Konstantin G. Kousoulas - US grants

Herpes viruses are important pathogens of humans and animals. They enter eukaryotic cells by fusion of their viral envelope with the cellular membrane, and spread partly by inducing fusion of adjacent cellular membranes. Our long term goal is to understand the mechanisms of viral-entry into cells and virus- induced cell fusion. HSV-1 glycoproteins B(gB), D(gD) and H(gH) and a hydrophobic protein with membrane spanning potential termed the Fusion Factor (FF) are involved in these fusion events. Viruses that carry single mutations in gB (gB-Syn3) and FF (FF- Syn1) cause extensive fusion of infected cells and enter faster than wild-type viruses. We propose to elucidate interactions among these proteins that are important for virus-entry and virus-induced cell fusion. Our approach is two-fold: first, a genetic component focuses on the identification of domains of viral proteins that interact with gB and FF via the isolation of intergenic and intragenic suppressor mutations of the gB-Syn1, and FF-Syn3 mutations and other mutations that enhance fusion at low pH conditions. Intragenic and intergenic suppressor mutations of the Syn1 and Syn3 loci will be isolated from viruses grown in the presence of mutagens. In addition, we will isolate mutations in HFEM(tsB5; gB-Syn3) and mP(MP; FF-Syn1) viruses that enable them to cause extensive fusion at pH 6.5. These mutations will be mapped on the viral genome in marker-rescue and marker- transfer experiments and mutations will be identified by DNA sequencing. Second, a physical component aims to assess the effect of co-expression of gB, gD, gH and FF on their synthesis, subcellular localization and transport to the cell surface and further aims to develop a virus-dependent model system for cell fusion to investigate physical interactions among these proteins. Glycoproteins gB, gB(Syn3), gD, gH, fusion factor(FF), and FF(Syn1) will be cloned into SV40 transient expression vectors and co-expressed in COS cells and Vero cells to define different combinations of these proteins that cause cell fusion. A permanent cell line will be constructed expressing a combination of proteins that cause cell fusion under inducible conditions. The synthesis, maturation and transport of each protein to the cell surface will be examined. Amino-terminal and carboxy- terminal truncations of each of the proteins will be used in complementation experiments to define interacting domains. A cell line transformed with a DNA fragment that codes for the FF protein will be used to isolate a FF-null virus and the intracellular distribution of the FF will be investigated.

tissue /cell culture

DESCRIPTION (provided by applicant): The LSU -Tulane Center for Experimental Infectious Diseases Research (CEIDR) was established as a NIH-NCRR-supported Center of Biomedical Research Excellence (COBRE) 1P20RR020159-01 in 2004, as a strategic alliance between LSU Baton Rouge (flagship campus of the LSU System) and the Tulane National Primate Research Center (TNPRC). The overall vision of the Center has been to establish a regional center in infectious disease research led by the LSU School of Veterinary Medicine (SVM) collaborating and influenced by the substantial NIH investment in infectious disease research carried out at TNPRC. Recently, LSU-SVM and TNPRC signed a memorandum of understanding for hiring a joint faculty member and negotiations are under way to appoint a TNPRC junior faculty member in this position. After nine years of COBRE support, we are now within reach to realize our goal to develop an interdisciplinary and collaborative program in infectious diseases that addresses fundamental questions about infectious disease agents and their host interactions. CEIDR's strong mentoring has enabled 12 junior faculty during the last nine years to obtain independent NIH funding (Apetrei, Cormier, Batra, Jeyaseelan, Maclean, Kaushal, Macaluso, Liang, Mores, Pahar, Sugimoto, Embers). Additionally CEIDR has recently recruited two senior NIH-funded faculty (Martinez-LSU and Panganiban (TNPRC), while one junior faculty was recruited by NIH for a permanent staff scientist position (Peterson). Inspite of significant progress, CEIDR has not yet become a fully independent center envisioned. Our vision will be realized by completing three specific aims:1)To provide administrative and scientific infrastructure and resources to CEIDR - affiliated investigators through continued support of four essential Cores: Administrative Core (AC), GeneLab Core (GeneLab), Protein Core Laboratory(PCL), Molecular Immunopathology and Microscopy Core (MIPC); 2) promote and evaluate career development and enhance research potential of CEIDR - affiliated investigators; 3)transition CEIDR to a nationally recognized, interdisciplinary Infectious Disease Center of Excellence by strengthening collaborative research and academic relationships between LSU and TNPRC.

