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Complexes of the element Re have recently been shown to possess promising anticancer activity through mechanisms of action that are distinct from the conventional metal-based drug cisplatin. In this study, we report our investigations on the anticancer activity of the complex [Re(CO)3 (dmphen)(p-tol-ICN)]+ (TRIP) in which dmphen=2,9-dimethyl-1,10-phenanthroline and p-tol-ICN=para-tolyl isonitrile. TRIP was synthesized by literature methods and exhaustively characterized. This compound exhibited potent in vitro anticancer activity in a wide variety of cell lines. Flow cytometry and immunostaining experiments indicated that TRIP induces intrinsic apoptosis. Comprehensive biological mechanistic studies demonstrated that this compound triggers the accumulation of misfolded proteins, which causes endoplasmic reticulum (ER) stress, the unfolded protein response, and apoptotic cell death. Furthermore, TRIP induced hyperphosphorylation of eIF2α, translation inhibition, mitochondrial fission, and expression of proapoptotic ATF4 and CHOP. These results establish TRIP as a promising anticancer agent based on its potent cytotoxic activity and ability to induce ER stress.

Multiple sclerosis (MS) is a neuroinflammatory multifocal disorder. Optic neuritis is common in MS and leads to visual disability. No current treatments repair this damage. Discerning gene expression changes within specific cell types in optic nerve (ON) may suggest new treatment targets for visual disability in MS. Astrocytes are pivotal regulators of neuroinflammation, playing either detrimental or beneficial roles. Here, we used RiboTag technology to characterize the astrocyte-specific transcriptome in ON in the experimental autoimmune encephalomyelitis (EAE) model of MS. RNA sequencing analysis showed the Complement Cascade and Cholesterol Biosynthesis Pathways as the most enriched and de-enriched pathways, respectively, in ON astrocytes in EAE. Expression of complement component 3 (C3) was confirmed to be increased in ON astrocytes at the protein level during EAE. A bigger increase in C3 expressing ON astrocytes was found in EAE females versus healthy females, as compared to that in EAE males versus healthy males. Also, there was worse retinal ganglion cell (RGC) and axonal loss in EAE females. Regression analyses showed a negative correlation between C3 expressing astrocytes and RGC density. This cell-specific and sex-specific investigation of the optic nerve provides targets for the development of therapeutic strategies tailored for optic neuritis in MS.

Myelodysplastic syndromes (MDS) with ring sideroblasts are hematopoietic stem cell disorders with erythroid dysplasia and mutations in the SF3B1 splicing factor gene. Patients with MDS with SF3B1 mutations often accumulate excessive tissue iron, even in the absence of transfusions, but the mechanisms that are responsible for their parenchymal iron overload are unknown. Body iron content, tissue distribution, and the supply of iron for erythropoiesis are controlled by the hormone hepcidin, which is regulated by erythroblasts through secretion of the erythroid hormone erythroferrone (ERFE). Here, we identified an alternative ERFE transcript in patients with MDS with the SF3B1 mutation. Induction of this ERFE transcript in primary SF3B1-mutated bone marrow erythroblasts generated a variant protein that maintained the capacity to suppress hepcidin transcription. Plasma concentrations of ERFE were higher in patients with MDS with an SF3B1 gene mutation than in patients with SF3B1 wild-type MDS. Thus, hepcidin suppression by a variant ERFE is likely responsible for the increased iron loading in patients with SF3B1-mutated MDS, suggesting that ERFE could be targeted to prevent iron-mediated toxicity. The expression of the variant ERFE transcript that was restricted to SF3B1-mutated erythroblasts decreased in lenalidomide-responsive anemic patients, identifying variant ERFE as a specific biomarker of clonal erythropoiesis.

Autophagy, a lysosomal degradation pathway, plays crucial roles in health and disease. p62/SQSTM1 (hereafter p62) is an autophagy adaptor protein that can shuttle ubiquitinated cargo for autophagic degradation. Here, we show that upregulating the Drosophila p62 homolog ref(2)P/dp62, starting in midlife, delays the onset of pathology and prolongs healthy lifespan. Midlife induction of dp62 improves proteostasis, in aged flies, in an autophagy-dependent manner. Previous studies have reported that p62 plays a role in mediating the clearance of dysfunctional mitochondria via mitophagy. However, the causal relationships between p62 expression, mitochondrial homeostasis, and aging remain largely unexplored. We show that upregulating dp62, in midlife, promotes mitochondrial fission, facilitates mitophagy, and improves mitochondrial function in aged flies. Finally, we show that mitochondrial fission is required for the anti-aging effects of midlife dp62 induction. Our findings indicate that p62 represents a potential therapeutic target to counteract aging and prolong health in aged mammals.

Mechanisms coordinating pancreatic β cell metabolism with insulin secretion are essential for glucose homeostasis. One key mechanism of β cell nutrient sensing uses the mitochondrial GTP (mtGTP) cycle. In this cycle, mtGTP synthesized by succinyl-CoA synthetase (SCS) is hydrolyzed via mitochondrial PEPCK (PEPCK-M) to make phosphoenolpyruvate, a high-energy metabolite that integrates TCA cycling and anaplerosis with glucose-stimulated insulin secretion (GSIS). Several strategies, including xenotopic overexpression of yeast mitochondrial GTP/GDP exchanger (GGC1) and human ATP and GTP-specific SCS isoforms, demonstrated the importance of the mtGTP cycle. These studies confirmed that mtGTP triggers and amplifies normal GSIS and rescues defects in GSIS both in vitro and in vivo. Increased mtGTP synthesis enhanced calcium oscillations during GSIS. mtGTP also augmented mitochondrial mass, increased insulin granule number, and membrane proximity without triggering de-differentiation or metabolic fragility. These data highlight the importance of the mtGTP signal in nutrient sensing, insulin secretion, mitochondrial maintenance, and β cell health.

Chronic exposure to copper and its dyshomeostasis have been linked to accelerated cognitive decline and potentially increasing risk for Alzheimer's disease (AD). We and others have previously demonstrated that exposure to copper through drinking water significantly increased parenchymal amyloid-beta (Aβ) plaques and decreased endothelial low-density lipoprotein receptor-related protein 1 (LRP1) in mouse models of AD. In this study, we determined the underlying mechanisms that microRNA critically mediated the copper-induced loss of endothelial LRP1. In human primary microvascular endothelial cells (MVECs), microRNA-200b-3p, -200c-3p, and -205-5p were significantly elevated within the 24-h exposure to copper and returned to baseline after 48-h postexposure, which corresponded with the temporal change of LRP1 expression in these cells. Transient expression of synthetic microRNA-200b-3p, -200c-3p, or -205-5p on MVECs significantly decreased endothelial LRP1, and cotreatment of synthetic antagomirs effectively prevented the loss of LRP1 during copper exposure, collectively supporting the key regulatory role of these microRNAs in copper-induced loss of LRP1. In mice, a significant reduction of LRP1 in cortical vasculature was evident following 9 months exposure to 1.3 ppm copper in drinking water, although the levels of cortical microRNA-205-5p, -200b-3p, and -200c-3p were only marginally elevated. This, however, correlated with increased vascular accumulation of Aβ and impairment of spatial memory, indicating that copper exposure has the pivotal role in the vascular damage and development of cognitive decline.

Pancreatic cancer typically spreads rapidly and has poor survival rates. Here, we report that the calcium-activated chloride channel TMEM16A is a biomarker for pancreatic cancer with a poor prognosis. TMEM16A is up-regulated in 75% of cases of pancreatic cancer and high levels of TMEM16A expression are correlated with low patient survival probability. TMEM16A up-regulation is associated with the ligand-dependent EGFR signaling pathway. In vitro, TMEM16A is required for EGF-induced store-operated calcium entry essential for pancreatic cancer cell migration. TMEM16A also has a profound impact on phosphoproteome remodeling upon EGF stimulation. Moreover, molecular actors identified in this TMEM16A-dependent EGFR-induced calcium signaling pathway form a gene set that makes it possible not only to distinguish neuro-endocrine tumors from other forms of pancreatic cancer, but also to subdivide the latter into three clusters with distinct genetic profiles that could reflect their molecular underpinning.

