Impact of the National Heart, Lung, and Blood Institute's Loan Repayment Program Funding on Retention of the National Institutes of Health Biomedical Workforce.

Background: The National Institutes of Health (NIH) Loan Repayment Programs (LRPs) were established by Congress in 2000 to help attract and retain highly qualified health professionals in biomedical careers by relieving financial pressure incurred from educational loans obtained during medical school and other advanced-degree clinical training programs. In 2019, the NIH LRP Program increased the maximum repayment from $35,000 per year to $50,000 per year for an individual's educational debt in return for two years of research performed in an NIH mission-relevant area (https://www.lrp.nih.gov/eligibility-programs). In addition, in 2020, the National Heart, Lung, and Blood Institute (NHLBI) increased its participation in the LRP by adding the Health Disparities Research Program to Clinical Research and Pediatric Research Programs. Objective: Before these substantive changes took effect, we sought to determine the impact of the NHLBI's participation in the LRP program on retention of scientists in the biomedical research workforce over the past 20 years. Methods: NHLBI LRP applicant cohorts from 2003 and 2008 were carefully examined with a 10-year follow-up period to measure the impact of applying for and obtaining NIH LRP funding on subsequent K- and R-level application and award rates, publication number, and average relative citation ratio as metrics to assess recruitment and retention of scientists in the biomedical research workforce. Results: Obtaining the LRP award was strongly associated with increased submission of and success in obtaining K- and RPG-grant funding and publications for both the 2003 and 2008 NHLBI LRP cohorts. An analysis of subgroups in the 2008 LRP cohort without prior F, K, or RPG funding revealed a consistently strong association between obtaining an LRP award and subsequent K- or RPG-award submission and success as well as potential synergy between obtaining an LRP award and participation on a T grant toward subsequent K- or RPG-award success rates. Conclusion: The LRP award appears to enhance retention in the biomedical research workforce when measured using metrics of grant application and award rates as well as research publications over a 10-year period.

Stable association of RNAi machinery is conserved between the cytoplasm and nucleus of human cells.

Argonaute 2 (AGO2), the catalytic engine of RNAi, is typically associated with inhibition of translation in the cytoplasm. AGO2 has also been implicated in nuclear processes including transcription and splicing. There has been little insight into AGO2's nuclear interactions or how they might differ relative to cytoplasm. Here we investigate the interactions of cytoplasmic and nuclear AGO2 using semi-quantitative mass spectrometry. Mass spectrometry often reveals long lists of candidate proteins, complicating efforts to rigorously discriminate true interacting partners from artifacts. We prioritized candidates using orthogonal analytical strategies that compare replicate mass spectra of proteins associated with Flag-tagged and endogenous AGO2. Interactions with TRNC6A, TRNC6B, TNRC6C, and AGO3 are conserved between nuclei and cytoplasm. TAR binding protein interacted stably with cytoplasmic AGO2 but not nuclear AGO2, consistent with strand loading in the cytoplasm. Our data suggest that interactions between functionally important components of RNAi machinery are conserved between the nucleus and cytoplasm but that accessory proteins differ. Orthogonal analysis of mass spectra is a powerful approach to streamlining identification of protein partners.

Regulation of mammalian transcription and splicing by Nuclear RNAi.

RNA interference (RNAi) is well known as a mechanism for controlling mammalian mRNA translation in the cytoplasm, but what would be the consequences if it also functions in cell nuclei? Although RNAi has also been found in nuclei of plants, yeast, and other organisms, there has been relatively little progress towards understanding the potential involvement of mammalian RNAi factors in nuclear processes including transcription and splicing. This review summarizes evidence for mammalian RNAi factors in cell nuclei and mechanisms that might contribute to the control of gene expression. When RNAi factors bind small RNAs, they form ribonucleoprotein complexes that can be selective for target sequences within different classes of nuclear RNA substrates. The versatility of nuclear RNAi may supply a previously underappreciated layer of regulation to transcription, splicing, and other nuclear processes.

Transcriptional silencing by hairpin RNAs complementary to a gene promoter.

Double-stranded RNAs can target gene promoters and inhibit transcription. To date, most research has focused on synthetic RNA duplexes. Transcriptional silencing by hairpin RNAs would facilitate a better understanding of endogenous RNA-mediated regulation of transcription within cells. Here we examine transcriptional silencing of progesterone receptor (PR) expression by hairpin RNAs. We identify the guide strand as the strand complementary to an antisense transcript at the PR promoter and that hairpin RNAs are active transcriptional silencing agents. The sequence of the hairpin loop affects activity, with the highest activity achieved when the loop has the potential for full complementarity to the antisense transcript target. Introduction of centrally mismatched bases relative to the target transcript does not prevent transcriptional silencing unless the mismatches are present on both the guide and passenger strands. These data demonstrate that hairpin RNAs can cause transcriptional silencing and offer insights into the mechanism of gene modulation by RNAs that target gene promoters.

Label-free voltammetric detection using individually addressable oligonucleotide microelectrode arrays.

The utility and performance of label-free, oligonucleotide probes for reagentless detection of dilute target analytes was examined using a voltammetric transduction principle in an array format. Multistep, solid-state fabrication yielded preproduction arrays of 16 individually addressable, 30 µm diameter microelectrodes in a 30 mm × 6.5 mm × 0.5 mm dipstick disposable device. The specificity of 16 nucleotide (nt) 2'-O-methylribonucleic acid and 22 nt DNA backbone probes bound through Mg(2+)-phosphonate bridges to polypyrrole films on the microelectrodes were studied using microbial target RNAs of various lengths. Probe-specific interactions with Escherichia coli O157 H7 23S rRNA (2907 nt) and Candida albicans 18S rRNA (1788 nt) were detected at 65 and 58 fmol/mL, respectively, in volumes as low as 0.5 mL. Specificity studies showed that, for a given probe, "nontarget" transcripts can contribute to changes in the voltammetric detection signal, though with responses that never exceed 70% of the detection signal acquired for specifically designed complementary targets. These results statistically validate the use of the voltammetric microelectrode array for obtaining a "yes-no" answer on complementary specific binding. The study also identifies challenges and pitfalls for the selection strategies of oligonucleotide probes.