Alphaherpesvirinae; glycoproteins; membrane fusion; host organism interaction; biological transport; Primates; animal colony;

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Administrative Core consists of Dr. Kousoulas and an administrative assistant. Due to the involvement in the COBRE on Experimental Infectious Disease Research of multiple TNPRC investigators as mentors as well as the inclusion of a research project (Apetrei) and the Non-Human Primate/Laboratory Core (Bohm), Dr. Lackner will also be a member of the Administrative Core. He will be directly involved in an advisory capacity in decisions with regard to administration of the COBRE including the resolution of fiscal, personnel and other matters affecting the operation of both LSU and TRPRC parts of the COBRE. The Administrative Core will be responsible for collecting and providing written reports to the External Advisory Committee (EAC) about the performance of individual projects including performance reports for both the LSU and Tulane Core Laboratories. The AC will organize and facilitate bi-yearly meetings of the EAC and IAC committees and will provide all subproject investigators with summaries of evaluations by both committees. The EAC and IAC committees will also evaluate the COBRE Core Laboratories (LSU-MBCL and TRPRC-NHPCL) and provide suggestions for improving their operations and services to the COBRE projects. The specific aims of the administrative core are: (1) To provide administrative supervision and monitoring of budgetary and infrastructure support operations for all COBRE activities;(2) To facilitate a continuous and vigorous review of all COBRE operations mediated by both the EAC and IAC;(3) To facilitate communication among individual laboratories and researchers;(4) To oversee the operation of the COBRE Core Laboratories and their service performance to the COBRE research projects;(5) To facilitate the review and selection of additional projects by the EAC for inclusion into the COBRE.