Trichomonas vaginalis, a prevalent sexually transmitted parasite, adheres to and induces cytolysis of human mucosal epithelial cells. We have characterized a hypothetical protein, TVAG_393390, with predicted tertiary structure similar to that of mammalian cadherin proteins involved in cell-cell adherence. TVAG_393390, renamed cadherin-like protein (CLP), contains a calcium-binding site at a position conserved in cadherins. CLP is surface localized, and its mRNA and protein levels are significantly upregulated upon parasite adherence to host cells. To test the roles of CLP and its calcium-binding dependency during host cell adherence, we first demonstrated that wild-type CLP (CLP) binds calcium with a high affinity, whereas the calcium-binding site mutant protein (CLP-mut) does not. CLP and CLP-mut constructs were then used to overexpress these proteins in T. vaginalis Parasites overexpressing CLP have ∼3.5-fold greater adherence to host cells than wild-type parasites, and this increased adherence is ablated by mutating the calcium-binding site. Additionally, competition with recombinant CLP decreased parasite binding to host cells. We also found that overexpression of CLP induced parasite aggregation which was further enhanced in the presence of calcium, whereas CLP-mut overexpression did not affect aggregation. Lastly, parasites overexpressing wild-type CLP induced killing of host cells ∼2.35-fold, whereas parasites overexpressing CLP-mut did not have this effect. These analyses describe the first parasitic CLP and demonstrate a role for this protein in mediating parasite-parasite and host-parasite interactions. T. vaginalis CLP may represent convergent evolution of a parasite protein that is functionally similar to the mammalian cell adhesion protein cadherin, which contributes to parasite pathogenesis.IMPORTANCE The adherence of pathogens to host cells is critical for colonization of the host and establishing infection. Here we identify a protein with no known function that is more abundant on the surface of parasites that are better at binding host cells. To interrogate a predicted function of this protein, we utilized bioinformatic protein prediction programs which allowed us to uncover the first cadherin-like protein (CLP) found in a parasite. Cadherin proteins are conserved metazoan proteins with central roles in cell-cell adhesion, development, and tissue structure maintenance. Functional characterization of this CLP from the unicellular parasite Trichomonas vaginalis demonstrated that the protein mediates both parasite-parasite and parasite-host adherence, which leads to an enhanced killing of host cells by T. vaginalis Our findings demonstrate the presence of CLPs in unicellular pathogens and identify a new host cell binding protein family in a human-infective parasite.

BYL719 is a PI3K inhibitor that has demonstrated efficacy in the treatment of head and neck squamous cell carcinoma. BYL719 exerts its therapeutic effect by suppressing AKT and other proliferative signaling mechanisms. Despite PI3K inhibition and AKT suppression, residual activity of protein S6, a core marker of proliferative activation, has been observed. HER3, either via dimerization or activation by its ligand neurgeulin (NRG), is known to activate PI3K. Thus, we hypothesized that co-targeting HER3 and PI3K would lead to greater suppression of the PI3K-AKT signaling pathway and greater tumor suppression than with BYL719 alone. We investigated biochemical expression and activation of the HER3-PI3K-AKT-S6 pathway in HNSCC cell lines and patient-derived xenografts (PDXs). Antitumor effects of HER3 and PI3K inhibitors alone and in combination were evaluated in cell culture and murine models. Treatment of HNSCC cell lines with BYL719 significantly reduced AKT activation and suppressed tumor growth. However, S6 was persistently activated despite suppression of AKT. Combination treatment with KTN3379, a monoclonal antibody targeted against HER3, and BYL719 led to enhanced suppression of in vitro and in vivo cancer growth and durable suppression of AKT and S6. Therefore, inhibition of HER3 with KTN3379 enhanced the effects of PI3K inhibition in pre-clinical HNSCC models. These data support co-targeting HER3 and PI3K for the treatment of HSNCC.

Right ventricular dysfunction is highly prevalent across cardiopulmonary diseases and independently predicts death in both heart failure (HF) and pulmonary hypertension (PH). Progression towards right ventricular failure (RVF) can occur in spite of optimal medical treatment of HF or PH, highlighting current insufficient understanding of RVF molecular pathophysiology. To identify molecular mechanisms that may distinctly underlie RVF, we investigated the cardiac ventricular transcriptome of advanced HF patients, with and without RVF. Using an integrated systems genomic and functional biology approach, we identified an RVF-specific gene module, for which WIPI1 served as a hub and HSPB6 and MAP4 as drivers, and confirmed the ventricular specificity of Wipi1, Hspb6, and Map4 transcriptional changes in adult murine models of pressure overload induced RV- versus LV- failure. We uncovered a shift towards non-canonical autophagy in the failing RV that correlated with RV-specific Wipi1 upregulation. In vitro siRNA silencing of Wipi1 in neonatal rat ventricular myocytes limited non-canonical autophagy and blunted aldosterone-induced mitochondrial superoxide levels. Our findings suggest that Wipi1 regulates mitochondrial oxidative signaling and non-canonical autophagy in cardiac myocytes. Together with our human transcriptomic analysis and corroborating studies in an RVF mouse model, these data render Wipi1 a potential target for RV-directed HF therapy.

Genetic variation in the NF-κB inhibitors, ABIN1 and A20, increase risk for psoriasis. While critical for hematopoietic immune cell function, these genes are believed to additionally inhibit psoriasis by dampening inflammatory signaling in keratinocytes. We dissected ABIN1 and A20s regulatory role in human keratinocyte inflammation using an RNA sequencing-based comparative genomic approach. Here we show subsets of the IL-17 and tumor necrosis factor-α signaling pathways are robustly restricted by A20 overexpression. In contrast, ABIN1 overexpression inhibits these genes more modestly for IL-17, and weakly for tumor necrosis factor-α. Our genome-scale analysis also indicates that inflammatory program suppression appears to be the major transcriptional influence of A20/ABIN1 overexpression, without obvious influence on keratinocyte viability genes. Our findings thus enable dissection of the differing anti-inflammatory mechanisms of two distinct psoriasis modifiers, which may be directly exploited for therapeutic purposes. Importantly, we report that IL-17-induced targets of A20 show similar aberrant epidermal layer-specific transcriptional upregulation in keratinocytes from diseases as diverse as psoriasis, atopic dermatitis, and erythrokeratodermia variabilis, suggesting a contributory role for epidermal inflammation in a broad spectrum of rashes.

Stem cell heterogeneity is recognized as functionally relevant for tissue homeostasis and repair. The identity, context dependence, and regulation of skeletal muscle satellite cell (SC) subsets remains poorly understood. We identify a minor subset of Pax7+ SCs that is indelibly marked by an inducible Mx1-Cre transgene in vivo, is enriched for Pax3 expression, and has reduced ROS (reactive oxygen species) levels. Mx1+ SCs possess potent stem cell activity upon transplantation but minimally contribute to endogenous muscle repair, due to their relative low abundance. In contrast, a dramatic clonal expansion of Mx1+ SCs allows extensive contribution to muscle repair and niche repopulation upon selective pressure of radiation stress, consistent with reserve stem cell (RSC) properties. Loss of Pax3 in RSCs increased ROS content and diminished survival and stress tolerance. These observations demonstrate that the Pax7+ SC pool contains a discrete population of radiotolerant RSCs that undergo clonal expansion under severe stress.

The use of biochemical signaling to derive smooth muscle cells (SMCs) from mesenchymal stem cells (MSCs) has been explored, but the induction of a fully functional SMC phenotype remains to be a major challenge. Cell morphology has been shown to regulate MSC differentiation into various lineages, including SMCs. We engineered substrates with microgrooves to induce cell elongation to study the mechanism underlying the MSC shape modulation in SMC differentiation. In comparison to those on flat substrates, MSCs cultured on engineered substrates were elongated with increased aspect ratios for both cell body and nucleus, as well as augmented cytoskeletal tensions. Biochemical studies indicated that the microgroove-elongated cells expressed significantly higher levels of SMC markers. MicroRNA analyses showed that up-regulation of miR-145 and the consequent repression of KLF4 in these elongated cells promoted MSC-to-SMC differentiation. Rho/ROCK inhibitions, which impair cytoskeletal tension, attenuated cell and nuclear elongations and disrupted the miR-145/KLF4 regulation for SMC differentiation. Furthermore, cell traction force measurements showed that miR-145 is essential for the functional contractility in the microgroove-induced SMC differentiation. Collectively, our findings demonstrate that, through a Rho-ROCK/miR-145/KLF4 pathway, the elongated cell shape serves as a decisive geometric cue to direct MSC differentiation into functional SMCs.

