ABSTRACT Cardiac fibrosis is a recognized cause of morbidity and mortality, yet effective pharmacological therapy that directly targets the fibrotic process remains lacking. Here we surveyed a group of methyltransferases known as protein arginine methyltransferases (PRMT) and demonstrated that PRMT1, which is the most highly expressed PRMT in the heart, was upregulated in activated cardiac fibroblasts, or myofibroblasts, in failing hearts. Deleting Prmt1 specifically in myofibroblasts or treating systemically with the PRMT1 inhibitor MS023 blocked myofibroblast formation, leading to a significant reduction in cardiac fibrosis and improvement in cardiac function in both acute and chronic heart injury models that manifest pervasive cardiac fibrosis. PRMT1 promoted the transition of cardiac fibroblasts to myofibroblasts by regulating transcription and epigenetic status. Additionally, PRMT1 methylated a key nucleolar protein fibrillarin 1 (FBL) and regulated nucleoli morphology and function during fibroblast fate transition. We further demonstrated a previously unrecognized requirement for FBL in myofibroblasts formation, by regulating myofibroblast gene induction and contractile force generation.

Vascular endothelial cells (ECs) perform key pleiotropic functions to maintain body homeostasis via the regulation of organ blood flow, vascular permeability, tissue growth and inflammation, and angiogenesis. Recent transcriptomic studies uncovered many EC subtypes across organs; however their specific functions are incompletely understood. Here we identified and characterized a novel, minority subtype of scattered ECs with a well-defined arteriovenous zonal localization exclusively in small resistance (strain) arterioles, and with the highest density in the brain>retina>kidney. Due to their expression of both endothelial and neuron-like functional and gene transcriptomic signatures, they were termed neuro-endothelial cells (NECs). High resolution single-cell transcriptome analysis of mouse brain and kidney ECs identified neuronal nitric oxide synthase (Nos1) and cytokine-like 1 (Cytl1) as top NEC biomarkers. Intravital multiphoton imaging of optogenetic mouse models with NEC gain/loss-of-function revealed NEC and Nos1-dependent vasodilation/vasoconstriction of intact brain and kidney arterioles and elevation/reduction in blood flow. Silencing NEC Nos1 and Cytl1 gene expression in vivo caused marked segmental arteriolar vasoconstrictions, reductions in vascular density and organ blood flow, increased vascular permeability and immune cell homing. Cytl1 administration triggered vasodilation and increased blood flow acutely, and increased capillary density and clonal EC remodeling chronically. NECs play major vasodilatory, angiogenic and anti-inflammatory functions that may be therapeutically targeted for vascular and inflammatory diseases. ### Competing Interest Statement The authors have declared no competing interest.

Tissue regeneration is limited in several organs, including the kidney, contributing to the high prevalence of kidney disease globally. However, evolutionary and physiological adaptive responses and the presence of renal progenitor cells suggest an existing remodeling capacity. This study uncovered endogenous tissue remodeling mechanisms in the kidney that were activated by the loss of body fluid and salt and regulated by a unique niche of a minority renal cell type called the macula densa (MD). Here, we identified neuronal differentiation features of MD cells that sense the local and systemic environment and secrete angiogenic, growth, and extracellular matrix remodeling factors, cytokines and chemokines, and control resident progenitor cells. Serial intravital imaging, MD nerve growth factor receptor and Wnt mouse models, and transcriptome analysis revealed cellular and molecular mechanisms of these MD functions. Human and therapeutic translation studies illustrated the clinical potential of MD factors, including CCN1, as a urinary biomarker and therapeutic target in chronic kidney disease. The concept that a neuronally differentiated key sensory and regulatory cell type responding to organ -specific physiological inputs controls local progenitors to remodel or repair tissues may be applicable to other organs and diverse tissue -regenerative therapeutic strategies.