DESCRIPTION (provided by applicant): Herpesviruses are etiologic agents of important human diseases including certain cancers. Herpes simplex virus type 1 (HSV-1) is a frequent cause of adult sporadic encephalitis, keratoconjunctivitis and oral mucocutaneous lesions;whereas, herpes simplex virus type 2 (HSV-2) causes genital lesions, meningitis and neonatal infections. HSV infects over 300,000 individuals annually, and they are second only to trauma as a cause of corneal blindness in the U.S.A. Herpes simplex virions acquire viral envelopes via a complicated, sequential de-envelopment and re-envelopment process, in which interactions between tegument proteins and viral glycoproteins embedded in intracellular membranes play important roles. The multi-membrane spanning proteins, UL20p and gK, have been directly implicated in intracellular virion envelopment, since deletion of either gene results in drastic accumulation of unenveloped capsids in the cytoplasm. In addition, both UL20p and gK harbor syncytial mutations suggesting that they are involved in membrane fusion phenomena during virion envelopment and egress, as well as virus-induced cell fusion. We have shown that HSV-1 gK and UL20p are co-dependent for cell-surface expression and TGN localization, and that they physically interact. In addition, we have found that UL20p can physically interact with gB. The proposed investigations focus on the role of herpes simplex virus (HSV) glycoprotein K (gK) and UL20 protein (UL20p) in virus-induced cell fusion and cytoplasmic virion envelopment. The main hypotheses of this grant application are: i) gK and UL20p interact and regulate gB-mediated cell fusion;ii) gK and UL20p function as virion structural components in cytoplasmic virion envelopment by binding to tegument proteins. The specific aims are: I) to characterize UL20p interactions with gK and investigate their coordinate transport to cell-surfaces, endocytosis and TGN co-localization;II) to investigate the role of UL20p interactions with gK and gB on gB-mediated virus-induced cell fusion;III) to investigate the role of UL20p in cytoplasmic virion envelopment. Elucidations of functional interrelationships between gK, UL20p and gB as well as between gK, UL20p and tegument proteins will help define viral protein-protein interactions involved in infectious virus production and may assist in the development of new antiviral strategies.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Louisiana State University COBRE Molecular Immunopathology (MIP) Core is a consortium arrangement of five existing Centralized Facilities and Service Units of the LSU-SVM working together to provide state-of-the art service, as well as to develop new technologies to detect, diagnose and analyze the effect of infectious disease pathogens in vitro and in vivo. Dr. Xavier Alvarez (TNPRC) and Dr. David Scollard (NHDP) will participate in the MIP Core. Their participation ensures technology transfer of state-of-the art confocal and molecular pathological technologies already implemented for non-human primate and other animal models to researchers at LSU-SVM and the COBRE-CEIDR. The MIP Consortium is composed of the following existing SVM Centralized Facilities: 1) BIOMMED concentrates on molecular biology and currently provides advanced recombinant DNA and cDNA library constructions, real-time PCR, DNA sequencing, BioPlex lymphokine profiling, protein production and purification, custom antibody production etc. 2) The Centralized Pathology Service Laboratory currently provides routine anatomical, clinical and molecular pathology, the later including immuno-histochemical approaches to detect pathogens and specialized cells. A specialized Unit wihin this Service Laboratory has been formed under the direction of Dr. Nobuko Wakamatsu (Board Certified Pathologist). This unit will be an intimate part of the MIP Core and will serve as liason to other pathology services. 3) the FACS Facility provides advanced immunological assays for lymphokine profiling, cell sorting, etc. 4) The Microscopy Center provides, transmission and electron microscopy, confocal microscopy and laser capture microscopy. New assays that will be developed by the MIP Core include the use of in situ hybridization to detect, viral , rickettsial and bacterial pathogens, tissue immunoprofiling to determine via confocal and immuno-histochemistry the nature of infiltrating immune cells within diseased tissues, etc. 5) The Biostatistics Service Unit provides statistical consultation and training to LSU-based researchers. It is expected that MIP will be a strong supporting Core of the LSU-SVM-based COBRE-CEIDR in future years receiving support from LSU-SVM, as well as other state and federal sources.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. MCBC is an infrastructure and coordination core of the INBRE, which is jointly operated with the Molecular Immunopathology Core (MIPC) of the LSU-Tulane Center for Experimental Infectious Disease Research (CEIDR) supported by a NCRR COBRE program grant. Both Cores are operated by the Division of Biotechnology and Molecular Medicine (BioMMED) of the LSU School of Veterinary Medicine. Dr. Kousoulas, Director of BioMMED serves also as director of both Cores. Molecular Cell Biology provides an essential linkage among important basic fields of biomedical sciences, such as genetics, developmental biology, structural biology, immunology, neurobiology and cancer biology. The elucidation of the human and other animal genomes in conjunction with analysis of all forms of the encoded proteins has enabled the identification of all the gene products that are potentially synthesized by an organism. The vast information generated by genomic sequencing and proteomic analysis is currently effectively mined by advanced biocomputation/bioinformatics approaches, which can connect sequence information (genomics) and protein identification (proteomics) to physiological function within the context of the cell. Despite these global genomic and proteomic advances molecular biology approaches and methodologies are still very much needed to understand the structure and function of individual proteins encoded by infectious disease pathogens (viruses, bacteria, parasites) as well as by different eukaryotic cell types. Recombinant DNA technologies are also increasingly utilized to produce large amounts of proteins with defined amino acids to facilitate elucidation of their three dimensional structure by physical means including x-ray crystallography and nuclear magnetic resonance. MCBC provides infrastructure and technical support to INBRE PUI campuses and individual investigators. Typically, PUI campuses do not have access to highly expensive equipment such as FACS, confocal and laser capture microscopy, electron microscopy, genomic and proteomic capabilities, etc. Furthermore, for those investigators that utilize experimental animals in research, resources for housing different types of experimental animals are very limited. Sophisticated imaging and other medical equipment that can be used to facilitate research with animals such as in vivo chemiluminescence and fluorescence imaging, x-ray tomography, etc. are typically highly expensive and absent from PUI campuses. Therefore the main goal of the MCBC core is to provide the sophisticated equipment and expertise of the LSU flagship campus in Baton Rouge to train and support all INBRE investigators'needs. Although the MCBC/MIPC provides the bulk of training and support to PUI investigators, this Core coordinates and facilitates research access and support provided by other centralized facilities in the LSU system and elsewhere in Louisiana to structure the greatest possible intellectual platform upon which innovative and creative ideas will merge with state-of-the-art technologies. The specific aims of the MCBC are: Specific Aim I: To support the research efforts and activities of all INBRE investigators by providing advanced molecular cell biology, immunology and pathology services and training and access to LSU system-wide state-of-the-art equipment and resources. Specific Aim II: To coordinate and facilitate interactions between INBRE and COBRE investigators and promote a mentoring environment for both INBRE and COBRE investigators led by NIH-funded investigators.

