Efficacy of Centers of Biomedical Research Excellence (CoBRE) grants to build research capacity in underrepresented states.

Federal funding for research has immediate and long-term economic impact. Since federal research funding is regionally concentrated and not geographically distributed, the benefits are not fully realized in some regions of the country. The Established (previously Experimental) Program to Stimulate Competitive Research (EPSCoR) programs at several agencies, for example, the National Science Foundation, and the Institutional Development Award (IDeA) program at the National Institutes of Health were created to increase competitiveness for funding in states with historically low levels of federal funding. The Centers of Biomedical Research Excellence (CoBRE) award program is a component of the IDeA program. The CoBRE grants support research core facilities to develop research infrastructure. These grants also support the research projects of junior investigators, under the guidance of mentoring teams of senior investigators, to develop human resources at these institutions. Few studies have assessed the effectiveness of these programs. This study examines the investment and outcomes of the CoBRE grants from 2000 through 2022. The maturation of junior investigators into independently funded principal investigators is comparable to other mentoring programs supported by NIH. The investment in research cores resulted in substantial research productivity, measured by publications. Despite the successes of individual investigators and increased research infrastructure and productivity, the geographic distribution of federal and NIH research dollars has not changed. These results will be informative in consideration of policies designed to enhance the geographic distribution of federal research dollars.

Training doctoral students in critical thinking and experimental design using problem-based learning.

BACKGROUND: Traditionally, doctoral student education in the biomedical sciences relies on didactic coursework to build a foundation of scientific knowledge and an apprenticeship model of training in the laboratory of an established investigator. Recent recommendations for revision of graduate training include the utilization of graduate student competencies to assess progress and the introduction of novel curricula focused on development of skills, rather than accumulation of facts. Evidence demonstrates that active learning approaches are effective. Several facets of active learning are components of problem-based learning (PBL), which is a teaching modality where student learning is self-directed toward solving problems in a relevant context. These concepts were combined and incorporated in creating a new introductory graduate course designed to develop scientific skills (student competencies) in matriculating doctoral students using a PBL format. METHODS: Evaluation of course effectiveness was measured using the principals of the Kirkpatrick Four Level Model of Evaluation. At the end of each course offering, students completed evaluation surveys on the course and instructors to assess their perceptions of training effectiveness. Pre- and post-tests assessing students' proficiency in experimental design were used to measure student learning. RESULTS: The analysis of the outcomes of the course suggests the training is effective in improving experimental design. The course was well received by the students as measured by student evaluations (Kirkpatrick Model Level 1). Improved scores on post-tests indicate that the students learned from the experience (Kirkpatrick Model Level 2). A template is provided for the implementation of similar courses at other institutions. CONCLUSIONS: This problem-based learning course appears effective in training newly matriculated graduate students in the required skills for designing experiments to test specific hypotheses, enhancing student preparation prior to initiation of their dissertation research.

Introducing conflict resolution and negotiation training into a biomedical sciences graduate curriculum.

BACKGROUND: Analysis of the biomedical workforce and graduate education have produced recommendations for modifications of pre-doctoral training to broadly prepare trainees for wider ranging scientific careers. Development of training in professional skills is widely recommended, but details of implementation are not widely available. In alignment with these recommendations, we have incorporated professional skills training into the biomedical science graduate curriculum at West Virginia University. An important component of the training is developing conflict resolution and negotiation skills. This training will provide useful skills for academic careers, non-academic careers and life situations outside of the workplace. Conflict resolution/negotiation skills are also relevant in managing issues in diversity, equity and inclusivity. We report our experience in developing this component of the training program, provide an overview of the approach to delivery and practice of skills, and provide an analysis of the reception and effectiveness of the training. METHODS: Evaluation of effectiveness of training used the principals of the Kirkpatrick Four Level Model of Evaluation. At the end of the course, students completed a questionnaire about their perceptions of training and were asked how they would respond to different scenarios requiring conflict resolution/negotiation skills. Several months later, students were surveyed to determine if they used some of these skills and/or witnessed situations where these skills would be useful. RESULTS: We report our experience in developing conflict resolution/negotiation training in our graduate curriculum, provide an overview of the approach to delivery and practice of skills, and provide an analysis of the reception and effectiveness of the training. The results suggest this training meets a need and is effective. Importantly, these materials provide a template for others wishing to implement similar training in their curricula. CONCLUSIONS: Conflict resolution and negotiation training meets a need in graduate education. A mixed approach using didactic and interactive components spaced out over time appears to be an effective method of training.