The evolutionarily conserved Target of Rapamycin (TOR) complex-2 (TORC2) is an essential regulator of plasma membrane homeostasis in budding yeast (Saccharomyces cerevisiae). In this yeast, TORC2 phosphorylates and activates the effector protein kinase Ypk1 and its paralog Ypk2. These protein kinases, in turn, carry out all the crucial functions of TORC2 by phosphorylating and thereby controlling the activity of at least a dozen downstream substrates. A previously uncharacterized interplay between the Rab5 GTPases and TORC2 signaling was uncovered through analysis of a newly suspected Ypk1 target. Muk1, one of two guanine nucleotide exchange factors for the Rab5 GTPases, was found to be a physiologically relevant Ypk1 substrate; and, genetic analysis indicates that Ypk1-mediated phosphorylation activates the guanine nucleotide exchange activity of Muk1. Second, it was demonstrated both in vivo and in vitro that the GTP-bound state of the Rab5 GTPase Vps21/Ypt51 physically associates with TORC2 and acts as a direct positive effector required for full TORC2 activity. These interrelationships provide a self-reinforcing control circuit for sustained up-regulation of TORC2-Ypk1 signaling. In this overview, we summarize the experimental basis of these findings, their implications, and speculate as to the molecular basis for Rab5-mediated TORC2 activation.

Previous studies have suggested a role for Tet1 in the pathogenesis of childhood asthma. However, how Tet1 contributes to asthma remains unknown. Here we used mice deficient for Tet1 in a well-established model of allergic airway inflammation and demonstrated that loss of Tet1 increased disease severity including airway hyperresponsiveness and lung eosinophilia. Increased expression of Muc5ac, Il13, Il33, Il17a, Egfr, and Tff2 were observed in HDM-challenged Tet1-deficient mice compared to Tet1+/+ littermates. Further, transcriptomic analysis of lung RNA followed by pathway and protein network analysis showed that the IFN signaling pathway was significantly upregulated and the aryl hydrocarbon receptor (AhR) pathway was significantly downregulated in HDM-challenged Tet1-/- mice. This transcriptional regulation of the IFN and AhR pathways by Tet1 was also present in human bronchial epithelial cells at base line and following HDM challenges. Genes in these pathways were further associated with changes in DNA methylation, predicted binding of transcriptional factors with relevant functions in their promoters, and the presence of histone marks generated by histone enzymes that are known to interact with Tet1. Collectively, our data suggest that Tet1 inhibits HDM-induced allergic airway inflammation by direct regulation of the IFN and AhR pathways.

Functional activation of the transforming growth factor-β (TGF-β) receptors (TGFBRs) is carefully regulated through integration of post-translational modifications, spatial regulation at the cellular level, and TGFBR availability at the cell surface. Although the bulk of TGFBRs resides inside the cells, AKT Ser/Thr kinase (AKT) activation in response to insulin or other growth factors rapidly induces transport of TGFBRs to the cell surface, thereby increasing the cell's responsiveness to TGF-β. We now demonstrate that TGF-β itself induces a rapid translocation of its own receptors to the cell surface and thus amplifies its own response. This mechanism of response amplification, which hitherto has not been reported for other cell-surface receptors, depended on AKT activation and TGF-β type I receptor kinase. In addition to an increase in cell-surface TGFBR levels, TGF-β treatment promoted TGFBR internalization, suggesting an overall amplification of TGFBR cycling. The TGF-β-induced increase in receptor presentation at the cell surface amplified TGF-β-induced SMAD family member (SMAD) activation and gene expression. Furthermore, bone morphogenetic protein 4 (BMP-4), which also induces AKT activation, increased TGFBR levels at the cell surface, leading to enhanced autocrine activation of TGF-β-responsive SMADs and gene expression, providing context for the activation of TGF-β signaling in response to BMP during development. In summary, our results indicate that TGF-β- and BMP-induced activation of low levels of cell surface-associated TGFBRs rapidly mobilizes additional TGFBRs from intracellular stores to the cell surface, increasing the abundance of cell-surface TGFBRs and cells' responsiveness to TGF-β signaling.

Glomeromycotina is a lineage of early diverging fungi that establish arbuscular mycorrhizal (AM) symbiosis with land plants. Despite their major ecological role, the genetic basis of their obligate mutualism remains largely unknown, hindering our understanding of their evolution and biology. We compared the genomes of Glomerales (Rhizophagus irregularis, Rhizophagus diaphanus, Rhizophagus cerebriforme) and Diversisporales (Gigaspora rosea) species, together with those of saprotrophic Mucoromycota, to identify gene families and processes associated with these lineages and to understand the molecular underpinning of their symbiotic lifestyle. Genomic features in Glomeromycotina appear to be very similar with a very high content in transposons and protein-coding genes, extensive duplications of protein kinase genes, and loss of genes coding for lignocellulose degradation, thiamin biosynthesis and cytosolic fatty acid synthase. Most symbiosis-related genes in R. irregularis and G. rosea are specific to Glomeromycotina. We also confirmed that the present species have a homokaryotic genome organisation. The high interspecific diversity of Glomeromycotina gene repertoires, affecting all known protein domains, as well as symbiosis-related orphan genes, may explain the known adaptation of Glomeromycotina to a wide range of environmental settings. Our findings contribute to an increasingly detailed portrait of genomic features defining the biology of AM fungi.

IntroductionPort wine stain (PWS) is characterized as a progressive dilatation of immature venule-like vasculatures which result from differentiation-impaired endothelial cells. In this study, we aimed to identify the major biological pathways accounting for the pathogenesis of PWS.MethodsSequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) was used to identify differentially expressed proteins in PWS lesions, followed by confirmative studies with immunohistochemistry, immunoblot and transmission electron microscopy (TEM).Results107 out of 299 identified proteins showed differential expressions in PWS lesions as compared to normal skin, mainly involving the functions of biosynthesis, membrane trafficking, cytoskeleton and cell adhesion/migration. The confirmative studies showed that expressions of membrane trafficking/exocytosis related proteins such as VAT1, IQGAP1, HSC70, clathrin, perlecan, spectrin α1 and GDIR1 were significantly increased in PWS blood vessels as compared to normal ones; while collagen subtypes 6A1 and 6A3 were decreased in PWS skin. Furthermore, TEM studies showed there is a significant upregulation of extracellular vesicle exocytosis from PWS blood vessels as compared to control.ConclusionsThe biological process of membrane trafficking and exocytosis is enhanced in PWS blood vessels. Our results imply that the extracellular vesicles released by lesional endothelial cells may act as potential intercellular signaling mediators to contribute to the pathogenesis of PWS.

