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Background & Aims Diarrhea is one of the most common illnesses and is often caused by bacterial infection. Recently, we have shown that human Na+/H+ exchanger NHE3 (hNHE3), but not non-human NHE3s, interacts with the E3 ubiquitin ligase Nedd4-2. We hypothesize that this property of hNHE3 contributes to the increased severity of diarrhea in humans. Methods We used humanized mice expressing hNHE3 in the intestine (hNHE3int) to compare the contribution of hNHE3 and mouse NHE3 to diarrhea induced by cholera toxin (CTX) and enteropathogenic Escherichia coli (EPEC). We measured Na+/H+ exchange activity and fluid absorption. The role of Nedd4-2 on hNHE3 activity and ubiquitination was determined by knockdown in Caco-2bbe cells. The effects of protein kinase A (PKA), the primary mediator of CTX-induced diarrhea, on Nedd4-2 and hNHE3 phosphorylation and their interaction were determined. Results The effects of CTX and EPEC were greater in hNHE3int mice than in control wild-type (WT) mice, resulting in greater inhibition of NHE3 activity and increased fluid accumulation in the intestine, the hallmark of diarrhea. Activation of PKA increased ubiquitination of hNHE3 and enhanced interaction of Nedd4-2 with hNHE3 via phosphorylation of Nedd4-2 at S342. S342A mutation mitigated the Nedd4-2–hNHE3 interaction and blocked PKA-induced inhibition of hNHE3. Unlike non-human NHE3s, inhibition of hNHE3 by PKA is independent of NHE3 phosphorylation, suggesting a distinct mechanism of hNHE3 regulation. Conclusions The effects of CTX and EPEC on hNHE3 are amplified, and the unique properties of hNHE3 may contribute to diarrheal symptoms occurring in humans., Graphical abstract

Sorafenib is a first-line molecular-target drug for advanced hepatocellular carcinoma (HCC), and reducing sorafenib resistance is an important issue to be resolved for the clinical treatment of HCC. In the current study, we identified that ABCC5 is a critical regulator and a promising therapeutic target of acquired sorafenib resistance in human hepatocellular carcinoma cells. The expression of ABCC5 was dramatically induced in sorafenib-resistant HCC cells and was remarkably associated with poor clinical prognoses. The down-regulation of ABCC5 expression could significantly reduce the resistance of sorafenib to HCC cells. Importantly, activation of PI3K/AKT/NRF2 axis was essential for sorafenib to induce ABCC5 expression. ABCC5 increased intracellular glutathione (GSH) and attenuated lipid peroxidation accumulation by stabilizing SLC7A11 protein, which inhibited ferroptosis. Additionally, the inhibition of ABCC5 enhanced the anti-cancer activity of sorafenib in vitro and in vivo. These findings demonstrate a novel molecular mechanism of acquired sorafenib resistance and also suggest that ABCC5 is a new regulator of ferroptosis in HCC cells.

Highlights • Elevated expression of Wnt pathway associated factors in HPV-positive penile cancer cells. • lacking nuclear beta-catenin translocation indicated an actively abrogated Wnt signaling. • elevated expression of the Wnt antagonist DKK1 in HPV-positive penile cancer cells. • DKK1-driven autocrine Wnt pathway inhibition in penile cancer cells. • DKK1+ penile cancers are with a higher frequency HPV+, less differentiated and grow more aggressively., Penile squamous cell cancer (PSCC) is the most frequent penile malignant disease. Infections with human papillomaviruses (HPV) are a major etiologic driver of PSCC. However, the molecular details of the underlying carcinogenesis are understudied because of rare clinical specimens and missing cell lines. Here, we investigated if the expression of high-risk HPV16 oncogenes causes an augmentation of the Wnt pathway using unique HPV-positive penile cancer (PeCa) cell lines in monolayer and organotypic 3D raft cultures as well as tissue micro arrays containing clinical tissue specimens. The HPV oncoproteins enhanced the expression of Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) and the HPV-positive PeCa cells expressed a signature of Wnt target and stemness-associated genes. However, the notable lack of nuclear β-catenin in vitro and in situ raised the question if the enhanced expression of Wnt pathway factors is tantamount to an active Wnt signaling. Subsequent TOP-flash reporter assays revealed Wnt signaling as absent and not inducible by respective Wnt ligands in PeCa cell lines. The HPV-positive PeCa cells and especially HPV-positive PeCa specimens of the tumor core expressed the Wnt antagonist and negative feedback-regulator Dickkopf1 (DKK1). Subsequent neutralization experiments using PeCa cell line-conditioned media demonstrated that DKK1 is capable to impair ligand-induced Wnt signaling. While gene expression analyses suggested an augmented and active canonical Wnt pathway, the respective signaling was inhibited due to the endogenous expression of the antagonist DKK1. Subsequent TMA stainings indicated Dkk1 as linked with HPV-positivity and metastatic disease progression in PeCa suggesting potential as a prognostic marker.

Background & Aims Epithelial cells form a monolayer at mucosal surface that functions as a highly selective barrier. Lysophosphatidic acid (LPA) is a bioactive lipid that elicits a broad range of biological effects via cognate G protein-coupled receptors. LPA receptor 5 (LPA5) is highly expressed in intestinal epithelial cells, but its role in the intestine is not well-known. Here we determined the role of LPA5 in regulation of intestinal epithelial barrier. Methods Epithelial barrier integrity was determined in mice with intestinal epithelial cell (IEC)-specific LPA5 deletion, Lpar5ΔIEC. LPA was orally administered to mice, and intestinal permeability was measured. Dextran sulfate sodium (DSS) was used to induce colitis. Human colonic epithelial cell lines were used to determine the LPA5-mediated signaling pathways that regulate epithelial barrier. Results We observed increased epithelial permeability in Lpar5ΔIEC mice with reduced claudin-4 expression. Oral administration of LPA decreased intestinal permeability in wild-type mice, but the effect was greatly mitigated in Lpar5ΔIEC mice. Serum lipopolysaccharide level and bacterial loads in the intestine and liver were elevated in Lpar5ΔIEC mice. Lpar5ΔIEC mice developed more severe colitis induced with DSS. LPA5 transcriptionally regulated claudin-4, and this regulation was dependent on transactivation of the epidermal growth factor receptor, which induced localization of Rac1 at the cell membrane. LPA induced the translocation of Stat3 to the cell membrane and promoted the interaction between Rac1 and Stat3. Inhibition of Stat3 ablated LPA-mediated regulation of claudin-4. Conclusions This study identifies LPA5 as a regulator of the intestinal barrier. LPA5 promotes claudin-4 expression in IECs through activation of Rac1 and Stat3., Graphical abstract

