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Monocytes (Mos)/macrophages (Mϕs) orchestrate biological processes critical for efficient skin wound healing. However, current understanding of skin wound Mo/Mϕ heterogeneity is limited by traditional experimental approaches such as flow cytometry and immunohistochemistry. Therefore, we sought to more fully explore Mo/Mϕ heterogeneity and associated state transitions during the course of excisional skin wound healing in mice using single-cell RNA sequencing. The live CD45+CD11b+Ly6G− cells were isolated from skin wounds of C57BL/6 mice on days 3, 6, and 10 postinjury and captured using the 10x Genomics Chromium platform. A total of 2813 high-quality cells were embedded into a uniform manifold approximation and projection space, and eight clusters of distinctive cell populations were identified. Cluster dissimilarity and differentially expressed gene analysis categorized those clusters into three groups: early-stage/proinflammatory, late-stage/prohealing, and Ag-presenting phenotypes. Signature gene and Gene Ontology analysis of each cluster provided clues about the different functions of the Mo/Mϕ subsets, including inflammation, chemotaxis, biosynthesis, angiogenesis, proliferation, and cell death. Quantitative PCR assays validated characteristics of early- versus late-stage Mos/Mϕs inferred from our single-cell RNA sequencing dataset. Additionally, cell trajectory analysis by pseudotime and RNA velocity and adoptive transfer experiments indicated state transitions between early- and late-state Mos/Mϕs as healing progressed. Finally, we show that the chemokine Ccl7, which was a signature gene for early-stage Mos/Mϕs, preferentially induced the accumulation of proinflammatory Ly6C+F4/80lo/− Mos/Mϕs in mouse skin wounds. In summary, our data demonstrate the complexity of Mo/Mϕ phenotypes, their dynamic behavior, and diverse functions during normal skin wound healing.

The hepatitis B virus (HBV) transgenic mouse model was used to interrogate the origins of HCC heterogeneity. HBV biosynthesis was used as a marker of liver tumor heterogeneity. Principal component and correlation analysis of HBV and cellular transcript levels demonstrated major differences within and between the gene expression profiles of Apc-deficient, Apc-deficient Pten-deficient, and Pten-deficient HCC. Hence, both oncogenic stimuli and zonal hepatocyte properties determine heterogeneous HCC phenotypes. Additionally, Apc-deficient HCC display decreased expression of Apob, Otc and Tet2 relative to Pten-deficient HCC and control liver tissue suggesting their gene products may represent markers of Apc-deficient HCC. A subset of human HCC with mutations in the β-catenin gene (CTNNB1) displayed a gene expression profile similar to that observed in the mouse Apc-deficient HCC indicating this model of liver cancer may be useful for interrogating the molecular properties of these tumors and their potential therapeutic vulnerabilities.

GO terms associated with upregulated genes bearing new ∙NO-induced H3K9ac peaks around promoter regions

Supplementary Materials and Methods. Figure S1. Nitric oxide induces significant changes in histone posttranslational modifications. Figure S2. NO-mediated gene expression changes are consistent with the induction of an oncogenic phenotype. Figure S3. Nitric oxide alters the distribution patterns of H3K9ac and H3K9me2 across the genome. Figure S4. NO-upregulated genes with a new promoter-associated H3K9ac peak are involved in critical tumorigenic pathways. Table S1. NO-mediated changes in global levels of histone acetylation. Table S2. List of NO-regulated miRNAs (1.5 Fold Change cut-off). Table S3. Control ChIP-PCR primer sequences. Table S4. List of transcription factors with binding motifs found exclusively around promoters of upregulated genes with a new NO-induced H3K9ac peak.