This CC-NIE Integration project performed at the Louisiana State University is developing a cyber-infrastructure integrating six different large scale scientific research groups with high performance computing (HPC) clusters at LSU using software defined network technology and Hadoop and MPI (Message Passing Interface) based parallel frameworks.

The project consists of three objectives: (i) Building 10Gbps software-defined network (SDN) with OpenFlow switches and controllers to provide multiple virtual network slices to each group; (ii) Transferring Big Data with automatically tuned operation through multiple optical paths over the SDN network achieving at most 20Gbps of aggregated disk-to-disk transfer rate; and (iii) Analyzing Big Data by developing data-intensive distributed computing frameworks with Hadoop and MPI technologies parallelizing large number of jobs over HPC clusters.

Those three components are integrated with a web portal service and a GENI-enabled network management system. To achieve high disk-to-disk transfer rate at 20Gbps, the project has an industrial partnership with Samsung Electronics that contributes 70TB Solid State Drive (SSD) storage and optimizes the I/O bandwidth.

The cyber-infrastructure accelerates the current Big Data research projects spanning a wide range of research areas including gene sequencing research at Biology and Vet School, computational chemistry, big data mining at Computer Science, coastal hazard simulation research at civil and environmental engineering. In the end, the project will establish a methodology to build integrated cyber-infrastructures consisting of high speed networks, high performance computing, and high speed storage for the Big Data Science and Engineering.

The two overlapping thematic research areas ofthe Centerfor Experimental Infectious Disease Research (CEIDR) have been vector-borne infectious diseases and immunopathogenesis of infectious diseases. We foresee that 5-7 additional faculty will be hired, including joint appointments between the LSU School of Veterinary Medicine (LSU-SVM) and the Tulane National Primate Research Center (TNPRC), during the next five years. To facilitate the main goal of achieving a critical mass of NIH-funded investigators, the Phase III COBRE application includes a Research Pilot Project Program that has a two-fold purpose. Firstly, it will provide funding for junior investigators who will pursue initial research efforts under the mentorship of two senior CEIDR investigators, as with the COBRE phases I and II. These pilot projects will be monitored and evaluated by the Intemal Scientific Advisory Committee (ISAC), the Extemal Advisory Committee (EAC) and the Administrative Core (AC). Secondly, the pilot project program will provide for additional collaborative research efforts between LSU and TNPRC-based scientists working in shared principal investigator arrangements. The main goal ofthe pilot research program is to rapidly increase the number of ROI applications and amount of NIH funding through strong mentoring of new investigators and financial support of collaborative projects that have high potential for NIH funding. All pilot projects w\\\ receive priority for core support services providing each project with strong research tools to help them succeed within the one year allotted to each pilot project. A second year of pilot funding will only be considered after review by the ISAC, EAC and AC for projects that accomplish outstanding results, as evidenced by recent publications on the subject matter of each pilot, or that have received review of a NIH-submitted application that scores within 10 percentile points of funding. We anticipate the funding of two collaborative projects at $75,000 each and two pilot projects at $50,000 annually. Core charges for each pilot project will be subsidized at 50% of cost. This strategy will sustain core equipment and services by involving new investigators who are expected to continue using these facilities as they succeed in obtaining extramural funding on a fee-for-service basis.