The bromodomain and extraterminal (BET) family member BRD4 is pivotal in the pathogenesis of cardiac hypertrophy. BRD4 induces hypertrophic gene expression by binding to the acetylated chromatin, facilitating the phosphorylation of RNA polymerases II (Pol II) and leading to transcription elongation. The present study identified a novel post-translational modification of BRD4: poly(ADP-ribosyl)ation (PARylation), that was mediated by poly(ADP-ribose)polymerase-1 (PARP1) in cardiac hypertrophy. BRD4 silencing or BET inhibitors JQ1 and MS417 prevented cardiac hypertrophic responses induced by isoproterenol (ISO), whereas overexpression of BRD4 promoted cardiac hypertrophy, confirming the critical role of BRD4 in pathological cardiac hypertrophy. PARP1 was activated in ISO-induced cardiac hypertrophy and facilitated the development of cardiac hypertrophy. BRD4 was involved in the prohypertrophic effect of PARP1, as implied by the observations that BRD4 inhibition or silencing reversed PARP1-induced hypertrophic responses, and that BRD4 overexpression suppressed the anti-hypertrophic effect of PARP1 inhibitors. Interactions of BRD4 and PARP1 were observed by co-immunoprecipitation and immunofluorescence. PARylation of BRD4 induced by PARP1 was investigated by PARylation assays. In response to hypertrophic stimuli like ISO, PARylation level of BRD4 was elevated, along with enhanced interactions between BRD4 and PARP1. By investigating the PARylation of truncation mutants of BRD4, the C-terminal domain (CTD) was identified as the PARylation modification sites of BRD4. PARylation of BRD4 facilitated its binding to the transcription start sites (TSS) of hypertrophic genes, resulting in enhanced phosphorylation of RNA Pol II and transcription activation of hypertrophic genes. The present findings suggest that strategies targeting inhibition of PARP1-BRD4 might have therapeutic potential for pathological cardiac hypertrophy., Graphical abstract BRD4 is PARylated by PARP1 in ISO-induced cardiac hypertrophy, facilitating the recruitment of BRD4 at the TSS of hypertrophic genes, then enhancing the phosphorylation of RNA pol II, thereby promoting the transcription of hypertrophic genes.Image 1

Graphical abstract, Lung adenocarcinoma (LUAD) has a high mortality rate and is difficult to diagnose and treat in its early stage. Previous studies have demonstrated that small nucleolar RNAs (snoRNAs) play a critical role in tumor immune infiltration and the development of a variety of solid tumors. However, there have been no studies on the correlation between tumor-infiltrating immune-related snoRNAs (TIISRs) and LUAD. In this study, we filtered six immune-related snoRNAs based on the tissue specificity index (TSI) and expression profile of all snoRNAs between all LUAD cell lines from the Cancer Cell Line Encyclopedia and 21 types of immune cells from the Gene Expression Omnibus database. Further, we performed real-time quantitative polymerase chain reaction (RT-qPCR) to validate the expression status of these snoRNAs on peripheral blood mononuclear cells (PBMCs) and lung cancer cell lines. Next, we developed a TIISR signature based on the expression profiles of snoRNAs from 479 LUAD patients filtered by the random survival forest algorithm. We then analyzed the value of this TIISR signature (TIISR risk score) for assessing tumor immune infiltration, immune checkpoint inhibitor (ICI) treatment response, and the prognosis of LUAD between groups with high and low TIISR risk score. Further, we found that the TIISR risk score groups showed significant differences in biological characteristics and that the risk score could be used to assess the level of tumor immune cell infiltration, thereby predicting prognosis and responsiveness to immunotherapy in LUAD patients.

Background & Aims Aryl hydrocarbon receptor (AhR) is a liver-enriched xenobiotic receptor that plays important role in detoxification response in liver. This study aimed to investigate how AhR signaling may impact the pathogenesis of alcohol-related liver disease (ALD). Methods Chronic alcohol feeding animal studies were conducted with mouse models of hepatocyte-specific AhR knockout (AhRΔhep) and NAD(P)H quinone dehydrogenase 1 (NQO1) overexpression, and dietary supplementation of the AhR ligand indole-3-carbinol. Cell studies were conducted to define the causal role of AhR and NQO1 in regulation of redox balance and apoptosis. Results Chronic alcohol consumption induced AhR activation and nuclear enrichment of NQO1 in hepatocytes of both alcoholic hepatitis patients and ALD mice. AhR deficiency exacerbated alcohol-induced liver injury, along with reduction of NQO1. Consistently, in vitro studies demonstrated that NQO1 expression was dependent on AhR. However, alcohol-induced NQO1 nuclear translocation was triggered by decreased cellular oxidized nicotinamide adenine dinucleotide (NAD+)-to-NADH ratio, rather than by AhR activation. Furthermore, both in vitro and in vivo overexpression NQO1 prevented alcohol-induced hepatic NAD+ depletion, thereby enhancing activities of NAD+-dependent enzymes and reversing alcohol-induced liver injury. In addition, therapeutic targeting of AhR in the liver with dietary indole-3-carbinol supplementation efficiently reversed alcoholic liver injury by AhR-NQO1 signaling activation. Conclusions This study demonstrated that AhR activation is a protective response to counteract alcohol-induced hepatic NAD+ depletion through induction of NQO1, and targeting the hepatic AhR-NQO1 pathway may serve as a novel therapeutic approach for ALD., Graphical abstract

Photodynamic therapy (PDT) is a well-known cancer therapy that utilizes light to excite a photosensitizer and generate cytotoxic reactive oxygen species (ROS). The efficacy of PDT primarily depends on the photosensitizer and oxygen concentration in the tumor. Hypoxia in solid tumors promotes treatment resistance, resulting in poor PDT outcomes. Hence, there is a need to combat hypoxia while delivering sufficient photosensitizer to the tumor for ROS generation. Here we showcase our unique theranostic perfluorocarbon nanodroplets as a triple agent carrier for oxygen, photosensitizer, and indocyanine green that enables light triggered spatiotemporal delivery of oxygen to the tumors. We evaluated the characteristics of the nanodroplets and validated their ability to deliver oxygen via photoacoustic monitoring of blood oxygen saturation and subsequent PDT efficacy in a murine subcutaneous tumor model. The imaging results were validated with an oxygen sensing probe, which showed a 9.1 fold increase in oxygen content inside the tumor, following systemic administration of the nanodroplets. These results were also confirmed with immunofluorescence. In vivo studies showed that nanodroplets held higher rates of treatment efficacy than a clinically available benzoporphyrin derivative formulation. Histological analysis showed higher necrotic area within the tumor with perfluoropentane nanodroplets. Overall, the photoacoustic nanodroplets can significantly enhance image-guided PDT and has demonstrated substantial potential as a valid theranostic option for patient-specific photodynamic therapy-based treatments., Graphical Abstract ga1

