Background The lack of race/ethnic and gender diversity in grants funded by the National Institutes of Health (NIH) is a persistent challenge related to career advancement and the quality and relevance of health research. We describe pilot programs at nine institutions supported by the NIH-sponsored Building Infrastructure Leading to Diversity (BUILD) program aimed at increasing diversity in biomedical research. Methods We collected data from the 2016-2017 Higher Education Research Institute survey of faculty and NIH progress reports for the first four years of the program (2015-2018). We then conducted descriptive analyses of data from the nine BUILD institutions that had collected data and evaluated which activities were associated with research productivity. We used Poisson regression and rate ratios of the numbers of BUILD pilots funded, students included, abstracts, presentations, publications, and submitted and funded grant proposals. Results Teaching workshops were associated with more abstracts (RR 4.04, 95% CI 2.21-8.09). Workshops on grant writing were associated with more publications (RR 2.64, 95% CI 1.644.34) and marginally with marginally more presentations. Incentives to develop courses were associated with more abstracts published (RR 4.33, 95% CI 2.56-7.75). Workshops on research skills and other incentives were not associated with any positive effects. Conclusions Pilot interventions show promise in supporting diversity in NIH-level research. Longitudinal modeling that considers time lags in career development in moving from project development to grants submissions can provide more direction for future diversity pilot interventions.

Background: Several mechanisms of action have been proposed to explain the apparent antineoplastic functions of metformin, many of which are observed at high concentrations that may not be reflective of achievable tissue concentrations. We propose that metformin at low concentrations functions to inhibit ROS production and inflammatory signaling in breast cancer, thereby reducing metastasis. Methods: Using the highly invasive MDA-MB-231 breast carcinoma model, we ascertained the impact of metformin on cell viability by DNA content analysis and fluorescent dye exclusion. Migration and invasion assays were performed using a modified Boyden chamber assay and metastasis was ascertained using the chorioallantoic membrane (CAM) assay. PGE2 production was measured by Enzyme-Linked Immunosorbent Assay (ELISA). COX2 and ICAM1 levels were determined by flow cytometry immunoassay. Results: Metformin acutely decreased cell viability and caused G2 cell cycle arrest only at high concentrations (10 mM). At 100 µM, however, metformin reduced ICAM1 and COX2 expression, as well as reduced PGE2 production and endogenous mitochondrial ROS production while failing to significantly impact cell viability. Consequently, metformin inhibited migration, invasion in vitro and PGE2-dependent metastasis in CAM assays. Conclusion: At pharmacologically achievable concentrations, metformin does not drastically impact cell viability, but inhibits inflammatory signaling and metastatic progression in breast cancer cells.

Patients with advanced epithelial ovarian cancer often experience disease recurrence after standard therapies, a critical factor in determining their five-year survival rate. Recent reports indicated that long-term or short-term survival is associated with varied gene expression of cancer cells. Thus, identification of novel prognostic biomarkers should be considered. Since the mouse genome is similar to the human genome, we explored potential prognostic biomarkers using two groups of mouse ovarian cancer cell lines (group 1: IG-10, IG-10pw, and IG-10pw/agar; group 2: IG-10 clones 2, 3, and 11) which display highly and moderately aggressive phenotypes in vivo. Mice injected with these cell lines have different survival time and rates, capacities of tumor, and ascites formations, reflecting different prognostic potentials. Using an Affymetrix Mouse Genome 430 2.0 Array, a total of 181 genes were differentially expressed (P < 0.01) by at least twofold between two groups of the cell lines. Of the 181 genes, 109 and 72 genes were overexpressed in highly and moderately aggressive cell lines, respectively. Analysis of the 109 and 72 genes using Ingenuity Pathway Analysis (IPA) tool revealed two cancer-related gene networks. One was associated with the highly aggressive cell lines and affiliated with MYC gene, and another was associated with the moderately aggressive cell lines and affiliated with the androgen receptor (AR). Finally, the gene enrichment analysis indicated that the overexpressed 89 genes (out of 109 genes) in highly aggressive cell lines had a function annotation in the David database. The cancer-relevant significant gene ontology (GO) terms included Cell cycle, DNA metabolic process, and Programmed cell death. None of the genes from a set of the 72 genes overexpressed in the moderately aggressive cell lines had a function annotation in the David database. Our results suggested that the overexpressed MYC and 109 gene set represented highly aggressive ovarian cancer potential biomarkers while overexpressed AR and 72 gene set represented moderately aggressive ovarian cancer potential biomarkers. Based on our knowledge, the current study is first time to report the potential biomarkers relevant to different aggressive ovarian cancer. These potential biomarkers provide important information for investigating human ovarian cancer prognosis.

