Background:Tenofovir-lamivudine-dolutegravir (TLD) is an effective, single-tablet ART regimen but the causes of virologic failure that can occur despite adherence to TLD are incompletely understood, especially when resistance mutations in integrase (IN) are absent. Noncanonical resistance mutations in nucleocapsid (NC) have been selected in cell culture and are associated with decreased dolutegravir (DTG) susceptibility in vitro . Samples from the ACTG A5381-Hakim study were examined to assess the potential contribution of NC mutations to virologic failure on TLD. Methods:A5381-Hakim was an observational cohort study that enrolled individuals with HIV-1 RNA >1000 copies/mL when initiating TLD as first line ART or switching from failing non-nucleoside reverse transcriptase inhibitor (NNRTI)-based or protease inhibitor (PI)-based ART. Whole HIV RNA genome next generation sequencing was performed on paired plasma samples from study entry and confirmed virologic failure in 56 participants receiving TLD for ≥six months. Mutations relative to the HIV HXB2 reference genome were identified with DeepChek® software using a mutation reporting threshold of five percent mixed-base frequency. Canonical drug resistance mutations (DRMs) were identified within the DeepChek® software using Stanford's HIVdb v9.8 algorithm. Site-directed NC and IN mutants were tested for susceptibility to DTG, raltegravir and cabotegravir in the TZM-bl HIV-1 indicator cell line and evaluated for infectivity and replication in multi-round assays. Results:Mutations in NC that emerged between TLD initiation and virologic failure were identified in 11 of 56 (20%) participants. Of these 11, three participants also had IN mutations at virologic failure but not at study entry, suggesting dual selection. Selected mutations in the NC zinc-finger domain included V13I, K20R, E21V, N27I/S, A30T, K34R, K38R, K41G/R/N, Q45R and/or M46I, alone or in combination with other NC and/or IN mutations. Specific NC mutations (K20R, N27I, A30T, K41N, M46I) conferred a significant decrease in DTG susceptibility. The combination of certain NC with IN mutations (e.g. NC N27I and IN R263K) conferred greater reduction in susceptibility to DTG in vitro than either mutation alone. Conclusions:This work provides the first clinical evidence of NC mutation selection in individuals on failing TLD ART and shows that NC mutations, combined with IN mutations, can further decrease susceptibility to DTG. Our findings support additional investigations of the contributions of mutations outside of IN to virologic failure of integrase inhibitor-containing ART regimens.

The American College of Medical Genetics and Genomics (ACMG) recommends Tier-3 reproductive carrier screening for 97 genes associated with autosomal recessive (AR) conditions (AR genes). Gene selection for screening is based on a gene carrier frequency (GCF) of ≥1/200 in the Genome Aggregation Database (gnomAD)v2 populations and disease severity. The utility of ACMG Tier 3 in the Middle Eastern population is unclear as this ancestral group was not represented in the gnomADv2. We utilized genome data from 14,392 individuals in the Qatar Genome Project to estimate the carrier frequency of AR conditions in the Middle Eastern population. The frequency of 136,624 pathogenic/likely pathogenic variants in 2,987 AR genes from ClinVar was analyzed to estimate the GCF in the Qatari cohort. Genes with GCF ≥ 1/200 were curated by an expert panel for the severity of corresponding conditions. We identified 69 genes with GCF ≥ 1/200 associated with moderate to profound clinical presentations, 53 of which were unique to the Middle Eastern population. Common variants were observed with high frequency for individual genes such as IL2RA that suggested the presence of founder effects not described previously. Simulation studies predicted that inclusion of the ancestry-specific genes increased the chance of detecting at-risk couples from 3.85% to 8.15% in the Middle Eastern population. This study highlights the limitations of relying on global datasets to accurately identify the candidate genes for carrier screening in different populations. Our findings provide a framework for targeted, population-specific carrier-screening programs in regions not represented in the large genome datasets.