Efficacy of Centers of Biomedical Research Excellence (CoBRE) Grants to Build Research Capacity in Underrepresented States.

Federal funding for research has immediate and long-term economic impact. Since federal research funding is regionally concentrated and not geographically distributed, the benefits are not fully realized in some regions of the country. The Established (previously Experimental) Program to Stimulate Competitive Research (EPSCoR) programs at several agencies, e.g. the National Science Foundation, and the Institutional Development Award (IDeA) program at the National Institutes of Health were created to increase competitiveness for funding in states with historically low levels of federal funding. The Centers of Biomedical Research Excellence (CoBRE) award program is a component of the IDeA program. The CoBRE grants support research core facilities to develop research infrastructure. These grants also support the research projects of junior investigators, under the guidance of mentoring teams of senior investigators, to develop human resources at these institutions. Few studies have assessed the effectiveness of these programs. This study examines the investment and outcomes of the CoBRE grants from 2000 through 2022. The maturation of junior investigators into independently funded principal investigators is comparable to other mentoring programs supported by NIH. The investment in research cores resulted in substantial research productivity, measured by publications. Despite the successes of individual investigators and increase research infrastructure and productivity, the geographic distribution of federal and NIH research dollars has not changed. These results will be informative in consideration of policies designed to enhance the geographic distribution of federal research dollars.

Research productivity and training support for doctoral students in the biological and biomedical sciences.

Training of doctoral students as part of the next generation of the biomedical workforce is essential for sustaining the scientific enterprise in the United States. Training primarily occurs at institutions of higher education, and these trainees comprise an important part of the workforce at these institutions. Federal investment in the support of doctoral students in the biological and biomedical sciences is distributed differently than the distribution of students across different types of institutions, for example, public vs private. Institutions in states that historically receive less federal support for research also receive less support for doctoral student training. Doctorates at different types of institution exhibit little difference in research productivity, with the exception of citations, and subsequent receipt of additional NIH awards. Thus, training outcomes, which are related to the quality of the student and training environment, are similar across different institutions. Research productivity of doctoral students does not correlate with the number of F31s awarded to an institution. Factors that correlate with F31 funding include R01 funding levels and program size. The findings suggest strategies for institutions to increase success at securing F31s and modification of policy to promote more equitable distribution of F31s across institutions.

In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase.

Focal adhesion kinase (FAK), a key regulator of cell adhesion and migration, is overexpressed in many types of cancer. The C-terminal focal adhesion targeting (FAT) domain of FAK is necessary for proper localization of FAK to focal adhesions and subsequent activation. Phosphorylation of Y926 in the FAT domain by the tyrosine kinase Src has been shown to promote metastasis and invasion in vivo by linking the FAT domain to the MAPK pathway via its interaction with growth factor receptor-bound protein 2. Several groups have reported that inherent conformational dynamics in the FAT domain likely regulate phosphorylation of Y926; however, what regulates these dynamics is unknown. In this paper, we demonstrate that there are two sites of in vitro Src-mediated phosphorylation in the FAT domain: Y926, which has been shown to affect FAK function in vivo, and Y1008, which has no known biological role. The phosphorylation of these two tyrosine residues is pH-dependent, but this does not reflect the pH dependence of Src kinase activity. Circular dichroism and nuclear magnetic resonance data indicate that the stability and conformational dynamics of the FAT domain are sensitive to changes in pH over a physiological pH range. In particular, regions of the FAT domain previously shown to regulate phosphorylation of Y926 as well as regions near Y1008 show pH-dependent dynamics on the microsecond to millisecond time scale.

