Biomedical Research Excellence Research Articles

Racial disparities in medication use: imperatives for managed care pharmacy.

The COVID-19 pandemic and the social unrest pervading U.S. cities in response to the killings of George Floyd and other Black citizens at the hands of police are historically significant. These events exemplify dismaying truths about race and equality in the United States. Racial health disparities are an inexcusable lesion on the U.S. health care system. Many health disparities involve medications, including antidepressants, anticoagulants, diabetes medications, drugs for dementia, and statins, to name a few. Managed care pharmacy has a role in perpetuating racial disparities in medication use. For example, pharmacy benefit designs are increasingly shifting costs of expensive medications to patients, creating affordability crises for lower income workers, who are disproportionally persons of color. In addition, the quest to maximize rebates serves to inflate list prices paid by the uninsured, among which Black and Hispanic people are overrepresented. While medication cost is a foremost barrier for many patients, other factors also propagate racial disparities in medication use. Even when cost sharing is minimal or zero, medication adherence rates have been documented to be lower among Blacks as compared with Whites. Deeper understandings are needed about how racial disparities in medication use are influenced by factors such as culture, provider bias, and patient trust in medical advice. Managed care pharmacy can address racial disparities in medication use in several ways. First, it should be acknowledged that racial disparities in medication use are pervasive and must be resolved urgently. We must not believe that entrenched health system, societal, and political structures are impermeable to change. Second, the voices of community members and their advocates must be amplified. Coverage policies, program designs, and quality initiatives should be developed in consultation with those directly affected by racial disparities. Third, the industry should commit to dramatically reducing patient cost sharing for essential medication therapies. Federal and state efforts to limit annual out-of-pocket pharmacy spending should be supported, even though increased premiums may be an undesirable (yet more equitable) consequence. Finally, information about race should be incorporated into all internal and external reporting and quality improvement activities. DISCLOSURES: No funding was received for the development of this manuscript. Kogut is partially supported by Institutional Development Award Numbers U54GM115677 and P20GM125507 from the National Institute of General Medical Sciences of the National Institutes of Health, which funds Advance Clinical and Translational Research (Advance-CTR), and the RI Lifespan Center of Biomedical Research Excellence (COBRE) on Opioids and Overdose, respectively. The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.

Neuroharmony: A new tool for harmonizing volumetric MRI data from unseen scanners