SARS-CoV-2 coronavirus emerged in 2019, leading to the Coronavirus disease 2019 (COVID-19). Expression of viral entry factors such as ACE2 and TMPRSS2 is higher in the testis, particularly in Sertoli cells, Leydig cells, and spermatogonia. To understand COVID-19's impact on testicular cell populations and gene expression, we analyzed testicular tissue samples from 28 COVID-19 patients and compared them with 23 non-diseased controls. COVID-19 samples showed increased immune cell infiltration, thrombosis, and reduced numbers of testicular cells. There was a significant decrease in Sertoli cells and spermatogonial stem cells (SSC) among COVID-19 patients, associated with high levels of DNA damage and apoptosis in these cell types. To explore the pathways through which the virus affects testicular function, we profiled 112,657 single-nucleus transcriptomes from the testes of 4 COVID-19 patients and 4 controls. We found that COVID-19 infection alters multiple transcriptome clusters and induces a new COVID-19-specific cluster. To confirm that these transcriptome changes are COVID-19-specific, we compared our results with those from the brains of patients with COVID-19 and influenza. We observed an average of 144 dysregulated genes unique to COVID-19, regardless of tissue type. Lastly, we examined whether the SSC phenotype seen in fatal COVID-19 cases was also present in recovered patients. Indeed, recovered patients also exhibited high DNA damage and a reduced SSC population at 3, 6, and 12 months post-infection. Additionally, embryos derived from recovered patients’ sperm showed lower fertilization rates compared to control-derived embryos and fewer live births. Overall, our findings demonstrate that SARS-CoV-2 disrupts spermatogenesis, alters the transcriptional landscape, and affects human testicular architecture and function well after the acute phase of infection, with potential long-term consequences for male fertility.

PURPOSE:The application of next-generation sequencing in prenatal and neonatal genomic medicine provides definite diagnosis and affects clinical decision making and reproductive planning. Despite recent advances, interpretation of variants identified by genome/exome sequencing in cases lacking obvious phenotypic abnormalities (stillbirth, miscarriage, and neonatal death) remains challenging. METHODS:To improve diagnostic accuracy, we created the Intolerome Database, a curated resource of 934 genes essential for viability in humans. This database has accumulated details on genes' mechanisms of action, phenotypes, inheritance, the mortality timing, and supporting publications. RESULTS:The Intolerome includes 59 (6.3%) genes linked to the first/second trimester miscarriages, 525 (56.2%) genes associated with stillbirth/neonatal death, and 350 (37.5%) genes with variants that can cause lethality at any prenatal/postnatal stage. De novo inheritance was documented for 159 autosomal-dominant genes. Heterozygous potentially lethal variants in 39 autosomal-recessive genes were present in all ancestries in gnomADv4.1.0 at a frequency of ≥1/100 individuals, underlining the major pregnancy loss contributors. CONCLUSION:The Intolerome serves as a comprehensive resource for identifying and interpreting genomic variants linked to fetal and neonatal mortality. It will support clinicians, laboratory professionals, and researchers in advancing the diagnosis and understanding of lethal genetic conditions, offering new insights into their clinical presentations and inheritance patterns.

Fetal Alcohol Spectrum Disorder, caused by prenatal alcohol exposure, remains a major public health concern, affecting up to 1 in 20 U.S. schoolchildren. Pregnancy is typically not recognized until six weeks of gestation, alcohol exposure often occurs unknowingly during the peri-conceptional period, a highly vulnerable developmental window of oocyte maturation. The placenta is increasingly recognized as a mediator of alcohol-induced neurodevelopmental risk through the placenta–brain axis. We characterize peri-conceptional alcohol exposure on placental development in a mouse PCA model mimicking human drinking patterns. Single-cell transcriptomic analyses on late gestation placentas revealed disruption of extracellular matrix and collagen-remodeling pathways within the labyrinth; processes essential for placental vascular development. Mitochondria isolated from E17.5 placentas showed compromised respiration. We assess placental vascular functions with non-invasive dynamic contrast-enhanced MRI, enabling analysis across placental compartments for longitudinal evaluation of perfusion, substrate delivery, and contractile function. We demonstrate that PCA exposure arrests placental vascularization, perfusion, and morphological maturation. PCA exposure before implantation altered long-term placental programming, resulting in impaired placental perfusion and mitochondrial functions across gestation. These findings implicate placental dysfunction as a critical mechanism in Fetal Alcohol Spectrum Disorder pathogenesis and identify extracellular matrix remodeling and mitochondria as potential therapeutic targets following PCA exposure.