A critical barrier to effective cancer therapy is the improvement of drug selectivity, toxicity, and reduced recurrence of tumors expanded from tumor-initiating stem-like cells (TICs). The aim is to identify circulating tumor cell (CTC)-biomarkers and to identify an effective combination of TIC-specific, repurposed federal drug administration (FDA)-approved drugs. Three different types of high-throughput screens targeting the TIC population are employed: these include a CD133 (+) cell viability screen, a NANOG expression screen, and a drug combination screen. When combined in a refined secondary screening approach that targets Nanog expression with the same FDA-approved drug library, histone deacetylase (HDAC) inhibitor(s) combined with all-trans retinoic acid (ATRA) demonstrate the highest efficacy for inhibition of TIC growth in vitro and in vivo. Addition of immune checkpoint inhibitor further decreases recurrence and extends PDX mouse survival. RNA-seq analysis of TICs reveals that combined drug treatment reduces many Toll-like receptors (TLR) and stemness genes through repression of the lncRNA MIR22HG. This downregulation induces PTEN and TET2, leading to loss of the self-renewal property of TICs. Thus, CTC biomarker analysis would predict the prognosis and therapy response to this drug combination. In general, biomarker-guided stratification of HCC patients and TIC-targeted therapy should eradicate TICs to extend HCC patient survival.

Programmed cell suicide of infected bacteria, known as abortive infection (Abi), serves as an immune defense strategy to prevent the propagation of bacteriophage viruses. Many Abi systems utilize bespoke cyclic nucleotide immune messengers generated upon infection to mobilize cognate death effectors. Here, we identify a family of bacteriophage nucleotidyltransferases (NTases) that synthesize competitor cyclic dinucleotide (CDN) ligands and inhibit TIR NADase effectors activated via a linked STING CDN sensor domain (TIR-STING). Through a functional screen of NTase-adjacent phage genes, we uncover candidate inhibitors of cell suicide induced by heterologous expression of tonically active TIR-STING. Among these, we demonstrate that a virus MazG-like nucleotide pyrophosphohydrolase, Atd1, depletes the starvation alarmone (p)ppGpp, revealing a potential role for the alarmone-activated host toxin MazF as an executioner of TIR-driven Abi. Phage NTases and counterdefenses like Atd1 preserve host viability to ensure virus propagation and represent tools to modulate TIR and STING immune responses.

Hepatocellular carcinoma (HCC) is the 3rd most deadly malignancy. Activated hepatic stellate cells (aHSC) give rise to cancer-associated fibroblasts in HCC and are considered a potential therapeutic target. Here we report that selective ablation of stearoyl CoA desaturase-2 (Scd2) in aHSC globally suppresses nuclear CTNNB1 and YAP1 in tumors and tumor microenvironment and prevents liver tumorigenesis in male mice. Tumor suppression is associated with reduced leukotriene B4 receptor 2 (LTB4R2) and its high affinity oxylipin ligand, 12-hydroxyheptadecatrienoic acid (12-HHTrE). Genetic or pharmacological inhibition of LTB4R2 recapitulates CTNNB1 and YAP1 inactivation and tumor suppression in culture and in vivo. Single cell RNA sequencing identifies a subset of tumor-associated aHSC expressing Cyp1b1 but no other 12-HHTrE biosynthetic genes. aHSC release 12-HHTrE in a manner dependent on SCD and CYP1B1 and their conditioned medium reproduces the LTB4R2-mediated tumor-promoting effects of 12-HHTrE in HCC cells. CYP1B1-expressing aHSC are detected in proximity of LTB4R2-positive HCC cells and the growth of patient HCC organoids is blunted by LTB4R2 antagonism or knockdown. Collectively, our findings suggest aHSC-initiated 12-HHTrE-LTB4R2-CTNNB1-YAP1 pathway as a potential HCC therapeutic target.

