Citizenship status and career self-efficacy: An intersectional study of biomedical trainees in the United States.

This study examines the intersectional role of citizenship and gender with career self-efficacy amongst 10,803 doctoral and postdoctoral trainees in US universities. These biomedical trainees completed surveys administered by 17 US institutions that participated in the National Institutes of Health Broadening Experiences in Scientific Training (NIH BEST) Programs. Findings indicate that career self-efficacy of non-citizen trainees is significantly lower than that of US citizen trainees. While lower career efficacy was observed in women compared with men, it was even lower for non-citizen female trainees. Results suggest that specific career interests may be related to career self-efficacy. Relative to US citizen trainees, both male and female non-citizen trainees showed higher interest in pursuing a career as an academic research investigator. In comparison with non-citizen female trainees and citizen trainees of all genders, non-citizen male trainees expressed the highest interest in research-intensive (and especially principal investigator) careers. The authors discuss potential causes for these results and offer recommendations for increasing trainee career self-efficacy which can be incorporated into graduate and postdoctoral training.

From goal to outcome: Analyzing the progression of biomedical sciences PhD careers in a longitudinal study using an expanded taxonomy.

Biomedical sciences PhDs pursue a wide range of careers inside and outside academia. However, there is little data regarding how career interests of PhD students relate to the decision to pursue postdoctoral training or to their eventual career outcomes. Here, we present the career goals and career outcomes of 1452 biomedical sciences PhDs who graduated from Vanderbilt University between 1997 and 2021. We categorized careers using an expanded three-tiered taxonomy and flags that delineate key career milestones. We also analyzed career goal changes between matriculation and doctoral defense, and the reasons why students became more- or less-interested in research-intensive faculty careers. We linked students' career goal at doctoral defense to whether they did a postdoc, the duration of time between doctoral defense and the first non-training position, the career area of the first non-training position, and the career area of the job at 10 years after graduation. Finally, we followed individual careers for 10 years after graduation to characterize movement between different career areas over time. We found that most students changed their career goal during graduate school, declining numbers of alumni pursued postdoctoral training, many alumni entered first non-training positions in a different career area than their goal at doctoral defense, and the career area of the first non-training position was a good indicator of the job that alumni held 10 years after graduation. Our findings emphasize that students need a wide range of career development opportunities and career mentoring during graduate school to prepare them for futures in research and research-related professions.

Angiopoietin-2 blockade suppresses growth of liver metastases from pancreatic neuroendocrine tumors by promoting T cell recruitment.

Improving the management of metastasis in pancreatic neuroendocrine tumors (PanNETs) is critical, as nearly half of patients with PanNETs present with liver metastases, and this accounts for the majority of patient mortality. We identified angiopoietin-2 (ANGPT2) as one of the most upregulated angiogenic factors in RNA-Seq data from human PanNET liver metastases and found that higher ANGPT2 expression correlated with poor survival rates. Immunohistochemical staining revealed that ANGPT2 was localized to the endothelial cells of blood vessels in PanNET liver metastases. We observed an association between the upregulation of endothelial ANGPT2 and liver metastatic progression in both patients and transgenic mouse models of PanNETs. In human and mouse PanNET liver metastases, ANGPT2 upregulation coincided with poor T cell infiltration, indicative of an immunosuppressive tumor microenvironment. Notably, both pharmacologic inhibition and genetic deletion of ANGPT2 in PanNET mouse models slowed the growth of PanNET liver metastases. Furthermore, pharmacologic inhibition of ANGPT2 promoted T cell infiltration and activation in liver metastases, improving the survival of mice with metastatic PanNETs. These changes were accompanied by reduced plasma leakage and improved vascular integrity in metastases. Together, these findings suggest that ANGPT2 blockade may be an effective strategy for promoting T cell infiltration and immunostimulatory reprogramming to reduce the growth of liver metastases in PanNETs.

Career self-efficacy disparities in underrepresented biomedical scientist trainees.

