The model student: GPT-4 performance on graduate biomedical science exams.

The GPT-4 large language model (LLM) and ChatGPT chatbot have emerged as accessible and capable tools for generating English-language text in a variety of formats. GPT-4 has previously performed well when applied to questions from multiple standardized examinations. However, further evaluation of trustworthiness and accuracy of GPT-4 responses across various knowledge domains is essential before its use as a reference resource. Here, we assess GPT-4 performance on nine graduate-level examinations in the biomedical sciences (seven blinded), finding that GPT-4 scores exceed the student average in seven of nine cases and exceed all student scores for four exams. GPT-4 performed very well on fill-in-the-blank, short-answer, and essay questions, and correctly answered several questions on figures sourced from published manuscripts. Conversely, GPT-4 performed poorly on questions with figures containing simulated data and those requiring a hand-drawn answer. Two GPT-4 answer-sets were flagged as plagiarism based on answer similarity and some model responses included detailed hallucinations. In addition to assessing GPT-4 performance, we discuss patterns and limitations in GPT-4 capabilities with the goal of informing design of future academic examinations in the chatbot era.

Circulating microRNA Biomarkers of Thiazide Response in Hypertension.

BACKGROUND: Thiazide diuretics are the second most frequently prescribed class of antihypertensives, but up to 50% of patients with hypertension have minimal antihypertensive response to thiazides. We explored circulating microRNAs (miRNAs) in search of predictive biomarkers of thiazide response. METHODS AND RESULTS: We profiled 754 miRNAs in baseline plasma samples of 36 hypertensive European American adults treated with hydrochlorothiazide, categorized into responders (n=18) and nonresponders (n=18) on the basis of diastolic blood pressure response to hydrochlorothiazide. miRNAs with ≥2.5-fold differential expression between responders and nonresponders were considered for validation in 3 cohorts (n=50 each): hydrochlorothiazide-treated European Americans, chlorthalidone-treated European Americans, and hydrochlorothiazide-treated Black individuals. Different blood pressure phenotypes including categorical (responder versus nonresponder) and continuous diastolic blood pressure and systolic blood pressure were tested for association with the candidate miRNA expression using multivariate regression analyses adjusting for age, sex, and baseline blood pressure. After quality control, 74 miRNAs were available for screening, 19 of which were considered for validation. In the validation cohort, miR-193b-3p and 30d-5p showed significant associations with continuous SBP or diastolic blood pressure response or both, to hydrochlorothiazide in European Americans at Benjamini-Hochberg adjusted P<0.05. In the combined analysis of validation cohorts, let-7g (odds ratio, 0.6 [95% CI, 0.4-0.8]), miR-142-3p (odds ratio, 1.1 [95% CI, 1.0, 1.2]), and miR-423-5p (odds ratio, 0.7 [95% CI, 0.5-0.9]) associated with categorical diastolic blood pressure response at Benjamini-Hochberg adjusted P<0.05. Predicted target genes of the 5 miRNAs were mapped to key hypertension pathways: lysine degradation, fatty acid biosynthesis, and metabolism. CONCLUSIONS: The above identified circulating miRNAs may have a potential for clinical use as biomarkers for thiazide diuretic selection in hypertension. REGISTRATION: URL: ClinicalTrials.gov. Unique identifiers: NCT00246519, NCT01203852, NCT00005520.

Identifying locally actionable strategies to increase participant acceptability and feasibility to participate in Phase I cancer clinical trials.

BACKGROUND: Recruitment of cancer clinical trial (CCT) participants, especially participants representing the diversity of the US population, is necessary to create successful medications and a continual challenge. These challenges are amplified in Phase I cancer trials that focus on evaluating the safety of new treatments and are the gateway to treatment development. In preparation for recruitment to a Phase I recurrent head and neck cancer (HNC) trial, we assessed perceived barriers to participation or referral and suggestions for recruitment among people with HNC and community physicians (oncologist, otolaryngologist or surgeon). METHODS: Between December 2020 and February 2022, we conducted a qualitative needs assessment via semistructured interviews with a race and ethnicity-stratified sample of people with HNC (n = 30: 12 non-Hispanic White, 9 non-Hispanic African American, 8 Hispanic and 1 non-Hispanic Pacific Islander) and community physicians (n = 16) within the University of Florida Health Cancer Center catchment area. Interviews were analyzed using a qualitative content analysis approach to describe perspectives and identify relevant themes. RESULTS: People with HNC reported thematic barriers included: concerns about side effects, safety and efficacy; lack of knowledge and systemic and environmental obstacles. Physicians identified thematic barriers of limited physician knowledge; clinic and physician barriers and structural barriers. People with HNC and physicians recommended themes included: improved patient education, dissemination of trial information and interpersonal communication between community physicians and CCT staff. CONCLUSIONS: The themes identified by people with HNC and community physicians are consistent with research efforts and recommendations on how to increase the participation of people from minoritized populations in CCTs. This community needs assessment provides direction on the selection of strategies to increase CCT participation and referral. PATIENT OR PUBLIC CONTRIBUTION: This study focused on people with HNC and community physicians' lived experience and their interpretations of how they would consider a future Phase I clinical trial. In addition to our qualitative data reflecting community voices, a community member reviewed the draft interview guide before data collection and both people with HNC and physicians aided interpretation of the findings.