Cellular functions of FAK kinases: insight into molecular mechanisms and novel functions.

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are related tyrosine kinases that have important cellular functions, primarily through regulation of the cytoskeleton. Recent studies have identified multiple molecular mechanisms that regulate cytoskeletal responses, and have provided important and exciting insights into how FAK and Pyk2 control cellular processes such as cell migration. Equally exciting are reports of novel and originally unanticipated functions of these kinases, providing the groundwork for future avenues of investigation. This Commentary summarizes some of these recent discoveries that are relevant to the control of biological responses of the cell.

The costs and benefits of a modified biomedical science workforce.

Analyses of the biomedical research workforce, the biomedical research enterprise, and its sustainability have identified a number of threats and offered many solutions to alleviate the problems. While a number of these solutions have been implemented, one solution that has not been broadly adopted, despite being widely recommended, is to increase the number of staff scientists and reduce dependency on trainees. The perceived impediment of this is the cost. This paper explores the costs associated with laboratory personnel and the benefits, in terms of productivity, associated with different positions in the workforce. The results of this cost-benefit analysis depend upon the values assigned to different metrics of productivity by individuals and institutions. If first and senior author publications are the most important metrics of productivity, a trainee-dependent workforce is much more cost effective. If total publications are the most valued metric of productivity, the cost effectiveness of trainee and staff scientists is reasonably equitable. This analysis provides data for consideration when making personnel decisions and for the continued discussion of modification of the biomedical research workforce. It also provides insight into the incentives for modification of the workforce at the grass roots, which must be considered by institutions genuinely committed to workforce modification to sustain the biomedical research enterprise.

FastTrack, a strategy to shorten time to degree.

Recent reports express concern about the sustainability of the biomedical research enterprise in its current form. Recurring concerns include the predictability and sustainability of funding for research, regulatory burden and training the next generation in the biomedical workforce. One specific concern is the duration of training periods during pre-doctoral and post-doctoral studies. This article addresses the issue of time-to-degree (TTD) for doctorates. Many reports stress the importance of shortening the TTD, but provide no recommendations to achieve this goal. Herein, factors potentially affecting TTD are discussed and one mechanism that harmonizes undergraduate and graduate programs is proposed as a strategy to reduce the TTD.

Functional Hierarchy and Cooperation of EMT Master Transcription Factors in Breast Cancer Metastasis.

Several master transcription factors (TF) can activate the epithelial-to-mesenchymal transition (EMT). However, their individual and combinatorial contributions to EMT in breast cancer are not defined. We show that overexpression of EMT-TFs individually in epithelial cells upregulated endogenous SNAI2, ZEB1/2, TCF4, and TWIST1/2 as a result of positive feedback mediated in part by suppression of their negative regulator miRNAs miR200s/203/205. We identified TCF4 as a potential new target of miR200s. Expression of ZEB1/2 strongly correlated with the mesenchymal phenotype in breast cancer cells, with the CD24-/CD44+ stemness profile, and with lower expression of core epithelial genes in human breast tumors. Knockdown of EMT-TFs identified the key role of ZEB1 and its functional cooperation with other EMT-TFs in the maintenance of the mesenchymal state. Inducible ZEB1+2 knockdown in xenograft models inhibited pulmonary metastasis, emphasizing their critical role in dissemination from primary site and in extravasation. However, ZEB1+2 depletion one-week after intravenous injection did not inhibit lung colonization, suggesting that ZEB1/2 and EMT are not essential for macrometastatic outgrowth. These results provide strong evidence that EMT is orchestrated by coordinated expression of several EMT-TFs and establish ZEB1 as a key master regulator of EMT and metastasis in breast cancer. IMPLICATIONS: The EMT program is orchestrated by coordinated expression of multiple EMT transcription factors, whereas ZEB1 integrates the EMT master regulatory network and plays the major role in promoting EMT and metastasis.

PTP-PEST controls motility, adherens junction assembly, and Rho GTPase activity in colon cancer cells.