The GeneLab Core Facility provides molecular biology support, reagents and services to all Centerfor Experimental Infectious Disease Research (CEIDR) investigators. GeneLab sen/ices include general support and state-of-the art next generation sequencing (NGS) and bioinformatics support services. Of primary importance to all CEIDR faculty at Louisiana State University (LSU) and the Tulane National Primate Research Center (TNPRC) is the relatively new (one year) GeneLab state-of-the-art services, which are devoted to NGS and bioinformatics. These activities were started after consensus decisions were reached among CEIDR researchers that they were critically needed by ongoing NIH-funded research projects, as well as the entire Louisiana-based NIH-funded community. NGS equipment includes a Life Technologies, Inc. equipment suite with an Ion PGM and Ion Proton Sequencing Machines, as well as a Quant Studio Flex system equipped with digital PCR capabilities. GeneLab NGS activities are focused on confirmation and sequencing of viral and bacterial genomes, as well as the determination of pathogen and host transcriptional changes during host-pathogen interactions. Bioinformatics support services are provided through a collaboration with the Center for Computation and Technology (CCT), and the Louisiana Biomedical Research Network (LBRN), which is supported by the NIH: NIGMS INBRE grant P20GM103424. GeneLab is currently staffed with 5 PhD-level scientists, one research associate, two computer science graduate students and two undergraduate student workers. In addition, James Cavalcoli, PhD, Director of the Bionformatics Core at the University of Michigan, Ann Arbor serves as a consultant providing expert advice and support, as well as a resource for state-of-the art bioinformatics knowledge. The GeneLab Core fulfills a highly unmet need in the State of Louisiana, since there is no other similar support Core laboratory in the state.GeneLab's efforts in NGS and bioinformatics are consistent with the NIH Director's roadmap, which emphasizes computational approaches to biology and in our infectious disease research.

The Administrative Core (AC) ofthe Centerfor Experimental Infectious Disease Research (CEIDR) consists of K. G. Kousoulas PhD, (Principal Investigator, Director), Andrew Lackner, DVM, PhD, Co-lnvestigator, CoDirector),Thomas R. Klei, PhD (Co-lnvestigator, Co-Director) and Christopher Mores, PhD, (Co-investigator; Associate Director). Drs. Kousoulas, Lackner and Klei have worked closely over the past nine years in managing all COBRE affairs including resolution of fiscal, personnel and other matters affecting operation ofall Center functions. Dr. Mores is a recent graduate ofthe phase II COBRE. He is the Principal Investigator of a U01 program project grant involving two other universities. Dr. Mores brings a new perspective and experience to CEIDR, and he will be specifically involved in overall core coordination and resolution of inter-institutional and inter-core issues. The COBRE phase III will continue to be administered by the Division of Biotechnology & Molecular Medicine (BIOMMED) ofthe LSU School of Veterinary Medicine (SVM), which is responsible for all fiscal and scientific matters pertaining to the COBRE. The day-to-day operations ofthe COBRE will be facilitated by Dr. Ramesh Subramanian (Coordinator) who will be spending 25% of his time on managing the COBRE, as well as assisting in GeneLab's technical operations. The AC will organize and facilitate annual meetings ofthe EAC, bi-annual meetings ofthe ISAC and a quarterly meeting of Core Directors in which each Core Director will present oral and written reports of current issues relating to the operations of each Core. A yearly workshop on technological aspects of Core sen/ices will be organized and will contain a scientific program, as well as Core presentations on state-of-the-art technologies. All CEIDR pilot projects will be mentored by two senior investigators and evaluated by both ISAC and EAC during their annual meeting. ISAC will lead efforts in organizing program projects or shared R01 applications to NIH in the thematic areas of vector bome diseases and immunopathogenesis of infectious diseases.