Irinotecan (CTP-11) is one of the standard therapies for colorectal cancer (CRC). CTP-11 is enzymatically converted to the hydrophobic 7-ethyl-10-hydroxycamptothecin (SN38), a one hundred-fold more active metabolite. Conjugation of hydrophobic anticancer drugs to nanomaterials is a strategy to improve their solubility, efficacy, and selectivity. Carbon dots (CDs) have garnered interest for their small sizes (10 ​nm), low toxicity, high water solubility, and bright fluorescence. This paper describes the use of CDs to improve drug vehiculation, stability, and chemotherapeutic efficiency of SN38 through a direct intracellular uptake in CRC. The covalent conjugation of SN38 to CDs via a carbamate bond provides a CD-SN38 hybrid material for slow, sustained, and pH-responsive drug release. CD-SN38 successfully penetrates the CRC cells with a release in the nucleus affecting first the cell cycle and then the cytoskeleton. Moreover, CD-SN38 leads to a deregulation of the extracellular matrix (ECM), one of the major components of the cancer niche considered a possible target therapy for reducing the cancer progression. This work shows the combined therapeutic and imaging potential of CD-based hybrid materials for the treatment of CRC. Future efforts for targeted therapy of chronic diseases characterized by altered ECM deposition, such as chronic kidney disease and chronic allograft nephropathy in kidney transplant patients are envisaged.

Testicular torsion and detorsion (TTD) is a serious urological condition affecting young males that is underlined by an ischemia reperfusion injury (tIRI) to the testis as the pathophysiological mechanism. During tIRI, uncontrolled production of oxygen reactive species (ROS) causes DNA damage leading to germ cell apoptosis (GCA). The aim of the study is to explore whether inhibition of NADPH oxidase (NOX), a major source of intracellular ROS, will prevent tIRI-induced GCA and its association with endoplasmic reticulum (ER) stress. Sprague-Dawley rats (n=36) were divided into three groups: sham, tIRI only and tIRI treated with apocynin (a NOX inhibitor). Rats undergoing tIRI endured an ischemic injury for 1 hour followed by 4 hours of reperfusion. Spermatogenic damage was evaluated histologically, while cellular damages were assessed using real time PCR, immunofluorescence staining, Western blot and biochemical assays. Disrupted spermatogenesis was associated with increased lipid and protein peroxidation and decreased antioxidant activity of the enzyme superoxide dismutase (SOD) as a result of tIRI. In addition, increased DNA double strand breaks and formation of 8-OHdG adducts associated with increased phosphorylation of the DNA damage response (DDR) protein H2AX. The ASK1/JNK apoptosis signaling pathway was also activated in response to tIRI. Finally, increased immuno-expression of the unfolded protein response (UPR) downstream targets: GRP78, eIF2-α1, CHOP and caspase 12 supported the presence of ER stress. Inhibition of NOX by apocynin protected against tIRI-induced GCA and ER stress. In conclusion, NOX inhibition minimized tIRI-induced intracellular oxidative damages leading to GCA and ER stress.

Background & Aims Although the healthy pancreas consists mostly of epithelial cells, pancreatic cancer and the precursor lesions known as pancreatic intraepithelial neoplasia, are characterized by an extensive accumulation of fibroinflammatory stroma that includes a substantial and heterogeneous fibroblast population. The cellular origin of fibroblasts within the stroma has not been determined. Here, we show that the Gli1 and Hoxb6 markers label distinct fibroblast populations in the healthy mouse pancreas. We then set out to determine whether these distinct fibroblast populations expanded during carcinogenesis. Methods We developed genetically engineered models using a dual-recombinase approach that allowed us to induce pancreatic cancer formation through codon-optimized Flp recombinase-driven epithelial recombination of Kirsten rat sarcoma viral oncogene homolog while labeling Gli1+ or Hoxb6+ fibroblasts in an inducible manner. By using these models, we lineage-traced these 2 fibroblast populations during the process of carcinogenesis. Results Although in the healthy pancreas Gli1+ fibroblasts and Hoxb6+ fibroblasts are present in similar numbers, they contribute differently to the stroma in carcinogenesis. Namely, Gli1+ fibroblasts expand dramatically, whereas Hoxb6+ cells do not. Conclusions Fibroblasts present in the healthy pancreas expand during carcinogenesis, but with a different prevalence for different subtypes. Here, we compared Gli1+ and Hoxb6+ fibroblasts and found only Gli1+ expanded to contribute to the stroma during pancreatic carcinogenesis., Graphical abstract

Visual Abstract, Highlights • Quantitative multiplexed immune-phenotyping of cardiac allograft biopsies provides novel diagnostic and prognostic information about allograft health. • Reduced proportions of cells expressing PD-L1 and FoxP3 are associated with clinical evidence of serious allograft injury, even when conventional histologic analysis provides falsely reassuring histologic rejection grades. • The proportions of PD-L1- and FoxP3-expressing cells are dynamic within cardiac allografts, and reduced levels can precede future rejection., Summary Recognizing that guideline-directed histologic grading of endomyocardial biopsy tissue samples for rejection surveillance has limited diagnostic accuracy, quantitative, in situ characterization was performed of several important immune cell types in a retrospective cohort of clinical endomyocardial tissue samples. Differences between cases were identified and were grouped by histologic grade versus clinical rejection trajectory, with significantly increased programmed death ligand 1+, forkhead box P3+, and cluster of differentiation 68+ cells suppressed in clinically evident rejections, especially cases with marked clinical-histologic discordance. Programmed death ligand 1+, forkhead box P3+, and cluster of differentiation 68+ cell proportions are also significantly higher in “never-rejection” when compared with “future-rejection.” These findings suggest that in situ immune modulators regulate the severity of cardiac allograft rejection.

Background & Aims The intestinal epithelium must be resilient to physiochemical stress to uphold the physiological barrier separating the systemic compartment from the microbial and antigenic components of the gut lumen. Identifying proteins that mediate protection and enhancing their expression is therefore a clear approach to promote intestinal health. We previously reported that oral ingestion of the probiotic Lactobacillus rhamnosus GG not only induced the expression of several recognized cytoprotective factors in the murine colon, but also many genes with no previously described function, including the gene encoding proline-rich acidic protein 1 (PRAP1). PRAP1 is a highly expressed protein in the epithelium of the gastrointestinal tract and we sought to define its function in this tissue. Methods Purified preparations of recombinant PRAP1 were analyzed biochemically and PRAP1 antisera were used to visualize localization in tissues. Prap1-/- mice were characterized at baseline and challenged with total body irradiation, then enteroids were generated to recapitulate the irradiation challenge ex vivo. Results PRAP1 is a 17-kilodalton intrinsically disordered protein with no recognizable sequence homology. PRAP1 expression levels were high in the epithelia of the small intestine. Although Prap1-/- mice presented only mild phenotypes at baseline, they were highly susceptible to intestinal injury upon challenge. After irradiation, the Prap1-/- mice showed accelerated death with a significant increase in apoptosis and p21 expression in the small intestinal epithelium. Conclusions PRAP1 is an intrinsically disordered protein highly expressed by the gastrointestinal epithelium and functions at exposed surfaces to protect the barrier from oxidative insult., Graphical abstract