An important step in carcinoma progression is loss of cell-cell adhesion leading to increased invasion and metastasis. We show here that the protein tyrosine phosphatase, PTP-PEST, is a critical regulator of cell-cell junction integrity and epithelial cell motility. Using colon carcinoma cells, we show that the expression level of PTP-PEST regulates cell motility. Either transient small interfering RNA or stable short hairpin RNA knockdown of PTP-PEST enhances haptotactic and chemotactic migration of KM12C colon carcinoma cells. Furthermore, KM12C cells with stably knocked down PTP-PEST exhibit a mesenchymal-like phenotype with prominent membrane ruffles and lamellae. In contrast, ectopic expression of PTP-PEST in KM20 or DLD-1 cells, which lack detectable endogenous PTP-PEST expression, suppresses haptotactic migration. Importantly, we find that PTP-PEST localizes in adherens junctions. Concomitant with enhanced motility, stable knockdown of PTP-PEST causes a disruption of cell-cell junctions. These effects are due to a defect in junctional assembly and not to a loss of E-cadherin expression. Adherens junction assembly is impaired following calcium switch in KM12C cells with stably knocked down PTP-PEST and is accompanied by an increase in the activity of Rac1 and a suppression of RhoA activity in response to cadherin engagement. Taken together, these results suggest that PTP-PEST functions as a suppressor of epithelial cell motility by controlling Rho GTPase activity and the assembly of adherens junctions.

JNK phosphorylates paxillin and regulates cell migration.

The c-Jun amino-terminal kinase (JNK) is generally thought to be involved in inflammation, proliferation and apoptosis. Accordingly, its substrates are transcription factors and anti-apoptotic proteins. However, JNK has also been shown to be required for Drosophila dorsal closure, and MAP kinase/ERK kinase kinase 1, an upstream kinase in the JNK pathway, has been shown to be essential for cell migration. Both results imply that JNK is important in cell migration. Here we show that JNK1 is required for the rapid movement of both fish keratocytes and rat bladder tumour epithelial cells (NBT-II). Moreover, JNK1 phosphorylates serine 178 on paxillin, a focal adhesion adaptor, both in vitro and in intact cells. NBT-II cells expressing the Ser 178 --> Ala mutant of paxillin (Pax(S178A)) formed focal adhesions and exhibited the limited movement associated with such contacts in both single-cell-migration and wound-healing assays. In contrast, cells expressing wild-type paxillin moved rapidly and retained close contacts as the predominant adhesion. Expression of Pax(S178A) also inhibited the migration of two other cell lines. Thus, phosphorylation of paxillin by JNK seems essential for maintaining the labile adhesions required for rapid cell migration.

Activation of Src kinase by protein-tyrosine phosphatase-PEST in osteoclasts: comparative analysis of the effects of bisphosphonate and protein-tyrosine phosphatase inhibitor on Src activation in vitro.

PTP-PEST is involved in the regulation of sealing ring formation in osteoclasts. In this article, we have shown a regulatory role for PTP-PEST on dephosphorylation of c-Src at Y527 and phosphorylation at Y418 in the catalytic site. Activation of Src in osteoclasts by over-expression of PTP-PEST resulted in the phosphorylation of cortactin at Y421 and WASP at Y294. Also enhanced as a result, is the interaction of Src, cortactin, and Arp2 with WASP. Moreover, the number of osteoclasts displaying sealing ring and bone resorbing activity was increased in response to PTP-PEST over-expression as compared with control osteoclasts. Cells expressing constitutively active-Src (527YDeltaF) simulate the effects mediated by PTP-PEST. Treatment of osteoclasts with a bisphosphonate alendronate or a potent PTP inhibitor PAO decreased the activity and phosphorylation of Src at Y418 due to reduced dephosphorylation state at Y527. Therefore, Src-mediated phosphorylation of cortactin and WASP as well as the formation of WASP.cortactin.Arp2 complex and sealing ring were reduced in these osteoclasts. Similar effects were observed in osteoclasts treated with an Src inhibitor PP2. We have shown that bisphosphonates could modulate the function of osteoclasts by inhibiting downstream signaling mediated by PTP-PEST/Src, in addition to its effect on the inhibition of the post-translational modification of small GTP-binding proteins such as Rab, Rho, and Rac as shown by others. The promising effects of the inhibitors PP2 and PAO on osteoclast function suggest a therapeutic approach for patients with bone metastases and osteoporosis as an alternative to bisphosphonates.