This research has been conducted using the UK Biobank Resource (Project Number 40323) and has been supported by a Wellcome Trust’s Innovator Award (208519/Z/17/Z) to Andrea Mechelli. The present work was carried out within the scope of the research program Dipartimenti di Eccellenza (art.1, commi 314-337 legge 232/2016), which was supported by a grant from MIUR to the Department of General Psychology, University of Padua. The data from UCLA, LOSS AVERSION, EMOTIONREGULATION, FALSEBELIEFS, MATURATIONAL CHANGES, ASSOCIATIVE LEARNING, HARMAVOIDANCE, PLACEBO, MORAL JUDGEMENT, CYBERBALL, ROUTE LEARNING, SEQUENTIAL INFERENCE VBM, WASHINGTON UNIVERSITY datasets were obtained from the OpenfMRI database. Their accession numbers are ds000030, ds000053, ds000108, ds000109, ds000119, ds000168, ds000202, ds000208, ds000212, ds000214, ds000217, ds000222, and ds000243, respectively. The acquisition of dataset HMRRC was supported by the National Natural Science Foundation of China to Prof. Qiyong Gong (81220108013, 8122010801, 81621003, 81761128023 and 81227002). Part of the data used in this article (NITRC) have been funded in whole or in part with Federal funds from the Department of Health and Human Services, National Institute of Biomedical Imaging and Bioengineering, the National Institute of Neurological Disorders and Stroke, under the following NIH grants: 1R43NS074540, 2R44NS074540, and 1U24EB023398and previously GSA Contract No. GS-00F-0034P, Order Number HHSN268200100090U. This research has been conducted using the UK Biobank Resource. Part of the data used in preparation of this article were obtained from the Alzheimer’s Disease Repository Without Borders (ARWiBo – www.arwibo.it). The overall goal of ARWiBo is to contribute, thorough synergy with neuGRID (https://neugrid2.eu), to global data sharing and analysis in order to develop effective therapies, prevention methods and a cure for Alzheimer’ and other neurodegenerative diseases. Part of the data used in this article was downloaded from the Collaborative Informatics and Neuroimaging Suite Data Exchange tool (COINS; http://coins.mrn.org/dx) and data collection was performed at the Mind Research Network and funded by a Center of Biomedical Research Excellence (COBRE) grant 5P20RR021938/P20GM103472 from the NIH to Dr. Vince Calhoun. Part of the data used for this study were downloaded from the Function BIRN Data Repository (http://fbirnbdr.birncommunity.org:8080/BDR/), supported by grants to the Function BIRN (U24-RR021992) Testbed funded by the National Center for Research Resources at the National Institutes of Health, U.S.A. Part of the data used in the preparation of this work were obtained from the Mind Clinical Imaging Consortium database through the Mind Research Network (www.mrn.org). The MCIC project was supported by the Department of Energy under Award Number DE-FG02-08ER64581. MCIC is the result of efforts of co-investigators from University of Iowa, University of Minnesota, University of New Mexico, Massachusetts General Hospital. CLING/HMS: The CliNG study sample was partially supported by the Deutsche Forschungsgemeinschaft (DFG) via the Clinical Research Group 241 ‘Genotype-phenotype relationships and neurobiology of the longitudinal course of psychosis’, TP2 (PI Gruber; http://www.kfo241.de; grant number GR 1950/5-1). Part of the data used in preparation of this article were obtained from the NU Schizophrenia Data and Software Tool (NUSDAST) database (http://central.xnat.org/REST/projects/NUDataSharing) As such, the investigators within NUSDAST contributed to the design and implementation of NUSDAST and/or provided data but did not participate in analysis or writing of this report. Part of the data used in the preparation of this article were obtained from the Parkinson’s Progression Markers Initiative (PPMI) database (www.ppmi-info.org/data). For up-to-date information on the study, visit www.ppmi-info.org. PPMI – a public-private partnership – is funded by the Michael J. Fox Foundation for Parkinson’s Research and funding partners, including [list the full names of all of the PPMI funding partners found at www.ppmi-info.org/fundingpartners]. Part of the data used in preparation of this article were obtained from the SchizConnect database (http://schizconnect.org). As such, the investigators within SchizConnect contributed to the design and implementation of SchizConnect and/or provided data but did not participate in analysis or writing of this report. Data collection and sharing for this project was funded by NIMH cooperative agreement 1U01 MH097435. Joao Sato is supported by Sao Paulo Research Foundation (FAPESP, Brazil) Grants 2018/04654-9 and 2018/21934-5.

Formative evaluation results of a phase 2 Center of Biomedical Research Excellence (COBRE).

Because a primary focus of Centers of Biomedical Research Excellence (COBRE) is the development of junior-level investigators into competent and successful research scientists, evaluation of their skills, mentoring experiences, and usefulness of COBRE services is paramount to the transition of the Center to a self-sustaining, collaborative, multidisciplinary research environment. A formative evaluation, focused on the processes of a COBRE, was undertaken and is presented here. Two instruments, one for completion by junior investigators and one for completion by mentors, were developed for the purpose of evaluating this COBRE. Areas of inquiry were relationships between junior investigators and mentors, research self-efficacy, mentee progress, and satisfaction with the COBRE. All eight of the COBRE's current junior investigators and six of their mentors completed the online questionnaires. Junior investigators were very positive about mentors and vice versa. Junior investigators were least positive about their progress as academicians and most positive about their abilities to develop collaborations with other scholars/professionals. Mentors felt as though junior investigators could benefit most by increasing the number of publications they had generated. Activities provided by the CardioPulmonary Vascular Biology (CPVB) COBRE were extremely positive. Junior investigators felt as though the scientific, academic, and professional development opportunities afforded by this COBRE were integral to their success as researchers; however they would like more assistance developing professional networks (i.e., serving on committees of professional societies). Leadership of the CPVB COBRE may consider expanding the role of their advisory committee to ensure these opportunities are provided.