Pancreatic invasive ductal adenocarcinoma (PDAC) is a representative intractable malignancy under the current cancer therapies, and is considered a scirrhous carcinoma because it develops dense stroma. Both PODXL1, a member of CD34 family molecules, and C5aR, a critical cell motility inducer, have gained recent attention, as their expression was reported to correlate with poor prognosis for patients with diverse origins including PDAC; however, previous studies reported independently on their respective biological significance. Here we demonstrate that PODXL1 is essential for metastasis of PDAC cells through its specific interaction with C5aR. In vitro assay demonstrated that PODXL1 bound to C5aR, which stabilized C5aR protein and recruited it to cancer cell plasma membranes to receive C5a, an inflammatory chemoattractant factor. PODXL1 knockout in PDAC cells abrogated their metastatic property in vivo, emulating the liver metastatic mouse model treated with anti-C5a neutralizing antibody. In molecular studies, PODXL1 triggered EMT on PDAC cells in response to stimulation by C5a, corroborating PODXL1 involvement in PDAC cellular invasive properties via specific interaction with the C5aR/C5a axis. Confirming the molecular assays, histological examination showed coexpression of PODXL1 and C5aR at the invasive front of primary cancer nests as well as in liver metastatic foci of PDAC both in the mouse metastasis model and patient tissues. Hence, the novel direct interaction between PODXL1 and the C5aR/C5a axis may provide a better integrated understanding of PDAC biological characteristics including its tumor microenvironment factors.

Background: Hepatic encephalopathy (HE) is a severe neuropsychiatric syndrome caused by various types of liver failure resulting in hyperammonemia-induced dysfunction of astrocytes. It is unclear whether autophagy, an important pro-survival pathway, is altered in the brains of ammonia-intoxicated animals as well as in HE patients. Methods: Using primary rat astrocytes, a co-culture model of primary mouse astrocytes and neurons, an in vivo rat HE model, and post mortem brain samples of liver cirrhosis patients with HE we analyzed whether and how hyperammonemia modulates autophagy. Findings: We show that autophagic flux is efficiently inhibited after administration of ammonia in astrocytes. This occurs in a fast, reversible, time-, dose-, and ROS-dependent manner and is mediated by ammonia-induced changes in intralysosomal pH. Autophagic flux is also strongly inhibited in the cerebral cortex of rats after acute ammonium intoxication corroborating our results using an in vivo rat HE model. Transglutaminase 2 (TGM2), a factor promoting autophagy, is upregulated in astrocytes of in vitro- and in vivo-HE models as well as in post mortem brain samples of liver cirrhosis patients with HE, but not in patients without HE. LC3, a commonly used autophagy marker, is significantly increased in the brain of HE patients. Ammonia also modulated autophagy moderately in neuronal cells. We show that taurine, known to ameliorate several parameters caused by hyperammonemia in patients suffering from liver failure, is highly potent in reducing ammonia-induced impairment of autophagic flux. This protective effect of taurine is apparently not linked to inhibition of mTOR signaling but rather to reducing ammonia-induced ROS formation. Interpretation: Our data support a model in which autophagy aims to counteract ammonia-induced toxicity, yet, as acidification of lysosomes is impaired, possible protective effects thereof, are hampered. We propose that modulating autophagy in astrocytes and/or neurons, e.g. by taurine, represents a novel strategy to treat liver diseases associated with HE. Funding: Supported by the DFG, CRC974 “Communication and Systems Relevance in Liver Injury and Regeneration“, Düsseldorf (Project number 190586431) Projects A05 (DH), B04 (BG), B05 (NK), and B09 (ASR). Keywords: Hepatic encephalopathy, Liver disease, Autophagy, Lysosome, Taurine

The G-quadruplex (G4) resolvase RNA helicase associated with AU-rich element (RHAU) possesses the ability to unwind G4 structures in both DNA and RNA molecules. Previously, we revealed that RHAU plays a critical role in embryonic heart development and postnatal heart function through modulating mRNA translation and stability. However, whether RHAU functions to resolve DNA G4 in the regulation of cardiac physiology is still elusive. Here, we identified a phenotype of noncompaction cardiomyopathy in cardiomyocyte-specific Rhau deletion mice, including such symptoms as spongiform cardiomyopathy, heart dilation, and death at young ages. We also observed reduced cardiomyocyte proliferation and advanced sarcomere maturation in Rhau mutant mice. Further studies demonstrated that RHAU regulates the expression levels of several genes associated with ventricular trabeculation and compaction, including the Nkx2-5 and Hey2 that encode cardiac transcription factors of NKX2-5 and Hey2, and the myosin heavy chain 7 (Myh7) whose protein product is MYH7. While RHAU modulates Nkx2-5 mRNA and Hey2 mRNA at the post-transcriptional level, we uncovered that RHAU facilitates the transcription of Myh7 through unwinding of the G4 structures in its promoter. These findings demonstrated that RHAU regulates ventricular chamber development through both transcriptional and post-transcriptional mechanisms. These results contribute to a knowledge base that will help to understand the pathogenesis of diseases such as noncompaction cardiomyopathy.

Previous studies suggested that cancer cells resemble neural stem/progenitor cells in regulatory network, tumorigenicity, and differentiation potential, and that neural stemness might represent the ground or basal state of differentiation and tumorigenicity. The neural ground state is reflected in the upregulation and enrichment of basic cell machineries and developmental programs, such as cell cycle, ribosomes, proteasomes, and epigenetic factors, in cancers and in embryonic neural or neural stem cells. However, how these machineries are concertedly regulated is unclear. Here, we show that loss of neural stemness in cancer or neural stem cells via muscle-like differentiation or neuronal differentiation, respectively, caused downregulation of ribosome and proteasome components and major epigenetic factors, including PRMT1, EZH2, and LSD1. Furthermore, inhibition of PRMT1, an oncoprotein that is enriched in neural cells during embryogenesis, caused neuronal-like differentiation, downregulation of a similar set of proteins downregulated by differentiation, and alteration of subcellular distribution of ribosome and proteasome components. By contrast, PRMT1 overexpression led to an upregulation of these proteins. PRMT1 interacted with these components and protected them from degradation via recruitment of the deubiquitinase USP7, also known to promote cancer and enriched in embryonic neural cells, thereby maintaining a high level of epigenetic factors that maintain neural stemness, such as EZH2 and LSD1. Taken together, our data indicate that PRMT1 inhibition resulted in repression of cell tumorigenicity. We conclude that PRMT1 coordinates ribosome and proteasome activity to match the needs for high production and homeostasis of proteins that maintain stemness in cancer and neural stem cells.

