris.

This work was supported by Sidney Kimmel Foundation for Cancer Research and NIH.


Genetic variant causes altered gut microbiome composition

An international team led by researchers at Pitt, Cedars-Sinai Medical Center and the University of California-Los Angeles discovered that a genetic variation previously linked to obesity, cholesterol levels, blood pressure and schizophrenia also is associated with Crohn’s disease, a chronic inflammatory condition of the gastrointestinal tract that is estimated to cost the U.S. $6 billion annually.

In addition, the genetic variant is associated with changes in the composition of the gut microbiome — which is made up of potentially billions of microbes that help people digest food, synthesize nutrients and perform myriad other essential functions — in healthy people, overweight people and people with Crohn’s disease. The findings were reported in Gastroenterology, and the research was funded by the National Institute of Diabetes and Digestive and Kidney Diseases and Helmsley Charitable Trust, among others.

Said co-senior and corresponding author Richard Duerr, School of Medicine faculty member and co-director and scientific director of the UPMC Inflammatory Bowel Disease Center: “We knew from previous studies that there is reduced diversity of the gut microbiome in patients with Crohn’s disease. But that left us with a question: Does Crohn’s disease alter the composition of the gut microbiota, or do pre-existing changes in the gut microbiota confer risk for Crohn’s disease? Our study found that there is a reduction in the abundance of hundreds of minor species of gut bacteria in healthy, overweight and Crohn’s disease-affected people who carry this genetic variant, suggesting that the genetic variant may increase risk for disease by altering the gut habitat. This is an important step toward understanding how the disease works so we can develop therapies or a cure in the future.”

Duerr’s team focused their analysis on 10,523 blood samples from people with inflammatory bowel disease (half of whom had been diagnosed with Crohn’s disease) and 5,726 samples from healthy people. They discovered that a variation in the SLC39A8 gene is associated with Crohn’s disease.

Taking it a step further, the team identified healthy people, overweight people and Crohn’s disease-affected people with the genetic variant and analyzed their gut microbiomes. That is how they discovered that the genetic variant is not just linked to Crohn’s and other conditions, but also to a reduction in hundreds of species of gut bacteria.

The findings have sparked additional questions and potential research avenues, but therapies are still quite a way off, said Duerr, who is also a public health faculty member in the Department of Human Genetics. However, the recent establishment of the Center for Medicine and the Microbiome will help accelerate this research and bring potential therapies — which may involve the center’s clinical fecal transplantation program — to patients.

Additional institutions with researchers who participated in this study are Cleveland Clinic; Yale University; Karolinska Institutet and Örebro University, both in Sweden; Biocruces Health Research Institute in Spain; University Hospital Munich-Grosshadern, University of Ulm, Krankenhaus Waldfriede and Ludwig-Maximilians-University, all in Germany; QIMR Berghofer Medical Research Institute, Royal Brisbane and Women’s Hospital and University of Queensland, all in Australia; Inselspital Bern and University Hospital Basel, both in Switzerland; Emory University; University of Chicago; Harvard University; Université de Montréal, Hôpital Maisonneuve-Rosemont, University of Toronto and Montreal Heart Institute, all in Canada; Icahn School of Medicine at Mount Sinai; University of California-Riverside; Children’s Hospital of Philadelphia; Massachusetts Institute of Technology; and Johns Hopkins University.


Small-molecule switch activates proteins

From growing teeth, bones and tissue in skin and organs to creating enzymes and hormones, proteins are one of the most diverse and important elements of living organisms. Those varied purposes, along with the interconnectivity of all systems in living cells, make it challenging for researchers to determine how they operate.

But recently, researchers led by chemistry faculty member Alexander Deiters in the Dietrich School of Arts and Sciences developed a technology that allows a small-molecule phosphine to act as an “off-to-on switch” to control protein activity, giving scientists more control over studies involving the molecular details of biological processes.

Said Deiters: “Being able to precisely control specific protein function in cells using a small, drug-like molecule as an external trigger reveals activities related to the protein in isolation and provides the kinetics of cellular processes.” Deiters added that their research could lead to applications in gene therapy and be used as a research tool to better understand disease processes. “Similar to turning on a light switch to see who is grabbing a late-night snack in the kitchen, a switch that rapidly activates proteins allows us to learn more about their behavior and function,” he said.