The complex involvement of neutrophils in inflammatory diseases makes them intriguing but challenging targets for therapeutic intervention. Here, we tested the hypothesis that varying endocytosis capacities would delineate functionally distinct neutrophil subpopulations that could be specifically targeted for therapeutic purposes. By using uniformly sized (∼120 nm in diameter) albumin nanoparticles (ANP) to characterize mouse neutrophils

Approximately 70% of breast cancers are estrogen receptor (ER) positive and can be treated with endocrine therapy. Unfortunately, 30-40% of tumors will recur, often as therapy-resistant metastatic disease. One contributor to disease recurrence is a population of cells with stem-like features (i.e. breast cancer stem cells, BCSCs). Previously, we have shown that positive crosstalk between two critical transcription factors, ER and NFκB, can contribute to the expansion of ER+ BCSCs, as indicated by increased mammosphere (MS) forming efficiency and BCSC markers. NFκB has been previously described as a major player in BCSC biology. However, the role of ER, as well as the interaction between ER and NFκB, in driving BCSCs remains unclear. We found in MS that both ER and NFκB are endogenously active and required for MS formation. To investigate whether ER and NFκB work together or independently, we made dual ERE and NFκB-RE reporter cell lines. We confirmed activation of both ER and NFκB in MS, but with a high degree of heterogeneity as indicated by 4 different cell populations (ERE+/NFκB-RE+, ERE+/NFκB-RE-, ERE-/NFκB-RE+ and ERE-/NFκB-RE-). Interestingly, we found >90% of MS arose from ERE+ cells while NFκB-RE+ cells failed to from MS, suggesting that ER but not NFκB activity was sufficient to initiate MS development. Moreover, ERE+ cells, with or without NFκB-RE activity, formed more secondary MS and expressed higher levels of stem cell-associated genes than ERE- cells, indicating ERE+ cells are more stem-like. To study ER and NFκB function in more detail, we utilized single cell RNA sequencing of MS. We identified 4 clusters with clusters 1 and 2 enriched for stem cell gene signatures. Bioinformatic analysis revealed Cluster 2 to be enriched for ER, proliferation, and DREAM complex signatures, and we confirmed these genes were enriched in ERE+ cells. However, we also found that ER activity is associated with activation of the DREAM complex in standard cell culture. This suggests that ER regulation of proliferation via the DREAM complex may not be specific for BCSCs. In contrast to Cluster 2, Cluster 1 was enriched with ER and NFκB signatures, as well as an ER-NFκB crosstalk signature. In addition, Cluster 1 was enriched with pathways related to adaptive stress response such as: oxidative phosphorylation, unfolded protein response, and protein secretion. As some studies have reported activation of stress pathways is associated with maintenance of stemness, we hypothesize that ER and NFκB may work together to promote stem features through activation of multiple stress pathways to increase survival and growth in an anchorage-independent environment. To determine whether either Cluster 1 or Cluster 2 derived signatures are associated with clinical outcome, we first interrogated single cell RNA sequencing data from a metastatic ER+ PDX model. The Cluster 2 signature was enriched in proliferative cell populations found in both primary and metastatic tumors. In contrast, the Cluster 1 signature was enriched only in a metastatic tumor cell population. We also showed that expression of signatures derived from Cluster 1 or 2 in ER+ breast cancer patient tumors predict poor relapse free survival in the METABRIC dataset. However, only co-expression of both signatures predicted poor disease-free survival. Taken together, our findings suggest there are two unique subpopulations of stem-like cells, one of which is dependent on ER driven proliferation, whereas the other population relies on an ER-NFkB mediated stress response for survival and self-renewal. Furthermore, both of these population signatures in patients’ tumors are associated with more aggressive disease and poor outcome. Citation Format: Svetlana E. Semina, Mark Maienschein-Cline, Elaine T. Alarid, Carol A. Sartorius, Jonna Frasor. Identification of novel ER and ER-NFκB driven stem-like cell populations in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-11-01.