Hybkit: a Python API and command-line toolkit for hybrid sequence data from chimeric RNA methods.

SUMMARY: Experimental methods using microRNA/target ligation have recently provided significant insights into microRNA functioning through generation of chimeric (hybrid) RNA sequences. Here, we introduce Hybkit, a Python3 API, and command-line toolkit for analysis of hybrid sequence data in the "hyb" file format to enable customizable evaluation and annotation of hybrid characteristics. The Hybkit API includes a suite of python objects for developing custom analyses of hybrid data as well as miRNA-specific analysis methods, built-in plotting of analysis results, and incorporation of predicted miRNA/target interactions in Vienna format. AVAILABILITY AND IMPLEMENTATION: Hybkit is provided free and open source under the GNU GPL license at github.com/RenneLab/hybkit and archived on Zenodo (doi.org/10.5281/zenodo.7834299). Hybkit distributions are also provided via PyPI (pypi.org/project/hybkit), Conda (bioconda.github.io/recipes/hybkit/README.html), and Docker (quay.io/repository/biocontainers/hybkit).

Sex-Biased Expression Is Associated With Chromatin State in Drosophila melanogaster and Drosophila simulans.

In Drosophila melanogaster and D. simulans head tissue, 60% of orthologous genes show evidence of sex-biased expression in at least one species. Of these, ∼39% (2,192) are conserved in direction. We hypothesize enrichment of open chromatin in the sex where we see expression bias and closed chromatin in the opposite sex. Male-biased orthologs are significantly enriched for H3K4me3 marks in males of both species (∼89% of male-biased orthologs vs. ∼76% of unbiased orthologs). Similarly, female-biased orthologs are significantly enriched for H3K4me3 marks in females of both species (∼90% of female-biased orthologs vs. ∼73% of unbiased orthologs). The sex-bias ratio in female-biased orthologs was similar in magnitude between the two species, regardless of the closed chromatin (H3K27me2me3) marks in males. However, in male-biased orthologs, the presence of H3K27me2me3 in both species significantly reduced the correlation between D. melanogaster sex-bias ratio and the D. simulans sex-bias ratio. Male-biased orthologs are enriched for evidence of positive selection in the D. melanogaster group. There are more male-biased genes than female-biased genes in both species. For orthologs with gains/losses of sex-bias between the two species, there is an excess of male-bias compared to female-bias, but there is no consistent pattern in the relationship between H3K4me3 or H3K27me2me3 chromatin marks and expression. These data suggest chromatin state is a component of the maintenance of sex-biased expression and divergence of sex-bias between species is reflected in the complexity of the chromatin status.

Investigating miR-9 as a mediator in laryngeal cancer health disparities.