Background & Aims Liver lobules are typically subdivided into 3 metabolic zones: zones 1, 2, and 3. However, the contribution of zonal differences in hepatocytes to liver regeneration, as well as to carcinogenic susceptibility, remains unclear. Methods We developed a new method for sustained genetic labelling of zone 3 hepatocytes and performed fate tracing to monitor these cells in multiple mouse liver tumour models. Results We first examined changes in the zonal distribution of the Wnt target gene Axin2 over time using Axin2-CreERT2;Rosa26-Lox-Stop-Lox-tdTomato mice (Axin2;tdTomato). We found that following tamoxifen administration at 3 weeks of age, approximately one-third of total hepatocytes that correspond to zone 3 were labelled in Axin2;tdTomato mice; the tdTomato+ cell distribution closely matched that of the zone 3 marker CYP2E1. Cell fate analysis revealed that zone 3 hepatocytes maintained their own lineage but rarely proliferated beyond their liver zonation during homoeostasis; this indicated that our protocol enabled persistent genetic labelling of zone 3 hepatocytes. Using this system, we found that zone 3 hepatocytes generally had high neoplastic potential, which was promoted by constitutive activation of Wnt/β-catenin signalling in the pericentral area. However, the frequency of zone 3 hepatocyte-derived tumours varied depending on the regeneration pattern of the liver parenchyma in response to liver injury. Notably, Axin2-expressing hepatocytes undergoing chronic liver injury significantly contributed to liver regeneration and possessed high neoplastic potential. Additionally, we revealed that the metabolic phenotypes of liver tumours were acquired during tumorigenesis, irrespective of their spatial origin. Conclusions Hepatocytes receiving Wnt/β-catenin signalling from their microenvironment have high neoplastic potential, and Wnt/β-catenin signalling is a potential drug target for the prevention of hepatocellular carcinoma. Lay summary Lineage tracing revealed that zone 3 hepatocytes residing in the pericentral niche have high neoplastic potential. Under chronic liver injury, hepatocytes receiving Wnt/β-catenin signalling broadly exist across all hepatic zones and significantly contribute to liver tumorigenesis as well as liver regeneration. Wnt/β-catenin signalling is a potential drug target for the prevention of hepatocellular carcinoma., Graphical abstract, Highlights • We developed a new method for sustained genetic labelling of Zone 3 hepatocytes. • Lineage tracing revealed that Zone 3 hepatocytes generally have high neoplastic potential. • The frequency of Zone 3 hepatocyte-derived tumours varied depending on the regeneration pattern of liver parenchyma. • Under chronic liver injury, hepatocytes receiving Wnt/β-catenin signalling significantly contributed to tumorigenesis. • Wnt/β-catenin signalling is a potential drug target for the prevention of HCC.

Lamin A/C provides a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. Lamin A/C dysfunction is associated with cancer, aging, and degenerative diseases. The mechanisms whereby lamin A/C regulates genome stability remain poorly understood. We demonstrate a crucial role for lamin A/C in DNA replication. Lamin A/C binds to nascent DNA, especially during replication stress (RS), ensuring the recruitment of replication fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, function in the stabilization, remodeling, and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamin A/C depletion elicits replication fork instability (RFI) characterized by MRE11 nuclease-mediated degradation of nascent DNA, RS-induced DNA damage, and sensitivity to replication inhibitors. Importantly, unlike homologous recombination-deficient cells, RFI in lamin A/C-depleted cells is not linked to replication fork reversal. Thus, the point of entry of nucleases is not the reversed fork but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamin A/C-depleted cells is rescued by exogenous overexpression of RPA or RAD51. These data unveil involvement of structural nuclear proteins in the protection of ssDNA from nucleases during RS by promoting recruitment of RPA and RAD51 to stalled forks. Supporting this model, we show physical interaction between RPA and lamin A/C. We suggest that RS is a major source of genomic instability in laminopathies and lamin A/C-deficient tumors.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Radiotherapy is an effective option for the treatment of TNBC; however, acquired radioresistance is a major challenge to the modality. In this study, we show that the integrated stress response (ISR) is the most activated signaling pathway in radioresistant TNBC cells. The constitutive phosphorylation of eIF2α in radioresistant TNBC cells promotes the activation of ATF4 and elicits the transcription of genes implicated in glutathione biosynthesis, including GCLC, SLC7A11, and CTH, which increases the intracellular level of reduced glutathione (GSH) and the scavenging of reactive oxygen species (ROS) after irradiation (IR), leading to a radioresistant phenotype. The cascade is significantly up-regulated in human TNBC tissues and is associated with unfavorable survival in patients. Dephosphorylation of eIF2α increases IR-induced ROS accumulation in radioresistant TNBC cells by disrupting ATF4-mediated GSH biosynthesis and sensitizes them to IR in vitro and in vivo. These findings reveal ISR as a vital mechanism underlying TNBC radioresistance and propose the eIF2α/ATF4 axis as a novel therapeutic target for TNBC treatment., Graphical abstract Image 1, Highlights • The eIF2α/ATF4 axis is constitutively activated in radioresistant TNBC. • Phosphorylated eIF2α increases the expression of ATF4 and GCLC at the translational level in TNBC. • The eIF2α/ATF4 axis activation causes radioresistance in TNBC by promoting GSH biosynthesis and ROS scavenging. • ATF4 promotes GSH biosynthesis in radioresistant TNBC by triggering the expression of GCLC, CTH, and SLC7A11. • Inhibition of the eIF2α/ATF4 axis can improve the sensitivity of TNBC to radiotherapy in vitro and in vivo.