Transcriptional profiling of mitochondrial metabolic regulatory genes during lipid absorption in larval Danio rerio (zebrafish).

Mitochondrial associated genes play an important role in metabolic processes that are linked to major diseases in the human body such as cardiovascular disease, diabetes, and cancer. Two such genes that relate to these processes are PCK1, linked to regulating gluconeogenesis working in conjunction with the citric acid cycle, and SLC25a33, a solute carrier associated with mitochondrial biogenesis and ROS levels. An initial RNAseq experiment comparing oleic acid and oleic acid plus cholesterol diets identified 57 genes responsive to cholesterol metabolism. Genes were then mapped using Cytoscape and literature databases to identify potential genes involved in cardiovascular disease. PCK1 and SLC25a33, were chosen due to their increased-up regulation in the initial RNAseq experiment and their association with mitochondrial functions. The original experiment was repeated with the three dietary challenges – 2% BSA, 2% BSA and Oleic Acid (OAD), and 2%BSA Oleic Acid and Cholesterol (OACD). Zebrafish larvae were used to model the human intestinal tract during dietary absorption. Intestinal dissections were performed after a 3-hour feeding period of each condition. Subsequently a cDNA library was made of each replicate and qRT-PCR was performed to assess the differential regulation of genes involved in metabolic adaption to the diets. We found PCK1 and Timm10b were up-regulated in the OAD diet as compared to OACD and SLC25a33 showed the opposite regulation with theses diets. This work was supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and NIH Grant P20GM103434 awarded to Bluefield State College.

Modeling the effect of a combinatorial anti-CTLA antibody and radiation therapy on metastatic lung cancer.

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is an inhibitory receptor that is upregulated after T-cell activation, and transmits signals that suppress T-cell activation and proliferation through the depletion of CD8+ T cells. Antibodies targeting CTLA-4 can selectively deplete regulatory T cells by antibody-mediated cellular cytotoxicity mechanisms. However, anti-CTLA-4 antibody therapy as a single treatment is limited on effectiveness to fight against immunogenic diseases like metastatic cancers. Radiation therapy (RT) on the other hand is a longstanding pillar of cancer treatment, historically utilized to treat a discrete target and provide local tumor control. Recent preclinical studies demonstrated that RT is synergistic with anti-CTLA antibody and induces systemic anti-tumor responses. In order to better understand the effect of the combination therapy on metastatic lung cancer, we developed a mathematical model using a system of impulsive differential equations. The objective of this study was to develop a predictive simulation platform to improve cancer management by manipulating dose and fractionation schedule of RT and dose and timing for the anti-CTLA-4 antibody therapy. This study is supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence.

OpiA, a Type Six Secretion System Substrate, localizes to the cell pole and plays a role in bacterial growth and viability in Francisella tularensis LVS.

Francisella tularensis is an intracellular pathogen and the causative agent of tularemia. The F. tularensis type six secretion system (T6SS) is required for a number of host-pathogen interactions including phagolysosomal escape and invasion of erythrocytes. One known effector of the T6SS, OpiA, has recently been shown to be a phosphatidylinositol-3 kinase. To investigate the role of OpiA in erythrocyte invasion, we constructed an opiA-null mutant in the live vaccine strain, F. tularensis LVS. OpiA was not required for erythrocyte invasion, however, deletion of opiA affected growth of F. tularensis LVS in broth cultures in a medium-dependent manner. We also found that opiA influenced cell size, gentamicin sensitivity, bacterial viability, and the lipid content of F. tularensis. A fluorescently tagged OpiA (OpiA-EmGFP) accumulated at the cell poles of F. tularensis which is consistent with the location of the T6SS. However, OpiA-EmGFP also exhibited a highly dynamic localization and this fusion protein was detected in erythrocytes and THP-1 cells in vitro further supporting that OpiA is secreted. Similar to previous reports using F. novicida, our data demonstrated that opiA had a minimal effect on intracellular replication of F. tularensis in host immune cells in vitro. However, THP-1 cells infected with the opiA mutant produced modestly (but significantly) higher levels of the pro-inflammatory cytokine TNF-α compared to these host cells infected with wild-type bacteria. Together, our results support previous observations that implicate opiA in infection and virulence, but also reveal an additional role for opiA central to the biology of F. tularensis bacteria. (This research was made possible by NASA West Virginia Space Grant Consortium Training Grant #NNX15A101H and by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).