Xavier University of Louisiana leads the nation in awarding BS degrees in the biological sciences to African-American students. In this multiyear study with ∼5500 participants, data-driven interventions were adopted to improve student academic performance in a freshman-level general biology course. The three hour-long exams were common and administered concurrently to all students. New exam questions were developed using Bloom’s taxonomy, and exam results were analyzed statistically with validated assessment tools. All but the comprehensive final exam were returned to students for self-evaluation and remediation. Among other approaches, course rigor was monitored by using an identical set of 60 questions on the final exam across 10 semesters. Analysis of the identical sets of 60 final exam questions revealed that overall averages increased from 72.9% (2010) to 83.5% (2015). Regression analysis demonstrated a statistically significant correlation between high-risk students and their averages on the 60 questions. Additional analysis demonstrated statistically significant improvements for at least one letter grade from midterm to final and a 20% increase in the course pass rates over time, also for the high-risk population. These results support the hypothesis that our data-driven interventions and assessment techniques are successful in improving student retention, particularly for our academically at-risk students.

The association of B7-1/CD28 between antigen presenting cells (APCs) and T-cells provides a second signal to proliferate and activate T-cell immunity at the induction phase. Many reports indicate that tumor cells transfected with B7-1 induced augmented antitumor immunity at the induction phase by mimicking APC function; however, the function of B7-1 on antitumor immunity at the effector phase is unknown. Here, we report direct evidence of enhanced T-cell antitumor immunity at the effector phase by the B7-1 molecule. Our experiments in vivo and in vitro indicated that reactivity of antigen-specific monoclonal and polyclonal T-cell effectors against a Lass5 epitope presented by RMA-S cells is increased when the cells expressed B7-1. Use of either anti-B7-1 or anti-CD28 antibodies to block the B7-1/CD28 association reduced reactivity of the T effectors against B7-1 positive RMA-S cells. Transfection of Lass5 cDNA into or pulse of Lass5 peptide onto B7-1 positive RMA-S cells overcomes the requirement of the B7-1/CD28 signal for T effector response. To our knowledge, the data offers, for the first time, strong evidence that supports the requirement of B7-1/CD28 secondary signal at the effector phase of antitumor T-cell immunity being dependent on the density of an antigenic peptide.

BACKGROUND:KSHV is a tumorigenic γ-herpesvirus that has been identified as the etiologic agent of Kaposi's sarcoma (KS), a multifocal highly vascularized neoplasm that is the most common malignancy associated with acquired immunodeficiency syndrome (AIDS). The virus encodes a constitutively active chemokine receptor homologue, vGPCR that possesses potent angiogenic and tumorigenic properties, and is critical for KSHV pathobiology. To date, a number of signaling pathways have been identified as key in mediating vGPCR oncogenic potential.FINDINGS:In this study, we identify a novel pathway, the Wnt/β-catenin pathway, which is dysregulated by vGPCR expression in endothelial cells. Expression of vGPCR in endothelial cells enhances the nuclear accumulation of β-catenin, that correlates with an increase in β-catenin transcriptional activity. Activation of β-catenin signaling by vGPCR is dependent on the PI3K/Akt pathway, as treatment of vGPCR-expressing cells with a pharmacological inhibitor of PI3K, leads to a decreased activation of a β-catenin-driven reporter, a significant decrease in expression of β-catenin target genes, and reduced endothelial tube formation.CONCLUSIONS:Given the critical role of Wnt/β-catenin signaling in angiogenesis and tumorigenesis, the findings from this study suggest a novel mechanism in KSHV-induced malignancies.