Dysregulations of epithelial-immune interactions frequently culminate in chronic inflammatory diseases of the skin, lungs, kidneys, and gastrointestinal tract. Yet, the intraepithelial processes that initiate and perpetuate inflammation in these organs are poorly understood. Here, by utilizing redox lipidomics we identified ferroptosis-associated peroxidation of polyunsaturated phosphatidylethanolamines in the epithelia of patients with asthma, cystic fibrosis, psoriasis, and renal failure. Focusing on psoriasis as a disease model, we used high-resolution mass spectrometry imaging and identified keratin 14-expressing (K14-expressing) keratinocytes executing a ferroptotic death program in human psoriatic skin. Psoriatic phenotype with characteristic Th1/Th17 skin and extracutaneous immune responses was initiated and maintained in a murine model designed to actuate ferroptosis in a fraction of K14+ glutathione peroxidase 4-deficient (Gpx4-deficient) epidermal keratinocytes. Importantly, an antiferroptotic agent, liproxstatin-1, was as effective as clinically relevant biological IL-12/IL-23/ TNF-alpha-targeting therapies or the depletion of T cells in completely abrogating molecular, biochemical, and morphological features of psoriasis. As ferroptosis in select epidermal keratinocytes triggers and sustains a pathological psoriatic multiorgan inflammatory circuit, we suggest that strategies targeting ferroptosis or its causes may be effective in preventing or ameliorating a variety of chronic inflammatory diseases.

Stroke is a leading cause of mortality and disability globally. Despite advancements in acute stroke therapies, patient outcomes with ischemic stroke remain suboptimal. Understanding its molecular mechanisms is crucial for developing effective treatments. Angiogenesis actively contributes to post-stroke functional recovery and improves long-term survival in stroke patients. Pericytes are essential for maintaining vascular stability and promoting angiogenesis. We hypothesized that microRNA-15a/16-1 in pericytes significantly modulates post-stroke angiogenesis and neurological recovery. Using a pericyte-specific miR-15a/16-1 conditional knockout (cKO) mouse model, we found that genetic deletion of miR-15a/16-1 in pericytes enhances angiogenesis, promotes cerebral blood flow recovery, and improves sensorimotor and cognitive outcomes following ischemic stroke. Mechanistically, RNA sequencing identified several novel targets of miR-15a/16-1, including Pappa2, Fgf9, Islr, and Ccr2. Interestingly, Pappa2, Fgf9, and Islr function as secreted proteins. Luciferase reporter assays demonstrated that miR-15a/16-1 directly binds and suppresses Pappa2, Fgf9, Islr, and Ccr2 activity in cultured pericytes. In vivo and in vitro assays further confirmed that miR-15a/16-1 silencing in pericytes significantly elevates the protein levels of Pappa2, Fgf9, Islr, and Ccr2 and enhances endothelial cell proliferation, migration, and tube formation under ischemic conditions. These findings suggest that targeting miR-15a/16-1 in pericytes offers a promising therapeutic strategy for enhancing stroke recovery by promoting neurovascular repair and reducing brain damage.

Bronchopulmonary dysplasia, the most prevalent chronic lung disease of prematurity, is often treated with glucocorticoids (GCs) such as dexamethasone (DEX), but their use is encumbered with several adverse somatic, metabolic, and neurologic effects. We previously reported that systemic delivery of the GC prodrug ciclesonide (CIC) in neonatal rats activated glucocorticoid receptor (GR) transcriptional responses in lung but did not trigger multiple adverse effects caused by DEX. To determine whether limited systemic metabolism of CIC was solely responsible for its enhanced safety profile, we treated neonatal rats with its active metabolite desisobutyryl-ciclesonide (Des-CIC). DEX but not Des-CIC caused a reduction in body weight as well as reduced insulin-like growth factor-1 serum levels and chronic hyperglycemia in neonatal rats. However, Des-CIC was as effective as DEX in reducing the expression of various bleomycin-induced proinflammatory cytokine mRNAs. In vitro studies with various cell types demonstrate the potent GR transactivation and transrepression activity of Des-CIC, although genome-wide transcriptomic analyses reveal differences in DEX vs. Des-CIC responses in neonatal rat lung and liver tissue. Des-CIC is a GR super-agonist as revealed by an in vitro coregulator peptide binding assay. In addition, molecular dynamics simulations revealed unique Des-CIC-dependent allosteric signaling pathways between specific residues in the GR ligand-binding domain and receptor surfaces interacting with coregulator peptides. Thus, Des-CIC is a potential novel selective GR modulator that could impart a favorable therapeutic index for CIC use for even modest durations of GC exposure which could have long-lasting adverse somatic, metabolic, or neurologic effects.