We previously discovered using transcriptomics that rats undergoing withdrawal after chronic ethanol exposure had increased expression of several genes involved in RNA splicing in the hippocampus. Here, we examined RNA splicing in the rat hippocampus during withdrawal from chronic ethanol exposure and in postmortem hippocampus of human subjects diagnosed with alcohol use disorder (AUD). We found that expression of the gene encoding the splicing factor, poly r(C) binding protein 1 (PCBP1), was elevated in the hippocampus of rats during withdrawal after chronic ethanol exposure and AUD subjects. We next analyzed the rat RNA-Seq data for differentially expressed (DE) exon junctions. One gene, Hapln2, had increased usage of a novel 3’ splice site in exon 4 during withdrawal. This splice site was conserved in human HAPLN2 and was used more frequently in the hippocampus of AUD compared to control subjects. To establish a functional role for PCBP1 in HAPLN2 splicing, we performed RNA immunoprecipitation with a PCBP1 antibody in rat and human hippocampus. PCBP1 association was enriched near the HAPLN2 exon 4 3’ splice site in the hippocampus of rats during ethanol withdrawal and AUD subjects. Our results indicate a conserved role for the splicing factor PCBP1 in aberrant splicing of HAPLN2 after chronic ethanol exposure. As the HAPLN2 gene encodes an extracellular matrix protein involved in nerve conduction velocity, use of this alternative splice site is predicted to result in loss of protein function that could negatively impact neurotransmission in AUD.

Hippocampal neurogenesis is impaired in Alzheimer’s disease (AD) patients and familial Alzheimer’s disease (FAD) mouse models. However, it is unknown whether new neurons play a causative role in memory deficits. Here, we show that immature neurons were actively recruited into the engram following a hippocampus-dependent task. However, their recruitment is severely deficient in FAD. Recruited immature neurons exhibited compromised spine density and altered transcript profile. Targeted augmentation of neurogenesis in FAD mice restored the number of new neurons in the engram, the dendritic spine density, and the transcription signature of both immature and mature neurons, ultimately leading to the rescue of memory. Chemogenetic inactivation of immature neurons following enhanced neurogenesis in AD, reversed mouse performance, and diminished memory. Notably, AD-linked App, ApoE, and Adam10 were of the top differentially expressed genes in the engram. Collectively, these observations suggest that defective neurogenesis contributes to memory failure in AD.

Introduction: The voltage-gated proton channel Hv1 is a member of the voltage-gated ion channel family. Hv1 channel controls acid extrusion and regulates pH homeostasis in various cell types. Recent evidence showed that the Hv1 were expressed in the heart. Hypothesis: We propose the Hv1 channel is associated with cardiac function. Methods: To investigate the role of Hv1 in cardiac myocytes, we performed a transcriptomic analysis of wild-type (WT) and Hv1-/- mouse hearts. Results: Hv1 null hearts exhibited a differential transcriptome profile compared with wild-type hearts. The RNA-seq data indicating impaired pH homeostasis in Hv1 null hearts were the downregulated NADPH oxidoreductases (NOXs) and decreased expression of Cl-/HCO3- exchanger. Additionally, Hv1 null hearts exhibited differential expression of cardiac ion channels, suggesting a potential role of Hv1 in cardiac electrophysiological remodeling. Conclusions: The data indicated that Hv1 was involved in cardiac pH homeostasis and showed that Hv1 was a regulator of gene transcriptional networks controlling NOX signaling and CO2 homeostasis in the heart. The study highlights the importance of Hv1 in cardiac function and may present a new target associated with heart diseases.

Acute respiratory distress syndrome (ARDS) is the acute onset of non-cardiogenic pulmonary edema, hypoxemia, and respiratory failure, conditions associated with a high mortality rate. ARDS is frequently caused by lung infections including SARS-CoV-2. In light of the COVID-19 pandemic, development of therapeutic approaches to combat ARDS have become an urgent unmet medical need. As increased vascular leakage is a culprit of alveolar damage and lung inflammation, we hypothesize that an effective therapy against vascular leakage should improve the clinical outcome of ARDS patients. We have identified a drug candidate Vascular Therapeutics (VT)-109, an allosteric inhibitor of End Binding protein 3, which contributes to vascular leakage by amplifying pathological calcium signaling in endothelial cells. Treatment of mice with VT-109 reduced the leakage of proteinous fluids in lungs to 55.4% (n=7, p2-fold) suggesting that it promotes endothelial cell regeneration by activating the FOXM1 pathway. Our data demonstrate that VT-109 successfully treats vascular leakage and is a promising drug candidate for future clinical studies. Funding for this work is provided by the Congressionally Directed Medical Research Programs through the Peer Reviewed Medical Research Program (PRMRP) under award No. W81XWH-21-1-0639. Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the Department of Defense.

