National Institute Of General Medical Sciences Research Articles

A Realistic View of Proteins : Solution Scattering at the Life Sciences X‐ray Scattering Beamline (LIX)

Small angle X‐ray scattering is an important tool that reveals the behavior of proteins in their native environment. The scattering pattern produced from a SAXS experiment represents an average of all conformations present in the solution. Since most proteins in our body function in a solution environment, SAXS data can reveal additional biologically relevant states of a molecule or molecular complex. Furthermore, SAXS provides data about the shape and size of the molecule of interest and is a complementary technique to other structural methods. Combining SAXS data with X‐ray crystallography, NMR or Cryo‐EM can lead to a more robust understanding of the species being studied. High quality SAXS data can be used to build ab initio low resolution bead models, which provide a 3D envelope of your molecule. Here at the life sciences X‐ray scattering beamline (LiX), we collect data by flowing sample through the X‐ray path in a 3 channel flow cell. As the sample enters the X‐ray path, the simultaneous collection of SAXS and wide angle X‐ray scattering (WAXS) is obtained via a 3 detector setup. The detector setup has a q‐range of 0.006Å−1 to 3.2 Å−1, primarily to obtain scattering data from the water peak to normalize buffer subtraction. Automated sample handling at LiX allows for collection of up to 360 samples, packaging of data and processing. One challenge in obtaining high quality SAXS data is preparation of pure, monodisperse samples. For these complicated samples that have multiple species, our in‐line HPLC system seamlessly integrates with our beamline for SEC‐SAXS (size‐exclusion chromatography‐SAXS), and increases the chances of obtaining a more pure, monodisperse sample immediately before data collection.Support or Funding InformationLiX beamline is part of the Life Science Biomedical Technology Research resource, co‐funded by the National Institute of General Medical Sciences (NIGMS) under grant P41 GM111244 and by the DOE Office of Biological and Environmental Research under grant KP1605010, with additional support from NIH under grant S10 OD012331. The operation of NSLS2 is supported by US Department of Energy, Office of Basic Energy Sciences, under contract No. DE‐SC0012704.

Assessing the Global Impact of an Adenylate Kinase Isoenzyme on AMPK and mTOR Signaling Pathways Through Metabolic Profiling in both Brain and Lung Cancer Cell Lines

In a variety of human cancers, it is well observed that specific altered bioenergetic programs must be sustained in order to maintain the cancer cell phenotype. Well‐described genes play a role in sustaining energy flux for the cancer cell and recent studies add the regulation of AK4 expression, an adenylate kinase localized to the mitochondrial matrix, to the list. Our results demonstrate that in response to altered AK4 expression in cancer cells, intracellular ATP levels drastically increase. Secondly, we demonstrate that in response to perturbed AK4 expression in cancer cells, two critical homeostatic signaling pathways, the 5′ AMP‐activated protein kinase (AMPK) and a mammalian target of rapamycin (mTOR) pathways, are significantly altered. Both mTOR and AMPK proteins govern a metabolic pathway in which they sense cellular energy levels and nutrient status in response to regulate cancer cell growth and/or proliferation. Lastly, AK4 also has an impact on other bioenergetic pathways. For instance, AK4 can catalyze the reaction GTP + AMP ↔ GDP + ADP24 and sustain critical energy flux of nucleotides in the cell. Because AK4 expression appears to regulate cellular metabolic responses at multiple levels, further analyses by obtaining a metabolomic profile is necessary to highlight the global impact AK4 exerts on all the metabolites in the cancer cell. To do this, cell lines will be engineered without the AK4 gene in glioblastomas, lung adenocarcinomas, and HeLa cells then analyzed to identify all known metabolites in the system. It is hypothesized that without the expression of an AK4 protein there will be indirect or direct disruptions in the global nucleotide pools by either increasing nucleotide biosynthesis or decreasing nucleotide consumption. The outcome of this study will add to the small library of research on AK4 as well as provide a comprehensive understanding on the enzymatic activity and interactions where AK4 exerts its biological effects on cancer cell metabolism.Support or Funding InformationFunded by National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) Grant 1R15GM123382‐01.