Developing Software to Numerically Solve a System of Impulsive Ordinary Differential Equations

Mathematical models can be used to simulate complex real-world behaviors and provide insights into how these behaviors work. Additionally, these models can be enhanced through complementary software that is able to better manipulate the large sample spaces their parameters tend to have. This project seeks to develop software to numerically solve multiple newly developed Cancer models. This is accomplished by using the Runge-Kutta method to approximate solutions to the models and leveraging computational power to achieve higher precision and speed. The result of this software will be numerical solutions of the new models that can be used to screen optimal treatment strategy based on calibrated cancer models using impulsive ODEs. The project was supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and the Research Challenge Fund through a Summer Undergraduate Research Experience Grant from the West Virginia Higher Education Policy Commission Division of Science and Research.

Modeling and Analyzing the Effect of a Radiation Therapy on Tumor Growth

Radiotherapy (RT) is one of the most common and effective cancer treatment options, even being used in more than half of all cases to cure cancer in high income countries. Due to the need for radiation therapy, research into maximizing the effectiveness of RT is critical. Mathematical modeling and computer simulations provide powerful tools to investigate potential optimal dosage and timing for tumor control. In this work, we have developed a mathematical model using a system of impulsive ordinary differential equations (IODE) to describe how RT interacts with other major players of the tumor microenvironment. Stability analysis was conducted for the tumor-free equilibrium. Future work includes analyzing ways to maximize the effects of RT using computer simulations. The objective of this study was to develop a platform to improve cancer management by manipulating dose and fractionation schedules of RT. This study is supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence.

Gene expression profiles T helper cell chemokines, cytokines and transcription factors in the genital tract of a stress mouse model during Chlamydia muridarum infection

A stress mouse model established in our laboratory showed that prolonged exposure of mice to cold water-induced stress results in elevated plasma norepinephrine levels, increased intensity of Chlamydia muridarum genital infection, and decreased functions of the immune system. The current study was designed to test whether Active Hexose Correlated Compound (AHCC) modulates the gene expression profiles of chemokines associated with either Thelper 1 (Th1) or Th2 in the stress model. We hypothesized that oral administration of AHCC feeding up-regulates the gene expression profiles of Th1 chemokines and Th1 transcription factors and increases the secretion of cytokines in Th cells and lysates of the genital tract. During the stressing period of 21 days, we mice either 300 mg/kg of AHCC or PBS per day using a gavage feeding technique. We used real PCR to determine gene expression of chemokines favoring Th1 or Th2 chemokines and cytokines. We used ELISA to assess cytokine production in the genital tract lysates. Oral administration of AHCC to stressed mice resulted in the up-regulation of Th1 chemokines. The level of mRNA fold-changes of CXCL10 (+2.2 fold), CXCL9 (+1.8 fold), and CCL5 (+2.fold) was observed. Moreover, AHCC feeding resulted in a significant increase in productions of interleukin-1b, and interleukin 10(IL-10) in the genital tract of stressed mice. However, the production of TNF-a and IL-6 were undetectable by ELISA. Although further study is needed, preliminary data suggest that AHCC feeding favors the production of protective cytokines in our stress mouse model. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and NIH Grant P20GM103434 awarded to Bluefield State College).

The Role of FTL_1229 during Infection and Erythrocyte Invasion by Francisella tularensis.