Fascin has recently emerged as a potential therapeutic target, as its expression in cancer cells is closely associated with tumor progression and metastasis. Following the initial discovery of a series of thiazole derivatives that demonstrated potent antimigration and antiinvasion activities via possible inhibition of fascin function, we report here the design and synthesis of 63 new thiazole derivatives by further structural modifications in search of more potent fascin inhibitors. The 5 series of analogues with longer alkyl chain substitutions on the thiazole nitrogen exhibited greater antimigration activities than those with other structural motifs. The most potent analogue, 5p, inhibited 50% of cell migration at 24 nM. Moreover, the thiazole analogues showed strong antiangiogenesis activity, blocking new blood vessel formation in a chicken embryo membrane assay. Finally, a functional study was conducted to investigate the mechanism of action via interaction with the F-actin bundling protein fascin.

A series of novel pyridine-bridged analogues of combretastatin-A4 (CA-4) were designed and synthesized. As expected, the 4-atom linker configuration retained little cytotoxicities in the compounds 2e, 3e, 3g, and 4i. Activities of the analogues with 3-atom linker varied widely depending on the phenyl ring substitutions, and the 3-atom linker containing nitrogen represents the more favorable linker structure. Among them, three analogues (4h, 4s, and 4t) potently inhibited cell survival and growth, arrested cell cycle, and blocked angiogenesis and vasculature formation in vivo in ways comparable to CA-4. The superposition of 4h and 4s in the colchicine-binding pocket of tubulin shows the binding posture of CA-4, 4h, and 4s are similar, as confirmed by the competitive binding assay where the ability of the ligands to replace tubulin-bound colchicine was measured. The binding data are consistent with the observed biological activities in antiproliferation and suppression of angiogenesis but are not predictive of their antitubulin polymerization activities.

BACKGROUND:There have been conflicting observations regarding the receptors utilized by human multipotent mesenchymal bone marrow stromal cells (hMSC) to adhere to endothelial cells (EC). To address the discrepancies, we performed experiments with cells prepared with a standardized, low-density protocol preserving a sub-population of small cells that are rapidly self-renewing.METHODS:Sialyl Lewis X (SLeX) and α4 integrin expression were determined by flow cytometry. Fucosyltransferase expression was determined by quantitative realtime RT-PCR. Cell adhesion assays were carried out with a panel of endothelial cells from arteries, veins and the microvasculature in vitro. In vivo experiments were performed to determine single cell interactions in the chick embryo chorioallantoic membrane (CAM). The CAM is a well-characterized respiratory organ allowing for time-lapse image acquisition of large numbers of cells treated with blocking antibodies against adhesion molecules expressed on hMSC.RESULTS:hMSC expressed α4 integrin, SLeX and fucosyltransferase 4 and adhered to human EC from arteries, veins and the microvasculature under static conditions in vitro. In vivo, hMSC rolled on and adhered to arterioles in the chick embryo CAM, whereas control melanoma cells embolized. Inhibition of α4 integrin and/or SLeX with blocking antibodies reduced rolling and adhesion in arterioles and increased embolism of hMSC.CONCLUSIONS:The results demonstrated that rapidly self-renewing hMSC were retained in the CAM because they rolled on and adhered to respiratory arteriolar EC in an α4 integrin- and SLeX-dependent manner. It is therefore important to select cells based on their cell adhesion receptor profile as well as size depending on the intended target of the cell and the injection route.