FAK and paxillin: regulators of N-cadherin adhesion and inhibitors of cell migration?

FAK and paxillin are important components in integrin-regulated signaling. New evidence suggests that these two proteins function in crosstalk between cell-matrix and cell-cell adhesions. Further, new insight suggests that under some conditions these proteins inhibit cell migration, in contrast to their established roles in several cell systems as positive regulators of cell adhesion and migration.

Phosphorylation of paxillin by p38MAPK is involved in the neurite extension of PC-12 cells.

Cell adhesions play an important role in neurite extension. Paxillin, a focal adhesion adaptor protein involved in focal adhesion dynamics, has been demonstrated to be required for neurite outgrowth. However, the molecular mechanism by which paxillin regulates neurite outgrowth is unknown. Here, we show that paxillin is phosphorylated by p38MAPK in vitro and in nerve growth factor (NGF)-induced PC-12 cells. Ser 85 (Ser 83 for endogenous paxillin) is identified as one of major phosphorylation sites by phosphopeptide mapping and mass spectrometry. Moreover, expression of the Ser 85 --> Ala mutant of paxillin (paxS85A) significantly inhibits NGF-induced neurite extension of PC-12 cells, whereas expression of wild-type (wt) paxillin does not influence neurite outgrowth. Further experiments indicate that cells expressing paxS85A exhibit small, clustered focal adhesions which are not normally seen in cells expressing wt paxillin. Although wt paxillin and paxS85A have the same ability to bind vinculin and focal adhesion kinase, wt paxillin more efficiently associates with Pyk2 than paxS85A. Thus, phosphorylation of paxillin is involved in NGF-induced neurite extension of PC-12 cells, probably through regulating focal adhesion organization.

Tyrosine phosphorylation of type Igamma phosphatidylinositol phosphate kinase by Src regulates an integrin-talin switch.

Engagement of integrin receptors with the extracellular matrix induces the formation of focal adhesions (FAs). Dynamic regulation of FAs is necessary for cells to polarize and migrate. Key interactions between FA scaffolding and signaling proteins are dependent on tyrosine phosphorylation. However, the precise role of tyrosine phosphorylation in FA development and maturation is poorly defined. Here, we show that phosphorylation of type Igamma phosphatidylinositol phosphate kinase (PIPKIgamma661) on tyrosine 644 (Y644) is critical for its interaction with talin, and consequently, localization to FAs. PIPKIgamma661 is specifically phosphorylated on Y644 by Src. Phosphorylation is regulated by focal adhesion kinase, which enhances the association between PIPKIgamma661 and Src. The phosphorylation of Y644 results in an approximately 15-fold increase in binding affinity to the talin head domain and blocks beta-integrin binding to talin. This defines a novel phosphotyrosine-binding site on the talin F3 domain and a "molecular switch" for talin binding between PIPKIgamma661 and beta-integrin that may regulate dynamic FA turnover.

CIB1 regulates endothelial cells and ischemia-induced pathological and adaptive angiogenesis.

Pathological angiogenesis contributes to various ocular, malignant, and inflammatory disorders, emphasizing the need to understand this process on a molecular level. CIB1 (calcium- and integrin-binding protein), a 22-kDa EF-hand-containing protein, modulates the activity of p21-activated kinase 1 in fibroblasts. Because p21-activated kinase 1 also contributes to endothelial cell function, we hypothesized that CIB1 may have a role in angiogenesis. We found that endothelial cells depleted of CIB1 by either short hairpin RNA or homologous recombination have reduced migration, proliferation, and tubule formation. Moreover, loss of CIB1 in these cells decreases p21-activated kinase 1 activation, downstream extracellular signal-regulated kinase 1/2 activation, and matrix metalloproteinase 2 expression, all of which are known to contribute to angiogenesis. Consistent with these findings, tissues derived from CIB1-deficient (CIB1-/-) mice have reduced growth factor-induced microvessel sprouting in ex vivo organ cultures and in vivo Matrigel plugs. Furthermore, in response to ischemia, CIB1-/- mice demonstrate decreased pathological retinal and adaptive hindlimb angiogenesis. Ischemic CIB1-/- hindlimbs also demonstrate increased tissue damage and significantly reduced p21-activated kinase 1 activation. These data therefore reveal a critical role for CIB1 in ischemia-induced pathological and adaptive angiogenesis.