Non-small cell lung cancer exhibits the highest rates of brain metastases (BMs) among all solid tumors, presenting a major clinical challenge. The development of novel therapeutic strategies targeting BMs is clearly needed. We identified a significant enrichment of MET amplification in lung adenocarcinoma (LUAD) BMs compared with primary LUAD and extracranial metastases in oncogene driver-negative patients. Of note, MET-amplified BMs were responsive to MET inhibitors in vivo, including models with acquired MET amplification at the time of metastasis. MET alterations (amplifications and/or mutations) were also more frequently detected in circulating tumor DNA from patients with LUAD BMs than in those without BMs. MET-altered BMs also demonstrated unique genomic features compared with non-MET-altered BMs. Transcriptomic analyses revealed that in contrast to MET WT BMs, MET-amplified BMs exhibited a more inflamed tumor microenvironment and displayed evidence of metabolic adaptation, particularly a reliance on glycolysis in contrast to OXPHOS in MET WT BMs. Furthermore, MET-amplified BMs demonstrated evidence of epithelial-mesenchymal transition signaling, including increased expression of TWIST1. Patients with MET-amplified BMs had significantly shorter overall survival. These findings highlight MET amplification as a critical driver of LUAD BMs, emphasizing its potential as a therapeutic target.

Declining mitochondrial function is an established feature of aging and contributes to most aging-related diseases through its impact on various pathologies such as chronic inflammation, fibrosis and cellular senescence. Our recent work suggests that benign prostatic hyperplasia, which is an aging-related disease frequently associated with inflammation, fibrosis and senescence, is characterized by a decline in mitochondrial function. Here, we utilize glycolytic restriction and pharmacologic inhibition of the mitochondrial electron transfer chain complex I to promote mitochondrial dysfunction and identify the cellular processes impacted by declining mitochondrial function in benign prostate stromal cells. Using this model, we show that mitochondrial dysfunction induced alterations in cell-cell and cell-matrix adhesion, elevated fibronectin expression, resistance to anoikis and stress-induced premature senescence (SIPS). We also showed that ablation of ZC3H4, a transcription termination factor implicated in anoikis-resistance and reduced in BPH relative to normal prostates, phenocopied various phenotypes in the human BHPrS1 prostate stromal cell line that resulted from inhibition of complex I. Furthermore, ZC3H4 ablation resulted in the elevation of mitochondrial superoxide (mtROS) and mitochondrial membrane potential, altered mitochondrial morphology and NAD+/NADH ratio, and reduced CI function in BHPrS1 cells. Thus, ZC3H4 loss promotes mitochondrial dysfunction to drive pathophysiologic changes in the stromal compartment that are features of the aging prostate.

Faithful meiotic segregation requires pairwise alignment of the homologous chromosomes and their synaptonemal complex (SC) mediated stabilization. Here, we investigate factors that promote and coordinate these events during C. elegans meiosis. We identify BRA-2 (BMP Receptor Associated family member 2) as an interactor of HIM-17, previously shown to promote double-strand break formation. We found that loss of bra-2 impairs synapsis elongation without affecting homolog recognition, chromosome movement or SC maintenance. Epistasis analyses reveal previously unrecognized activities for HIM-17 in regulating homolog pairing and SC assembly in a partially overlapping manner with BRA-2. We show that removing bra-2 or him-17 restores nuclear clustering, recruitment of PLK-2 at the nuclear periphery, and abrogation of ectopic synapsis in htp-1 mutants, suggesting intact CHK-2-mediated signaling and presence of a barrier that prevents SC polymerization in the absence of homology. Our findings shed light on the regulatory mechanisms ensuring faithful pairing and synapsis.