Summary Background & Aims Cholangiocyte senescence is important in the pathogenesis of primary sclerosing cholangitis (PSC). We found that CDKN2A (p16), a cyclin-dependent kinase inhibitor and mediator of senescence, was increased in cholangiocytes of patients with PSC and from a PSC mouse model (multidrug resistance 2; Mdr2-/-). Given that recent data suggest that a reduction of senescent cells is beneficial in different diseases, we hypothesised that inhibition of cholangiocyte senescence would ameliorate disease in Mdr2-/- mice. Methods We used 2 novel genetic murine models to reduce cholangiocyte senescence: (i) p16Ink4a apoptosis through targeted activation of caspase (INK-ATTAC)xMdr2-/-, in which the dimerizing molecule AP20187 promotes selective apoptotic removal of p16-expressing cells; and (ii) mice deficient in both p16 and Mdr2. Mdr2-/- mice were also treated with fisetin, a flavonoid molecule that selectively kills senescent cells. p16, p21, and inflammatory markers (tumour necrosis factor [TNF]-α, IL-1β, and monocyte chemoattractant protein-1 [MCP-1]) were measured by PCR, and hepatic fibrosis via a hydroxyproline assay and Sirius red staining. Results AP20187 treatment reduced p16 and p21 expression by ~35% and ~70% (p >0.05), respectively. Expression of inflammatory markers (TNF-α, IL-1β, and MCP-1) decreased (by 60%, 40%, and 60%, respectively), and fibrosis was reduced by ~60% (p >0.05). Similarly, p16-/-xMdr2-/- mice exhibited reduced p21 expression (70%), decreased expression of TNF-α, IL-1β (60%), and MCP-1 (65%) and reduced fibrosis (~50%) (p >0.05) compared with Mdr2-/- mice. Fisetin treatment reduced expression of p16 and p21 (80% and 90%, respectively), TNF-α (50%), IL-1β (50%), MCP-1 (70%), and fibrosis (60%) (p >0.05). Conclusions Our data support a pathophysiological role of cholangiocyte senescence in the progression of PSC, and that targeted removal of senescent cholangiocytes is a plausible therapeutic approach. Lay summary Primary sclerosing cholangitis is a fibroinflammatory, incurable biliary disease. We previously reported that biliary epithelial cell senescence (cell-cycle arrest and hypersecretion of profibrotic molecules) is an important phenotype in primary sclerosing cholangitis. Herein, we demonstrate that reducing the number of senescent cholangiocytes leads to a reduction in the expression of inflammatory, fibrotic, and senescence markers associated with the disease., Graphical abstract, Highlights • p16 and p21 are major mediators of cellular senescence and are highly expressed in cholangiocytes in a Mdr2-/- murine model of PSC. • The senescence-associated secretory phenotype markers are all increased in cholangiocytes of Mdr2-/- mice. • Genetic and pharmacological elimination of senescent cholangiocytes reduces peribiliary inflammation and fibrosis in Mdr2-/- mice. • Preclinical work suggests that fisetin, a naturally occurring and safe senolytic flavonoid, has the potential to be tested in patients with PSC.

Human cell division is a highly regulated process that relies on the accurate capture and movement of chromosomes to the metaphase plate. Errors in the fidelity of chromosome congression and alignment can lead to improper chromosome segregation, which is correlated with aneuploidy and tumorigenesis. These processes are known to be regulated by extracellular signal-regulated kinase 2 (ERK2) in other species, but the role of ERK2 in mitosis in mammals remains unclear. Here, we have identified the dual-specificity phosphatase 7 (DUSP7), known to display selectivity for ERK2, as important in regulating chromosome alignment. During mitosis, DUSP7 bound to ERK2 and regulated the abundance of active phospho-ERK2 through its phosphatase activity. Overexpression of DUSP7, but not catalytically inactive mutants, led to a decrease in the levels of phospho-ERK2 and mitotic chromosome misalignment, while knockdown of DUSP7 also led to defective chromosome congression that resulted in a prolonged mitosis. Consistently, knockdown or chemical inhibition of ERK2 or chemical inhibition of the MEK kinase that phosphorylates ERK2 led to chromosome alignment defects. Our results support a model wherein MEK-mediated phosphorylation and DUSP7-mediated dephosphorylation regulate the levels of active phospho-ERK2 to promote proper cell division.

Background Abnormal construction of the extracellular matrix (ECM) is intimately linked with carcinogenesis and the development of solid tumours, especially hepatocellular carcinoma (HCC). As the major component of the ECM, collagen plays a pivotal role in carcinogenesis. P4HA2, the essential enzyme during collagen formation, becomes an important target in HCC treatment. Here, we tried to decipher whether aspirin (ASA), a classic anti-inflammatory drug, could improve the prognosis of HCC through targeting P4HA2. Methods Western blotting, qRT-PCR assay, immunofluorescence staining, luciferase reporter gene assay, and ChIP assay were applied to demonstrate the molecular mechanism of the regulation of P4HA2 expression by aspirin. A mouse xenograft model, cell viability assay, colony formation assay, and immunohistochemistry analysis were used to evaluate the anti-fibrosis effect of aspirin through targeting the NF-κB/P4HA2 axis and LMCD1-AS1/let-7g/P4HA2 axis in vitro and in vivo. The TCGA database was used to evaluate the correlation among P4HA2, let-7g, LMCD1-AS1 and overall survival of HCC patients. Findings In xenograft mice, aspirin was capable of targeting P4HA2 to decrease collagen deposition, resulting in the inhibition of liver tumour growth. TCGA database analysis revealed the close association between a higher P4HA2 concentration in HCC patients and shorter overall survival or a higher cancer stage and the pathological grade. Mechanistically, NF-κB can bind to the promoter of P4HA2 to activate its transcription. Moreover, lncRNA LMCD1-AS1 functions as a molecular sponge of let-7g to post-transcriptionally induce the target gene of let-7g, namely, P4HA2. Interpretation Our findings disclose the novel role and regulatory mechanism of aspirin in the suppression of HCC by disrupting abnormal collagen deposition. Funds 973 Program, National Natural Scientific Foundation of China, Fundamental Research Funds for the Central Universities, Project of Prevention and Control of Key Chronic Non-Infectious Diseases.

Background DNA damage repair (DDR) alterations are important events in cancer initiation, progression, and therapeutic resistance. However, the involvement of DDR alterations in glioma malignancy needs further investigation. This study aims to characterize the clinical and molecular features of gliomas with DDR alterations and elucidate the biological process of DDR alterations that regulate the cross talk between gliomas and the tumor microenvironment. Methods Integrated transcriptomic and genomic analyses were undertaken to conduct a comprehensive investigation of the role of DDR alterations in glioma. The prognostic DDR-related cytokines were identified from multiple datasets. In vivo and in vitro experiments validated the role of p53, the key molecule of DDR, regulating M2 polarization of microglia in glioma. Findings DDR alterations are associated with clinical and molecular characteristics of glioma. Gliomas with DDR alterations exhibit distinct immune phenotypes, and immune cell types and cytokine processes. DDR-related cytokines have an unfavorable prognostic implication for GBM patients and are synergistic with DDR alterations. Overexpression of MDK mediated by p53, the key transcriptional factor in DDR pathways, remodels the GBM immunosuppressive microenvironment by promoting M2 polarization of microglia, suggesting a potential role of DDR in regulating the glioma microenvironment. Interpretation Our work suggests that DDR alterations significantly contribute to remodeling the glioma microenvironment via regulating the immune response and cytokine pathways. Fund This study was supported by: 1. The National Key Research and Development Plan (No. 2016YFC0902500); 2. National Natural Science Foundation of China (No. 81702972, No. 81874204, No. 81572701, No. 81772666); 3. China Postdoctoral Science Foundation (2018M640305); 4. Special Fund Project of Translational Medicine in the Chinese-Russian Medical Research Center (No. CR201812); 5. The Research Project of the Chinese Society of Neuro-oncology, CACA (CSNO-2016-MSD12); 6. The Research Project of the Health and Family Planning Commission of Heilongjiang Province (2017–201); and 7. Harbin Medical University Innovation Fund (2017LCZX37, 2017RWZX03)., Highlights • Gliomas with DNA damage repair alterations had distinct genomic variation spectrum. • DDR alterations exhibit distinct immune phenotypes, cytokine processes and immune cell types in glioma. • DDR-related cytokines in GME have an unfavorable prognostic implication for GBM patients. • P53-mediated midkine expression derived from glioma cells promotes M2 polarization of microglia.