Glycogen synthase kinase 3- and extracellular signal-regulated kinase-dependent phosphorylation of paxillin regulates cytoskeletal rearrangement.

Paxillin is a 68-kDa focal adhesion-associated protein that plays an important role in controlling cell spreading and migration. Phosphorylation of paxillin regulates its biological activity and thus has warranted investigation. Serine 126 and serine 130 were previously identified as two major extracellular signal-regulated kinase (ERK)-dependent phosphorylation sites in Raf-transformed fibroblasts. Here serine 126 is identified as a phosphorylation site induced by lipopolysaccharide (LPS) stimulation of RAW264.7 cells. A number of other stimuli, including adhesion and colony-stimulating factor, induce serine 126 phosphorylation in RAW264.7 cells, and nerve growth factor (NGF) treatment induces serine 126 phosphorylation in PC12 cells. The kinase responsible for phosphorylation of this site is identified as glycogen synthase kinase 3 (GSK-3). Interestingly, this GSK-3-dependent phosphorylation is regulated via an ERK-dependent priming mechanism, i.e., phosphorylation of serine 130. Phosphorylation of S126/S130 was required to promote spreading in paxillin null cells, and LPS-induced spreading of RAW264.7 cells was inhibited by expression of the paxillin S126A/S130A mutant. Furthermore, this mutant also retarded NGF-induced PC12 cell neurite outgrowth. Hence, phosphorylation of paxillin on serines 126 and 130, which is mediated by an ERK/GSK-3 dual-kinase mechanism, plays an important role in cytoskeletal rearrangement.

Focal adhesion kinase regulates cell-cell contact formation in epithelial cells via modulation of Rho.

Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that plays a key role in cellular processes such as cell adhesion, migration, proliferation and survival. Recent studies have also implicated FAK in the regulation of cell-cell adhesion. Here, evidence is presented showing that siRNA-mediated suppression of FAK levels in NBT-II cells and expression of dominant negative mutants of FAK caused loss of epithelial cell morphology and inhibited the formation of cell-cell adhesions. Rac and Rho have been implicated in the regulation of cell-cell adhesions and can be regulated by FAK signaling. Expression of active Rac or Rho in NBT-II cells disrupted formation of cell-cell contacts, thus promoting a phenotype similar to FAK-depleted cells. The loss of intercellular contacts in FAK-depleted cells is prevented upon expression of a dominant negative Rho mutant, but not a dominant negative Rac mutant. Inhibition of FAK decreased tyrosine phosphorylation of p190RhoGAP and elevated the level of GTP-bound Rho. This suggests that FAK regulates cell-cell contact formation by regulation of Rho.

Calcium-dependent Pyk2 activation: a role for calmodulin?

Pyk2 (proline-rich tyrosine kinase 2) and FAK (focal adhesion kinase) are highly related tyrosine kinases. One distinguishing feature is the differential regulation of the two enzymes in response to elevation of cytoplasmic calcium. In the latest issue of the Biochemical Journal, Sasaki and co-workers have provided insight into the calcium-dependent regulation of Pyk2. The findings suggest that calmodulin may bind the FERM (4.1/ezrin/radixin/moesin) domain to promote Pyk2 activation in response to calcium signals triggered by vasopressin. While the molecular details of the protein-protein interaction and mechanism of activation remain to be firmly established, this study is the first to provide mechanistic insight into the regulation of Pyk2 by calcium.