? DESCRIPTION (provided by applicant): overall goal of this INBRE renewal application is to continue the facilitation of quality biomedical research activities throughout the State of Louisiana with an emphasis on building research capacity and workforce development. The Louisiana Biomedical Research Network (LBRN) has established a strong foundation for interdisciplinary and interinstitutional research, education, training, and mentoring programs focused on seven primarily undergraduate institutions (PUIs). These PUIs have been chosen as partners based on their interest and their missions' fit with one or more of the three thematic areas of the LBRN: Computational Biology, Molecular Mechanisms of Disease, and Therapeutic and Preventive Medicine. Undergraduate students from seventeen other PUIs have the opportunity to participate in the research outreach program. Faculty and students at PUIs will be mentored and supported by scientists in the flagship, LSU, and five biomedical- research-intensive institutions. A leading-edge cyberinfrastructure connects all of these units. Overlaying all of our activities will be regular metric-driven assessment of all programs, including cores, using internal and external reviewers. Major changes have been made to improve the currently successful LBRN program. These include: improvement and expansion of our cyberinfrastructure; increased interaction with existing state COBREs; and the expansion of the number of PUIs from five to seven, adding one additional historically black college. We will also partner with Louisiana's new IDeA CTR (LA CaTS) to provide more translational science research experiences for PUI faculty and students. Undergraduate research experiences will be expanded to include opportunities during the academic year. As a demonstration of their recognition of the LBRNs importance to the state, the Louisiana Board of Regents has agreed to continue to provide a match of $1.14 million for the new application. Other support from universities brings this total match to more than $2.93 million. With these improvements and this match it is clear that the LBRN will meet its goals and those of the national INBRE program.

PROJECT SUMMARY Molecular and cell biology provide an essential linkage among important basic fields of biomedical science, such as genetics, developmental biology, structural biology, immunology, neurobiology, and cancer biology. In recent years, the application of molecular biological principles, methods, and advances in next generation sequencing (NGS) to characterize genomes and transcriptomes has greatly improved our understanding of various cellular mechanisms. Newly developed methodologies often require access to state-of-the-art equipment and technical expertise. Centralized core facilities such as the Molecular and Cell Biology Resources Core (MCBRC) that offer specialized equipment and services in molecular and cell biology, immunology, immunopathology, and microscopy are often called on to provide critical support for biomedical research. The specific aims of the MCBRC are: To support the research efforts and activities of all LBRN investigators by providing advanced molecular cell biology, microscopy, immunology, and pathology services, and access to state-of-the-art equipment, expertise, and resources. To organize, coordinate, and facilitate interactions among Louisiana Biomedical Research Network (LBRN) investigators, mentors, and students. To promote the development and use of new technologies and assays. And to train primarily undergraduate institution (PUI) faculty, staff, and students in advanced molecular and cellular biology technologies. The MCBRC takes advantage of existing highly organized, centralized services and equipment facilities located primarily at the LSU flagship institution in Baton Rouge, effectively uniting these units toward the common goal of supporting biomedical research performed by PUI investigators. The MCBRC will provide technical and logistical support, enabling the ready exchange of information, ideas, technology, and research capabilities among PUI investigators. MCBRC will ensure that PUI researchers have full access to state-of-the-art equipment and modern research techniques and services. The MCBRC will be administered by the Division of Biotechnology and Molecular Medicine (BioMMED) at the LSU School of Veterinary Medicine.

The overall goal of this INBRE renewal application is to continue the facilitation of quality biomedical research activities throughout the State of Louisiana with an emphasis on building research capacity and workforce development. The Louisiana Biomedical Research Network (LBRN) has established a strong foundation for interdisciplinary and interinstitutional research, education, training, and mentoring programs focused on seven primarily undergraduate institutions (PUIs). These PUIs have been chosen as partners based on their interest and their missions? fit with one or more of the three thematic areas of the LBRN: Computational Biology, Molecular Mechanisms of Disease, and Therapeutic and Preventive Medicine. Undergraduate students from seventeen other PUIs have the opportunity to participate in the research outreach program. Faculty and students at PUIs will be mentored and supported by scientists in the flagship, LSU, and five biomedicalresearch- intensive institutions. A leading-edge cyberinfrastructure connects all of these units. Overlaying all of our activities will be regular metric-driven assessment of all programs, including cores, using internal and external reviewers. Major changes have been made to improve the currently successful LBRN program. These include: improvement and expansion of our cyberinfrastructure; increased interaction with existing state COBREs; and the expansion of the number of PUIs from five to seven, adding one additional historically black college. We will also partner with Louisiana?s new IDeA CTR (LA CaTS) to provide more translational science research experiences for PUI faculty and students. Undergraduate research experiences will be expanded to include opportunities during the academic year. As a demonstration of their recognition of the LBRNs importance to the state, the Louisiana Board of Regents has agreed to continue to provide a match of $1.14 million for the new application. Other support from universities brings this total match to more than $2.93 million. With these improvements and this match it is clear that the LBRN will meet its goals and those of the national INBRE program.