Introduction: Angiogenesis actively contributes to post-stroke functional recovery and improves long-term survival in stroke patients. Previously, we demonstrated that endothelium-targeted deletion of miR-15a/16-1 promotes post-stroke angiogenesis by enhancing classic pro-angiogenic factors and their receptors, and genetic deletion of miR-15a/16-1 in pericytes likewise promotes post-stroke functional recovery by stimulating cerebral angiogenesis. Here, we further investigate the underlying mechanisms and downstream targets of pericytic miR-15a/16-1 in cerebral angiogenesis after ischemic stroke. Methods: Pericyte-miR15a/16-1 conditional knockout mice (Pericyte-miR-15a/16-1 cKO) and wild-type (WT) controls were subjected to 1h MCAO followed by 7d reperfusion. Cerebral microvessels were extracted and subjected to RNA-seq to identify the significant differentially expressed genes (DEGs) that related to miR-15a/16-1 and angiogenesis among experimental groups. TargetScan and miRDB were used to predict potential miR-15a/16-1 downstream targets. Primary mouse brain vascular pericytes (mBVPs) were treated with lentivirus to achieve loss- or gain-of-miR-15a/16-1 function, and subjected to OGD 8h and reperfusion 24h. Pro-angiogenic factors were further verified by dual-luciferase reporter assay, qPCR, and western blotting. Results: RNA-seq analysis showed 289 upregulated genes (P < 0.05 & at least one CPM > 1) and 399 upregulated genes from 100 positive enriched gene sets (GESA) in cerebral microvessels of pericyte-miR-15a/16-1 cKO mice compared to WT controls 7d after MCAO. By comparing to miR-15a/16-1 potential target database from TargetScan and miRDB, 9 angiogenic genes were identified. Further biochemical analysis revealed that Pappa2, Fgf9, Islr, and Ccr2 mRNA and protein expression are significantly increased in cerebral microvessels of cKO mice and mBVPs with loss-of-miR-15a/16-1 function. Furthermore, dual-luciferase assay results verified that loss- or gain-of-miR-15a/16-1 function significantly increased or decreased luciferase reporter activity of these four genes, respectively. Conclusion: Our findings reveal novel pericytic miR-15a/16-1 mediated angiogenic targets in ischemic stroke.

CRISPR screen results for human SKMEL28 cells

De novo purine synthesis and one carbon metabolism genes are associated with worse overall survival in metastatic melanomas. Data from TCGA Skin Cutaneous Melanoma PanCancer Atlas (367 metastatic melanomas). Raw data can be found in Supplementary Table S8. A,De novo purine synthesis and one carbon metabolism genes in TCGA metastatic melanoma samples. Red indicates increased mRNA expression. Blue indicates decreased mRNA expression. Overall survival probability of patients with alterations in de novo purine synthesis and one carbon metabolism genes (B), de novo pyrimidine genes (C), overall nucleotide biosynthesis genes (D), and nucleotide salvage genes (E). log-rank P-value and 95% confidence interval.

shp16 tumor bearing mice treated with methotrexate have a trend towards a survival advantage; and methotrexate does not affect body weight or blood cell counts. Related to Figure 5.

mRNA expression of nucleotide metabolism genes

The oral mucosa is the interface between the host immune response and the oral microbiota. In periodontal disease, the microbial plaque elicits a tissue-destructive immune response. Removal of the microbial stimulus initiates active resolution of inflammatory. Here, we use single-cell RNA- sequencing (scRNA-seq) to characterize the immune response within the oral mucosa across three distinct conditions of periodontal health, disease, and resolution in mice. We report gene expression shifts across the three conditions are driven by macrophage and neutrophils and identify a unique gene signature that characterizes resolution of disease. Macrophage subgroups are identified that demonstrate differential expansion across conditions, including a subgroup that expands during resolution with an immunoregulatory gene signature and enriched for surface marker Cd74. We validate expansion of this subgroup during resolution via flow cytometry. This work presents a robust single- cell dataset of immunological changes in the oral mucosa and identifies a resolution-associated macrophage phenotype in mucosal immunity.