Background and Aims: Relative roles of HSCs and portal fibroblasts in alcoholic hepatitis (AH) are unknown. We aimed to identify subpopulations of collagen type 1 alpha 1 (Col1a1)–expressing cells in a mouse AH model by single‐cell RNA sequencing (scRNA‐seq) and filtering the cells with the HSC (lecithin retinol acyltransferase [Lrat]) and portal fibroblast (Thy‐1 cell surface antigen [Thy1] and fibulin 2 [Fbln2]) markers and vitamin A (VitA) storage. Approach and Results: Col1a1–green fluorescent protein (GFP) mice underwent AH, CCl 4 , and bile duct ligation (BDL) procedures to have comparable F1‐F2 liver fibrosis. Col1a1‐expressing cells were sorted via FACS by VitA autofluorescence and GFP for single‐cell RNA sequencing. In AH, approximately 80% of Lrat+Thy1−Fbln2− activated HSCs were VitA‐depleted (vs. ~13% in BDL and CCl 4 ). Supervised clustering identified a subset co‐expressing Lrat and Fbln2 (Lrat+Fbln2+), which expanded 44‐fold, 17‐fold, and 1.3‐fold in AH, BDL, and CCl 4 . Lrat+Fbln2+ cells had 3–15‐times inductions of profibrotic, myofibroblastic, and immunoregulatory genes versus Lrat+Fbln2− cells, but 2–4‐times repressed HSC‐selective genes. AH activated HSCs had up‐regulated inflammatory (chemokine [C‐X‐C motif] ligand 2 [Cxcl2], chemokine [C‐C motif] ligand 2), antimicrobial (Il‐33, Zc3h12a), and antigen presentation (H2‐Q6, H2‐T23) genes versus BDL and CCl 4 . Computational deconvolution of AH versus normal human bulk‐liver RNA‐sequencing data supported an expansion of LRAT+FBLN2+ cells in AH; AH patient liver immunohistochemistry showed FBLN2 staining along fibrotic septa enriched with LRAT+ cells; and in situ hybridization confirmed co‐expression of FBLN2 with CXCL2 and/or human leukocyte antigen E in patient AH. Finally, HSC tracing in Lrat‐Cre;Rosa26mTmG mice detected GFP+FBLN2+ cells in AH. Conclusion: A highly profibrotic, inflammatory, and immunoregulatory Lrat+Fbln2+ subpopulation emerges from HSCs in AH and may contribute to the inflammatory and immunoreactive nature of AH.

PDF file - 48K, A, performing a cell growth assay, it was observed that the knockdown of -catulin does not affect cell growth in USC-HN1 cells. B, AnnexinV analysis was performed to look at cell apoptosis. Cells were either left untreated or treated with TNF- (10ng/mL) and CHX (10g/mL) for 30 hrs to induce apoptosis. There was no considerable difference in apoptosis between the control and -catulin knockdown USC-HN1 cells, either untreated or treated with TNF- and CHX. One representative experimental result is shown

Objective: Vascular endothelial cells (ECs) play important roles in the physiological maintenance of organ blood flow and in the development of renal and cardiovascular diseases. Tissue-specific EC dysfunction can contribute to several different diseases including hypertension. Recent transcriptomic studies identified many EC subtypes in multiple organs including the brain and the kidneys, however, their functions are incompletely understood. The present study aimed to explore physiological functional significance and cardiovascular disease and hypertension relevance of a newly discovered minority subtype of scattered ECs expressing neuronal nitric oxide synthase (Nos1). Design and method: A comprehensive research toolbox was applied in this study including transgenic mouse models (Nos1-GFP, GCaMP6, mTORgof/lof), intravital multiphoton imaging of calcium dynamics of Nos1+ endothelial cells in the brain and the kidney, genetic cell fate tracking, two-kidney one-clip (2K1C) model of renovascular (Goldblatt) hypertension (RVHT), whole mount organ imaging for 3D vascular density measurements, and single-cell transcriptomic analysis. Results: Our studies identified and characterized, for the first time, a new endothelial cell type expressing both endothelial and neuron-like functional and gene transcriptomic signatures, therefore we named them neuroendothelial cells (NECs). NECs exhibit a well-defined arteriovenous zonal localization exclusively to small resistance arterioles. NECs are found only in the three organs that exhibit the best blood flow autoregulation capacity, with the highest density in the brain>kidney>heart (NEC/EC (%) 8.42+/-0.79, 3.21+/−0.33, 1.73+/−0.07, respectively). NEC density is reduced with aging in the brain and the kidney (NEC/EC (%) 4.675, and 1.850, respectively, p < 0.01, 2.5 years old compared to 2-month-old). The number of NECs increased significantly in the hypo-perfused, hypoxic clipped (CK) kidney, but reduced in the hyperperfused non-clipped (NCK) kidney in RVHT. Intravital multiphoton microscopy (MPM) of intact brain and kidney arterioles in vivo revealed regular, autonomous NEC calcium transients with blood pressure-dependent frequency alterations. Newly established NEC gain-of-function mouse models exhibited increased endothelium-dependent vasodilation and diminished agonist-induced vascular contractility in brain and kidney resistance arterioles compared to controls. In addition, preliminary single-cell RNA sequencing and transcriptomic analyses showed that, in contrast to other ECs, NECs highly express several traditional (e.g., Nos1, Klotho) and novel (e.g., Aard) tissue trophic factors that are known to play important roles in angiogenesis, aging, vascular (dys)function, and chronic vascular diseases. Conclusions: These new vascular anatomy and hemodynamic findings strongly suggest sensory, blood flow and/or baroreceptor functions of NECs as well as a role in the autoregulation of organ blood flow and hypertension pathogenesis.