PROJECT SUMMARY The Molecular Biology (MB) Core has been created to meet the need for advanced molecular biology support by PJIs. In particular, access to and experience in utilizing advanced molecular biology that are limited for most PJIs and established NIH-funded investigators. Specifically, MB Core will leverage and expand the resources and expertise of the Genelab Core facility which is comprised of three existing Core Facilities operated by the Division of Biotechnology and Molecular Medicine (BioMMED) at the LSU School of Veterinary Medicine (SVM): Gene expression and sequencing Laboratory (GeneLab), Protein characterization and purification laboratory (PCPL), which is also responsible for generation of monospecific antibodies to proteins of interest, and the Molecular Immunopathology laboratory (MIPL) that provides expertise and access to bioimaging and advanced immunofluorescence and electron microscopy. These three laboratories are currently co-supported by the NIGMS-funded CEIDR-COBRE (Phase III) as well as the Louisiana Biomedical Research Network (LBRN/INBRE). The goal of the MB Core Facility of the Lung COBRE is to provide high quality instrumentation, sample preparation and training in a broad spectrum of fundamental and advanced molecular biology techniques to all COBRE PJIs and other NIH-funded investigators to achieve their research objectives. The MB Core has established 2 specific aims. Aim 1 is to provide the COBRE and other NIH-funded investigators with resources and expertise in molecular and cell biology, including instrumentation, technical support, consultation, and training. Aim 2 is to organize, coordinate, and facilitate interactions among COBRE investigators, mentors, and students, promote the development and use of new technologies and assays, and train PJI faculty, staff, and students in advanced molecular biology technologies.

Alterations and Renovations Project Summary SUBR Vivarium: The Southern University and A&M College, Baton Rouge (SUBR) has the distinction of being seventh in the country for serving minority students and producing BS graduates with PhD degree. In the last five years, SUBR has successfully produced around 1000 BS-degree science graduates (STEM related). However, from the students who enroll in graduate programs, about 12% complete their PhDs. Enhancing both retention and graduation can be strengthened by improving the research infrastructure at SUBR. The Southern University Health Research Center (HRC) was established in 1969 through a matching grant from the Health Research Facilities of the Division of Research Resources of the National Institutes of Health (NIH). It consists of 35 rooms, of these 7 rooms have been dedicated for the vivarium in the HRC. The vivarium facility has not been used in over 10 years, necessitating revival, and the current proposal seeks to reestablish the vivarium facility in the existing space. We are confident that once the animal house in HRC becomes operational again, the SUBR faculty from various departments will be able to engage in research activities and work towards obtaining research support and further enhance biomedical research portfolio. Concomitantly, the animal experimentations at HRC will help in attracting more students and provide greater diversity of faculty, staff and students. The planned renovation will be executed according to the following aims: Specific Aim 1: To upgrade the infrastructure of vivarium facility at SUBR. Specific Aim 2: To develop/enhance a pipeline of investigators that utilize the proposed animal facility at Southern University. GSU Carver Core Laboratory: The overall goal of this project is to modernize the Biological Research Core Laboratory (Room185) in the Carver Hall Science Building Annex through substantial renovation. This space was originally built as a teaching laboratory in 1970 and has never been renovated. The classroom-style laboratory floorplan and casework severely restrict floor space for freestanding equipment and bench space for benchtop instruments. This core laboratory was first established in 2015 to support research through grant funding received from the Louisiana Board of Regents Support Fund. The equipment housed in this room has been enhanced using funding received from the Louisiana Biomedical Research Network. The laboratory currently supports the research and training activities of several faculty members (including two INBRE/LBRN principal investigators) and their students. We propose four specific aims in this renovation: Specific Aim 1: Improve workflow by replacing the existing casework with modern workstations connected to power and Ethernet. Specific Aim 2: Enhance laboratory safety and functionality by installing a new fume hood and solvent cabinet. Specific Aim 3: Boost the recruitment and retention of underrepresented minorities in biological research by providing an up-to-date collaborative research environment. Specific Aim 4: Create a new research space for faculty engaged in biomedical research who are not yet INBRE PIs