Francisella tularensis is a gram-negative bacterium that causes the disease, tularemia. Due to the highly infectious nature of F. tularensis, the Centers for Disease Control and Prevention classified this bacterium as a category A bioterrorism agent. To combat this threat, understanding the pathogenesis of F. tularensis is necessary. FTL_1229 encodes a protein homologous to the ATP binding protein portion of an ABC transporter. This gene is of interest because its expression is induced in the presence of human red blood cells. Therefore, we hypothesized that FTL_1229 may be involved during invasion of these host cells. However, we and others have produced evidence that this gene is essential which means that producing a null mutation is unfeasible. Therefore, we utilized a Tet-responsive promoter system to control expression of FTL_1229 in trans and are attempting to delete the chromosomal copy of this gene. By modulating expression of FTL_1229, we will be able to evaluate the role of this gene in erythrocyte invasion and during infection. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and funding from the WV Research Challenge Fund [HEPC.dsr.14.13] and NIH grant 1R15HL147135-01 and This research was made possible by NASA West Virginia Space Grant Consortium, Training Grant # NNX15AI01H.).

Optimizing a micro-fluidic slide for in vivo imaging of larval Zebrafish.

Larval zebrafish (Danio rerio) are optically transparent at six- and seven-days post fertilization (dpf) which makes them useful research models for the study of metabolic and physiological processes. Previous methods to image zebrafish were limited to embryogenesis and early larval stages. Our goal was to design a novel micro-fluidic slide for mounting zebrafish that greatly extends the duration of time for in vivo imaging of zebrafish larvae that are six to seven days post fertilization (dpf). The ideal mounting slide will have channels to provide media exchange, nourishment, and/or drug treatments and channels to remove waste and flush the system of previous test conditions. We had a microfluidic slide manufactured to our specifications. Initial testing revealed that the larval bed was too large. To optimize our previous prototype, we used the Zeiss Discovery V8 microscope to recalculate our measurements for the larvae bed. We have now re-imaged five each of six dpf larvae and five each of seven dpf larvae in order to adjust the measurements to the new average larval size. This work was supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and NIH Grant P20GM103434 awarded to Bluefield State College

Gene Expression of Transcription factors and Secretion of Cytokines in Co-culturing of Dendritic Cells and Naïve T cells in Stressed AHCC-fed Mice Courtney Rolen1, Elisha Martin1, Rajnish Sahu2Tesfaye Belay1, 1Bluefield State College, Bluefield, WV; 2Alabama State University, Montgomery, AL.

Cold-induced stress is known to inhibit the activity of immune cells and has been shown to cause different expression of cytokine profiles during Chlamydia muridarum genital infection. Active Hexose Correlated Compound (AHCC) is a mushroom extract known to modulate the immune system, the mechanism is not well known. The purpose of this study was to investigate if AHCC feeding to stressed mice would affect the pattern of surface marker expression of transcription factors and CD4+ T cell cytokines. We hypothesized that AHCC feeding to stressed mice decreases the expression and secretion of GATA-3 and IL-4 in stressed mice compared to PBS-fed stressed mice. We used magnet enrichment protocols to isolate DCs from bone marrow and naïve T cells from the spleen. Naive T cells and matured DCs were co-cultured for 24 and 48 hours. A quantitative RT-PCR and ELISA were used to determine gene expression and secretion of cytokines, respectively. Our data show downregulation of transcription factor GATA-3 in AHCC-fed mice (-0.45 ) and upregulation of GATA-3 in PBS-fed stressed mice (+1.5). In contrast, transcription factors T-bet was upregulated in stress AHCC-fed (0.9) and non-stressed (+2.0) during infection. Moreover, non-stressed (+2.9) and AHCC-fed (+1.85) mice showed a significant upregulation of T-bet during Chlamydia muridarum genital infection. Increased secretion of IL-12 and IFN-g and decreased secretion of IL-4 and IL-23 was observed in AHCC-fed mice compared to PBS-fed mice. Overall, our results show that AHCC-feeding leads to the restoration of the function of the protective immune system against chlamydia genital infection. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and NIH Grant P20GM103434 awarded to Bluefield State College).