PDF file - 44K, shRNA lentiviral system was used to generate stable MDA-MB-231 cell lines that were -catulin-deficient. The specific knockdown of -catulin in MDA-MB-231 cells (G85) compared to the non-silencing control (GNS) was confirmed by RT-PCR (A), real-time qPCR (A'), and western blot (B). In vitro cell migration (C) and invasion (D) was decreased in -catulin-ablated MDA-MB-231 cells; cells were visualized with toluidine blue. Standard error bars are shown (n=4 for cell migration and n=4 for invasion assay). *** statistical significance was determined using t-test (p < 0.001) or * analysis of variant (ANOVA) (p <0.02)

PDF file - 1MB, 3 days after isolation, metastatic hSCC USC-HN1 GFP-labeled cells from the lymphatic vessels (A) show a mesenchymal morphology, compared to USC-HN1 cells from the primary SCC tumor (C). 10 days after isolation, the metastatic cells from the lymphatic vessels (B) revert to an epithelial morphology, typical of USC-HN1 cells, as compared to the cells from the primary SCC tumor (D)

A concise summary of the publically available transcriptome data sets that were used to generate Figure 1 in the main text.

Drug-induced nephrotoxicity is a leading cause of drug attrition, partly due to the limited relevance of pre-clinical models of the proximal tubule. Culturing proximal tubule epithelial cells (PTECs) under fluid flow to mimic physiological shear stress has been shown to improve select phenotypes, but existing flow systems are expensive and difficult to implement by non-experts in microfluidics. Here, we designed and fabricated an accessible and modular flow system for culturing PTECs under physiological shear stress, which induced native-like cuboidal morphology, downregulated pathways associated with hypoxia, stress, and injury, and upregulated xenobiotic metabolism pathways. We also compared the expression profiles of shear-dependent genes in our in vitro PTEC tissues to that of ex vivo proximal tubules and observed stronger clustering between ex vivo proximal tubules and PTECs under physiological shear stress relative to PTECs under negligible shear stress. Together, these data illustrate the utility of our user-friendly flow system and highlight the role of shear stress in promoting native-like morphological and transcriptomic phenotypes in PTECs in vitro, which is critical for developing more relevant pre-clinical models of the proximal tubule for drug screening or disease modeling.

PDF file - 4.7MB, We further analyzed the tumors that formed from the injection of control and -catulin-deficient USC-HN1 cell lines. We can observe and track injected cells by the tGFP reporter. A and B, checking for proliferation rate, we do not see a considerable difference between the two tumors, as assessed by the proliferative marker, Ki67. C and D, when analyzing the tumors for human-specific lymphatic vessel marker, lyve-1, the control tumor (C) had collective groups of cells that were able to invade the surrounding stroma and form their own lymphatic vessels, whereas -catulin-deficient tumors (D) were unable to do so. E-F, IHC staining using phospho-JNK antibody show an increase in activated phospho-JNK in control tumor (E) compared to -catulin-deficient tumor (F). Arrows indicate invasive tumor fronts in control tumors, whereas dashed lines indicate a less invasive tumor margin in -catulin-deficient tumors

