The Association Between NRMN STAR Grantsmanship Self-Efficacy and Grant Submission.

BACKGROUND: Eliminating the NIH funding gap among underrepresented minorities (URMs) remains a high priority for the National Institutes of Health. In 2014, the National Research Mentoring Network1 Steps Toward Academic Research (NRMN STAR) program recruited postdoctoral, early-stage and junior faculty to participate in a 12-month grant writing and professional development program. The expectation of the program was to increase the number of grant submissions and awards to URM researchers. Although receiving a grant award is the gold standard of NRMN STAR, instilling confidence for postdocs and early-stage faculty to submit an application is a critical first step. Based on our previous study, a sustained increase in trainee self-efficacy score over a 24-month period was observed after completing NRMN STAR. METHODS: The current study sought to determine the association between self-efficacy score and grant submissions among two cohorts of trainees. Grantsmanship Self-Efficacy was measured using a 19-item questionnaire previously described by and used in our own work, which was originally adapted from an 88-item Clinical Research Appraisal Inventory.2 A binary variable was created to identify trainees who submitted an initial or revised proposal vs those who abandoned their proposal or were still writing. Trainees were assessed prior to and following program completion with subsequent assessments at 6 and 12 months beyond participation. RESULTS: As of June 20, 2019, 12 of the 21 (57%) trainees had submitted a grant proposal (eg, NIH, other federal or non-federal grant). For every point increase in 12-month post assessments, Grantsmanship Self-Efficacy scores across all domains had a 44% higher prevalence of submitting a grant after controlling for race, sex, education level, academic rank, research experience, duration of postdoctoral training, institution type, and NRMN STAR cohort. CONCLUSIONS: Our findings demonstrate that NRMN STAR had a positive impact on trainees' confidence in grant writing and professional development activities, which resulted in higher grant submission rates.

The Impact of Grantsmanship Self-Efficacy on Early Stage Investigators of The National Research Mentoring Network Steps Toward Academic Research (NRMN STAR).

The NRMN STAR program was created to address the persistent underrepresentation in grant submissions and receipt of National Institutes of Health (NIH) awards by racial/ethnic minority groups. In our current study, we assessed program impact on trainees' self-efficacy related to grant writing. The program was conducted with two cohorts: one in June 2014 and one in June 2015. We used a 19-item grant writing self-efficacy scale drawn from the 88-item Clinical Research Assessment Inventory of three domains (conceptualizing, designing, and funding a study) to predict whether self-efficacy influences researchers' grant submissions. Trainees were assessed prior to and following program completion with subsequent assessments at 6 and 12 months beyond participation. The majority of trainees were Black (62%), female (62%), and had obtained a PhD (90%). More than half (52%) were assistant professors and 57% had none or <1 year of research experience beyond postdoctoral training. However, 24% of trainees reported no postdoctoral research training. NRMN STAR trainees' self-efficacy significantly improved on all three domains exhibiting a 2.0-point mean change score on two domains (conceptualizing and design) and 3.7 point mean change score on the domain, funding a study. Findings suggest that NRMN's STAR provides impactful, confidence-building training for diverse, early stage investigators with little-to-no skills, experiences, or low self-efficacy in writing research grants.

Use of Proteomic Imaging Coupled With Transcriptomic Analysis to Identify Biomolecules Responsive to Cochlear Injury.

Exposure to noise or ototoxic agents can result in degeneration of cells in the sensory epithelium and auditory nerve, as well as non-sensory cells of the cochlear lateral wall. However, the molecular mechanisms underlying this pathology remain unclear. The purpose of this study was to localize and identify proteins in the cochlea that are responsive to noise or ototoxic exposure using a complementary proteo-transcriptomic approach. MALDI imaging of cochlear sections revealed numerous protein signals with distinct cochlear localization patterns in both cochlear injury models, of which six were chosen for further investigation. A query of proteomic databases identified 709 candidates corresponding to m/z values for the six proteins. An evaluation of mRNA expression data from our previous studies of these injured models indicated that 208 of the candidates were affected in both injury models. Downstream validation analyses yielded proteins with confirmatory distributions and responses to injury. The combined analysis of MALDI imaging with gene expression data provides a new strategy to identify molecular regulators responsive to cochlear injury. This study demonstrates the applicability of MALDI imaging for investigating protein localization and abundance in frozen sections from animals modeling cochlear pathology.

Linear array of multi-substrate tracts for simultaneous assessment of cell adhesion, migration, and differentiation.

Cell migration, which is central to a wide variety of life processes, involves integration of the extracellular matrix (ECM) with the internal cytoskeleton and motor proteins via receptors spanning the plasma membrane. Cell migration can be induced by a variety of signals, including gradients of external soluble molecules, differences in ECM composition, or electrical gradients. Current in vitro methods to study cell migration only test one substrate at a time. Here, we present a method for assessing cell adhesion, migration, and differentiation in up to 20 different test conditions simultaneously, using only minute amounts of target substrate. Our system, which we call the linear array of multi-substrate cell migration assay (LAMA), has two configurations for direct comparison of one or two cell types in response to an array of ECM constituents under the same culture conditions. This culture model utilizes only nanogram amounts of test substrates and a minimal number of cells, which maximizes the use of limited and expensive test reagents. Moreover, LAMA can also be used for high-throughput screening of potential pharmaceuticals that target ECM-dependent cell behavior and differentiation.