The antimicrobial activity of Serena repens (Saw Palmetto) extract against Burkholderia cepacia.

UMESH NEPALI & JOSEPH HORZEMPA, Department of Mathematics and Natural Sciences, West Liberty University, West Liberty, WV, 26074.The antimicrobial activity of Serena repens (Saw Palmetto) extract against Burkholderia cepacia. Burkholderia cepacia is a gram-negative bacterium and opportunistic human pathogen that most often causes pneumonia in individuals with weak immunity or underlying lung diseases such as Cystic Fibrosis. B cepacia is naturally drug resistant to numerous antibiotics. Therefore, to potentially identify novel antibiotics that inhibit the growth of B. cepacia, we previously screened a library of nearly 4000 extracts of plants, marine life, and fungi from the National Center for Natural Products Research. From this screen, Serena repens (Saw Palmetto) extract significantly inhibited the growth of B. cepacia. The purpose of this work is to validate the antimicrobial activity exhibited by Saw Palmetto extract, and to isolate and identify the compound(s) responsible for this activity. Both disk diffusion and time-to-kill assays will be used to test the antibiotic properties of Saw Palmetto extract. I have conducted pilot studies to optimize experimental conditions that will be used in these assays. Further, we will extract organic compounds from different parts of the Saw Palmetto plant. These extracts will be lyophilized, reconstituted, and then utilized in the bioassays to test antibiotic activity. Subsequently, extracts with activity will be separated by chromatography. To identify the active compounds, pure molecules showing antibiotic activity will be subjected to NMR and Mass Spectrometry. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence)

Modeling the Effects of An Anti-CTLA-4 Antibody Therapy on Metastatic Melanoma.

CTLA-4 is a co-inhibitory molecule that functions to regulate T cell activation. Antibodies that block the interaction of CTLA-4 with its ligands B7.1 and B7.2 can enhance immune responses, including anti-tumor immunity. CTLA-4 blockade has demonstrated benefits in treatment of metastatic melanoma. Prior research has found that antitumor immunity can be enhanced through the blocking of CLTA-4. More recently, efforts to combine anti-CTLA-4 antibodies with other forms of cancer treatment like radiotherapy and chemotherapy have proven to be successful. This study has thus focused on the modeling and analyzing the effects of anti-CTLA-4 antibody therapy on tumor growth using impulsive differential equations and mathematics analysis. This project is supported by the NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence.

Investigation of the role of Band 3 in red blood cell invasion by Francisella tularensis.

Francisella tularensis is a pathogenic gram-negative bacterium and the causative agent of the disease tularemia. As an intracellular pathogen, F. tularensis can infect and replicate within cells of the immune system, such as macrophages as well as non-immune cells including hepatocytes and epithelial cells. In all of the aforementioned cases, uptake is facilitated by the endocytic machinery and cytoskeletal rearrangements of the host cell. F. tularensis is also capable of invading erythrocytes - cells that are incapable of endocytosis or phagocytosis. Therefore, it quite likely that red blood cell invasion occurs through a distinct mechanism. Unlike the cytoskeleton of the nucleated host cells, erythrocytes are supported through a meshwork of spectrin filaments that are tethered by small actin bundles. We previously showed that actin rearrangement is not required for invasion. However, treatment of red blood cells with a snake venom that inhibits the activity of spectrin abrogates erythrocyte invasion. This venom acts through binding and inactivating Band 3, an anion transporter that complexes with spectrin. We therefore hypothesized that interaction with Band 3 is required for erythrocyte invasion. To test this, we generated Fab fragments from monoclonal and polyclonal antibodies specific to Band 3. These fragments will be used to treat erythrocytes during in vitro assays to determine the role of Band 3 during red blood cell invasion by F. tularensis. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and 1R15HL147135).