Capsaicin enhances the pro‐apoptotic activity of Camptothecin in human small cell lung cancer

Small cell lung cancer (SCLC) is characterized by early dissemination, aggressive clinical course and a dismal survival rate. The first line therapy for SCLC patients is cisplatin‐based combination chemotherapy along with radiation. Initially, cisplatin shows excellent results causing remission in 60–80% of the SCLC patients but within a year the tumor inevitably relapses and this relapsed tumor is resistant to cisplatin. A subset of SCLC tumors are unresponsive to cisplatin from the outset. These patients are said to have platinum‐refractory SCLC tumors. Patients with platinum resistant or refractory disease have very limited options, as the only standard chemotherapy with an FDA‐approved drug, camptothecin (and its derivations), has an objective response rate of approximately 3% and little or no survival benefit. Such sobering statistics define the arena where there is an urgent need for drugs which enhance the pro‐apoptotic activity of camptothecin in human SCLC. The present study tests the hypothesis that capsaicin can sensitize human SCLC cells to the apoptotic effects of camptothecin. Caspase‐3 activity assays reveal that the combination of camptothecin and capsaicin displayed greater apoptotic activity than the drugs used alone. We verified these results using a second apoptosis assay, namely the Cell Death ELISA and obtained similar results. Chou‐Talalay isobologram analysis showed the interaction between camptothecin and capsaicin is synergistic. Chicken chorioallantoic membrane (CAM) assays confirmed the combinatorial apoptotic effects of capsaicin and camptothecin in human SCLC. The co‐operative effects of capsaicin and camptothecin were also confirmed in human SCLCs xenografted on athymic mice. The results of our experiments pave the way to novel combination therapies for human small cell lung cancer.Support or Funding InformationSupport or Funding Information Funding for our study was supported by a NIH R15‐AREA Grant (2R15CA161491‐02). Furthermore, this study was supported in part by a Center for Natural Products pilot grant from Institutional Development Award (IDeA) Grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS), National Institutes of Health (NIH).

The Effects of Rasagiline on Glucose Metabolism and Cognition and Their Relationship to Tau Burden in a Double-Blind, Placebo-Controlled Phase Ii Clinical Trial of Participants with Alzheimer's Dementia

Background: A Phase II proof of concept (POC) randomized clinical trial was conducted to evaluate the effects of rasagiline, a MAO-B inhibitor, in mild to moderate Alzheimer’s disease. The primary objective was to determine if 1 mg of rasagiline daily for 24 weeks is associated with improved regional brain metabolism (FDG PET) compared to placebo. Secondary objectives included use of flortaucipir PET to assess effects on tau pathology and determination of directional consistency of clinical endpoints with FDG PET. Methods: This was a double-blind, parallel group, placebo-controlled, community-based, three-site trial of 50 participants randomized 1:1 to receive oral rasagiline or placebo (NCT02359552). FDG PET was analyzed at screening for the presence of an AD-like pattern as an inclusion criterion and longitudinally using prespecified regions of interest and image classification. Participants who completed baseline and 24-week FDG PET scans and passed quality control were included in the primary analysis; secondary clinical outcomes were analyzed using an ITT model. Findings: Between May 19, 2015 and January 26, 2018, 96 participants were screened, 50 randomized, and 43 completed treatment. The study met its primary endpoint, demonstrating favorable change in FDG PET differences in rasagiline vs. placebo in middle frontal (2·5%,95%CI 0·3–4·7%,p<0·025), anterior cingulate (2·2%,0·1–4·3%,p<0·041), and striatal (2·4%,0·2–4·3%,p<0·023) regions. Tau PET cortical region secondary endpoints did not differ between study arms. Clinical secondary endpoint measures of Quality of Life (p<0·04) and COWAT favored rasagiline. Rasagiline was generally well tolerated with low rates of both serious and other adverse events and notably less neuropsychiatric symptoms reported with rasagiine. Interpretation: These study outcomes illustrate the potential benefits of rasagiline on clinical and neuroimaging measures in AD, with positive effects on frontostriatal pathways. Strengths are the FDG PET results coupled with the directional consistency of clinical outcomes. This data adds important converging evidence for POC of rasagiline in patients well characterized using FDG and tau PET. Limitations include the study size and duration. Further investigation of rasagiline in AD is warranted. Funding: Alzheimer’s Drug Discovery Foundation (ADDF) Declaration of Interest: Dawn Matthews is an employee of ADM Diagnostics, Inc., which was compensated to provide image analysis for the study. Dr. Aaron Ritter has nothing to disclose. Dr. Ronald Thomas has nothing to disclose. Randolph Andrews is an employee of ADM Diagnostics, Inc., which was compensated to provide image analysis for the study. Dr. Ana Lukic is an employee of ADM Diagnostics, Inc., which was compensated to provide image analysis for the study. Carolyn Revta has received grants from Toyama Pharmaceuticals, Biohaven Pharmaceuticals, and Vivoryon (Probiodrug) during the conduct of the study. Dr. Babak Tousi has received a grant from the Alzheimer’s Drug Discovery Foundation during the study. Dr. James Leverenz has received research support from the Alzheimer’s Association, Avid Radiopharmaceuticals, Department of Defense, GE Healthcare, Lewy Body Dementia Association, Michael J Fox Foundation, National Institute of Health and Sanofi/Genzyme. Dr. Howard Fillit is founding Executive Director and Chief Science Officer of the Alzheimer’s Drug Discovery Foundation which funded the rasagiline clinical trial, and has provided consulting to the following pharmaceutical companies: Axovant, vTv, Lundbeck, Otsuka, Lilly, Biogen (RTI), Roche, Genentech, Merck, Samus, Pfizer, and Alector. Dr. Kate Zhong has nothing to disclose. Dr. Howard Feldman reports a service agreement through UCSD with the Cleveland Clinic for data management and biostatistics during the conduct of this study; grants from Toyama Pharmaceuticals , Biohaven Pharmaceuticals , and Vivoryon (Probiodrug). Dr. Feldman also reports service agreements through UCSD for consulting with Eisai Pharmaceuticals, Merck Pharmaceuticals, Tau RX , Samus Therapeutics, Arkuda Therapeutics, Samumed, Axon Neurosciences Roche/Genentech Pharmaceuticals for DMC and DSMB activities; Tau Consortium for Scientific Advisory Board; Novo Nordisk for Advisory Board. He reports travel expenses Samus, Samumed, Axox and Alion Pharmaceuticals and speaker fees to UCSD from World Events Forum, Optum Health, and Medscape. Dr. Jeffrey Cummings reports grants from National Institute of General Medical Sciences (NIGMS) COBRE grant #P20GM109025, during the conduct of the study; personal fees from Acadia, Actinogen, AgenBio, Alkahest, Alzheon, Avanir, Axsome, Biogen, Cassava, Cerecin, Cerevel, Cognoptix, Cortexyme, EIP, Eisai, Foresight, Green Valley, Grifols, Idorsia, Karuna, Nutricia, Orion, Otsuka, Probiodrug, QR Pharma, ReMYND, Resverlogix, Roche, Samumed, Samus Therapeutics, Third Rock, Signant Health, Sunovion, Suven, and United Neuroscience pharmaceutical and assessment companies;other from Alzheimer Drug Discovery Foundation; other from ADAMAS, BioAsis, MedAvante, QR Pharma, and United Neuroscience,;personal fees from Neuropsychiatric Inventory (NPI), outside the submitted work; and Dr. Cummings is the Chief Scientific Advisor for CNS Innovations. Ethical Approval: The study was conducted under Institutional Review Board approval.