Summary Mutations in SCN5A, encoding the cardiac sodium channel, are linked with familial atrial fibrillation (AF) but the underlying pathophysiologic mechanisms and implications for therapy remain unclear. To characterize the pathogenesis of AF-linked SCN5A mutations, we generated patient-specific induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) from two kindreds carrying SCN5A mutations (E428K and N470K) and isogenic controls using CRISPR-Cas9 gene editing. We showed that mutant AF iPSC-aCMs exhibited spontaneous arrhythmogenic activity with beat-to-beat irregularity, prolonged action potential duration, and triggered-like beats. Single-cell recording revealed enhanced late sodium currents (INa,L) in AF iPSC-aCMs that were absent in a heterologous expression model. Gene expression profiling of AF iPSC-aCMs showed differential expression of the nitric oxide (NO)-mediated signaling pathway underlying enhanced INa,L. We showed that patient-specific AF iPSC-aCMs exhibited striking in vitro electrophysiological phenotype of AF-linked SCN5A mutations, and transcriptomic analyses supported that the NO signaling pathway modulated the INa,L and triggered AF., Highlights • Mutations in SCN5A, the gene encoding the cardiac sodium channel, have been linked with familial atrial fibrillation (AF) • SCN5A mutant iPSC-derived atrial cardiomyocytes (aCMs) exhibited arrhythmogenic activity with an AF-like phenotype • AF iPSC-aCMs carrying SCN5A mutation exhibited enhanced late sodium currents (INa,L) • Nitric oxide-mediated signaling pathway modulated enhanced INa,L in AF iPSC-aCMs, Hong et al. showed that patient-specific iPSC-aCMs exhibited striking electrophysiological phenotype of an AF-linked mutation that produced minimal changes in heterologous expression system, and discovered a link between enhanced late sodium currents (INa,L), transcriptional regulation of NO signaling, and triggered AF. These findings not only provide insights into underlying cellular mechanisms of AF-linked mutations but also highlight potential mechanism-based therapies for this common arrhythmia.

Introduction: Numerous studies over the past 30 years have shown that bioactive compounds present in functional foods, including vitamins, minerals and phytochemicals reduce cancer risk. For example, vitamins A and D derivatives found in fruits, vegetables, and dairy products, reduce the growth of breast, bladder, head, neck, lung, prostate, and skin cancers. However, the effects of these combined vitamins have not been previously reported for breast cancer.Aims: To investigate the activities of vitamin A (all-trans-retinoic acid; ATRA), as well as vitamins D2 and D3 in combination in the breast epithelial cancer cell lines T47D:A18, MCF-7, and SK-BR-3 and perform whole genome analysis in MCF-7 cells using RNA-seq.Methods: Breast cancer cells were cultured in appropriate media and treated with ATRA, D2 or D3 alone in concentrations from 1-10 μg/ml, or in combination at 1, 5, and 10 μg/ml. The CellTiter-Glo® 2.0 assay, Caspase-Glo®3/7, Caspase®8, and ApoTox-Glo™ Triplex assays measured cell viability and apoptosis. The effect of treatment on autophagy in MCF-7 cells was measured with a CYTO-ID® Autophagy Detection Kit 2.0. The whole transcriptome analysis was assessed using mRNA-seq and qPCR.Results: Separately, ATRA, D2, and D3 all reduced the viability of all breast cancer cell lines tested, with median inhibitory concentrations (IC50) between 2.1 to 31.7 μg/ml. However, when breast cancer cells were treated with combinations of ATRA+D2+D3, the IC50 was reduced indicating synergism. In MCF-7 cells, 5-flurouracil (5-FLU) had an IC50 of 1.37 µg/ml, while the vitamin A and D combination had an IC50 of 1.5 µg/ml, indicating the combination was ~90% as effective as 5-FLU. Treatment of MCF-7 cells with ATRA+D2+D3 enhanced caspase 3/7 activity, as well as the expression of Bax, BAD, PTEN and p53 (apoptosis canonical pathway), as well as induced autophagy. Whole genome analysis of treated MCF-7 cells showed a significant upregulation in gene expression in the autophagy, endoplasmic reticulum stress and the unfolded protein response apoptosis canonical pathways. Furthermore, MCF-7 cells treated with ATRA+D2+D3 demonstrated significant downregulation of gene expression in estrogen-mediated S phase entry and estrogen signaling canonical pathways suggesting antiestrogenic effects. Conclusions: Vitamins A and D combinations had synergistic effects in breast cancer cells and induced both apoptosis and autophagy. Transcriptional profiling showed significant alterations in gene expression patterns and upregulation of multiple cancer signaling pathways supporting the hypothesis that combining vitamins A and D is a more effective treatment than either vitamin alone. Keywords: all-trans-retinoic acid (ATRA), Vitamin D2, Vitamin D3, breast cancer cells, apoptosis, estrogen receptors