Effects of CHD8 mutations on the intestinal dysmotility and peristalsis rate in a larval zebrafish (Danio rerio) model of autism

Autism Spectrum Disorder (ASD) is one of the fastest-growing disorders in America today. While ASD is primarily known as a developmental disorder that affects communication and behavior, comorbidities like constipation from restrictive gut movement also exist. Our work explores gut motility and quantifies the difference in the rate of flow of food in the gut and the smooth muscle movement. Many genes have been associated with ASD our lab is focused on CHD8 as it is one of the prevalent mutated genes. CHD8 is a chromatin helicase involves in brain development and nerve cells, which affects communication and proliferation. To study the CHD8 mutation, we are using Zebrafish to model Autism Disorder. With an intestinal transit assay, 7 days post-fertilization larvae are fed with a diet containing fluorescent beads for 1.5 hours and imaged it for five different time points over the next 24hours. With videography, we blindly recorded the larvae for 5 min at one frame/sec and capture the peristaltic events in the gut. Intestinal measurements of regions of interest were quantified for peristalsis timing and sequence. Larvae were then genotyped. This work was supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and NIH Grant P20GM103434 awarded to Bluefield State College.

Sequencing of a npc1L1 mutation in larval zebrafish

NPC1L1 transport protein is located in the lumen of the intestines and is critical for cholesterol absorption. The reason why we study NPC1L1 is because cholesterol absorption in the intestines affect obesity and diabetes which is still under investigation. Our goal is to investigate the mechanism by which the protein NPC1L1 absorbs cholesterol we are creating an npc1l1 -/- (knockout) line of zebrafish. We hypothesize that the npc1l1 -/- larval zebrafish will show reduced intestinal cholesterol absorption when feed a high fat diet compared to wild type zebrafish larvae. We are screening two zebrafish lines that carry mutations within the NPC1L1 sequence. These zebrafish lines were obtained from the Zebrafish International Resource Center. Parent fish carrying the mutation are identified by fin clipping, DNA extraction, and DNA sequencing of the potential mutant locus within NPC1L1.After identifying the mutation we will amplify the DNA fragments. The DNA fragments will then be sent off to Marshall Genomics Core for sequencing. The mutation sequence will be compared to a wild type sequence. Then the zebrafish that has been confirmed a carrier will be outcrossed with a wild type to eliminate background mutations and then intercrossed to make a homozygous carrier. This work was supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and NIH Grant P20GM103434 awarded to Bluefield State College.

Role of the Center of Excellence in fostering Health based Research in Africa

In recent decades, several global health challenges have been emerged to face both developing and developed countries. The latest challenge was the outbreak of the new coronavirus (SARS-Cov-19) which caused severe acute respiratory syndrome (SARS), which affected more than 3,212,993 infected cases with more about 227,784 deaths. Although most of the cases of SARS-Cov-19 are distributed in developed countries, this pandemic pointed to the necessity to establish new health centers and upgrade the existed ones in the developing counties to combat the consequences of the diseases. In addition, these centers should be equipped with the research core facilities that can perform research in a controlled biosafety environment. Indeed, cutting-edge research requires state-of-art infrastructure as well as a well-trained junior researcher under the supervision of a senior researcher. Therefore, we do believe that center of excellence is a must to establish in developing countries especially in Africa. In this opinion paper, we highlight the importance of the center of excellence in biomedical research.

In‐frame Genetic Disruption of SPECC1L Microtubule‐Interaction Domain Causes Embryonic Tissue Movement and Fusion Defects