Neural stem/progenitor cell properties of glial cells in the adult mouse auditory nerve.

The auditory nerve is the primary conveyor of hearing information from sensory hair cells to the brain. It has been believed that loss of the auditory nerve is irreversible in the adult mammalian ear, resulting in sensorineural hearing loss. We examined the regenerative potential of the auditory nerve in a mouse model of auditory neuropathy. Following neuronal degeneration, quiescent glial cells converted to an activated state showing a decrease in nuclear chromatin condensation, altered histone deacetylase expression and up-regulation of numerous genes associated with neurogenesis or development. Neurosphere formation assays showed that adult auditory nerves contain neural stem/progenitor cells (NSPs) that were within a Sox2-positive glial population. Production of neurospheres from auditory nerve cells was stimulated by acute neuronal injury and hypoxic conditioning. These results demonstrate that a subset of glial cells in the adult auditory nerve exhibit several characteristics of NSPs and are therefore potential targets for promoting auditory nerve regeneration.

Imbalanced expression of Vcan mRNA splice form proteins alters heart morphology and cellular protein profiles.

The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart's pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican's expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan ((tm1Zim)), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan ((tm1Zim)) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles.

Sox2 up-regulation and glial cell proliferation following degeneration of spiral ganglion neurons in the adult mouse inner ear.

In the present study, glial cell responses to spiral ganglion neuron (SGN) degeneration were evaluated using a murine model of auditory neuropathy. Ouabain, a well-known Na,K-ATPase inhibitor, has been shown to induce SGN degeneration while sparing hair cell function. In addition to selectively removing type I SGNs, ouabain leads to hyperplasia and hypertrophy of glia-like cells in the injured auditory nerves. As the transcription factor Sox2 is predominantly expressed in proliferating and undifferentiated neural precursors during neurogenesis,we sought to examine Sox2 expression patterns following SGN injury by ouabain. Real-time RT-PCR and Western blot analyses of cochlea indicated a significant increase in Sox2 expression by 3 days posttreatment with ouabain. Cells incorporating bromodeoxyuridine(BrdU) and expressing Sox2 were counted in the auditory nerves of control and ouabain-treated ears. The glial phenotype of Sox2+cells was identified by two neural glial markers: S100 and Sox10. The number of Sox2+ glial cells significantly increased at 3 days post-treatment and reached its maximum level at 7 days post-treatment. Similarly,the number of BrdU+ cells increased at 3 and 7 days post-treatment in the injured nerves. Quantitative analysis with dual-immunostaining procedures indicated that about 70% of BrdU+ cells in the injured nerves were Sox2+ glial cells. These results demonstrate that up-regulation of Sox2 expression is associated with increased cell proliferation in the auditory nerve after injury.

Molecular morphology of the chick heart visualized by MALDI imaging mass spectrometry.

Utilization of MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry) for tissue imaging is a relatively new proteomic technique that simultaneously maps the spatial distribution of multiple proteins directly within a single frozen tissue section. Here, we report the development of a methodology to apply MALDI tissue imaging to chick heart tissue sections acquired from fixed and paraffin-embedded samples. This protocol produces molecular images that can be related to the high-quality histological tissue sections. Perfused term chick hearts were fixed in acidic ethanol and embedded in paraffin wax. Tissue sections (15 microm) were collected onto conductive slides, deparaffinized with xylene, and transitioned into water with graded ethanol washes and allowed to air dry. In separate experiments, three different MALDI matrices were applied to chick heart tissue sections through repeated cycles from a glass nebulizer. Tissue sections were then analyzed by MALDI mass spectrometry using a raster step-size of 75-100 microm, and molecular images for specific m/z ratios reconstituted. MALDI tissue imaging revealed spatially resolved protein signals within single heart sections that are specific to structures or regions of the heart, for example, vessels, valves, endocardium, myocardium, or septa. Moreover, no prior knowledge of protein expression is required as is the case for immunohistochemistry and in situ hybridization methodologies. The ability to simultaneously localize a large number of unique protein signals within a single tissue section, with good preservation of histological features, provides cardiovascular researchers a new tool to give insight into the molecular mechanisms underlying normal and pathological conditions.

iQuantitator: a tool for protein expression inference using iTRAQ.