Objective- The topographical distribution of atherosclerosis in vasculature underscores the importance of shear stress in regulating endothelium. With a systems approach integrating sequencing data, the current study aims to explore the link between shear stress-regulated master transcription factor and its regulation of endothelial cell (EC) function via epigenetic modifications. Approach and Results- H3K27ac (acetylation of histone 3 lysine 27)-ChIP-seq (chromatin immunoprecipitation followed by high throughput sequencing), ATAC-seq (an assay for transposase-accessible chromatin-sequencing), and RNA-seq (RNA-sequencing) were performed to investigate the genome-wide epigenetic regulations in ECs in response to atheroprotective pulsatile shear stress (PS). In silico prediction revealed that KLF4 binding motifs were enriched in the PS-enhanced H3K27ac regions. By integrating PS- and KLF4-modulated H3K27ac, we identified 18 novel PS-upregulated genes. The promoter regions of these genes showed an overlap between the KLF4-enhanced assay for transposase-accessible chromatin signals and the PS-induced H3K27ac peaks. Experiments using ECs isolated from mouse aorta, lung ECs from EC-KLF4-TG versus EC-KLF4-KO mice, and atorvastatin-treated ECs showed that ITPR3 (inositol 1,4,5-trisphosphate receptor 3) was robustly activated by KLF4 and statins. KLF4 ATAC-qPCR (quantitative polymerase chain reaction) and ChIP-qPCR further demonstrated that a specific locus in the promoter region of the ITPR3 gene was essential for KLF4 binding, H3K27ac enrichment, chromatin accessibility, RNA polymerase II recruitment, and ITPR3 transcriptional activation. Deletion of this KLF4 binding locus in ECs by using CRISPR-Cas9 resulted in blunted calcium influx, reduced expression of endothelial nitric oxide synthase, and diminished nitric oxide bioavailability. Conclusions- These results from a novel multiomics study suggest that KLF4 is crucial for PS-modulated H3K27ac that allow the transcriptional activation of ITPR3. This novel mechanism contributes to the Ca2+-dependent eNOS (endothelial nitric oxide synthase) activation and EC homeostasis.

DC, through the uptake, processing, and presentation of antigen, are responsible for activation of T cell responses to defend the host against infection, yet it is not known if they can directly kill invading bacteria. Here, we studied in human leprosy, how Langerhans cells (LC), specialized DC, contribute to host defense against bacterial infection. IFN-γ treatment of LC isolated from human epidermis and infected with Mycobacterium leprae (M. leprae) activated an antimicrobial activity, which was dependent on the upregulation of the antimicrobial peptide cathelicidin and induction of autophagy. IFN-γ induction of autophagy promoted fusion of phagosomes containing M. leprae with lysosomes and the delivery of cathelicidin to the intracellular compartment containing the pathogen. Autophagy enhanced the ability of M. leprae-infected LC to present antigen to CD1a-restricted T cells. The frequency of IFN-γ labeling and LC containing both cathelicidin and autophagic vesicles was greater in the self-healing lesions vs. progressive lesions, thus correlating with the effectiveness of host defense against the pathogen. These data indicate that autophagy links the ability of DC to kill and degrade an invading pathogen, ensuring cell survival from the infection while facilitating presentation of microbial antigens to resident T cells.

ST3Gal1 is a key sialyltransferase which adds α2,3-linked sialic acid to substrates and generates core 1 O-glycan structure. Upregulation of ST3Gal1 has been associated with worse prognosis of breast cancer patients. However, the protein substrates of ST3Gal1 implicated in tumor progression remain elusive. In our study, we demonstrated that ST3GAL1-silencing significantly reduced tumor growth along with a notable decrease in vascularity of MCF7 xenograft tumors. We identified vasorin (VASN) which was shown to bind TGF-β1, as a potential candidate that links ST3Gal1 to angiogenesis. LC-MS/MS analysis of VASN secreted from MCF7, revealed that more than 80% of its O-glycans are sialyl-3T and disialyl-T. ST3GAL1-silencing or desialylation of VASN by neuraminidase enhanced its binding to TGF-β1 by 2- to 3-fold and thereby dampening TGF-β1 signaling and angiogenesis, as indicated by impaired tube formation of HUVECs, suppressed angiogenesis gene expression and reduced activation of Smad2 and Smad3 in HUVEC cells. Examination of 114 fresh primary breast cancer and their adjacent normal tissues showed that the expression levels of ST3Gal1 and TGFB1 were high in tumor part and the expression of two genes was positively correlated. Kaplan Meier survival analysis showed a significantly shorter relapse-free survival for those with lower expression VASN, notably, the combination of low VASN with high ST3GAL1 yielded even higher risk of recurrence (p = 0.025, HR = 2.967, 95% CI = 1.14-7.67). Since TGF-β1 is known to transcriptionally activate ST3Gal1, our findings illustrated a feedback regulatory loop in which TGF-β1 upregulates ST3Gal1 to circumvent the negative impact of VASN.

Growing evidence suggests that long non-coding RNAs NORAD and miR-205 play a significant role in regulating cancer progression and metastasis. In this study, high expression of NORAD was firstly observed in melanoma tissues and human malignant melanoma cell lines, our aim was to study the interaction of them in the process of invasion and migration of malignant melanoma cells. NORAD, miR-205, and EGLN2 mRNA level in MM cells was detected by qRT-PCR. In situ hybridization (ISH) was performed to detect NORAD expression in MM tissues specimens. Effects of NORAD and miR-205 on Prolyl hydroxylase 2 (EGLN2) expression was explored by western blot in MM cells line. Dual-luciferase reporter assay was performed to verify the interaction relationship between NORAD and miR-205, as well as, miR-205 and EGLN2. Transwell assay was conducted to explore the effects of NORAD and miR-205 in vitro. Xenografts in nude mice experiment were used to confirm the role of NORAD and miR-205 in vivo. In vitro, NORAD knockdown significantly inhibited migration and invasion of malignant melanoma cells and elevated the expression of miR-205, there was an interaction between miR-205 and NORAD in the RNA-induced silencing complex. Upregulation of miR-205 induced significant inhibition of migratory and invasive ability compared with the scrambled control. However, downregulating NORAD largely reversed this effect. Furthermore, the regulatory effects of miR-205 on EGLN2 levels and the induction of endoplasmic reticulum stress were reversed by NORAD. In vivo, deletion of miR-205 induced tumor growth in nude mice. NORAD may play critical roles in tumorigenesis and progression of malignant melanoma by regulating of the miR-205-EGLN2 pathway, and may serve as a new therapeutic target.

Many viruses shut off host gene expression to inhibit antiviral responses. Viral proteins and host proteins required for viral replication are typically spared in this process, but the mechanisms of target selectivity during host shutoff remain poorly understood. Using transcriptome-wide and targeted reporter experiments, we demonstrate that the influenza A virus endoribonuclease PA-X usurps RNA splicing to selectively target host RNAs for destruction. Proximity-labeling proteomics reveals that PA-X interacts with cellular RNA processing proteins, some of which are partially required for host shutoff. Thus, PA-X taps into host nuclear pre-mRNA processing mechanisms to destroy nascent mRNAs shortly after their synthesis. This mechanism sets PA-X apart from other viral host shutoff proteins that target actively translating mRNAs in the cytoplasm. Our study reveals a unique mechanism of host shutoff that helps us understand how influenza viruses suppress host gene expression.

Medulloblastoma is a brain tumor that arises predominantly in infants and children. It is the most common pediatric brain malignancy. Around 25% of medulloblastomas are driven by constitutive activation of the Hedgehog signaling pathway. Hedgehog-driven medulloblastoma is often studied in the laboratory using genetic mouse models with overactive Hedgehog signaling, which recapitulate many of the pathological features of human Hedgehog-dependent tumors. However, we show here that on a molecular level the human and mouse HH-dependent MB are quite distinct, with human, but not mouse, tumors characterized by the presence of markers of increased oxidative phosphorylation and mitochondrial biogenesis. The latter suggests that, unlike for many other types of tumors, a switch to glycolytic metabolism might not be co-opted by human SHH-MB to perpetuate their survival and growth. This needs to be taken into consideration and could potentially be exploited in the design of therapies.