The present study examines racial, ethnic, and gender disparities in career self-efficacy amongst 6077 US citizens and US naturalized graduate and postdoctoral trainees. Respondents from biomedical fields completed surveys administered by the National Institutes of Health Broadening Experiences in Scientific Training (NIH BEST) programs across 17 US institutional sites. Graduate and postdoctoral demographic and survey response data were examined to evaluate the impact of intersectional identities on trainee career self-efficacy. The study hypothesized that race, ethnicity and gender, and the relations between these identities, would impact trainee career self-efficacy. The analysis demonstrated that racial and ethnic group, gender, specific career interests (academic principal investigator vs. other careers), and seniority (junior vs. senior trainee level) were, to various degrees, all associated with trainee career self-efficacy and the effects were consistent across graduate and postdoctoral respondents. Implications for differing levels of self-efficacy are discussed, including factors and events during training that may contribute to (or undermine) career self-efficacy. The importance of mentorship for building research and career self-efficacy of trainees is discussed, especially with respect to those identifying as women and belonging to racial/ethnic populations underrepresented in biomedical sciences. The results underscore the need for change in the biomedical academic research community in order to retain a diverse biomedical workforce.

Biomedical graduate student experiences during the COVID-19 university closure.

COVID-19-associated university closures moved classes online and interrupted ongoing research in universities throughout the US. In Vanderbilt University, first year biomedical sciences PhD students were in the middle of their spring semester coursework and in the process of identifying a thesis research lab, while senior students who had already completed the first year were at various stages of their graduate training and were working on their thesis research projects. To learn how the university closure and resulting interruptions impacted our students' learning and well-being, we administered two surveys, one to the first year students and the other to the senior students. Our main findings show that the university closure negatively impacted the overall psychological health of about one-third of the survey respondents, time management was the aspect of remote learning that caused the highest stress for close to 50% of the students, and interaction with their peers and in-person discussions were the aspects of on-campus learning that students missed the most during the remote learning period. Additionally, survey responses also show that students experienced positive outcomes as a result of remote learning that included spending increased time on additional learning interests, with family, on self-care, and for dissertation or manuscript writing. Though a variety of supportive resources are already available to students in our institution, results from our survey suggest enhancing these measures and identifying new ones targeted to addressing the academic and emotional needs of PhD students would be beneficial. Such support measures may be appropriate for students in other institutions as well.

The Host Cell Metabolite Inositol Hexakisphosphate Promotes Efficient Endogenous HIV-1 Reverse Transcription by Stabilizing the Viral Capsid.

A defining activity of retroviruses is reverse transcription, the process by which the viral genomic RNA is converted into the double-stranded DNA required for virus replication. Reverse transcriptase (RT), the viral enzyme responsible for this process, was identified in 1970 by assaying permeabilized retrovirus particles for DNA synthesis in vitro Such reactions are inefficient, with only a small fraction of viral genomes being converted to full-length double-stranded DNA molecules, possibly owing to disruption of the structure of the viral core. Here, we show that reverse transcription in purified HIV-1 cores is enhanced by the addition of the capsid-binding host cell metabolite inositol hexakisphosphate (IP6). IP6 potently enhanced full-length minus-strand synthesis, as did hexacarboxybenzene (HCB), which also stabilizes the HIV-1 capsid. Both IP6 and HCB stabilized the association of the viral CA and RT proteins with HIV-1 cores. In contrast to the wild type, cores isolated from mutant HIV-1 particles containing intrinsically hyperstable capsids exhibited relatively efficient reverse transcription in the absence of IP6, further indicating that the compound promotes reverse transcription by stabilizing the viral capsid. We also observed that the capsid-destabilizing antiviral compound PF74 inhibited endogenous reverse transcription with a potency that mirrors its ability to inhibit reverse transcription during infection. Our results show that the stabilization of the HIV-1 capsid permits efficient reverse transcription in HIV-1 cores, providing a sensitive experimental system for analyzing the functions of viral and host cell molecules and the role of capsid disassembly (uncoating) in the process.IMPORTANCE HIV-1 infection requires reverse transcription of the viral genome. While much is known about the biochemistry of reverse transcription from simplified biochemical reactions, reverse transcription during infection takes place within a viral core. However, endogenous reverse transcription reactions using permeabilized HIV-1 virions or purified viral cores have been inefficient. Using viral cores purified from infectious HIV-1 particles, we show that efficient reverse transcription is achieved in vitro by addition of the capsid-stabilizing metabolite inositol hexakisphosphate. The enhancement of reverse transcription was linked to the capsid-stabilizing effect of the compound, consistent with the known requirement for an intact or semi-intact viral capsid for HIV-1 infection. Our results establish a biologically relevant system for dissecting the function of the viral capsid and its disassembly during reverse transcription. The system should also prove useful for mechanistic studies of capsid-targeting antiviral drugs.

Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly.

The HIV-1 core consists of the viral genomic RNA and several viral proteins encased within a conical capsid. After cell entry, the core disassembles in a process termed uncoating. Although HIV-1 uncoating has been linked to reverse transcription of the viral genome in target cells, the mechanism by which uncoating is initiated is unknown. Using time-lapse atomic force microscopy, we analyzed the morphology and physical properties of isolated HIV-1 cores during the course of reverse transcription in vitro We found that, during an early stage of reverse transcription the pressure inside the capsid increases, reaching a maximum after 7 h. High-resolution mechanical mapping reveals the formation of a stiff coiled filamentous structure underneath the capsid surface. Subsequently, this coiled structure disappears, the stiffness of the capsid drops precipitously to a value below that of a pre-reverse transcription core, and the capsid undergoes partial or complete rupture near the narrow end of the conical structure. We propose that the transcription of the relatively flexible single-stranded RNA into a more rigid filamentous structure elevates the pressure within the core, which triggers the initiation of capsid disassembly.IMPORTANCE For successful infection, the HIV-1 genome, which is in the form of a single-stranded RNA enclosed inside a capsid shell, must be reverse transcribed into double-stranded DNA and released from the capsid (in a process known as uncoating) before it can be integrated into the target cell genome. The mechanism that triggers uncoating is a pivotal question of long standing. By using atomic force microscopy, we found that during reverse transcription the pressure inside the capsid increases until the internal stress exceeds the strength of the capsid structure and the capsid breaks open. The application of AFM technologies to study purified HIV-1 cores represents a new experimental platform for elucidating additional aspects of capsid disassembly and HIV-1 uncoating.

Drug resistant integrase mutants cause aberrant HIV integrations.

BACKGROUND: HIV-1 integrase is the target for three FDA-approved drugs, raltegravir, elvitegravir, and dolutegravir. All three drugs bind at the active site of integrase and block the strand transfer step of integration. We previously showed that sub-optimal doses of the anti-HIV drug raltegravir can cause aberrant HIV integrations that are accompanied by a variety of deletions, duplications, insertions and inversions of the adjacent host sequences. RESULTS: We show here that a second drug, elvitegravir, also causes similar aberrant integrations. More importantly, we show that at least two of the three clinically relevant drug resistant integrase mutants we tested, N155H and G140S/Q148H, which reduce the enzymatic activity of integrase, can cause the same sorts of aberrant integrations, even in the absence of drugs. In addition, these drug resistant mutants have an elevated IC50 for anti-integrase drugs, and concentrations of the drugs that would be optimal against the WT virus are suboptimal for the mutants. CONCLUSIONS: We previously showed that suboptimal doses of a drug that binds to the HIV enzyme integrase and blocks the integration of a DNA copy of the viral genome into host DNA can cause aberrant integrations that involve rearrangements of the host DNA. We show here that suboptimal doses of a second anti-integrase drug can cause similar aberrant integrations. We also show that drug-resistance mutations in HIV integrase can also cause aberrant integrations, even in the absence of an anti-integrase drug. HIV DNA integrations in the oncogenes BACH2 and MKL2 that do not involve rearrangements of the viral or host DNA can stimulate the proliferation of infected cells. Based on what is known about the association of DNA rearrangements and the activation of oncogenes in human tumors, it is possible that some of the deletions, duplications, insertions, and inversions of the host DNA that accompany aberrant HIV DNA integrations could increase the chances that HIV integrations could lead to the development of a tumor.

Treatment with suboptimal doses of raltegravir leads to aberrant HIV-1 integrations.