The amphoteric C31G solution contains equimolar alkyl dimethlyglycine and alkyl dimethyl amine oxide buffered with citric acid. C31G acts as a broad spectrum antiviral and an antibacterial. No previous in vivo studies have been done to test C31G in an animal model of HSV-1 ocular keratitis. We assessed the anti-herpetic activity of C31G in the rabbit eye model using three treatment groups: (1) 1% trifluorothymidine (TFT); (2) 0.25% C31G plus 0.5% hydroxypropyl methylcellulose (HPMC); and (3) vehicle, 0.5% HPMC. Scarified rabbit corneas were inoculated with the HSV-1 strain McKrae. On post inoculation (PI) day 3, rabbits were placed in three balanced groups based on slit-lamp examination (SLE) scores. Treatment began on PI day 3, five times a day for five consecutive days. In addition to the daily, masked SLE scoring, the eyes were assessed daily for stromal opacity, scleral inflammation, neovascularization, eyelid inflammation, inflammatory discharge, and epiphora. C31G and TFT were very effective in reducing the lesions and pathogenesis associated with HSV-1 ocular keratitis. The vehicle control scores were significantly higher and did not effectively treat HSV-1 keratitis. C31G has the potential to be used to treat herpetic keratitis as well as other herpetic topical lesions in humans.

Corneal neovascularization (CNV) is a complication of inflammatory and hypoxic ocular pathologies or immune response to ocular infections. CNV is a manifestation of an imbalance between pro-angiogenic and anti-angiogenic signaling molecules. Despite a magnitude of evidence suggesting angiogenic signaling molecules play a pivotal role in modulating CNV, there are insufficient current pharmacologic treatment options for CNV. Thus, targeting signaling molecules appears to be one promising treatment. Multiple drugs have been used off-label and have shown success in treating CNV, most notably the anti-VEGF medications. In addition, novel drugs are being developed and studied for CNV. This article will describe various molecular mechanisms that induce angiogenesis in the cornea, in addition to potential chemotherapies for CNV that inhibit these molecular mechanisms. This review is pertinent but not all-inclusive. The authors will give examples of publications that have reported novel discoveries related to potential future anti-CNV therapies.

BACKGROUND:Kaposi sarcoma-associated herpesvirus (KSHV) encoded G protein-coupled receptor (vGPCR) is a constitutively active lytic phase protein with significant homology to the human interleukin-8 receptor. vGPCR is necessary and sufficient to induce angiogenesis as well as the spindle cell proliferation characteristic of Kaposi sarcoma (KS) lesions. We previously demonstrated that Bcl-2, an antiapoptotic protein, is upregulated in KS lesions. The aim of this study was to determine if vGPCR enhances endothelial cell survival through upregulation of Bcl-2 expression and to elucidate the signaling pathways involved.METHODS:Primary human umbilical vein endothelial cells were transduced with a recombinant retrovirus expressing vGPCR and then subjected to serum starvation. Cell viability and apoptosis were analyzed by fluorescence-activated cell sorting. Bcl-2 expression was determined by real-time quantitative reverse transcription polymerase chain reaction and immunoblotting. Specific pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) were employed to elucidate the signaling pathways involved. Bcl-2 expression was knocked down using small interfering RNA (siRNA).RESULTS:Endothelial cells expressing vGPCR showed increased survival after serum starvation and upregulation of Bcl-2 messenger RNA (mRNA) and protein. The vGPCR-induced increases in both Bcl-2 mRNA and protein levels were dependent on PI3K signaling but not on mTOR. Moreover, siRNA inhibition of Bcl-2 resulted in significant abrogation of the observed vGPCR-mediated cell survival advantage.CONCLUSIONS:Taken together, the results demonstrate that Bcl-2 is a mediator of vGPCR-induced endothelial cell survival and is a downstream effector of Akt in this process.

Rabbits latent with HSV-1 strain McKrae spontaneously shed infectious virus and viral DNA into their tears and develop recurrent herpetic-specific corneal lesions. The rabbit eye model has been used for many years to assess acute ocular infections and pathogenesis, antiviral efficacy, as well as latency, reactivation, and recurrent eye diseases. This study used real-time PCR to quantify HSV-1 DNA in the saliva and tears of rabbits latent with HSV-1 McKrae.