Ligand-dependent oligomerization of receptor tyrosine kinases (RTKs) results in their activation through highly specific conformational changes in the extracellular and intracellular receptor domains. These conformational changes are unique for each RTK subfamily, limiting cross-activation between unrelated RTKs. The proto-oncogene MET receptor tyrosine kinase overcomes these structural constraints and phosphorylates unrelated RTKs in numerous cancer cell lines. The molecular basis for these interactions is unknown. We investigated the mechanism by which MET phosphorylates the human epidermal growth factor receptor-3 (HER3 or ERBB3), a catalytically impaired RTK whose phosphorylation by MET has been described as an essential component of drug resistance to inhibitors targeting EGFR and HER2. We find that in untransformed cells, HER3 is not phosphorylated by MET in response to ligand stimulation, but rather to increasing levels of MET expression, which results in ligand-independent MET activation. Phosphorylation of HER3 by its canonical co-receptors, EGFR and HER2, is achieved by engaging an allosteric site on the HER3 kinase domain, but this site is not required when HER3 is phosphorylated by MET. We also observe that HER3 preferentially interacts with MET during its maturation along the secretory pathway, before MET is post translationally processed by cleavage within its extracellular domain. This results in accumulation of phosphorylated HER3 in the Golgi apparatus. We further show that in addition to HER3, MET phosphorylates other RTKs in the Golgi, suggesting that this mechanism is not limited to HER3 phosphorylation. These data demonstrate a link between MET overexpression and its aberrant activation in the Golgi endomembranes and suggest that non-canonical interactions between MET and other RTKs occur during maturation of receptors. Our study highlights a novel aspect of MET signaling in cancer that would not be accessible to inhibition by therapeutic antibodies.

Cancer cells heavily depend on the amino acid glutamine to meet the demands associated with growth and proliferation. Due to the rapid consumption of glutamine, cancer cells frequently undergo glutamine starvation in vivo. We and others have shown that p53 is a critical regulator in metabolic stress resistance. To better understand the molecular mechanisms by which p53 activation promotes cancer cell adaptation to glutamine deprivation, we identified p53-dependent genes that are induced upon glutamine deprivation by using RNA-seq analysis. We show that Slc7a3, an arginine transporter, is significantly induced by p53. We also show that increased intracellular arginine levels following glutamine deprivation are dependent on p53. The influx of arginine has minimal effects on known metabolic pathways upon glutamine deprivation. Instead, we found arginine serves as an effector for mTORC1 activation to promote cell growth in response to glutamine starvation. Therefore, we identify a p53-inducible gene that contributes to the metabolic stress response.

The Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions are highly dependent on the specific cellular context. Gain-of-function NOTCH1 mutations are prevalent in human T-cell leukemia, while loss of Notch signaling is reported in myeloid leukemias. Here, we report a novel oncogenic function of Notch signaling in oncogenic Kras-induced myeloproliferative neoplasm (MPN). We find that downregulation of Notch signaling in hematopoietic cells via DNMAML expression or Pofut1 deletion significantly blocks MPN development in KrasG12D mice in a cell-autonomous manner. Further mechanistic studies indicate that inhibition of Notch signaling upregulates Dusp1, a dual phosphatase that inactivates p-ERK, and downregulates cytokine-evoked ERK activation in KrasG12D cells. Moreover, mitochondrial metabolism is greatly enhanced in KrasG12D cells but significantly reprogrammed by DNMAML close to that in control cells. Consequently, cell proliferation and expanded myeloid compartment in KrasG12D mice are significantly reduced. Consistent with these findings, combined inhibition of the MEK/ERK pathway and mitochondrial oxidative phosphorylation effectively inhibited the growth of human and mouse leukemia cells in vitro. Our study provides a strong rational to target both ERK signaling and aberrant metabolism in oncogenic Ras-driven myeloid leukemia.

Dilated cardiomyopathy (DCM) is a leading cause of morbidity and mortality worldwide; yet how genetic variation and environmental factors impact DCM heritability remains unclear. Here, we report that compound genetic interactions between DNA sequence variants contribute to the complex heritability of DCM. By using genetic data from a large family with a history of DCM, we discovered that heterozygous sequence variants in the TROPOMYOSIN 1 (TPM1) and VINCULIN (VCL) genes cose-gregate in individuals affected by DCM. In vitro studies of patient-derived and isogenic human-pluripotent-stem-cell-derived cardio-myocytes that were genome-edited via CRISPR to create an allelic series of TPM1 and VCL variants revealed that cardiomyocytes with both TPM1 and VCL variants display reduced contractility and sarcomeres that are less organized. Analyses of mice genetically engineered to harbour these human TPM1 and VCL variants show that stress on the heart may also influence the variable penetrance and expressivity of DCM-associated genetic variants in vivo. We conclude that compound genetic variants can interact combinatorially to induce DCM, particularly when influenced by other disease-provoking stressors.

Cancer cells develop mechanisms to escape immunosurveillance, among which modulating the expression of immune suppressive messenger RNAs is most well-documented. However, how this is molecularly achieved remains largely unresolved. Here, we develop an in vivo mouse model of liver cancer to study oncogene cooperation in immunosurveillance. We show that MYC overexpression (MYCTg) synergizes with KRASG12D to induce an aggressive liver tumor leading to metastasis formation and reduced mouse survival compared with KRASG12D alone. Genome-wide ribosomal footprinting of MYCTg;KRASG12 tumors compared with KRASG12D revealed potential alterations in translation of mRNAs, including programmed-death-ligand 1 (PD-L1). Further analysis revealed that PD-L1 translation is repressed in KRASG12D tumors by functional, non-canonical upstream open reading frames in its 5' untranslated region, which is bypassed in MYCTg;KRASG12D tumors to evade immune attack. We show that this mechanism of PD-L1 translational upregulation was effectively targeted by a potent, clinical compound that inhibits eIF4E phosphorylation, eFT508, which reverses the aggressive and metastatic characteristics of MYCTg;KRASG12D tumors. Together, these studies reveal how immune-checkpoint proteins are manipulated by distinct oncogenes at the level of mRNA translation, which can be exploited for new immunotherapies.

Endogenous hydrogen sulfide (H2S) is a potent vasodilator and proangiogenic second messenger synthesized from L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). Estrogens are potent vasodilators that stimulate H2S biosynthesis in uterine arteries (UA) in vivo; however, the underlying mechanisms are unknown. We hypothesized that estrogens stimulate H2S biosynthesis in UA endothelial cells (UAEC) via specific estrogen receptor (ER)-dependent mechanisms. In cultured primary UAEC, treatment with estradiol-17β (E2β) stimulated CBS and CTH mRNAs and proteins in a time- and concentration-dependent fashion. As little as 0.1 nM E2β was effective in increasing CBS and CTH expressions and these stimulatory effects maximized with 10-100 nM E2β at 48-72 h. E2β also activated CBS and CTH promoters in UAEC, leading to CBS and CTH expression. Treatment with E2β stimulated H2S production, which was blocked by specific inhibitors of either CBS or CTH and their combination and the ER antagonist ICI 182780. Treatment with either specific agonist of ERα or ERβ stimulated both CBS and CTH mRNA and protein expressions and H2S production to levels similar to that of E2β. Specific antagonist of either ERα or ERβ blocked E2β-stimulated CBS and CTH mRNA and protein expressions and H2S production. Combinations of either ERα or ERβ agonists or their antagonists had no additive effects. Thus, E2β stimulates H2S production by upregulating CBS and CTH mRNA and protein expressions through specific ERα or ERβ-dependent CBS and CTH transcription in UAEC in vitro.

Background and aimsFibroblast growth factor 21 (FGF21) plays an important role in glucose and lipid metabolism. We have investigated the relationship of plasma FGF21 levels with both prevalent and incident metabolic syndrome (MetS) in participants from the Multi-Ethnic Study of Atherosclerosis (MESA).Methods5783 participants from four major ethnic groups (non-Hispanic white, African American, Hispanic American, and Chinese American) were included in the cross-sectional analysis. Longitudinal analysis involved 3479 participants without MetS at baseline, of whom 1100 participants developed incident MetS over 9.2 years.ResultsElevated FGF21 levels were found in participants with prevalent MetS (median [interquartile range] = 189.4 [114.4-302.1] vs. 123.7 [65.9-210.3] pg/mL, p

The peripartum period is associated with the onset of behaviors that shelter, feed and protect young offspring from harm. The neural pathway that regulates caregiving behaviors has been mapped in female rats and is conserved in mice. However, rats rely on late gestational hormones to shift their perception of infant cues from aversive to attractive, whereas laboratory mice are "spontaneously" maternal, but their level of responding depends on experience. For example, pup-naïve virgin female mice readily care for pups in the home cage, but avoid pups in a novel environment. In contrast, pup-experienced virgin mice care for pups in both contexts. Thus, virgin mice rely on experience to shift their perception of infant cues from aversive to attractive in a novel context. We hypothesize that alterations in immediate early gene activation may underlie the experience-driven shift in which neural pathways (fear/avoidance versus maternal/approach) are activated by pups to modulate context-dependent changes in maternal responding. Here we report that the effects of sodium butyrate, a drug that allows for an amplification of experience-induced histone acetylation and gene expression in virgins, are comparable to the natural onset of caregiving behaviors in postpartum mice and induce postpartum-like patterns of immediate early gene expression across brain regions. These data suggest that pups can activate a fear/defensive circuit in mice and experience-driven improvements in caregiving behavior could be regulated in part through decreased activation of this pathway.