Strategic planning for research grant applications.

I am honored to receive the 2019 ASCB Public Service Award. It has been a true privilege to work with the cell biology community; I thank them for their collegiality in their interactions with me over the years. In this essay I offer some perspectives on constructing goals and strategy for research grant and fellowship applications in basic cell biology. They are based on my observations as a program officer for grants and applications in cell biology in the National Institute of General Medical Sciences (NIGMS) from 1991 to 2019.

Signal Processing Techniques for Removal of Various Artifacts from Obstructive Sleep Apnea Signals

Sleep is judgmental to health and well-being. Deficient quality sleep is similar with a wide range of negative outcomes varies from schizophrenia to cardiovascular disorders. Obstructive sleep apnea is one of the sleep disorders. In order to identify the various syndromes the signals are need to record by using the sensors. Sleep signals are recorded by using the polysomnography (PSG) labs which is the old traditional and gold standard for recording the sleep signals. PhysioNet is a large online medical database that consists of a large collection of recordings of various physiological signals. PhysioNet database consist of sleep apnea database available. Physionet website is a universal service, physionet resource supported by the national institute of health’s National Institute of Biomedical Imaging and Bioengineering (NIBIB) and National Institute of General Medical Sciences (NIGMS). This survey paper aims to bring the different Signal Processing Techniques for Removal of Various Artifacts from Obstructive Sleep Apnea Signals to identify sleep apnea syndrome, because pre-processing is most effective and efficient to reduce unwanted signals from the original sleep signals. While recording the sleep apnea signals various artifacts records along with raw signals either directly or indirectly due to the internal and external sources like Power line interference, Muscle contractions, Electrode contact noise, Motion Artifacts, Baseline wandering, Noise generated by electronic circuits, while breathing and coughing, body position movements etc, and they need to be eliminated in order to acquire genuine health information. So in order to remove there artificats from the sleep signals the signal processing techniques (filtering techniques) are predominantly used for pre-processing of the sleep signals and have been executed in a wide variety of systems for analysis. Filtering of the sleep signal is contingent and should be implemented only when the required statistics remains cryptic.