IL-4 plays an important role in the pathogenesis of atopic dermatitis (AD). Previously we showed that the expression of genes in chemotaxis, angiogenesis, inflammation and barrier functions is dysregulated in IL-4 transgenic (Tg) mice, a well-characterized AD mouse model. In this study, we aim to study differential expression of microRNAs in IL-4 Tg mice. As compared with wild-type mice, we found that 10 and 79 microRNAs are dysregulated in the skin of IL-4 mice before and after the onset of skin lesions, respectively. Bioinformatic analysis and previous reports show that these dysregulated microRNAs may be involved in the NF-κB, TLRs, IL-4/IL-13, MAPK and other pathways. We also found that miR-139-5p and miR-196b-3p are significantly up-regulated in the peripheral blood of IL-4 Tg mice. Taken together, our data have identified many dysregulated microRNAs in IL-4 Tg mice, which may play important roles in AD pathogenesis and pathophysiology.

Combination antiretroviral therapy (cART) dramatically changed the face of the HIV/AIDS pandemic, making it one of the most prominent medical breakthroughs of the past 3 decades. However, as the life span of persons living with HIV (PLWH) continues to approach that of the general population, the same cannot be said regarding their quality of life. PLWH are affected by comorbid conditions such as high blood pressure, diabetes, and neurocognitive impairment at a higher rate and increased severity than their age-matched counterparts. PLWH also have higher levels of inflammation, the drivers of which are not entirely clear. As cART treatment is lifelong, we assessed here the effects of cART, independent of HIV, on primary human monocyte-derived macrophages (MDMs). MDMs were unskewed or skewed to an alternative phenotype and treated with Atripla or Triumeq, two first-line cART treatments. We report that Triumeq skewed alternative MDMs toward an inflammatory nonsenescent phenotype. Both Atripla and Triumeq caused mitochondrial dysfunction, specifically efavirenz and abacavir. Additionally, transcriptome sequencing (RNA-seq) demonstrated that both Atripla and Triumeq caused differential regulation of genes involved in immune regulation and cell cycle and DNA repair. Collectively, our data demonstrate that cART, independent of HIV, alters the MDM phenotype. This suggests that cART may contribute to cell dysregulation in PLWH that subsequently results in increased susceptibility to comorbidities.

Data on transmission of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), among college athletes are limited. In August 2020, the Chicago Department of Public Health (CDPH) was notified of a cluster of COVID-19 cases among a university's men's and women's soccer teams. CDPH initiated an investigation, interviewed members of both teams, and collated laboratory data to understand transmission of SARS-CoV-2 within the teams. Numerous social gatherings with limited mask use or social distancing preceded the outbreak. Transmission resulted in 17 laboratory-confirmed COVID-19 cases across both teams (n = 45), likely from a single source introduction of SARS-CoV-2 (based on whole genome sequencing) and subsequent transmission during multiple gatherings. Colleges and universities are at risk for COVID-19 outbreaks because of shared housing and social gatherings where recommended prevention guidance is not followed. Improved strategies to promote mask use and social distancing among college-aged adults need to be implemented, as well as periodic repeat testing to identify asymptomatic infections and prevent outbreaks among groups at increased risk for infection because of frequent exposure to close contacts in congregate settings on and off campus.