ObjectiveResistin is a hormone that has been associated with metabolic syndrome and cardiovascular disease. The role of resistin in patients with peripheral artery disease (PAD) has not been fully explored. This study seeks to understand the relationship between serum resistin, vascular function, and cardiovascular outcomes in patients with PAD.MethodsThere were 106 patients with PAD who were recruited between 2011 and 2016. Patients attended a baseline visit during which a comprehensive vascular physiology assessment including medical and surgical history, radial artery tonometry, and flow mediated-vasodilation (FMD) was completed. A blood sample was drawn, and serum resistin was assayed using enzyme-linked immunosorbent assay kits. Using the time of study enrollment as the time of origin, incident major adverse cardiac events (MACEs) were identified by subsequent chart review and defined as a composite end point of myocardial infarction, coronary revascularization, transient ischemic attack, stroke, or death from a cardiac cause.ResultsPatients had a mean age of 68 ± 8 years, were largely white (75%), and had comorbidities commonly associated with PAD including hypertension (92%), hyperlipidemia (87%), coronary artery disease (37%), and diabetes mellitus (38%). After stratification by resistin quartile, higher resistin quartiles were significantly associated with an older age, a greater number of pack-years smoked, and a lower estimated glomerular filtration rate. Despite similar comorbidities and medication use, endothelial function, as measured by FMD, was significantly lower with increasing resistin quartile (I, 9.1% ± 3.3%; II, 7.1% ± 3.5%; III, 5.8% ± 4.0%; IV, 5.6% ± 3.5%; P = .002). In multivariable linear regression, higher resistin quartiles (III and IV) were associated with lower FMD relative to quartile I after adjusting for several patient characteristics, medications, and comorbidities (III, -2.26 [95% confidence interval (CI), -4.51 to -0.01; P = .05]; IV, -2.53 [95% CI, -4.87 to -0.20; P = .03]). During a median follow-up period of 36 months (interquartile range, 29-45 months), 21 patients experienced the primary end point. In a Cox proportional hazards model adjusted for smoking status, coronary artery disease, and age, each 1 ng/mL increase in resistin was associated with a 10% increased risk of MACEs (hazard ratio, 1.10; 95% CI, 1.00-1.20; P = .04).ConclusionsIn patients with PAD, higher levels of resistin were associated with impaired endothelial function and an increased rate of MACEs. These results suggest that resistin may be a marker or effector of impaired vascular physiology and adverse cardiac outcomes in patients with PAD. Further research is needed to determine the potential mechanisms by which resistin may impair endothelial function and increase MACEs in this population.

Cell-to-cell communication in skin participates to the maintenance of homeostatic responses to foreign substances. Certain strains of Staphylococcus (S) aureus are vicious pathogens that cause deleterious effects in host cells and tissues. Both secreted toxins and structural components of S. aureus trigger an immune response, though how S. aureus stimulates host immune responses is poorly understood. We explored here how keratinocytes and fibroblasts initiate the first steps of an immune response by activating dendritic cells (DCs) through recognition of structural components of S. aureus. We treated monocyte-derived Langerhans cells (moLCs) and monocyte-derived DCs (moDCs) with conditioned media from keratinocytes (K-CM) and fibroblasts (F-CM) treated with heat-killed S. aureus (HKSA) respectively, or directly with HKSA. Immune and inflammatory responses from keratinocytes, fibroblasts, moLCs and moDCs were assessed by analysis of cell surface markers and cytokine production using flow cytometry, real-time PCR and ELISA assays. K-CM and F-CM increased the expression of CD86 and HLA-DR on moLCs and moDCs, in association with a specific cytokine profile. K-CM upregulated TNFA, IL-1B and GM-CSF mRNA expression in moLCs, while F-CM upregulated IL-12 and downregulated TNFA and TGFB mRNA expression in moDCs. Additionally, F-CM attenuated the induction of an inflammatory profile in monocytes. The recognition of structural components from S. aureus by cutaneous microenvironment induces the activation and the expression of specific cytokines from LCs and DCs.

Tissue fibrosis is a major cause of mortality that results from the deposition of matrix proteins by an activated mesenchyme. Macrophages accumulate in fibrosis, but the role of specific subgroups in supporting fibrogenesis has not been investigated in vivo. Here, we used single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of macrophages in bleomycin-induced lung fibrosis in mice. A novel computational framework for the annotation of scRNA-seq by reference to bulk transcriptomes (SingleR) enabled the subclustering of macrophages and revealed a disease-associated subgroup with a transitional gene expression profile intermediate between monocyte-derived and alveolar macrophages. These CX3CR1+SiglecF+ transitional macrophages localized to the fibrotic niche and had a profibrotic effect in vivo. Human orthologs of genes expressed by the transitional macrophages were upregulated in samples from patients with idiopathic pulmonary fibrosis. Thus, we have identified a pathological subgroup of transitional macrophages that are required for the fibrotic response to injury.

A wide range of human diseases result from haploinsufficiency, where the function of one of the two gene copies is lost. Here, we targeted the remaining functional copy of a haploinsufficient gene using CRISPR-mediated activation (CRISPRa) in Sim1 and Mc4r heterozygous mouse models to rescue their obesity phenotype. Transgenic-based CRISPRa targeting of the Sim1 promoter or its distant hypothalamic enhancer up-regulated its expression from the endogenous functional allele in a tissue-specific manner, rescuing the obesity phenotype in Sim1 heterozygous mice. To evaluate the therapeutic potential of CRISPRa, we injected CRISPRa-recombinant adeno-associated virus into the hypothalamus, which led to reversal of the obesity phenotype in Sim1 and Mc4r haploinsufficient mice. Our results suggest that endogenous gene up-regulation could be a potential strategy to treat altered gene dosage diseases.

Neuroendocrine prostate cancer (NEPC), a lethal form of the disease, is characterized by loss of androgen receptor (AR) signaling during neuroendocrine transdifferentiation, which results in resistance to AR-targeted therapy. Clinically, genomically and epigenetically, NEPC resembles other types of poorly differentiated neuroendocrine tumors (NETs). Through pan-NET analyses, we identified ONECUT2 as a candidate master transcriptional regulator of poorly differentiated NETs. ONECUT2 ectopic expression in prostate adenocarcinoma synergizes with hypoxia to suppress androgen signaling and induce neuroendocrine plasticity. ONEUCT2 drives tumor aggressiveness in NEPC, partially through regulating hypoxia signaling and tumor hypoxia. Specifically, ONECUT2 activates SMAD3, which regulates hypoxia signaling through modulating HIF1α chromatin-binding, leading NEPC to exhibit higher degrees of hypoxia compared to prostate adenocarcinomas. Treatment with hypoxia-activated prodrug TH-302 potently reduces NEPC tumor growth. Collectively, these results highlight the synergy between ONECUT2 and hypoxia in driving NEPC, and emphasize the potential of hypoxia-directed therapy for NEPC patients.