Proteins are composed of long chains of amino acids and the researchers found that adding an unnatural amino acid called ortho-azidobenzyloxycarbonyl lysine to a specific site in proteins “protected” them, or rendered them inactive. When the researchers treated cells expressing the protected protein with a phosphine reagent, the unnatural amino acid was converted back to natural lysine, “deprotecting” it and forming an active, wild-type protein.

Having a triggered on-switch enables researchers to observe the protein’s activity in isolation. It also helps to separate it from its interaction with other parts of the cell, leading to a better understanding of the protein’s role as well as its relationship and interaction with other components of the cell.

In their study, the researchers used the small molecule switch in four cellular processes:

• bioluminescence — by deprotecting the enzyme luciferase, the researchers were able to trigger cells to emit light;
• fluorescence — by activating a protein originally found in jellyfish, the researchers were able to convert blue light into green light;
• protein translocation — the researchers were able to use the small molecule switch to assist in moving proteins between different cellular compartments; and
• gene editing and DNA recombination — the switch enabled researchers to control the insertion and removal of genetic information.
The paper, “Small-Molecule Control of Protein Function Through Staudinger Reduction,” was published in Nature Chemistry by Deiters, along with graduate assistants Ji Luo and Qingyang Liu and postdoctoral student Kunihiko Morihiro.
The research was supported in part by NIH, NSF and the Charles E. Kaufman Foundation of The Pittsburgh Foundation.


Sampling method may underestimate cancer risk

Not only is breast cancer more than one disease, but a single breast cancer tumor can vary within itself, a finding that UPCI researchers discovered has the potential to lead to very different patient treatment plans depending on the tumor sample and diagnostic testing used.

The research results, reported in Clinical Cancer Research, demonstrate that tumor sampling techniques used with newly developed personalized-medicine gene expression profile tests may need to be refined to ensure that the most appropriate tumor sections are selected for testing.

Said Adrian V. Lee, faculty member in pharmacology and chemical biology at UPCI, partner with UPMC CancerCenter: “These tests are a good thing — they’ve done an incredible job identifying women with breast cancers that have a low risk of recurrence who don’t need chemotherapy, saving them from the toxicity and discomfort of unnecessary treatment. However, as with any new technology, we need to understand how these tests work, and we’re finding that the sampling process, which involves liquefying tumors, loses information that could be important in determining the best treatment plan for patients with more aggressive tumors.”

Gene expression profiling is an increasingly popular type of test that tells doctors what certain genes are doing in a tissue sample, such as causing the cells to actively divide and multiply. Several tests have been developed in recent years to aid oncologists in developing breast cancer treatment plans. They involve taking a small bit of the tumor — or multiple small bits mixed together — and testing it.

The tests can tell oncologists if the cancer has a low, intermediate or high risk of recurring. The level of risk can help doctors and patients decide whether an aggressive treatment plan involving chemotherapy is beneficial or likely to do more harm than good.

Lee and his team examined 71 cases of a type of breast cancer called “estrogen-receptor-positive” that was caught early and hadn’t yet spread to other parts of the body. In all cases, the tumor had been removed and samples taken for gene expression profiling. A total of 181 samples were taken from various parts of the tumors, and the researchers measured the expression of 141 different genes from five different types of gene expression profile tests commonly used for breast cancer tumors.

For 25 percent of the patients, their tumors received a different risk of recurrence score depending on which sample was processed.

“This indicates that one part of the tumor is more aggressive than another part,” said Lee. “If an oncologist were to know this, he or she would likely recommend a treatment plan tailored to destroy the most aggressive section of the tumor.”

Because the patients in this study all were caught early, their risk of recurrence was low to begin with, and there weren’t enough recurrences to make a meaningful determination on whether they would have done better if more samples had been tested from their tumors.

Additional Pitt researchers on this study were Rekha Gyanchandani, Yan Lin, Hui-Min Lin, Kristine Cooper, Daniel P. Normolle, Adam Brufsky, Michael Fastuca, Whitney Crosson, Steffi Oesterreich, Nancy E. Davidson, Rohit Bhargava and David J. Dabbs.

This work was supported by the Breast Cancer Research Foundation, NCI, Fashion Footwear of New York and UPMC.


Stolen guns dominate guns recovered by police

Nearly 80 percent of perpetrators carrying a gun recovered by Pittsburgh Police were not the lawful owners, a strong indication that theft and trafficking are significant sources of firearms involved in crimes in southwest Pennsylvania, a new Graduate School of Public Health analysis reveals.