Using database linkages to measure innovation, commercialization, and survival of small businesses

Here, we report the results of an outcomes evaluation of the Small Business Innovation Research (SBIR) and Small Business Technology Transfer (STTR) Programs at the National Institute of General Medical Sciences (NIGMS). Since the programs’ inception, assessments of the SBIR/STTR programs at several federal agencies have utilized surveys of former grantees as the primary source of data. Response rates have typically been low, making non-response bias a potential threat to the validity of some of these studies’ results. Meanwhile, the availability of large publicly-available datasets continues to grow and methods of text mining and linking databases continue to improve. By linking NIGMS grant funding records, U.S. Patent and Trademark Office data, and business intelligence databases, we explored innovation, commercialization and survival for recipients of NIGMS SBIR/STTR funding. In doing so, we were able to more completely assess several key outcomes of the NIGMS SBIR/STTR program. Our evaluation demonstrated that the NIGMS program performed above baseline expectations along all dimensions, and comparably to other federal agency SBIR/STTR grant programs. In addition, we show that the use of extant data increasingly is a viable, less expensive, and more reliable approach to gathering data for evaluation studies.

Cell scientists to watch – Kassandra Ori-McKenney and Richard McKenney

ABSTRACT Kassandra Ori-McKenney received her Bachelor's degree in Neuroscience and Behavior from Vassar College, Poughkeepsie, New York, before joining the laboratory of Richard Vallee at Columbia University, New York, for her PhD. She moved to the University of California, San Francisco, for her postdoctoral training with Yuh-Nung Jan on the role of the microtubule cytoskeleton in neuronal development in Drosophila with the aid of a Jane Coffin Childs fellowship. In 2016, Kassandra became an Assistant Professor at the Department of Molecular and Cellular Biology at the University of California, Davis (UC Davis). Richard McKenney completed his PhD on regulatory proteins that influence the activity of the motor protein dynein in the laboratory of Richard Vallee after a Bachelor's degree at Xavier University, Cincinnati, Ohio. For his postdoctoral work, Richard moved to the laboratory of Ron Vale at the University of California, San Francisco, to continue his work on the regulation of dynein motor activity. Richard joined the Department of Molecular and Cellular Biology at UC Davis in 2016 as an Assistant Professor. Kassandra and Richard share their laboratory space, as well as equipment, ideas and meetings. Their research groups are interested in the microtubule cytoskeleton, microtubule-associated proteins (MAPs), activities of the motor proteins kinesin and dynein, and their roles and functions in neuronal transport and human diseases. Kassandra is funded by grants from the Pew Foundation, Simons Foundation, the March of Dimes and the National Institutes of Health (NIH). Richard is funded by grants from the March of Dimes and the National Institute of General Medical Sciences (NIGMS).

Geographically-related outcomes of U.S. funding for small business research and development: Results of the research grant programs of a component of the National Institutes of Health

This article examines the geographic distribution of funding for the U.S. Small Business Innovation Research (SBIR) and Small Business Technology Transfer (STTR) programs sponsored by the National Institute of General Medical Sciences (NIGMS). Despite a significant investment in SBIR/STTR and an interest in increasing geographic diversity in the institute’s research portfolio, there has not been an assessment of the distribution of NIGMS’s SBIR/STTR funding, outcomes associated with that investment, and relationships between the two. The geographic distribution of NIGMS’ SBIR/STTR funding was highly concentrated in a small number of states, with a high correlation between each state’s funding and its number of small scientific research and development businesses. Affiliation with a major research university was correlated with several measures of innovation and firm success. Our findings are consistent with earlier research showing that economic activity in research and development and research output tend to cluster in geographic regions where knowledge can be generated and shared more efficiently. These findings lend support to an investment strategy for small business research and development that creates networks between major research universities and small businesses.

On Using Local Ancestry to Characterize the Genetic Architecture of Human Traits: Genetic Regulation of Gene Expression in Multiethnic or Admixed Populations

Understanding the nature of the genetic regulation of gene expression promises to advance our understanding of the genetic basis of disease. However, the methodological impact of the use of local ancestry on high-dimensional omics analyses, including, most prominently, expression quantitative trait loci (eQTL) mapping and trait heritability estimation, in admixed populations remains critically underexplored. Here, we develop a statistical framework that characterizes the relationships among the determinants of the genetic architecture of an important class of molecular traits. We provide a computationally efficient approach to local ancestry analysis in eQTL mapping while increasing control of type I and type II error over traditional approaches. Applying our method to National Institute of General Medical Sciences (NIGMS) and Genotype-Tissue Expression (GTEx) datasets, we show that the use of local ancestry can improve eQTL mapping in admixed and multiethnic populations, respectively. We estimate the trait variance explained by ancestry by using local admixture relatedness between individuals. By using simulations of diverse genetic architectures and degrees of confounding, we show improved accuracy in estimating heritability when accounting for local ancestry similarity. Furthermore, we characterize the sparse versus polygenic components of gene expression in admixed individuals. Our study has important methodological implications for genetic analysis of omics traits across a range of genomic contexts, from a single variant to a prioritized region to the entire genome. Our findings highlight the importance of using local ancestry to better characterize the heritability of complex traits and to more accurately map genetic associations.