Gene exchange via genetic transformation makes major contributions to antibiotic resistance of the human pathogen, Streptococcus pneumoniae (pneumococcus). The transfers begin when a pneumococcal cell, in a transient specialized physiological state called competence, attacks and lyses another cell, takes up fragments of the liberated DNA, and integrates divergent genes into its genome. Recently, it has been demonstrated that the pneumococcal cells can be enclosed in femtoliter-scale droplets for study of the transformation mechanism, offering the ability to characterize individual cell–cell interactions and overcome the limitations of current methods involving bulk mixed cultures. To determine the relevance and reliability of this new method for study of bacterial genetic transformation, we compared recombination events occurring in 44 recombinants recovered after competence-mediated gene exchange between pairs of cells confined in femtoliter-scale droplets vs. those occurring in exchanges in parallel bulk culture mixtures. The pattern of recombination events in both contexts exhibited the hallmarks of the macro-recombination exchanges previously observed within the more complex natural contexts of biofilms and long-term evolution in the human host.

Chronic HBV infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The phenotypes of HCC are diverse, in part, due to mutations in distinct oncogenes and/or tumor suppressor genes. These genetic drivers of HCC development have generally been considered as major mediators of tumor heterogeneity. Using the liver-specific Pten-null HBV transgenic mouse model of chronic viral infection, a critical role for liver lobule zone-specific gene expression patterns in determining HCC phenotype and β-catenin-dependent HBV biosynthesis is demonstrated. These observations suggest that the position of the hepatocyte within the liver lobule, and hence its intrinsic gene expression pattern at the time of cellular transformation, make critical contributions to the properties of the resulting liver tumor. These results may explain why therapies targeting pathways modulated by specific identified tumor driver genes display variable treatment efficacy.

Introduction: The limited success of pharmacological approaches to atrial fibrillation ( AF ) is due to limitations of in vitro and in vivo models and inaccessibility of human atrial tissue. Patient-specific induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) are a robust platform to model the heterogeneous myocardial substrate of AF, but their immaturity limits their fidelity. Objective: We hypothesized that a combinatorial approach of biochemical (triiodothyronine [ T3 ], insulin-like growth factor-1 [ IGF-1 ], and dexamethasone; collectively TID ), bioenergetic (fatty acids [ FA ]), and electrical stimulation ( ES ) will enhance electrophysiological ( EP ), structural, and metabolic maturity of iPSC- a CMs. Methods: We assessed maturation with whole cell patch clamping, calcium transients, immunofluorescence (IF), Seahorse Analyzer, contractility assay, RT-PCR, Western Blotting, and RNA sequencing (RNAseq). Using a time series with RNAseq we identified signaling pathways and transcriptional regulation that drive EP, structural, and metabolic atrial development and compared iPSC-aCM maturity with human aCMs (haCMs) obtained from the same patient. Results: TID+FA+ES significantly improved structural organization and cell morphology ( Fig. 1a ), enhanced membrane potential stability and improved depolarization ( Fig. 1b ), improved Ca 2+ kinetics with faster and increased Ca 2+ release from sarcoplasmic reticulum ( Fig. 1c ), and increased expression of Na + , Ca 2+ , and K + channels, markers of structural maturity, FA metabolism, and oxidative phosphorylation ( Fig. 1d ). There was no difference in each parameter between TID+FA+ES iPSC-aCMs and haCMs from the same patient. Conclusion: Our optimized, combinatorial TID+FA+ES approach markedly enhanced EP, structural, and metabolic maturity of human iPSC-aCMs, which will be useful for elucidating the genetic basis of AF developing precision drug therapies.

Positive effects of alcohol drinking such as anxiolysis and euphoria appear to be a crucial factor in the initiation and maintenance of alcohol use disorder (AUD). However, the mechanisms that lead from chromatin reorganization to transcriptomic changes after acute ethanol exposure remain unknown. Here, we used Assay for Transposase-Accessible Chromatin followed by high throughput sequencing (ATAC-seq) and RNA-seq to investigate epigenomic and transcriptomic changes that underlie anxiolytic effects of acute ethanol using an animal model. Analysis of ATAC-seq data revealed an overall open or permissive chromatin state that was associated with transcriptomic changes in the amygdala after acute ethanol exposure. We identified a candidate gene, Hif3a (Hypoxia-inducible factor 3, alpha subunit), that had ‘open’ chromatin regions (ATAC-seq peaks), associated with significantly increased active epigenetic histone acetylation marks and decreased DNA methylation at these regions. The mRNA levels of Hif3a were increased by acute ethanol exposure, but decreased in the amygdala during withdrawal after chronic ethanol exposure. Knockdown of Hif3a expression in the central nucleus of amygdala attenuated acute ethanol-induced increases in Hif3a mRNA levels and blocked anxiolysis in rats. These data indicate that chromatin accessibility and transcriptomic signatures in the amygdala after acute ethanol exposure underlie anxiolysis and possibly prime the chromatin for the development of AUD.