The finding suggests a timely opportunity for collaboration between public health and law enforcement officials to better understand and reduce violent crimes involving firearms. The results were published in Social Medicine and funded by the former Falk Foundation.

Said lead author Anthony Fabio, epidemiology faculty member: “Homicide by firearms continues to rank among the leading causes of death for young people in the U.S. Given the pandemic threat in the United States of firearm violence, immediate improvement in firearm surveillance is needed to save lives. It is estimated that there are more than 300 million guns in the U.S. And we know that firearm production is increasing. In 2013, nearly 11 million firearms were manufactured in the U.S., more than double the number produced in 2008.”

Fabio and his team analyzed 762 cases in which a gun was recovered by the Pittsburgh Bureau of Police firearm tracking unit in 2008.

In 44.3 percent of the cases where the perpetrator was not the owner of the firearm, the police could not get in contact with the owner to find out how they lost possession of it. In cases where police made contact with the original owner, more than 30 percent said the firearms were stolen, but only 57.9 percent of people had officially reported the theft prior to recovery by police.

Firearms reported stolen before recovery by police were owned by women 16.6 percent of the time. However, that number climbs to 19.3 percent for firearms reported stolen only after recovery by police.
“Owners who have illegally transferred their firearm, perhaps as a straw purchase where they buy the gun for someone who otherwise would not be able to legally obtain one, may be more likely to resist attempts by police to contact them or claim the firearm was stolen after police contact them,” said Fabio. “The disparity we found in firearms reported stolen by women may be due to girlfriends and spouses making straw purchases for their male partners. But the overriding issue here is that these numbers are just estimates. Even police departments do not have the resources to accurately and consistently track firearms used in illegal activities.”

Fabio and his co-authors recommend that more efforts be made to educate the public about safe storage of firearms and injury prevention, as well as encourage ongoing, systemic collaboration between public health and law enforcement experts to better understand and reduce violent crime and improve access to data collection on firearms.

Additional Pitt authors on the research were Jessica Duell, Kathleen Creppage and Ron Laporte. A colleague from the former Falk Foundation also contributed.


New kidney stone prevention proposed

A natural citrus fruit extract has been found to dissolve calcium oxalate crystals, the most common component of human kidney stones, in a finding that could lead to significantly improving kidney stone treatment, according to researchers at Pitt, the University of Houston and Litholink Corp.

In a study published in Nature, the researchers offer evidence that the compound hydroxycitrate (HCA) effectively inhibits calcium oxalate crystal growth and, under certain conditions, is able to dissolve the crystals and shows promise as a potential therapy to prevent kidney stones.

Kidney stones are small mineral pellets that form in the kidneys and may be found throughout the urinary tract. Frequently painful, kidney stones each year cause more than 300,000 patients to visit emergency rooms, NIH estimates. Though it’s the most frequent urinary tract ailment, little has changed in preventative treatments for kidney stones in the past 30 years. Most patients at risk for kidney stones are instructed to drink water, reduce the amount of foods high in oxalates such as leafy green vegetables and nuts in their diet and take citrate (CA) in the form of a potassium citrate supplement to slow crystal growth.

HCA, which is chemically similar to potassium citrate, is found in several tropical plants including garcinia cambogia, commonly known as Malabar tamarind.

The researchers found that the HCA inhibits growth of the crystals by binding to them and that even in very small concentrations it actually can dissolve those crystals.

Giannis Mpourmpakis, faculty member in chemical and petroleum engineering at the Swanson school, was joined in the research by graduate student Michael G. Taylor.

Mpourmpakis and Taylor applied density functional theory, a highly accurate computational method used to study the structure and properties of materials, to discover how HCA and CA bind to calcium and to calcium oxalate crystals. They found that HCA formed a stronger bond with crystal surfaces, inducing a strain that appears to be relieved by the release of calcium and oxalate, thus dissolving the crystal.

Their colleagues at other institutions studied interactions between the crystals CA and HCA under realistic growth conditions, allowing the researchers to record crystal growth in real time with near-molecular resolution. They noted that the images recorded the crystal actually shrinking when exposed even to supersaturated concentrations of calcium oxalate. Other co-authors tested HCA in human subjects, allowing researchers to determine that HCA is excreted through urine, a requirement for the supplement to work as a treatment.

The authors say more work is needed, including additional human studies, to address the longterm safety and dosage.

—Compiled by Marty Levine

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