MON-538 Ca2+Allostery in PTH-Receptor Signaling

The parathyroid hormone (PTH) type 1 receptor (PTHR) serves as the cognate receptor for two endogenous ligands, PTH and its related peptide (PTHrP). Binding of PTHrP to the PTHR results in only transient cAMP production that is limited to the plasma membrane, while PTH induces sustained cAMP responses derived from endosomes following internalization of the ligand-receptor complex. The underlying mechanism and regulation of these kinetically-distinct signaling events are poorly understood. Here we show that extracellular Ca2+ acts as a positive allosteric modulator of the PTHR that promotes sustained cAMP production. Equilibrium and kinetic analysis of ligand binding and receptor activation revealed that extracellular Ca2+ increases the residence time of ligands on the receptor, consequently increasing the duration of receptor activation and cAMP production. Additionally, we found that this sensitivity to Ca2+ is lost for the PTH mutant R25C, identified as a cause of hypocalcemia in human patients. Using mass spectrometry (MS) approaches, we identified acidic clusters in the first extracellular loop of PTHR that serve a critical role in mediating Ca2+ allostery and sustained cAMP signaling. These findings provide insights toward understanding the determinants of PTHR-mediated signaling events and how these processes are regulated. Sources of Research Support: the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) and the National Institute of General Medical Sciences (NIGMS) of the US National Institutes of Health (NIH)

Non‐Pungent Capsaicin Analogs: Potential Applications in Lung Cancer Therapy

Capsaicin is the spicy pungent ingredient of chili peppers. Although traditionally associated with analgesic activity, recent studies have shown that capsaicin has profound anti‐neoplastic effects in several types of human cancers. However, the applications of capsaicin as a clinically viable drug are limited by its unpleasant side effects, such as gastric irritation, stomach cramps and burning sensation. This has led to extensive research focused on the identification and rational design of second‐generation capsaicin analogs, which possess greater bioactivity than capsaicin. Previous studies have shown that addition of long‐chain unsaturated groups after the amide group of capsaicin were non‐pungent and retained the bioactivity of capsaicin. These chemical nature of these compounds are unsaturated N‐acylvanillamides (uN‐AVAMs). However, a majority of these uN‐AVAMs have been studied for their pain‐relieving activity. We synthesized a panel of uN‐AVAMs with 0–4 double bonds in the side chain of capsaicin. We investigated the growth‐inhibitory activity of these compounds with an MTT‐based screening assay. We selected our “hit compound” Arvanil for further studies. Next, we compared the apoptotic activity of arvanil and capsaicin in a panel of human small cell lung cancer (SCLC) cells. We observed that the non‐pungent capsaicin‐analog arvanil displayed greater magnitude of apoptosis than capsaicin. Most interestingly, arvanil did not display apoptotic activity in normal lung epithelial cells. The pro‐apoptotic activity of arvanil and capsaicin was mediated by the intracellular calcium pathway. We measured the uN‐AVAM‐induced levels of intracellular calcium in SCLC cells. The pattern of uN‐AVAM‐induced intracellular calcium was analogous to the results obtained in the MTT assay. Therefore, the measurement of intracellular calcium may be used as a screening tool for capsaicin‐mimetics with anti‐cancer activity.Support or Funding InformationFunding for our study was supported by a NIH R15‐AREA Grant (2R15CA161491‐02). Furthermore, this study was supported in part by an Institutional Development Award (IDeA) Grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS) and the Research Core B of COBRE, a component of the National Institutes of Health (NIH).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fenugreek Saponins Lower HOMA‐IR Estimate in Insulin‐Resistant Subjects: A Randomized, Placebo‐Controlled, Study