Background: For several decades, Black patients have carried a higher burden of laryngeal cancer among all races. Even when accounting for sociodemographics, a disparity remains. Differentially expressed microRNAs have been linked to racially disparate clinical outcomes in breast and prostate cancers, yet an association in laryngeal cancer has not been addressed. In this study, we present our computational analysis of differentially expressed miRNAs in Black compared with White laryngeal cancer and further validate microRNA-9-5p (miR-9-5p) as a potential mediator of cancer phenotype and chemoresistance. Methods: Bioinformatic analysis of 111 (92 Whites, 19 Black) laryngeal squamous cell carcinoma (LSCC) specimens from the TCGA revealed miRNAs were significantly differentially expressed in Black compared with White LSCC. We focused on miR-9-5 p which had a significant 4-fold lower expression in Black compared with White LSCC (p<0.05). After transient transfection with either miR-9 mimic or inhibitor in cell lines derived from Black (UM-SCC-12) or White LSCC patients (UM-SCC-10A), cellular migration and cell proliferation was assessed. Alterations in cisplatin sensitivity was evaluated in transient transfected cells via IC50 analysis. qPCR was performed on transfected cells to evaluate miR-9 targets and chemoresistance predictors, ABCC1 and MAP1B. Results: Northern blot analysis revealed mature miR-9-5p was inherently lower in cell line UM-SCC-12 compared with UM-SCC-10A. UM -SCC-12 had baseline increase in cellular migration (p < 0.01), proliferation (p < 0.0001) and chemosensitivity (p < 0.01) compared to UM-SCC-10A. Increasing miR-9 in UM-SCC-12 cells resulted in decreased cellular migration (p < 0.05), decreased proliferation (p < 0.0001) and increased sensitivity to cisplatin (p < 0.001). Reducing miR-9 in UM-SCC-10A cells resulted in increased cellular migration (p < 0.05), increased proliferation (p < 0.05) and decreased sensitivity to cisplatin (p < 0.01). A significant inverse relationship in ABCC1 and MAP1B gene expression was observed when miR-9 levels were transiently elevated or reduced in either UM-SCC-12 or UM-SCC-10A cell lines, respectively, suggesting modulation by miR-9. Conclusion: Collectively, these studies introduce differential miRNA expression in LSCC cancer health disparities and propose a role for low miR-9-5p as a mediator in LSCC tumorigenesis and chemoresistance.

Sex-biased expression is associated with chromatin state in D. melanogaster and D. simulans.

We propose a new model for the association of chromatin state and sex-bias in expression. We hypothesize enrichment of open chromatin in the sex where we see expression bias (OS) and closed chromatin in the opposite sex (CO). In this study of D. melanogaster and D. simulans head tissue, sex-bias in expression is associated with H3K4me3 (open mark) in males for male-biased genes and in females for female-biased genes in both species. Sex-bias in expression is also largely conserved in direction and magnitude between the two species on the X and autosomes. In male-biased orthologs, the sex-bias ratio is more divergent between species if both species have H3K27me2me3 marks in females compared to when either or neither species has H3K27me2me3 in females. H3K27me2me3 marks in females are associated with male-bias in expression on the autosomes in both species, but on the X only in D. melanogaster . In female-biased orthologs the relationship between the species for the sex-bias ratio is similar regardless of the H3K27me2me3 marks in males. Female-biased orthologs are more similar in the ratio of sex-bias than male-biased orthologs and there is an excess of male-bias in expression in orthologs that gain/lose sex-bias. There is an excess of male-bias in sex-limited expression in both species suggesting excess male-bias is due to rapid evolution between the species. The X chromosome has an enrichment in male-limited H3K4me3 in both species and an enrichment of sex-bias in expression compared to the autosomes.

DExD/H Box Helicases DDX24 and DDX49 Inhibit Reactivation of Kaposi's Sarcoma Associated Herpesvirus by Interacting with Viral mRNAs.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that is the causative agent of primary effusion lymphoma and Kaposi's sarcoma. In healthy carriers, KSHV remains latent, but a compromised immune system can lead to lytic viral replication that increases the probability of tumorigenesis. RIG-I-like receptors (RLRs) are members of the DExD/H box helicase family of RNA binding proteins that recognize KSHV to stimulate the immune system and prevent reactivation from latency. To determine if other DExD/H box helicases can affect KSHV lytic reactivation, we performed a knock-down screen that revealed DHX29-dependent activities appear to support viral replication but, in contrast, DDX24 and DDX49 have antiviral activity. When DDX24 or DDX49 are overexpressed in BCBL-1 cells, transcription of all lytic viral genes and genome replication were significantly reduced. RNA immunoprecipitation of tagged DDX24 and DDX49 followed by next-generation sequencing revealed that the helicases bind to mostly immediate-early and early KSHV mRNAs. Transfection of expression plasmids of candidate KSHV transcripts, identified from RNA pull-down, demonstrated that KSHV mRNAs stimulate type I interferon (alpha/beta) production and affect the expression of multiple interferon-stimulated genes. Our findings reveal that host DExD/H box helicases DDX24 and DDX49 recognize gammaherpesvirus transcripts and convey an antiviral effect in the context of lytic reactivation.