Adipose tissue maintains a subpopulation of cells, referred to as adipose-derived stromal/stem cells (ASCs), which have been associated with increased breast cancer tumorigenesis and metastasis. For ASCs to affect breast cancer cells, it is necessary to delineate how they mobilize and home to cancer cells, which requires mobilization and invasion through extracellular matrix barriers. In this study, ASCs were separated into four different categories based on the donor's obesity status and depot site of origin. ASCs isolated from the subcutaneous abdominal adipose tissue of obese patients (Ob(+)Ab(+)) demonstrated increased invasion through Matrigel as well as a chick chorioallantoic membrane, a type I collagen-rich extracellular matrix barrier. Detailed mRNA and protein analyses revealed that calpain-4, calpastatin, and MMP-15 were associated with increased invasion, and the silencing of each protease or protease inhibitor confirmed their role in ASC invasion. Thus, the data indicate that both the donor's obesity status and depot site of origin distinguishes the properties of subcutaneous-derived ASCs with respect to enhanced invasion and this is associated with the dysregulation of calpain-4, calpastatin, and MMP-15.

Although the importance of human apolipoprotein E (apoE) in vascular diseases has clearly been established, most of the research on apoE has focused on its role in cholesterol metabolism. In view of the observation that apoE and its functional domains impact extracellular matrix (ECM) remodeling, we hypothesized that apoE could also confer protection against ECM degradation by mechanisms independent of its role in cholesterol and lipoprotein transport. The ECM degrading enzyme, heparanase, is secreted by cells as pro-heparanase that is internalized through low-density lipoprotein (LDL) receptor-related protein-1 (LRP-1) to become enzymatically active. Both apoE and pro-heparanase bind the LRP-1. We further hypothesized that an apoE mimetic peptide (apoEdp) would inhibit the production of active heparanase by blocking LRP-1-mediated uptake of pro-heparanase and thereby decrease degradation of the ECM. To test this hypothesis, we induced the expression of heparanase by incubating human retinal endothelial cells (hRECs) with high glucose (30 mM) for 72 hours. We found that elevated expression of heparanase by high glucose was associated with increased shedding of heparan sulfate (ΔHS) and the tight junction protein occludin. Treatment of hRECs with 100 µM apoEdp in the presence of high glucose significantly reduced the expression of heparanase, shedding of ΔHS, and loss of occludin as detected by Western blot analysis. Either eye drop treatment of 1% apoEdp topically 4 times a day for 14 consecutive days or intraperitoneal injection (40 mg/kg) of apoEdp daily for 14 consecutive days in an in vivo mouse model of streptozotocin-induced diabetes inhibited the loss of tight junction proteins occludin and zona occludin- 1 (ZO-1). These findings imply a functional relationship between apoE and endothelial cell matrix because the deregulation of these molecules can be inhibited by a short peptide derived from the receptor-binding region of apoE. Thus, strategies targeting ECM-degrading enzymes could be therapeutically beneficial for treating diabetic retinopathy.

The exact mechanisms of HSV-1 establishment, maintenance, latency, reactivation, and also the courses of recurrent ocular infections remain a mystery. Comprehensive understanding of the HSV-1 disease process could lead to prevention of HSV-1 acute infection, reactivation, and more effective treatments of recurrent ocular disease. Animal models have been used for over sixty years to investigate our concepts and hypotheses of HSV-1 diseases. In this paper we present descriptions and examples of rabbit and mouse eye models of HSV-1 latency, reactivation, and recurrent diseases. We summarize studies in animal models of spontaneous and induced HSV-1 reactivation and recurrent disease. Numerous stimuli that induce reactivation in mice and rabbits are described, as well as factors that inhibit viral reactivation from latency. The key features, advantages, and disadvantages of the mouse and rabbit models in relation to the study of ocular HSV-1 are discussed. This paper is pertinent but not intended to be all inclusive. We will give examples of key papers that have reported novel discoveries related to the review topics.