BackgroundType 2 Diabetes Mellitus (T2DM) is growing at epidemic proportions worldwide. Insulin resistance, defined as an impaired responsiveness of the body to insulin, is a pre‐diabetic stage in the transition from obesity to full‐blown diabetes. Recent studies from our lab demonstrated that furostanolic saponins (FS) isolated from the spice fenugreek improved glucose tolerance and reduced hepatic steatosis in high‐fat diet‐fed, obese mice.ObjectiveThe objective of this study was to evaluate the effect of supplementation of fenugreek FS on cardiometabolic parameters in insulin resistant, obese subjects.MethodsThis was a single center, double‐blind, randomized, placebo‐controlled, parallel‐arm, pilot study in overweight/obese (body mass index&gt;25Kg/m2) young (mean age 27.1±6.1y), otherwise healthy, male and female subjects who satisfied any two of the following three inclusion criteria: i) hemoglobin A1c &gt;5.6% ii) a two‐hour post‐glucose challenge blood‐glucose level &gt; 125 mg/dL and iii) Homeostatic Model Assessment‐insulin resistance (HOMA‐IR) value of &gt;2.5 (fasting insulin (microU/L) x fasting glucose (nmol/L)/22.5). Following informed consent, health‐screening and satisfaction of the inclusion/exclusion criterial, 28‐participants were randomly assigned to receive either placebo (microcrystalline cellulose) or FS (500 mg twice daily) capsules for a period of 12‐weeks. Anthropometric measurements, blood pressure, fasting blood glucose, fasting serum insulin, hemoglobin A1c, lipids, ALT, AST, C‐reactive proteins were determined at baseline and following 12‐week intervention. Body fat composition was determined by dual energy X‐ray absorptiometry. Serum cytokine levels were determined by a Bio‐Plex Pro Human Cytokine 8‐Plex assay. Data were analyzed by a nonparametric bootstrap analysis and the difference of simulated data from treatment and placebo were compared to test for significance. All runs were included in the calculation of 95% confidence interval.ResultsOf the 28 recruited, 25 subjects completed the study. No adverse events were reported, except for one subject who dropped form the study complaining of mild stomach discomfort. No changes were observed between pre‐ and post‐intervention in mean body mass, systolic or diastolic blood pressure, fasting glucose, hemoglobin A1c, lipid‐panel, serum transaminases, and cytokine levels in subjects receiving either the placebo or FS. In contrast, a significant reduction in HOMA‐IR was observed in subjects who received FS compared to those who received the placebo. Additionally, a trend towards lowering of serum insulin levels was observed in the group that received FS.ConclusionTwelve‐week supplementation with fenugreek saponins resulted in reduction in markers of insulin resistance in obese, insulin‐resistant subjects, warranting future studies with FS in overt‐diabetic subjects.Support or Funding InformationSupported by the NIH through the National Institute of General Medical Sciences (NIGMS) #1U54GM104944‐01A1 under the Institutional Development Award (IDeA) program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Analysis of Genes that Mediate Persistence in Halophilic Microbes Subjected to Osmotic Shock

All organisms are subject to stress from environmental changes, and understanding the specific molecular responses to allow cells to maintain viability is a fundamental biological problem. We explore this process in the halophilic (salt‐loving) microbe Haloferax volcanii. This species was originally isolated from the Dead Sea and grows optimally at 12–14% sodium chloride. However, these organisms are regularly subjected to rapid changes in their osmotic environment during rainfall or flooding events, and we hypothesize that they have evolved so that a subset of their population consists of cells prepared to survive dramatic stress events. As a first step to examine how these ‘persisting’ cells endure, we used RNA sequencing to examine transcriptome differences in cells that survived osmotic shock compared to unstressed cells. Our data revealed that approximately half of the protein‐coding genes exhibited statistically significant differences in transcriptome abundance with a nearly equal distribution of more abundant and less abundant transcripts. There were about twice the number of transcripts exhibiting a 10‐fold or greater decrease (5.3% of protein‐coding genes) in abundance than those with the same magnitude of increased abundance (2.7% of protein‐coding genes). This result may indicate that cells primed for survival reduce production of proteins required for active metabolism. We are currently conducting experiments to determine if deletion or overexpression of some of these genes affects susceptibility of H. volcanii to osmotic shock. Our work may provide insight into why some cells in a microbial population survive stress events despite being genetically identical to cells that are killed. Similar mechanisms may also allow disease‐causing bacteria that persist even after being exposed to antibiotic agents. Potentially, the factors that carry out these responses could be targeted to enable antibiotics to effectively eliminate persister cells.Support or Funding InformationThis project was supported by an Institutional Development Award from the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under grant P20GM103423. Additional funding was provided by the Colby College Natural Science Division.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ca2+ allostery in PTH‐receptor signaling