Metabolic signatures associated with oncolytic myxoma viral infections.

Oncolytic viral therapy is a recent advance in cancer treatment, demonstrating promise as a primary treatment option. To date, the secondary metabolic effects of viral infection in cancer cells has not been extensively studied. In this work, we have analyzed early-stage metabolic changes in cancer cells associated with oncolytic myxoma virus infection. Using GC-MS based metabolomics, we characterized the myxoma virus infection induced metabolic changes in three cancer cell lines-small cell (H446) and non-small cell (A549) lung cancers, and glioblastoma (SFxL). We show that even at an early stage (6 and 12 h) myxoma infection causes profound changes in cancer cell metabolism spanning several important pathways such as the citric acid cycle, fatty acid metabolism, and amino acid metabolism. In general, the metabolic effects of viral infection across cell lines are not conserved. However, we have identified several candidate metabolites that can potentially serve as biomarkers for monitoring oncolytic viral action in general.

Sequencing of Argonaute-bound microRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a.

MicroRNAs (miRNA) are short non-coding RNAs widely implicated in gene regulation. Most metazoan miRNAs utilize the RNase III enzymes Drosha and Dicer for biogenesis. One notable exception is the RNA polymerase II transcription start sites (TSS) miRNAs whose biogenesis does not require Drosha. The functional importance of the TSS-miRNA biogenesis is uncertain. To better understand the function of TSS-miRNAs, we applied a modified Crosslinking, Ligation, and Sequencing of Hybrids on Argonaute (AGO-qCLASH) to identify the targets for TSS-miRNAs in HCT116 colorectal cancer cells with or without DROSHA knockout. We observed that miR-320a hybrids dominate in TSS-miRNA hybrids identified by AGO-qCLASH. Targets for miR-320a are enriched for the eIF2 signaling pathway, a downstream component of the unfolded protein response. Consistently, in miR-320a mimic- and antagomir- transfected cells, differentially expressed gene products are associated with eIF2 signaling. Within the AGO-qCLASH data, we identified the endoplasmic reticulum (ER) chaperone calnexin as a direct miR-320a down-regulated target, thus connecting miR-320a to the unfolded protein response. During ER stress, but not amino acid deprivation, miR-320a up-regulates ATF4, a critical transcription factor for resolving ER stress. In summary, our study investigates the targetome of the TSS-miRNAs in colorectal cancer cells and establishes miR-320a as a regulator of unfolded protein response.

Modified Cross-Linking, Ligation, and Sequencing of Hybrids (qCLASH) to Identify MicroRNA Targets.

This protocol was designed to identify microRNA (miRNA) targetomes from smaller-input samples by performing a simplified workflow of the Cross-Linking and Sequencing of Hybrids (CLASH) technique developed in the Tollervey group. In this ribonomics-based technique, Cross-Linking and Immunoprecipitation (CLIP) of Argonaute (Ago) is combined with an RNA ligase reaction that yields covalently bound "hybrids" between miRNAs and their target RNAs. While this iteration of CLIP identifies "high-confidence" or "unambiguous" miRNA targets, the added ligation step is highly inefficient and therefore requires large numbers of cultured cells. To make this powerful approach applicable to smaller cell numbers, we created qCLASH, incorporating a workflow that performs all enzymatic reactions on bead-bound complexes and omits gel purification of immunoprecipitated Ago complexes associated with major loss of RNA. At a sequencing depth of 100 million reads per library, which is highly feasible with rapidly decreasing sequencing costs, qCLASH, when used with three biological replicates, results in thousands of high-confidence miRNA targets. qCLASH was first developed to identify viral miRNA targetomes of endothelial cells infected with Kaposi's sarcoma-associated herpesvirus. Since then, qCLASH has been applied to Epstein-Barr virus- and MHV68-infected cells, and more recently to metastatic melanoma and breast cancer cells. Currently, protocols are under development to apply qCLASH to human solid tumor specimens. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Quick Cross-Linking and Sequencing of Hybrids (qCLASH) Support Protocol: Optimization of Ago immunoprecipitation.

EBV miRNAs are potent effectors of tumor cell transcriptome remodeling in promoting immune escape.