Movement and fusion events are critical during embryonic development; defects in these events lead to common birth anomalies. For example, failure of neural tube, secondary palate and ventral body wall closure leads to exencephaly, cleft palate, and omphalocele, respectively. Autosomal‐dominant SPECC1L mutations have been identified in patients with syndromic malformations, including hypertelorism, cleft palate and omphalocele. These mutations cluster in the second coiled‐coil (CCD2) and calponin homology (CHD) domains, which interact with microtubule and actin, respectively. To study SPECC1L function in mice, we first generated genomic deletions that resulted in out‐of‐frame truncations. Homozygous mutants for these truncations, including those lacking CCD2 and CHD, died shortly after birth without cleft palate or omphalocele. We then generated a series of targeted in‐frame deletions in CCD2, homozygotes for which also exhibited perinatal lethality but now with ~50% exencephaly, ~50% cleft palate, and ~50% omphalocele. While the omphalocele phenotype segregated equally with the other two phenotypes, exencephaly and cleft palate were never observed in the same embryo, suggesting antagonistic tissue mechanics between neural tube and palate closure. Consistently, we found that the oral cavity was narrower in mutant embryos with exencephaly, allowing for the palatal shelves to close eventually. We have shown previously that SPECC1L deficiency leads to impaired cell speed and directionality. We now show that subcellular SPECC1L‐ΔCCD2 protein distribution was abnormal, accumulating asymmetrically and associating almost exclusively with filamentous actin. Thus, loss of SPECC1L interaction with microtubules – while maintaining actin association – was more detrimental to cell and tissue movement than loss of SPECC1L protein entirely. Taken together, our results showed that in‐frame perturbations of CCD2 in SPECC1L protein specifically affect embryonic closure of several tissues, suggesting that human SPECC1L CCD2 missense mutations are gain‐of‐function. Our data also revealed a novel role for SPECC1L cross‐linking of actin and microtubule cytoskeletons in embryonic tissue movement and fusion events.Support or Funding InformationThis project was supported in part by the National Institutes of Health grants DE026172 (I.S.). Core support and pilot funding was also provided by National Institutes of Health Center of Biomedical Research Excellence (COBRE) grant (P20 GM104936), Kansas IDeA Network for Biomedical Research Excellence (INBRE) grant (P20 GM103418), and Kansas Intellectual and Developmental Disabilities Research Center (IDDRC) grant (P30 HD 002528).

Mouse Trophoblasts Can Provide Antiviral Protection to Embryonic Stem Cells

The blastocyst is the preimplantation embryo that consists of two major components: the inner cell mass (ICM) and the trophectoderm, which give rise to the fetus and placenta, respectively. We have previously reported that embryonic stem cells (ESCs), the major component of the ICM of blastocyst, do not express type I interferons (IFNs) and lack response to inflammatory cytokines. One would assume that this could make ESCs susceptible to infection. Surprisingly ESCs are less susceptible to immunological cytotoxicity and are not hypersensitive to viral infection. These findings led to our hypothesis that the lack of antiviral responses could be a protective mechanism that allows ESCs to avoid negative impact of immune responses, but how ESCs deal with pathogen infection and immunological challenges is not known. In this study, we investigated IFN‐based antiviral innate immunity of the trophectoderm by using mouse trophoblast stem cells (TSCs) and their in vitro differentiated trophoblasts (TSC‐TBs). Unlike ESCs, our data indicated that both TSCs and TSC‐TBs have a functional IFN system as indicated by their ability to express IFNα, IFNβ, and IFNλ in respond to viral stimuli. They express IFN‐stimulated genes when treated with IFNα and IFNβ. We further showed that the conditioned medium from TSCs and TSC‐FBs stimulated with polyinosinic:polycytidylic acid (poly I:C), a synthetic viral RNA analog, contains antiviral activity that could inhibit infection of ESCs by Chikungunya and Zika virus, similar to the effect of purified IFNβ. These findings indicate that embryonic cell lineages in the blastocyst have different immunological properties at the same developmental stage. Although ESCs are deficient in producing IFNs, they can gain IFN antiviral protection from the trophectoderm via a paracrine signaling mechanism in the blastocyst. Therefore, lacking the production of IFN in ESCs could be a protective mechanism that allows ESCs to avoid the potential harmful impact of immune responses without compromising their antiviral innate immunity.Support or Funding InformationThis work was in part supported by the National Institute of General Medical Sciences (R15GM128196‐01). We thank Mississippi‐IDeA Network of Biomedical Research Excellence for the use of the imaging facility (funded by the National Institute of General Medical Sciences P20 GM103476‐11).