The parathyroid hormone (PTH) type 1 receptor (PTHR) serves as the cognate receptor for two endogenous ligands, PTH and its related peptide (PTHrP). Binding of PTHrP to the PTHR results in only transient cAMP production that is limited to the plasma membrane, while PTH induces sustained cAMP responses derived from endosomes following internalization of the ligand‐receptor complex. The underlying mechanism and regulation of these kinetically‐distinct signaling events are poorly understood. Here we show that extracellular Ca2+ acts as a positive allosteric modulator of the PTHR that promotes sustained cAMP production. Equilibrium and kinetic analysis of ligand binding and receptor activation revealed that extracellular Ca2+ increases the residence time of ligands on the receptor, consequently increasing the duration of receptor activation and cAMP production. Additionally, we found that this sensitivity to Ca2+ is lost for the PTH mutant R25C, identified as a cause of hypocalcemia in human patients. Using mass spectrometry (MS) approaches, we identified acidic clusters in the first extracellular loop of PTHR that serve a critical role in mediating Ca2+ allostery and sustained cAMP signaling. These findings provide insights toward understanding the determinants of PTHR‐mediated signaling events and how these processes are regulated.Support or Funding InformationNothing to Disclose: AW, IS, FGJ, KX, JPVSources of Research Support: the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) and the National Institute of General Medical Sciences (NIGMS) of the US National Institutes of Health (NIH) under grant Awards Number R01‐DK102495, R01‐DK111427, and R01‐DK116780 (to J.P.V.), and T32‐GM008424 (to A.D.W.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Covalent intermediates in flavoenzyme catalysis

Flavoenzymes catalyze C-H bond cleavage via a hydride transfer mechanism and single electron transfer in numerous cellular reactions. Flavoenzymes are also capable of activating molecular oxygen to catalyze hydroxylation, epoxidation, and Baeyer-Villiger reactions. These oxygenation reactions require formation of a C4a-(hydro)peroxyflavin (FlOO(H)) intermediate. In certain cases, this covalent intermediate is very stable, with reported half-lives of several minutes. Covalent intermediates have also been shown to occur in non-redox reactions where the reduced flavin is required for activity, such as in the isomerization of UDP-galactopyranose to UDP-galactofuranose by UDP-galactopyranose mutase (UGM) and in the dehalogenation reaction catalyzed by 2-haloacrylate hydratase (2HAH). Our mechanistic, structural, and computational data describing the mechanism of stabilization of the FlOO(H), with a focus on the role of NADP+, will be presented. Results of our studies on the UGM and 2HAH reactions will highlight the multiple functionalities of flavins, which include acting as covalent catalysts, nucleophile, radical chemistry, stabilization of protein conformations, and as a proton shuttle. Support or Funding Information The research reported here was supported by grants from the National Institute of General Medical Sciences (NIGMS) award R01GM094469 and the National Science Foundation award CHE-1506206. The major reactive centers of flavin cofactors This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Niakhar Social Networks and Health Project

This paper presents details of the design and implementation of the Niakhar Social Networks and Health Project (NSNHP), a large, mixed-methods project funded by the U.S. National Institute of General Medical Sciences (NIGMS). By redressing fundamental problems in conventional survey network data collection methods, the project is aimed at improving inferences concerning the association between social network structures and processes and health behaviors and outcomes. Fielded in collaboration with an ongoing demographic and health surveillance system in rural Senegal, the NSNHP includes qualitative data concerning the dimensions of social association and health ideologies and behaviors in the study zone, two panels of a new social network survey, and several supplementary and affiliated data sets.•Longitudinal social network survey linked to pre-existing surveillance data•Addresses fundamental methodological constraints in previous social network data•Enables social network analyses of health beliefs, behaviors, and outcomes

Acetylcholine signaling pathway: A novel target for lung cancer in smokers

The clinicopathological features of lung cancer in smokers is completely different from never‐smokers. Whereas lung cancer patients (who are never smokers) respond well to targeted therapies involving EGFR inhibitors and ALK kinase inhibitors, the response of these drugs in lung cancer patients who are smokers is poor. Nicotine is the addictive component of cigarette smoke. Nicotine (at concentrations present in the plasma of moderate smokers) accelerates the growth, angiogenesis and metastasis of lung cancers. The biological activity of nicotine is mediated by nicotinic acetylcholine receptors (nAChRs). The endogenous ligand for nAChRs in the lung is the neurotransmitter acetylcholine (ACh). ACh is known to be an autocrine growth factor for both SCLC and NSCLC. One of the ways that nicotine and cigarette smoke promote the growth of lung cancers is by promoting the production of ACh, which binds to its cognate receptor (on lung cancer cells) in an autocrine manner and induces the proliferation of lung cancer cells. The objective of the present study is to investigate the whether the ACh‐mitogenic signaling pathway are altered in human lung cancer. We show that levels three acetylcholine‐signaling proteins namely ChAT, VAChT and AChE are significantly altered in lung cancer occurring in active smokers. Small‐molecule regulators of these proteins suppress the growth of human lung cancer cells. Our studies will facilitate the identification of targeted therapies for the majority of lung cancer patients who are exposed to tobacco smoke.Support or Funding InformationFunding for our study was supported by an NIH R15‐AREA Grant (2R15CA161491‐02) and the West Virginia IDeA Network of Biomedical Research Excellence (WV‐INBRE) grant GM103434 (PI: Dr. G. Rankin). Furthermore, this study was supported in part by an Institutional Development grant (IDeA) Grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS) and the Research Core B of COBRE, a component of the National Institutes of Health (NIH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pasteurella multocida PfhB2 toxin displays a novel unconventional cysteine protease fold