The Epstein Barr virus (EBV) contributes to the tumor phenotype through a limited set of primarily non-coding viral RNAs, including 31 mature miRNAs. Here we investigated the impact of EBV miRNAs on remodeling the tumor cell transcriptome. Strikingly, EBV miRNAs displayed exceptionally abundant expression in primary EBV-associated Burkitt's Lymphomas (BLs) and Gastric Carcinomas (GCs). To investigate viral miRNA targeting, we used the high-resolution approach, CLASH in GC and BL cell models. Affinity constant calculations of targeting efficacies for CLASH hits showed that viral miRNAs bind their targets more effectively than their host counterparts, as did Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) miRNAs. Using public BL and GC RNA-seq datasets, we found that high EBV miRNA targeting efficacies translates to enhanced reduction of target expression. Pathway analysis of high efficacy EBV miRNA targets showed enrichment for innate and adaptive immune responses. Inhibition of the immune response by EBV miRNAs was functionally validated in vivo through the finding of inverse correlations between EBV miRNAs and immune cell infiltration and T-cell diversity in BL and GC datasets. Together, this study demonstrates that EBV miRNAs are potent effectors of the tumor transcriptome that play a role in suppressing host immune response.

A noncanonical microRNA derived from the snaR-A noncoding RNA targets a metastasis inhibitor.

MicroRNAs (miRNAs) are small noncoding RNAs that function as critical posttranscriptional regulators in various biological processes. While most miRNAs are generated from processing of long primary transcripts via sequential Drosha and Dicer cleavage, some miRNAs that bypass Drosha cleavage can be transcribed as part of another small noncoding RNA. Here, we develop the target-oriented miRNA discovery (TOMiD) bioinformatic analysis method to identify Drosha-independent miRNAs from Argonaute crosslinking and sequencing of hybrids (Ago-CLASH) data sets. Using this technique, we discovered a novel miRNA derived from a primate specific noncoding RNA, the small NF90 associated RNA A (snaR-A). The miRNA derived from snaR-A (miR-snaR) arises independently of Drosha processing but requires Exportin-5 and Dicer for biogenesis. We identify that miR-snaR is concurrently up-regulated with the full snaR-A transcript in cancer cells. Functionally, miR-snaR associates with Ago proteins and targets NME1, a key metastasis inhibitor, contributing to snaR-A's role in promoting cancer cell migration. Our findings suggest a functional link between a novel miRNA and its precursor noncoding RNA.

Cross-Linking Ligation and Sequencing of Hybrids (qCLASH) Reveals an Unpredicted miRNA Targetome in Melanoma Cells.

MicroRNAs are key post-transcriptional gene regulators often displaying aberrant expression patterns in cancer. As microRNAs are promising disease-associated biomarkers and modulators of responsiveness to anti-cancer therapies, a solid understanding of their targetome is crucial. Despite enormous research efforts, the success rates of available tools to reliably predict microRNAs (miRNA)-target interactions remains limited. To investigate the disease-associated miRNA targetome, we have applied modified cross-linking ligation and sequencing of hybrids (qCLASH) to BRAF-mutant melanoma cells. The resulting RNA-RNA hybrid molecules provide a comprehensive and unbiased snapshot of direct miRNA-target interactions. The regulatory effects on selected miRNA target genes in predicted vs. non-predicted binding regions was validated by miRNA mimic experiments. Most miRNA-target interactions deviate from the central dogma of miRNA targeting up to 60% interactions occur via non-canonical seed pairing with a strong contribution of the 3' miRNA sequence, and over 50% display a clear bias towards the coding sequence of mRNAs. miRNAs targeting the coding sequence can directly reduce gene expression (miR-34a/CD68), while the majority of non-canonical miRNA interactions appear to have roles beyond target gene suppression (miR-100/AXL). Additionally, non-mRNA targets of miRNAs (lncRNAs) whose interactions mainly occur via non-canonical binding were identified in melanoma. This first application of CLASH sequencing to cancer cells identified over 8 K distinct miRNA-target interactions in melanoma cells. Our data highlight the importance non-canonical interactions, revealing further layers of complexity of post-transcriptional gene regulation in melanoma, thus expanding the pool of miRNA-target interactions, which have so far been omitted in the cancer field.

Kaposi's Sarcoma-associated Herpesvirus microRNA mutants modulate cancer hallmark phenotypic differences in human endothelial cells.