BACKGROUND: Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) [Applied Biosystems] have seen increased application in differential protein expression analysis. To facilitate the growing need to analyze iTRAQ data, especially for cases involving multiple iTRAQ experiments, we have developed a modeling approach, statistical methods, and tools for estimating the relative changes in protein expression under various treatments and experimental conditions. RESULTS: This modeling approach provides a unified analysis of data from multiple iTRAQ experiments and links the observed quantity (reporter ion peak area) to the experiment design and the calculated quantity of interest (treatment-dependent protein and peptide fold change) through an additive model under log transformation. Others have demonstrated, through a case study, this modeling approach and noted the computational challenges of parameter inference in the unbalanced data set typical of multiple iTRAQ experiments. Here we present the development of an inference approach, based on hierarchical regression with batching of regression coefficients and Markov Chain Monte Carlo (MCMC) methods that overcomes some of these challenges. In addition to our discussion of the underlying method, we also present our implementation of the software, simulation results, experimental results, and sample output from the resulting analysis report. CONCLUSION: iQuantitator's process-based modeling approach overcomes limitations in current methods and allows for application in a variety of experimental designs. Additionally, hypertext-linked documents produced by the tool aid in the interpretation and exploration of results.

Is the training of biomedical scientists at a crossroads?

In this commentary, the authors respond to the allegation that the title "scientist" has lost much of its classical meaning because of the highly specialized nature of biomedical graduate training programs that produce "researchers" and "supertechnologists." Scientists, by this definition, have a firm grasp of the historical, philosophical, and biological contexts in which their work exists, whereas their researcher and supertechnologist counterparts are limited by narrowly focused, technologically driven experimentation and data collection with little knowledge or appreciation of the integrated nature of biological systems and the historical basis of discovery. With these definitions in mind, the authors discuss how to ensure that innovative thinking and the ability to integrate molecular knowledge into a higher-order context remain alive and well, complementing today's highly technological environment. In this regard, examples of new emphasis from both scientific societies and funding agencies are provided. However, effective mentoring strategies, practiced on a daily basis, remain the best means for assuring that narrowly focused researchers and supertechnologists do not become the norm of the future. Technological innovation is critical for acquiring new insight into fundamental questions, but using that information for a greater understanding will always favor the prepared intellect. Multidisciplinary teams are emerging as the future of biomedical research. The authors propose a course of action to ensure that trainees are given the necessary opportunities and guidance to help them function effectively in a contemporary teamwork environment with scientific reasoning and logic at its core.

Chick embryos exposed to trichloroethylene in an ex ovo culture model show selective defects in early endocardial cushion tissue formation.

BACKGROUND: Formation of the primitive heart is a critical step for establishing a competent circulatory system necessary for continued morphogenesis, and as such has significant potential as a target for environmental insult. The goal of this study was to identify the initial cellular events that precede more superficially observable abnormalities resulting from exposing early chick embryos to trichloroethylene (TCE). METHODS: A whole embryo culture method was used to assess the susceptibility of endocardial epithelial-mesenchymal transformation in the early chick heart to TCE. This method has the benefits of maintaining the anatomical relationships of developing tissues and organs, instantaneously exposing precisely staged embryos to quantifiable levels of TCE in a protein-free medium, and the ability to directly monitor developmental morphology. RESULTS: A minority of embryos (Hamburger and Hamilton [HH] stage 13-14) exposed to TCE (10-80 ppm) were not viable after 24 hr in culture and exhibited a variety of gross malformations in a dose-dependent fashion. However, the majority of treated embryos remained viable and developed into HH stage 17 embryos that were superficially indistinguishable from vehicle-treated controls. Further analysis of the hearts of these superficially normal embryos by whole-mount confocal microscopy revealed selective reduction in the number of atrioventricular canal mesenchymal cells. Additionally, those mesenchymal cells that did develop migrated abnormally as long thin cords of adherent cells. CONCLUSIONS: The regional selectivity of these effects in the chick heart suggests a critical window of susceptibility to TCE in the epithelial-mesenchymal transformation of atrioventricular canal endocardium.

Immunolocalization of chick periostin protein in the developing heart.

The process that cardiac cushions undergo to form the mature septa and valves of the adult heart is poorly understood. Periostin is an extracellular molecule that is expressed during cushion mesenchyme formation and throughout valvulogenesis. Once thought to be an osteoblast-specific factor, studies have shown this molecule is antiosteogenic. We have produced an antibody to chicken periostin and examined periostin's localization in the developing avian heart. This antibody recognized proteins from chick heart lysates around 90 kD molecular weight as predicted from the chick periostin mRNA and other periostin orthologs. Periostin immunolocalization was first evident as fibrous strands in the cushion mesenchyme. At HH25, periostin was detected on the basal surface of the trabecular endothelium and also on the endocardial epithelium of the atrioventricular cushion. We hypothesize that periostin may function in the organization of extracellular matrix molecules, providing cues necessary for attachment and spreading during the epithelial-to-mesenchymal transitions of the endocardial epithelium. Enhanced secretion of periostin in the region of delamination may directly or indirectly promote change in the myocardium that precedes or mediates delamination of the leaflet. At later stages of development (HH34-38), periostin was seen predominantly in the fibrous regions of the heart, such as the left atrioventricular valve (LAV), annulus, cardiac skeleton, and adventitia. We propose that periostin is induced by sheer stress and may be an essential molecular component for structures of the heart that undergo mechanical stress or tension during the cardiac cycle.