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are diseases of abnormal hematopoietic differentiation with aberrant epigenetic alterations. Azacitidine (AZA) is a DNA methyltransferase inhibitor widely used to treat MDS and AML, yet the impact of AZA on the cell-surface proteome has not been defined. To identify potential therapeutic targets for use in combination with AZA in AML patients, we investigated the effects of AZA treatment on four AML cell lines representing different stages of differentiation. The effect of AZA treatment on these cell lines was characterized at three levels: the DNA methylome, the transcriptome, and the cell-surface proteome. Untreated AML cell lines showed substantial overlap at all three omics levels; however, while AZA treatment globally reduced DNA methylation in all cell lines, changes in the transcriptome and surface proteome were subtle and differed among the cell lines. Transcriptome analysis identified five commonly up-regulated coding genes upon AZA treatment in all four cell lines, TRPM4 being the only gene encoding a surface protein, and surface proteome analysis found no commonly regulated proteins. Gene set enrichment analysis of differentially regulated RNA and surface proteins showed a decrease in metabolic pathways and an increase in immune defense response pathways. As such, AZA treatment led to diverse effects at the individual gene and protein levels but converged to common responses at the pathway level. Given the heterogeneous responses in the four cell lines, we discuss potential therapeutic strategies for AML in combination with AZA.

The complement system is an innate immune pathway typically thought of as part of the first line of defence against "non-self" species. In the context of cancer, complement has been described to have an active role in facilitating cancer-associated processes such as increased proliferation, angiogenesis and migration. Several cellular members of the tumour microenvironment express and/or produce complement proteins locally, including tumour cells. Dysregulation of the complement system has been reported in numerous tumours and increased expression of complement activation fragments in cancer patient specimens correlates with poor patient prognosis. Importantly, genetic or pharmacological targeting of complement has been shown to reduce tumour growth in several cancer preclinical models, suggesting that complement could be an attractive therapeutic target. Hypoxia (low oxygen) is frequently found in solid tumours and has a profound biological impact on cellular and non-cellular components of the tumour microenvironment. In this review, we focus on hypoxia since this is a prevailing feature of the tumour microenvironment that, like increased complement, is typically associated with poor prognosis. Furthermore, interesting links between hypoxia and complement have been recently proposed but never collectively reviewed. Here, we explore how hypoxia alters regulation of complement proteins in different cellular components of the tumour microenvironment, as well as the downstream biological consequences of this regulation.

Ectopic epigenetic mechanisms play important roles in facilitating tumorigenesis. Here, we first demonstrated that ANKDD1A is a functional tumor suppressor gene, especially in the hypoxia microenvironment. ANKDD1A directly interacts with FIH1 and inhibits the transcriptional activity of HIF1α by upregulating FIH1. In addition, ANKDD1A decreases the half-life of HIF1α by upregulating FIH1, decreases glucose uptake and lactate production, inhibits glioblastoma multiforme (GBM) autophagy, and induces apoptosis in GBM cells under hypoxia. Moreover, ANKDD1A is highly frequently methylated in GBM. The tumor-specific methylation of ANKDD1A indicates that it could be used as a potential epigenetic biomarker as well as a possible therapeutic target.

Bronchial premalignant lesions (PMLs) are precursors of lung squamous cell carcinoma, but have variable outcome, and we lack tools to identify and treat PMLs at risk for progression to cancer. Here we report the identification of four molecular subtypes of PMLs with distinct differences in epithelial and immune processes based on RNA-Seq profiling of endobronchial biopsies from high-risk smokers. The Proliferative subtype is enriched with bronchial dysplasia and exhibits up-regulation of metabolic and cell cycle pathways. A Proliferative subtype-associated gene signature identifies subjects with Proliferative PMLs from normal-appearing uninvolved large airway brushings with high specificity. In progressive/persistent Proliferative lesions expression of interferon signaling and antigen processing/presentation pathways decrease and immunofluorescence indicates a depletion of innate and adaptive immune cells compared with regressive lesions. Molecular biomarkers measured in PMLs or the uninvolved airway can enhance histopathological grading and suggest immunoprevention strategies for intercepting the progression of PMLs to lung cancer.

Multipotent mesenchymal stem cells (MSCs) maintain the ability to differentiate into adipogenic, chondrogenic, or osteogenic cell lineages. There is increasing concern that exposure to environmental agents such as aryl hydrocarbon receptor (AhR) ligands, may perturb the osteogenic pathways responsible for normal bone formation. The objective of the current study was to evaluate the potential of the prototypic AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to disrupt osteogenic differentiation of human bone-derived MSCs (hBMSCs) in vitro. Primary hBMSCs from three donors were exposed to 10 nM TCDD and differentiation was interrogated using select histological, biochemical, and transcriptional markers of osteogenesis. Exposure to 10 nM TCDD resulted in an overall consistent attenuation of alkaline phosphatase (ALP) activity and matrix mineralization at terminal stages of differentiation in primary hBMSCs. At the transcriptional level, the transcriptional regulator DLX5 and additional osteogenic markers (ALP, OPN, and IBSP) displayed attenuated expression; conversely, FGF9 and FGF18 were consistently upregulated in each donor. Expression of stem cell potency markers SOX2, NANOG, and SALL4 decreased in the osteogenic controls, whereas expression in TCDD-treated cells resembled that of undifferentiated cells. Coexposure with the AhR antagonist GNF351 blocked TCDD-mediated attenuation of matrix mineralization, and either fully or partially rescued expression of genes associated with osteogenic regulation, extracellular matrix, and/or maintenance of multipotency. Thus, experimental evidence from this study suggests that AhR transactivation likely attenuates osteoblast differentiation in multipotent hBMSCs. This study also underscores the use of primary human MSCs to evaluate osteoinductive or osteotoxic potential of chemical and pharmacologic agents in vitro.

BackgroundHigher fibroblast growth factor-23 (FGF23) concentrations have been found to be associated with incident heart failure (HF). Experimental data suggest FGF23 directly stimulates myocardial hypertrophy. FGF23 may also enhance renin-angiotensin-aldosterone system activity. Whether FGF23 is associated with increased HF risk in populations with hypertension and whether this association is weaker in the presence of angiotensin-converting enzyme inhibitor (ACEI) or angiotensin II receptor blocker (ARB) therapy is unknown.MethodsWe studied 2,858 adults with hypertension free of cardiovascular disease at baseline (65.6 ± 9.5 years, 46.2% male) participating in the Multi-Ethnic Study of Atherosclerosis (MESA). We investigated the association of baseline serum intact FGF23 with incident HF over a 14-year median follow-up and whether ACEI/ARB therapy modified this risk. We also investigated the relationship of FGF23 with aldosterone and plasma renin activity in a random subgroup of the entire MESA cohort with available assays (N = 1,642).ResultsIn adjusted Cox regression models, higher FGF23 was associated with a 63% greater hazard of incident HF (hazard ratio: 1.63, 95% confidence interval: [1.13-2.36] per 1-unit increase in log-transformed FGF23), which persisted after exclusion of participants with chronic kidney disease (hazard ratio: 1.94 [1.10-3.43]). There was no heterogeneity by ACEI/ARB use (Pinteraction = 0.438). FGF23 improved model fit over covariables (likelihood ratio χ2 = 6.67, P = 0.010). In multivariable linear regression models, there was no association between FGF23 and aldosterone or plasma renin activity.ConclusionsHigher FGF23 concentrations are associated with a significantly increased risk of HF in hypertension but this risk did not differ by ACEI/ARB treatment status. FGF23 may be a useful biomarker for HF risk in hypertensive populations.

BackgroundCyclin-dependent protein kinase 9 (CDK9) has been shown to play an important role in the pathogenesis of malignant tumors. However, the expression and function of CDK9 remain unknown in osteosarcomas. The purpose of this study is to assess the expression, function and clinical prognostic relationship of CDK9 in osteosarcomas.MethodsA tissue microarray of 70 patient specimens was analyzed by immunohistochemistry to measure CDK9 expression, which was further investigated for correlation with patient clinical characteristics. CDK9 expression in osteosarcoma cell lines and patient tissues was also evaluated by Western blotting. CDK9-specific siRNA and the CDK9 inhibitor were applied to determine the effect of CDK9 inhibition on osteosarcoma cell proliferation and anti-apoptotic activity. The clonogenicity and migration activity were also examined using clonogenic and wound healing assays. A 3D cell culture model was performed to mimic the in vivo osteosarcoma environment to further validate the effect of CDK9 inhibition on osteosarcoma cells.FindingsWe demonstrated that higher CDK9-expression is associated with significantly shortened patient survival by immunohistochemistry. Expression of CDK9 is inversely correlated to the percent of tumor necrosis post-neoadjuvant chemotherapy, which is the most important predictive factor of disease outcome for osteosarcoma patients. Knockdown of CDK9 with siRNA and inhibition of CDK9 activity with inhibitor decreased cell proliferation and induced apoptosis in osteosarcoma.InterpretationHigh expression of CDK9 is an independent predictor of poor prognosis in osteosarcoma patients. Our results suggest that CDK9 is a novel prognostic marker and a promising therapeutic target for osteosarcomas.