Supplemental Digital Content is available in the text., Background. T lymphocyte–mediated acute rejection is a significant complication following solid organ transplantation. Standard methods of monitoring for acute rejection rely on assessing histological tissue damage but do not define the immunopathogenesis. Additionally, current therapies for rejection broadly blunt cellular immunity, creating a high risk for opportunistic infections. There is, therefore, a need to better understand the process of acute cellular rejection to help develop improved prognostic tests and narrowly targeted therapies. Methods. Through next-generation sequencing, we characterized and compared the clonal T-cell receptor (TCR) repertoires of graft-infiltrating lymphocytes (GILs) and blood-derived lymphocytes from a hand transplant recipient over 420 days following transplantation. We also tracked the TCR clonal persistence and V beta (BV) gene usage, evaluating overlap between these 2 compartments. Results. TCR repertoires of blood and GIL populations remained distinct throughout the sampling period, and differential BV usage was consistently seen between these compartments. GIL TCR clones persisted over time and were seen in only limited frequency in the blood T-lymphocyte populations. Conclusions. We demonstrate that blood monitoring of TCR clones does not reveal the pathogenic process of acute cellular rejection in transplanted tissue. GILs show clonal persistence with biased BV usage, suggesting that tissue TCR clonal monitoring could be useful, although a deeper understanding is necessary to prognosticate rejection based on TCR clonal repertoires. Finally, the distinct TCR BV usage bias in GILs raises the possibility for prevention and therapy of acute cellular rejection based on targeting of specific TCR clones.

In B lymphopoiesis, activation of the pre-B cell antigen receptor (pre-BCR) is associated with both cell cycle exit and Igk recombination. Yet how the pre-BCR mediates these functions remains unclear. Here, we demonstrate that the pre-BCR initiates a feed-forward amplification loop mediated by the transcription factor interferon regulatory factor 4 and the chemokine receptor C-X-C motif chemokine receptor 4 (CXCR4). CXCR4 ligation by C-X-C motif chemokine ligand 12 activates the mitogen-activated protein kinase extracellular-signal-regulated kinase, which then directs the development of small pre- and immature B cells, including orchestrating cell cycle exit, pre-BCR repression, Igk recombination and BCR expression. In contrast, pre-BCR expression and escape from interleukin-7 have only modest effects on B cell developmental transcriptional and epigenetic programs. These data show a direct and central role for CXCR4 in orchestrating late B cell lymphopoiesis. Furthermore, in the context of previous findings, our data provide a three-receptor system sufficient to recapitulate the essential features of B lymphopoiesis in vitro.

Here we describe a novel method for studying the protein “interactome” in primary human cells and apply this method to investigate the effect of posttranslational protein modifications (PTMs) on the protein’s functions. We created a novel “biomimetic microsystem platform” (Bio-MSP) to isolate the protein complexes in primary cells by covalently attaching purified His-tagged proteins to a solid microscale support. Using this Bio-MSP, we have analyzed the interactomes of unphosphorylated and phosphomimetic end-binding protein-3 (EB3) in endothelial cells. Pathway analysis of these interactomes demonstrated the novel role of EB3 phosphorylation at serine 162 in regulating the protein’s function. We showed that phosphorylation “switches” the EB3 biological network to modulate cellular processes such as cell-to-cell adhesion whereas dephosphorylation of this site promotes cell proliferation. This novel technique provides a useful tool to study the role of PTMs or single point mutations in activating distinct signal transduction networks and thereby the biological function of the protein in health and disease.