Supplemental Figures. Fig S1:Expression of HEY1 and HEY2 in thyroid cancers vs. normal thyroid tissues. Fig S2:PROX1 mislocalization in TPC-1 cells does not depend on CRM1, energy or temperature. Fig S3: T3 promotes PROX1 nuclear exclusion in a dose-dependent manner. Fig S4: Engineered BCPAP cells conditionally express FLAG-tagged PROX1 by doxycycline administration. Fig S5: Wnt/β-catenin signaling is suppressed in PTC cells. Fig S6: PROX1-mediated regulation of thyroid cancer-associated genes in TPC1 and 8505c cells. Fig S7: Single cell motility assay. Fig S8: Reinstatement of PROX1 expression in TPC1 and 8505c thyroid cancer cells. Fig S9: Dox-induced expression of PROX1 in BCPAP tumor cells in mouse. Fig S10: PROX1 re-expression inhibits tumor growth and aggressive tumor cell morphology of TPC1 thyroid carcinoma cells. Fig S11: PROX1 re-expression suppressed tumor growth and aggressive morphology of 8505c thyroid cancer cells. Fig S12: Cytoplasmic localization of PROX1 in other cancer cells.

We constructed and analyzed the whole transcriptome in leukocytes of healthy adult vapers (with/without a history of smoking), ‘exclusive’ cigarette smokers, and controls (non-users of any tobacco products). Furthermore, we performed single-gene validation of expression data, and biochemical validation of vaping/smoking status by plasma cotinine measurement. Computational modeling, combining primary analysis (age- and sex-adjusted limmaVoom) and sensitivity analysis (cumulative e-liquid- and pack-year modeling), revealed that ‘current’ vaping, but not ‘past’ smoking, is significantly associated with gene dysregulation in vapers. Comparative analysis of the gene networks and canonical pathways dysregulated in vapers and smokers showed strikingly similar patterns in the two groups, although the extent of transcriptomic changes was more pronounced in smokers than vapers. Of significance is the preferential targeting of mitochondrial genes in both vapers and smokers, concurrent with impaired functional networks, which drive mitochondrial DNA-related disorders. Equally significant is the dysregulation of immune response genes in vapers and smokers, modulated by upstream cytokines, including members of the interleukin and interferon family, which play a crucial role in inflammation. Our findings accord with the growing evidence on the central role of mitochondria as signaling organelles involved in immunity and inflammatory response, which are fundamental to disease development.

Monoamine oxidase B (MAO B) oxidizes trace amine phenylethylamine (PEA), and neurotransmitters serotonin and dopamine in the brain. We reported previously that PEA levels increased significantly in all brain regions, but serotonin and dopamine levels were unchanged in MAO B knockout (KO) mice. PEA and dopamine are both synthesized from phenylalanine by aromatic L-amino acid decarboxylase in dopaminergic neurons in the striatum. A high concentration of PEA in the striatum may cause dopaminergic neuronal death in the absence of MAO B. We isolated the RNA from brain tissue of MAO B KO mice (2-month old) and age-matched wild type (WT) male mice and analyzed the altered genes by Affymetrix microarray. Differentially expressed genes (DEGs) in MAO B KO compared to WT mice were analyzed by Partek Genomics Suite, followed by Ingenuity Pathway Analysis (IPA) to assess their functional relationships. DEGs in MAO B KO mice are involved in brain inflammation and the genesis of GABAnergic neurons. The significant DEGs include four brain injury or inflammation genes (upregulated: Ido1, TSPO, AVP, Tdo2), five gamma-aminobutyric acid (GABA) receptors (down-regulated: GABRA2, GABRA3, GABRB1, GABRB3, GABRG3), five transcription factors related to adult neurogenesis (upregulated: Wnt7b, Hes5; down-regulated: Pax6, Tcf4, Dtna). Altered brain injury and inflammation genes in MAO B knockout mice are involved in various neurological disorders: attention deficit hyperactive disorder, panic disorder, obsessive compulsive disorder, autism, amyotrophic lateral sclerosis, Parkinson's diseases, Alzheimer's disease, bipolar affective disorder. Many were commonly involved in these disorders, indicating that there are overlapping molecular pathways.