Selenium-binding protein 1 (SELENBP1) has recently been reported to catalyse the oxidation of methanethiol, an organosulfur compound produced by gut microbiota. Two of the reaction products of methanethiol oxidation, hydrogen peroxide and hydrogen sulphide, serve as signalling molecules for cell differentiation. Indeed, colonocyte differentiation has been found to be associated with SELENBP1 induction. Here, we show that SELENBP1 is induced when 3T3-L1 preadipocytes undergo terminal differentiation and maturation to adipocytes. SELENBP1 induction succeeded the up-regulation of known marker proteins of white adipocytes and the intracellular accumulation of lipids. Immunofluorescence microscopy revealed predominant cytoplasmic localisation of SELENBP1 in 3T3-L1 adipocytes, as demonstrated by co-staining with the key lipogenic enzyme, acetyl-CoA-carboxylase (ACC), located in cytosol. In differentiating 3T3-L1 cells, the mTOR inhibitor rapamycin and the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-α) likewise suppressed SELENBP1 induction, adipocyte differentiation and lipid accumulation. However, lipid accumulation per se is not linked to SELENBP1 induction, as hepatic SELENBP1 was down-regulated in high fructose-fed mice despite increased lipogenesis in the liver and development of non-alcoholic fatty liver disease (NAFLD). In conclusion, SELENBP1 is a marker of cell differentiation/maturation rather than being linked to lipogenesis/lipid accumulation., Graphical abstract fx1, Highlights • SELENBP1 is a novel marker of mature adipocytes. • SELENBP1 was induced during terminal adipocyte differentiation. • Anti-adipogenic factors suppressed SELENBP1 induction. • Despite increased lipogenesis, hepatic SELENBP1 was down-regulated in NAFLD mice.

Background Epidermal growth factor receptor (EGFR) signalling is critical in epithelial cancer development. Human rhomboid family-1 (RHBDF1) facilitates the secretion of TGFα, an EGFR ligand, in breast cancer; however, the underlying mechanism remains unclear. We evaluated the role for RHBDF1 in clathrin-coated vesicle (CCV)-dependent pro-TGFα membrane trafficking in breast cancer cells upon stimulation by G-protein coupled receptor (GPCR) agonists. Methods RHBDF1 was silenced in various breast cancer cells using shRNA. TGFα levels, subcellular localization, and secretion were evaluated using ELISA, immunofluorescent staining, and coimmunoprecipitation. Phosphorylation and expression of relevant proteins were measured by western blotting. RHBDF1-dependent cell viability and invasion were measured. Findings RHBDF1 mediates GPCR agonist-induced EGFR phosphorylation by promoting TGFα secretion in various types of breast cancer cells. RHBDF1 not only mediates ADAM17-dependent shedding of TGFα, but is essential in membrane trafficking of pro-TGFα. RHBDF1 silencing results in blocking of clathrin uncoating from CCV, a crucial step for the plasma membrane release of pro-TGFα. Interaction of RHBDF1 with auxilin-2, a CCV protein, determines the recruitment of HSC70 to CCV to facilitate clathrin uncoating. RHBDF1 function is required for the proliferation and mobility of breast cancer cells upon stimulation by Sphingosine 1 Phosphate (S1P), a GPCR agonist. We demonstrate a significant correlation between RHBDF1 overexpression and EGFR activation in breast cancer tissues. Interpretation RHBDF1 is an indispensable component of the protein trafficking machinery involved in GPCR-mediated EGFR transactivation, and is an attractive therapeutic target for cancer. Fund National Natural Science Foundation of China (81,672,740 to ZSZ, 81,272,356 and 81,330,029 to LYL).

Highlights • Angiogenesis is critical to the development and survival of solid tumors. • Vanucizumab inhibits both vascular endothelial growth factor and angiopoietin-2. • Vanucizumab exhibits anti-tumor, anti-angiogenic and anti-metastatic effects. • It markedly reduced blood-vessel markers in tumor/skin samples in cancer patients. • Skin biopsies are a valuable surrogate for studying angiogenesis-related mechanisms., Vanucizumab is a novel bispecific antibody inhibiting vascular endothelial growth factor (VEGF-A) and angiopoietin-2 (Ang-2) that demonstrated safety and anti-tumor activity in part I of a phase I study of 42 patients with advanced solid tumors. Part II evaluated the pharmacodynamic effects of vanucizumab 30 or 15 mg/kg every 2 weeks in 32 patients. Serial plasma samples, paired tumor, and skin-wound-healing biopsies were taken over 29 days to evaluate angiogenic markers. Vanucizumab was associated with marked post-infusion reductions in circulating unbound VEGF-A and Ang-2. By day 29, tumor samples revealed mean reductions in density of microvessels (−32.2%), proliferating vessels (−47.9%) and Ang-2 positive vessels (−62.5%). Skin biopsies showed a mean reduction in density of microvessels (−49.0%) and proliferating vessels (−25.7%). Gene expression profiling of tumor samples implied recruitment and potential activation of lymphocytes. Biopsies were safely conducted. Vanucizumab demonstrated a consistent biological effect on vascular-related biomarkers, confirming proof of concept. Skin-wound-healing biopsies were a valuable surrogate for studying angiogenesis-related mechanisms.

Mantle cell lymphoma (MCL) is an especially aggressive and highly heterogeneous mature B-cell lymphoma. Heat shock protein 90 (HSP90) is considered an attractive therapeutic target in a variety of cancers, including MCL, but no HSP90 inhibitors have succeeded in the clinical trials to date. Exploring fine mechanisms of HSP90 inhibition in cancer cells may shed light on novel therapeutic strategies. Here, we found that HSP90 knockdown and continuous inhibition with ganetespib inhibited growth of MCL cells in vitro and in vivo. To our surprise, transient exposure over 12 h was almost as efficient as continuous exposure, and treatment with ganetespib for 12 h efficiently inhibited growth and induced G1 cell cycle arrest and apoptosis of MCL cells. Transcriptome analysis complemented by functional studies was performed to define critical MCL signaling pathways that are exceptionally sensitive to HSP90 inhibition and vital to cell fate. Six genes (cell division cycle 6, cell division cycle 45, minichromosome maintenance 4, minichromosome maintenance 7, RecQ-mediated genome instability 2, and DNA primase polypeptide 1) involved in DNA replication and repair were identified as consistently downregulated in three MCL cell lines after transient ganetespib treatment. E2F1, an important transcription factor essential for cell cycle progression, was identified as a ganetespib target mediating transcriptional downregulation of these six genes, and its stability was also demonstrated to be maintained by HSP90. This study identifies E2F1 as a novel client protein of HSP90 that is very sensitive and worthy of targeting and also finds that HSP90 inhibitors may be useful in combination therapies for MCL.