Integration of the DNA copy of the HIV-1 genome into a host chromosome is required for viral replication and is thus an important target for antiviral therapy. The HIV-encoded enzyme integrase (IN) catalyzes two essential steps: 3' processing of the viral DNA ends, followed by the strand transfer reaction, which inserts the viral DNA into host DNA. Raltegravir binds to IN and blocks the integration of the viral DNA. Using the Rous sarcoma virus-derived vector RCAS, we previously showed that mutations that cause one viral DNA end to be defective for IN-mediated integration led to abnormal integrations in which the provirus had one normal and one aberrant end, accompanied by rearrangements in the host genome. On the basis of these results, we expected that suboptimal concentrations of IN inhibitors, which could block one of the ends of viral integration, would lead to similar aberrant integrations. In contrast to the proviruses from untreated cells, which were all normal, ∼10-15% of the proviruses isolated after treatment with a suboptimal dose of raltegravir were aberrant. The aberrant integrations were similar to those seen in the RCAS experiments. Most of the aberrant proviruses had one normal end and one aberrant end and were accompanied by significant rearrangements in the host genome, including duplications, inversions, deletions and, occasionally, acquisition of sequences from other chromosomes. The rearrangements of the host DNA raise concerns that these aberrant integrations might have unintended consequences in HIV-1-infected patients who are not consistent in following a raltegravir-containing treatment regimen.

ATR3 encodes a diflavin reductase essential for Arabidopsis embryo development.

*The Arabidopsis genome possesses two confirmed Cytochrome P450 Reductase (CPR) genes, ATR1 and ATR2, together with a third putative homologue, ATR3, which annotation is questionable. *Phylogenetic analysis classified ATR3 as a CPR-like protein sharing homologies with the animal cytosolic dual flavin reductases, NR1 and Fre-1, distinct from the microsomal CPRs, ATR1 and ATR2. Like NR1 and Fre-1, ATR3 lacks the N-terminal endoplasmic reticulum (ER) anchor domain of CPRs and is localized in the cytoplasm. Recombinant ATR3 in plant soluble extracts was able to reduce cytochrome c but failed to reduce the human P450 CYP1A2. *Loss of ATR3 function resulted in early embryo lethality indicating that this reductase activity is essential. A yeast 2-hybrid screen identified a unique interaction of ATR3 with the homologue of the human anti-apoptotic CIAPIN1 and the yeast Dre2 protein. *This interaction suggests two possible roles for ATR3 in the control of cell death and in chromosome segregation at mitosis. Consistent with these results, the promoter of ATR3 is activated during cell cycle progression. Together these results demonstrated that ATR3 belongs to the NR1 subfamily of diflavin reductases whose characterized members are involved in essential cellular functions.

Differential regulation of the inhibitor of apoptosis ch-IAP1 by v-rel and the proto-oncogene c-rel.

The v-rel oncogene encoded by reticuloendotheliosis virus is the acutely transforming member of the Rel/NF-kappaB family of transcription factors. v-Rel is a truncated and mutated form of c-Rel and transforms cells by inducing the aberrant expression of genes regulated by Rel/NF-kappaB proteins. The expression of ch-IAP1, a member of the inhibitor-of-apoptosis family, is highly elevated in cells expressing v-Rel and contributes to the immortalization of cells transformed by this oncoprotein. In this study we demonstrate that the elevated expression of ch-IAP1 in v-Rel-expressing cells is due to an increased rate of transcription. The ch-IAP1 promoter was isolated, and four Rel/NF-kappaB binding sites were identified upstream of the transcription start site. Two kappaB sites proximal to the transcription start site were required for v-Rel to activate the ch-IAP1 promoter. While c-Rel also utilized these sites, a third more-distal kappaB site was required for its full activation of the ch-IAP1 promoter. Differences in the transactivation domains of v-Rel and c-Rel are responsible for their different abilities to utilize these sites and account for their differential activation of the ch-IAP1 promoter. Although c-Rel was a more potent activator of the ch-IAP1 promoter than v-Rel in transient reporter assays, cells stably overexpressing c-Rel failed to maintain high levels of ch-IAP1 expression. The reduction of ch-IAP1 expression in these cells correlated with the efficient regulation of c-Rel by IkappaBalpha. The ability of v-Rel to escape IkappaBalpha regulation allows for the gradual and sustained elevation of ch-IAP1 expression directly contributing to the transforming properties of v-Rel.