Kaposi's sarcoma (KS) results from the transformation of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected endothelial cells. The contribution of the KSHV microRNAs (miRNAs) to the process of oncogenesis in endothelial cells has not been fully elucidated. To better understand the contributions of individual miRNAs to oncogenesis-related cellular phenotypes, we used KSHV miRNA knockout mutants, each one lacking one of the twelve miRNA genes. An additional mutant lacked all miRNAs. Since KSHV infection causes a variety of phenotypic changes in endothelial cells, we tested the mutants for their ability to effect such changes in Telomerase-Immortalized Vein Endothelial (TIVE) cells infected with each of the mutant viruses. Wild type- and mutant-infected as well as uninfected cells were evaluated for perturbations to proliferation, migration, tubule formation, and glycolysis. We found broad variation between the different viruses in these aspects. With respect to proliferation rate, ΔmiR-K12-3, ΔmiR-K12-8, and ΔmiR-K12-11 showed significant impairment. Cells infected with ΔmiR-K12-11 had reduced migration. In tubule formation, the ΔmiR-K12-5, -6, and -7 viruses were deficient. At the same time, cells infected with the ΔmiR-K12-10 virus showed dysregulated glycolysis. By combining these observations with previously published KSHV miRNA targetome lists from ribonomics data, we were able to functionally validate a number of new miRNA targets in specific pathways. As proof of concept, miR-K12-3 was shown to target Cathepsin D, a strong promoter of apoptosis. Taken together, the results demonstrate that KSHV miRNAs play different roles in inducing the phenotypic changes which are characteristic of transformed cells.Importance: Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma (KS). The contribution of KSHV microRNAs (miRNAs) to oncogenesis is not fully understood. This is particularly true for human endothelial cells, the cell type from which KS tumors are derived. Here we used a panel of KSHV miRNA knockout viruses in order to shed light on the roles of individual miRNAs in the process of transformation. Latently infected endothelial cells were studied for phenotypic changes related to cancer, including proliferation, migration, angiogenesis, glycolysis, and apoptosis. The mutant-infected cell lines displayed a wide range of phenotypes in these selected measures of oncogenesis which differed from wild type-infected cells and from each other. These results indicate that KSHV miRNAs contribute to different aspects of oncogenesis, and that each one has a unique role to play.

Age-Related Changes in miRNA Expression Influence GSTZ1 and Other Drug Metabolizing Enzymes.

Previous work has shown that hepatic levels of human glutathione transferase zeta 1 (GSTZ1) protein, involved in tyrosine catabolism and responsible for metabolism of the investigational drug dichloroacetate, increase in cytosol after birth before reaching a plateau around age 7. However, the mechanism regulating this change of expression is still unknown, and previous studies showed that GSTZ1 mRNA levels did not correlate with GSTZ1 protein expression. In this study, we addressed the hypothesis that microRNAs (miRNAs) could regulate expression of GSTZ1. We obtained liver samples from donors aged less than 1 year or older than 13 years and isolated total RNA for use in a microarray to identify miRNAs that were downregulated in the livers of adults compared with children. From a total of 2578 human miRNAs tested, 63 miRNAs were more than 2-fold down-regulated in adults, of which miR-376c-3p was predicted to bind to the 3' untranslated region of GSTZ1 mRNA. There was an inverse correlation of miR-376c-3p and GSTZ1 protein expression in the liver samples. Using cell culture, we confirmed that miR-376c-3p could downregulate GSTZ1 protein expression. Our findings suggest that miR-376c-3p prevents production of GSTZ1 through inhibition of translation. These experiments further our understanding of GSTZ1 regulation. Furthermore, our array results provide a database resource for future studies on mechanisms regulating human hepatic developmental expression. SIGNIFICANCE STATEMENT: Hepatic glutathione transferase zeta 1 (GSTZ1) is responsible for metabolism of the tyrosine catabolite maleylacetoacetate as well as the investigational drug dichloroacetate. Through examination of microRNA (miRNA) expression in liver from infants and adults and studies in cells, we showed that expression of GSTZ1 is controlled by miRNA. This finding has application to the dosing regimen of the drug dichloroacetate. The miRNA expression profiles are provided and will prove useful for future studies of drug-metabolizing enzymes in infants and adults.

Human Cytomegalovirus Latency and Myelosuppression: A microRNA-Dependent Yin and Yang Regulatory Loop.

In this issue of Cell Host & Microbe, Hancock et al., 2020 show that latent HCMV infection, and specifically miR-US5-2, induces TGF-ß secretion, which inhibits myelopoiesis in uninfected HPCs. They also show that HCMV-infected cells become resistant to TGF-ß signaling through targeting of SMAD3 by miR-UL22-A-3p and -5p.