Dengue virus (DENV) is the most prevalent mosquito-borne virus in the world and a major cause of morbidity in the tropics and subtropics. Upregulation of HLA class I molecules has long been considered a feature of DENV infection, yet this has not been evaluated in the setting of natural infection. Natural killer (NK) cells, an innate immune cell subset critical for mounting an early response to viral infection, are inhibited by self HLA class I, suggesting that upregulation of HLA class I during DENV infection could dampen the NK cell response. Here we addressed whether upregulation of HLA class I molecules occurs during in vivo DENV infection and, if so, whether this suppresses the NK cell response. We found that HLA class I expression was indeed upregulated during acute DENV infection across multiple cell lineages in vivo. To better understand the role of HLA class I upregulation, we infected primary human monocytes, a major target of DENV infection, in vitro. Upregulation of total HLA class I is dependent on active viral replication and is mediated in part by cytokines and other soluble factors induced by infection, while upregulation of HLA-E occurs in the presence of replication-incompetent virus. Importantly, blocking DENV-infected monocytes with a pan-HLA class I Fab nearly doubles the frequency of degranulating NK cells, while blocking HLA-E does not significantly improve the NK cell response. These findings demonstrate that upregulation of HLA class I during DENV infection suppresses the NK cell response, potentially contributing to disease pathogenesis.

BackgroundUptake of riboflavin (RF) by intestinal epithelial cells occurs via a specific carrier-mediated process that involves the apically localized RF transporter-3 (RFVT3). Previous studies have shown that sodium butyrate (NaB) affects intestinal uptake of other substrates and expression of their membrane transporters, but its effect on intestinal uptake of RF and expression of RFVT3 has not been examined.AimsTo investigate the effect of NaB on intestinal RF uptake process and expression of the RFVT3.MethodsTwo experimental models were used in this study: Human-derived intestinal epithelial Caco-2 cells and ex vivo mouse colonoids. 3H-RF uptake assay, Western blot, RT-qPCR, and chromatin immunoprecipitation assay were performed.ResultsTreating Caco-2 cells with NaB led to a significant increase in carrier-mediated RF uptake. This increase was associated with a significant induction in the level of expression of the hRFVT3 protein, mRNA, and heterogenous nuclear RNA (hnRNA). Similarly, treating mouse colonoids with NaB led to a marked increase in the level of expression of the mRFVT3 protein, mRNA, and hnRNA. NaB did not affect hRFVT3 mRNA stability, rather it caused significant epigenetic changes (histone modifications) in the SLC52A3 gene where an increase in H3Ac and a reduction in H3K27me3 levels were observed in the NaB-treated Caco-2 cells compared to untreated controls.ConclusionThese findings demonstrate that NaB up-regulates intestinal RF uptake and that the effect appears to be mediated, at least in part, at the level of transcription of the SLC52A3 gene and may involve epigenetic mechanism(s).

Triclosan (TCS) is a prevalent anthropogenic contaminant in aquatic environments and its chronic exposure can lead to a series of neurotoxic effects in zebrafish. Both qRT-PCR and W-ISH identified that TCS exposure resulted in significant up-regulation of miR-137, but downregulation of its regulatory genes (bcl11aa, MAPK6 and Runx1). These target genes are mainly associated with neurodevelopment and the MAPK signaling pathway, and showed especially high expression in the brain. After overexpression or knockdown treatments by manual intervention of miR-137, a series of abnormalities were induced, such as ventricular abnormality, bent spine, yolk cyst, closure of swim sac and venous sinus hemorrhage. The most sensitive larval toxicological endpoint from intervened miR-137 expression was impairment of the central nervous system (CNS), ventricular abnormalities and notochord curvature. Microinjection of microRNA mimics or inhibitors of miR-137 both caused zebrafish malformations. The posterior lateral line neuromasts became obscured and decreased in number in intervened miR-137 groups and TCS-exposure groups. Up-regulation of miR-137 led to more severe neurotoxic effects than its down-regulation. Behavioral observations demonstrated that both TCS exposure and miR-137 over-expression led to inhibited hearing or vision sensitivity. HE staining indicated that hearing and vision abnormalities induced by long-term TCS exposure originated from CNS injury, such as reduced glial cells and loose and hollow fiber structures. The findings of this study enhance our mechanistic understanding of neurotoxicity in aquatic animals in response to TCS exposure. These observations provide theoretical guidance for development of early intervention treatments for nervous system diseases.

Inflammation plays a critical role in initiation of adaptive immunity, pathogen clearance and tissue repair. Interleukin (IL)-1β is a potent pro-inflammatory cytokine and therefore its production is tightly regulated: its secretion requires the assembly of a macromolecular protein complex, termed the inflammasome. Aberrant activation of the inflammasome has been linked to debilitating human diseases including chronic inflammatory and autoimmune diseases. Thus, there is a great interest in understanding how inflammasomes are regulated. Here we show that Dicer, an enzyme necessary for the production of mature micro-RNAs (miRNAs), is required for optimal activation of NLRP3 inflammasomes in bone marrow macrophages. Our data indicate that miRNAs may play an important role in promoting inflammasome activation.

Capicua (Cic) is a transcriptional repressor mutated in the brain cancer oligodendroglioma. Despite its cancer link, little is known of Cic's function in the brain. We show that nuclear Cic expression is strongest in astrocytes and neurons but weaker in stem cells and oligodendroglial lineage cells. Using a new conditional Cic knockout mouse, we demonstrate that forebrain-specific Cic deletion increases proliferation and self-renewal of neural stem cells. Furthermore, Cic loss biases neural stem cells toward glial lineage selection, expanding the pool of oligodendrocyte precursor cells (OPCs). These proliferation and lineage effects are dependent on de-repression of Ets transcription factors. In patient-derived oligodendroglioma cells, CIC re-expression or ETV5 blockade decreases lineage bias, proliferation, self-renewal, and tumorigenicity. Our results identify Cic as an important regulator of cell fate in neurodevelopment and oligodendroglioma, and suggest that its loss contributes to oligodendroglioma by promoting proliferation and an OPC-like identity via Ets overactivity.

Mechanical loading is essential for the maintenance of musculoskeletal homeostasis. Cartilage has been demonstrated to be highly mechanoresponsive, but the mechanisms by which chondrocytes respond to mechanical stimuli are not clearly understood. The goal of the study was to determine how LRP4, LRP5, and LRP6 within canonical Wnt-signaling are regulated in simulated microgravity and cyclic hydrostatic pressure, and to investigate the potential role of LRP 4/5/6 in cartilage degeneration. Rat chondrosacroma cell (RCS) pellets were stimulated using either cyclic hydrostatic pressure (1Hz, 7.5 MPa, 4hr/day) or simulated microgravity in a rotating wall vessel (RWV) bioreactor (11RPM, 24hr/day). LRP4/5/6 mRNA expression was assessed by RT-qPCR and LRP5 protein expression was determined by fluorescent immunostaining. To further evaluate our in vitro findings in vivo, mice were subjected to hindlimb suspension for 14 days and the femoral heads stained for LRP5 expression. We found that, in vitro, LRP4/5/6 mRNA expression is modulated in a time-dependent manner by mechanical stimulation. Additionally, LRP5 protein expression is upregulated in response to both simulated microgravity and cyclic hydrostatic pressure. LRP5 is also upregulated in vivo in the articular cartilage of hindlimb suspended mice. This is the first study to examine how LRP4/5/6, critical receptors within musculoskeletal biology, respond to mechanical stimulation. Further elucidation of this mechanism could provide significant clinical benefit for the identification of pharmaceutical targets for the maintenance of cartilage health.