Pasteurella multocida causes respiratory tract infections in a broad range of animals, as well as opportunistic infections in humans. P. multocida secretes a multi‐domain toxin, called PfhB2, which contains a YopT‐like putative cysteine protease domain at its C‐terminus. PfhB2's YopT domain contains a well‐conserved Cys‐His‐Asp catalytic triad that defines YopT family members, and shares high sequence similarity with the prototype YopT from Yersinia sp. PfhB2 is implicated in virulence. Its FHA (filamentous hemagglutinin) and Fic (filamention induced by cAMP) domains have been shown to provide protective immunity in turkeys and to adenylylate Rho‐GTPases in vitro, respectively. In contrast, little is known about PfhB2's YopT‐like domain. Here, we show that PfhB2‐YopT does not display cysteine protease activity in a manner similar to Yersinia YopT or any other characterized YopT family members. We, therefore, opted to characterize PfhB2‐YopT structurally. Using a catalytically inactive C3733S mutant of PfhB2‐YopT that provides enhanced protein stability, we determined the crystal structure of PfhB2‐YopTC3733S solved by experimental phasing with Selenomethionine derivative crystals at 2.2Å resolution. Our structure shows that PfhB2‐YopT exists as four molecules arranged as an asymmetric unit consisting of two dimers. Importantly, the Cys from each molecule of the dimer coordinates with the His and Asp from the other molecule to complete the catalytic triad. Such an unconventional orientation of the catalytic triad has never been observed for any class of Cysteine proteases. Thus, our PfhB2‐YopT structure represents a novel cysteine protease fold, and provides critical insights for the function of Cys‐His‐Asp containing domains that are often associated with large, multi‐domain bacterial toxins.Support or Funding InformationThis work was supported by funding from 1) an Indiana CTSI (Clinical and Translational Sciences Institute) Project Development Team Award and 2) the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under Award Number R01GM100092.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Anti‐cancer activity of non‐pungent capsaicin analogs: A Structure‐Activity Study

The nutritional compound capsaicin is the major spicy ingredient of chili peppers. Although traditionally associated with analgesic activity, recent studies have shown that capsaicin has profound anti‐neoplastic effects in several types of human cancers. However, the applications of capsaicin as a clinically viable drug are limited by its unpleasant side effects, such as gastric irritation, stomach cramps and burning sensation. This has led to extensive research focused on the identification and rational design of second‐generation capsaicin analogs. Previous studies have shown that addition of long‐chain unsaturated groups after the amide group of capsaicin were non‐pungent and retained the bioactivity of capsaicin. The chemical nature of these compounds are unsaturated N‐acylvanillamides (uN‐AVAMs). However, a majority of these uN‐AVAMs have been studied for their pain‐relieving activity. We synthesized a panel of uN‐AVAMs with 0–4 double bonds in the side chain of capsaicin. We investigated the growth‐inhibitory activity of these compounds with an MTT‐based screening assay. We selected our “hit compound” Arvanil for further studies. Next, we compared the apoptotic activity of arvanil and capsaicin in a panel of human small cell lung cancer (SCLC) cells. We observed that the non‐pungent capsaicin‐analog arvanil displayed greater magnitude of apoptosis than capsaicin. The pro‐apoptotic activity of arvanil and capsaicin was mediated by the intracellular calcium pathway. We measured the uN‐AVAM‐induced levels of intracellular calcium in SCLC cells. The pattern of uN‐AVAM‐induced intracellular calcium was analogous to the results obtained in the MTT assay. Therefore, the measurement of intracellular calcium may be used as a screening tool for capsaicin‐mimetics with anti‐cancer activity.Support or Funding InformationFunding for our study was supported by a NIH R15‐AREA Grant (2R15CA161491‐02). Furthermore, this study was supported in part by an Institutional Development Award (IDeA) Grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS) and the Research Core B of COBRE, a component of the National Institutes of Health (NIH).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.