PDGFRA defines the mesenchymal stem cell Kaposi's sarcoma progenitors by enabling KSHV oncogenesis in an angiogenic environment.

Kaposi's sarcoma (KS) is an AIDS-defining cancer caused by the KS-associated herpesvirus (KSHV). Unanswered questions regarding KS are its cellular ontology and the conditions conducive to viral oncogenesis. We identify PDGFRA(+)/SCA-1(+) bone marrow-derived mesenchymal stem cells (Pα(+)S MSCs) as KS spindle-cell progenitors and found that pro-angiogenic environmental conditions typical of KS are critical for KSHV sarcomagenesis. This is because growth in KS-like conditions generates a de-repressed KSHV epigenome allowing oncogenic KSHV gene expression in infected Pα(+)S MSCs. Furthermore, these growth conditions allow KSHV-infected Pα(+)S MSCs to overcome KSHV-driven oncogene-induced senescence and cell cycle arrest via a PDGFRA-signaling mechanism; thus identifying PDGFRA not only as a phenotypic determinant for KS-progenitors but also as a critical enabler for viral oncogenesis.

Identification of murine gammaherpesvirus 68 miRNA-mRNA hybrids reveals miRNA target conservation among gammaherpesviruses including host translation and protein modification machinery.

Gammaherpesviruses, including the human pathogens Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), establish lifelong latent infection in B cells and are associated with a variety of tumors. In addition to protein coding genes, these viruses encode numerous microRNAs (miRNAs) within their genomes. While putative host targets of EBV and KSHV miRNAs have been previously identified, the specific functions of these miRNAs during in vivo infection are largely unknown. Murine gammaherpesvirus 68 (MHV68) is a natural pathogen of rodents that is genetically related to both EBV and KSHV, and thus serves as an excellent model for the study of EBV and KSHV genetic elements such as miRNAs in the context of infection and disease. However, the specific targets of MHV68 miRNAs remain completely unknown. Using a technique known as qCLASH (quick crosslinking, ligation, and sequencing of hybrids), we have now identified thousands of Ago-associated, direct miRNA-mRNA interactions during lytic infection, latent infection and reactivation from latency. Validating this approach, detailed molecular analyses of specific interactions demonstrated repression of numerous host mRNA targets of MHV68 miRNAs, including Arid1a, Ctsl, Ifitm3 and Phc3. Notably, of the 1,505 MHV68 miRNA-host mRNA targets identified in B cells, 86% were shared with either EBV or KSHV, and 64% were shared among all three viruses, demonstrating significant conservation of gammaherpesvirus miRNA targeting. Pathway analysis of MHV68 miRNA targets further revealed enrichment of cellular pathways involved in protein synthesis and protein modification, including eIF2 Signaling, mTOR signaling and protein ubiquitination, pathways also enriched for targets of EBV and KSHV miRNAs. These findings provide substantial new information about specific targets of MHV68 miRNAs and shed important light on likely conserved functions of gammaherpesvirus miRNAs.

Downregulation of the human peripheral myelin protein 22 gene by miR-29a in cellular models of Charcot-Marie-Tooth disease.

The majority of hereditary neuropathies are caused by duplication of the peripheral myelin protein 22 (PMP22) gene. Therefore, mechanisms to suppress the expression of the PMP22 gene have high therapeutic significance. Here we asked whether the human PMP22 gene is a target for regulation by microRNA 29a (miR-29a). Using bioinformatics, we determined that the human PMP22 gene contains the conserved seed sequence of the miR-29a binding site and this regulatory motif is included in the duplicated region in neuropathic patients. Using luciferase reporter assays in HEK293 cells, we demonstrated that transient transfection of a miR-29a mimic is associated with reduction in PMP22 3'UTR reporter activity. Transfecting normal and humanized transgenic neuropathic mouse Schwann cells with a miR-29a expression plasmid effectively lowered both the endogenous mouse and the transgenic human PMP22 transcripts compared with control vector. In dermal fibroblasts derived from neuropathic patients, ectopic expression of miR-29a led to ~50% reduction in PMP22 mRNA, which corresponded to ~20% decrease in PMP22 protein levels. Significantly, miR-29a-mediated reduction in PMP22 mitigated the reduced mitotic capacity of the neuropathic cells. Together, these results support further testing of miR-29a and/or PMP22-targeting siRNAs as therapeutic agents for correcting the aberrant expression of PMP22 in neuropathic patients.