Summary Molecular Biology Immunopathology Core. The proposed Center for Pre-Clinical Cancer Research (Cancer COBRE) at Louisiana State University (LSU) focuses on elucidating molecular and cellular mechanisms of human cancer using specialized disease models that are predictive of disease in humans. The Cancer COBRE will focus on devastating human cancers that have poor treatment outcome in patients in need of novel therapeutic strategies. The proposed projects led by highly Promising Junior Investigators (PJIs) require the application of advanced molecular biological and immunopathological principles, methods and particularly advances in genomics, transcriptomics and proteomics, immunology and pathology to delineate key cellular mechanisms for each specific cancer necessitating access to state-of-the-art equipment and technical expertise. The Molecular Biology and Immunopathology Core (MBIPC) has been created to leverage and expand the resources and expertise of the Division of Biotechnology and Molecular Medicine (BioMMED) at the LSU School of Veterinary Medicine (SVM), which administers six Core Facilities: Gene Expression and Sequencing Laboratory (GeneLab), Viral Vectors Laboratory, Protein Characterization and Purification laboratory (PCPL), which is also generates monospecific and monoclonal antibodies to proteins of interest, the Small Animal Imaging Laboratory (SAIL) and the Molecular Immunopathology laboratory (MIPL) that provides expertise in immunopathology and access to the SVM Centralized Facilities (FACS and Pathology Cost Centers), and also provides expertise and access to bioimaging and advanced immunofluorescence and electron microscopy. These laboratories are currently co-supported by the Louisiana Biomedical Research Network (LBRN/INBRE), which also operates the LBRN Bioinformatics Core in close collaboration with the LSU Center for Computation & Technology (CCT). The goal of MBIPC is to provide and coordinate access to state of the advanced instrumentation and services and provide training in a broad spectrum of fundamental and advanced molecular biology and immunopathology to all PJIs and other NIH- funded investigators to achieve their research objectives. MBIPC has identified two specific aims: Aim 1. To provide the Cancer COBRE and other NIH-funded investigators with resources and expertise in molecular and cell biology and immunopathology including instrumentation, technical support, consultation and training, and assistance in developing new methodologies. Aim 2: To organize, coordinate, and facilitate interactions among COBRE investigators and work collaboratively to develop and use new technologies and assays, and train PJIs, faculty, staff, and students in advanced molecular biology and immunopathology technologies.

Project Summary The endothelium barrier regulates the exchange of blood fluid, electrolytes and proteins across the vascular wall, and it is subjected to dynamic remodeling. The function of this barrier is affected by various conditions, including inflammation, diabetes and sepsis. Endothelial barrier dysfunction (EBD) is the hallmark of severe respiratory disease, such as Acute Lung Injury (ALI), Acute Respiratory Distress Syndrome (ARDS) and sepsis. The development of novel therapeutic strategies against these devastating pathologies is of the utmost need. Our endeavors to expose new therapeutic avenues towards EBD-related diseases revealed that Hsp90 inhibitors and GHRH antagonists protect against endothelial barrier dysfunction, and induce the Unfolded Protein Response (UPR). This is a highly conserved molecular machinery, able to initiate protective and repairing cellular responses in human tissues, including the lungs. It consists of three ER transmembrane proteins: IRE1? (inositol-requiring enzyme 1?), PERK (pancreatic endoplasmic reticulum kinase), and ATF6 (activating transcription factor 6). UPR induction due to heat shock protein 90 inhibition or growth hormone releasing hormone antagonists counteracted the Kifunensine (UPR suppressor) ? induced endothelial hyperpermeability in vitro. Our proposal will focus on the effects of ATF6 in lung endothelial barrier function. Specific Aim 1 will associate ATF6 activation with endothelial barrier function, to demonstrate the supportive role of ATF6 against EBD in vitro. Specific Aim 2 will focus on an in vivo model of LPS-induced ALI, to substantiate our in vitro findings. To do so, we will generate ATF6 null ATF6 endothelial specific knock out mice. The outcomes of our study will enrich our current understanding on endothelial barrier regulation, to provide new targets for the management of diseases related to EBD. 1