Cardiac progenitor cells (CPCs) being multipotent offer a promising source for cardiac repair due to their ability to proliferate and multiply into cardiac lineage cells. Here, we explored a novel strategy for human CPCs generation from human induced pluripotent stem cells (hiPSCs) using a cardiogenic small molecule, isoxazole (ISX-9) and their ability to grow in the scar tissue for functional improvement in the infarcted myocardium. CPCs were induced from hiPSCs with ISX-9. CPCs were characterized by immunocytochemistry and RT-PCR. The CPC survival and differentiation in the infarcted hearts were determined by in vivo transplantation in immunodeficient mice following left anterior descending artery ligation and their effects were determined on fibrosis and functional improvement. ISX-9 simultaneously induced expression of cardiac transcription factors, NK2 homeobox 5, islet-1, GATA binding protein 4, myocyte enhancer factor-2 in hiPSCs within 3 days of treatment and successfully differentiated into three cardiac lineages in vitro. Messenger RNA and microRNA-sequencing results showed that ISX-9 targeted multiple cardiac differentiation, proliferation signaling pathways and upregulated myogenesis and cardiac hypertrophy related-microRNA. ISX-9 activated multiple pathways including transforming growth factor β induced epithelial-mesenchymal transition signaling, canonical, and non-canonical Wnt signaling at different stages of cardiac differentiation. CPCs transplantation promoted myoangiogenesis, attenuated fibrosis, and led to functional improvement in treated mice.

[Figure: see text].

ID2 is an essential transcription factor for natural killer cell development. Through epistasis analysis, Li et al. show that ID2 controls the amplitude of TCF1 and allows for its temporal downregulation, thereby supporting natural killer cell maturation., Gaining a mechanistic understanding of the expansion and maturation program of natural killer (NK) cells will provide opportunities for harnessing their inflammation-inducing and oncolytic capacity for therapeutic purposes. Here, we demonstrated that ID2, a transcriptional regulatory protein constitutively expressed in NK cells, supports NK cell effector maturation by controlling the amplitude and temporal dynamics of the transcription factor TCF1. TCF1 promotes immature NK cell expansion and restrains differentiation. The increased TCF1 expression in ID2-deficient NK cells arrests their maturation and alters cell surface receptor expression. Moreover, TCF1 limits NK cell functions, such as cytokine-induced IFN-γ production and the ability to clear metastatic melanoma in ID2-deficient NK cells. Our data demonstrate that ID2 sets a threshold for TCF1 during NK cell development, thus controlling the balance of immature and terminally differentiated cells that support future NK cell responses., Graphical Abstract

Alcohol use disorder (AUD) has a complex pathogenesis, making it a difficult disorder to treat. Identifying relevant signaling pathways in the brain may be useful for finding new pharmacological targets to treat AUD. The receptor tyrosine kinase anaplastic lymphoma kinase (ALK) activates the transcription factor STAT3 in response to ethanol in cell lines. Here, we show ALK activation and upregulation of known STAT3 target genes (Socs3, Gfap and Tnfrsf1a) in the prefrontal cortex (PFC) and ventral hippocampus (HPC) of mice after 4 days of binge-like ethanol drinking. Mice treated with the STAT3 inhibitor stattic drank less ethanol than vehicle-treated mice, demonstrating the behavioral importance of STAT3. To identify novel ethanol-induced target genes downstream of the ALK and STAT3 pathway, we analyzed the NIH LINCS L1000 database for gene signature overlap between ALK inhibitor (alectinib and NVP-TAE684) and STAT3 inhibitor (niclosamide) treatments on cell lines. These genes were then compared with differentially expressed genes in the PFC of mice after binge-like drinking. We found 95 unique gene candidates, out of which 57 had STAT3 binding motifs in their promoters. We further showed by qPCR that expression of the putative STAT3 genes Nr1h2, Smarcc1, Smarca4 and Gpnmb were increased in either the PFC or HPC after binge-like drinking. Together, these results indicate activation of the ALK-STAT3 signaling pathway in the brain after binge-like ethanol consumption, identify putative novel ethanol-responsive STAT3 target genes, and suggest that STAT3 inhibition may be a potential method to reduce binge drinking in humans.