Background Autophagosome, the central organelle in autophagy process, can assemble via canonical pathway mediated by LC3-II, the lipidated form of autophagy-related protein LC3/ATG8, or noncanonical pathway mediated by the small GTPase Rab9. Canonical autophagy is essential for exocrine pancreas homeostasis, and its disordering initiates and drives pancreatitis. The involvement of noncanonical autophagy has not been explored. We examine the role of Rab9 in pancreatic autophagy and pancreatitis severity. Methods We measured the effect of Rab9 on parameters of autophagy and pancreatitis responses using transgenic mice overexpressing Rab9 (Rab9TG) and adenoviral transduction of acinar cells. Effect of canonical autophagy on Rab9 was assessed in ATG5-deficient acinar cells. Results Pancreatic levels of Rab9 and its membrane-bound (active) form decreased in rodent pancreatitis models and in human disease. Rab9 overexpression stimulated noncanonical and inhibited canonical/LC3-mediated autophagosome formation in acinar cells through up-regulation of ATG4B, the cysteine protease that delipidates LC3-II. Conversely, ATG5 deficiency caused Rab9 increase in acinar cells. Inhibition of canonical autophagy in Rab9TG pancreas was associated with accumulation of Rab9-positive vacuoles containing markers of mitochondria, protein aggregates, and trans-Golgi. The shift to the noncanonical pathway caused pancreatitis-like damage in acinar cells and aggravated experimental pancreatitis. Conclusions The results show that Rab9 regulates pancreatic autophagy and indicate a mutually antagonistic relationship between the canonical/LC3-mediated and noncanonical/Rab9-mediated autophagy pathways in pancreatitis. Noncanonical autophagy fails to substitute for its canonical counterpart in protecting against pancreatitis. Thus, Rab9 decrease in experimental and human pancreatitis is a protective response to sustain canonical autophagy and alleviate disease severity., Graphical abstract

Before we have reported lamprey PHB2 could enhance the cellular oxidative-stressed tolerance, here the aim was to explore its mechanisms. We used flow cytometry analysis to identify a Lampetra morii homologue of PHB2 (Lm-PHB2) that could significantly decrease the levels of ROS generation in HEK293T cells. According to confocal microscopy observations, Lm-PHB2 contributed to maintain the mitochondrial morphology of HEK293T cells, and then both cellular nuclear location and translocation from the nucleus to mitochondria of Lm-PHB2 were also examined in HEK293T cells under oxidative stress. We also examined the expressions and locations of various Lm-PHB2 deletion mutants and the amino acid mutant by confocal microscopy and the results showed that the translocation of Lm-PHB2 into mitochondria was dependent on the Lm-PHB21-50aa region and the 17th, 48th and 57th three arginines (R) of N-terminal were very critical. In addition, the analyses of QRT-PCR and Western blot demonstrated that Lm-PHB2 increased the expression levels of OPA1 and HAX1 in HEK293T cells treated with H2O2. The analyses of immunofluorescence and immunoprecipitation showed that Lm-PHB2 could interact with OPA1 and HAX1, respectively. The above mentioned results indicate that Lm-PHB2 could assist OPA1 and HAX1 to maintain mitochondrial morphology and decrease ROS levels by the translocation from the nucleus to mitochondria under oxidative stress., Highlights • Lamprey, as one of the most primitive jawless vertebrate, the PHB2 protein of lamprey can translocalize from the nucleus to mitochondria in HEK293T cell lines under oxidative stress. • Under oxidative stress lamprey PHB2 protein maintains mitochondrial stability by interacting with OPA1 and HAX1 in mitochondria.

Azurin protein of Pseudomonas aeruginosa is an anti-tumor agent against breast cancer and mammaglobin-A (MAM-A) protein is a specific antigen on the surface of MCF-7 for induction of cellular immune. The purpose of the present study was to investigate the effects of simultaneous expression of azurin and human MAM-A genes on the mRNA expression level of apoptosis-related and cell cycle genes in MCF-7 breast cancer cell line. The recombinant or empty plasmids were separately transferred into MCF-7 cells using Lipofectamine reagent. Flow cytometry was done to detect cell death and apoptosis. The expression of azurin and MAM-A genes were evaluated by IF assay, RT-PCR and western blot methods. Finally, apoptosis-related and cell cycle genes expression was examined in transformed and non-transformed MCF-7 cells by qPCR method. The successful expression of azurin and MAM-A genes in the MCF-7 cell were confirmed by RT-PCR, IF and western blotting. The apoptosis assay was showed a statistically significant (p 0.05). Co-expression of azurin and MAM-A genes could induce apoptosis and necrosis in human MCF-7 breast cancer cells by up-regulation of BAK, FAS, and BAX genes. In future researches, it must be better the immune stimulation of pBudCE4.1-azurin-MAM-A recombinant vector in animal models and therapeutic approaches will be evaluated.

Hepatocellular carcinoma (HCC) is one of the most common cancers and it usually develops from a background of liver fibrosis or inflammation. The crosstalk between tumor cells and stromal cells plays an important and stimulating role during tumor progression. Previously we found in a krasV12–induced zebrafish HCC model that oncogenic hepatocytes activate hepatic stellate cells (HSCs) by up-regulation of serotonin and activate neutrophils and macrophages by up-regulation of cortisol. In the present study, we found a novel signaling transduction mechanism between oncogenic hepatocytes and HSCs. After krasV12 induction, fibrinogen was up-regulated in oncogenic hepatocytes. We reasoned that fibrinogen may bind to integrin αvβ5 on HSCs to activate HSCs. Consistent with this notion, pharmaceutical treatment using an antagonist of integrin αvβ5, cilengitide, significantly blocked HSC activation and function, accompanied by attenuated proliferation of oncogenic hepatocytes and progression of liver fibrosis. On the contrary, adenosine 5’-diphosphate, an agonist of αvβ5, activated HSCs significantly that further stimulated the tumor progression and liver fibrosis. Interestingly, in human liver disease samples, we detected an increased level of fibrinogen during tumor progression which indicated the potential role of fibrinogen signaling in HCC progression. Thus, we concluded a novel interaction between oncogenic hepatocytes and HSCs through the fibrinogen related pathway in both the zebrafish HCC model and human liver disease samples.

Aged tissues usually show a decreased regenerative capacity accompanied by a decline in functionality. During aging pancreatic islets also undergo several morphological and metabolic changes. Besides proliferative and regenerative limitations, endocrine cells lose their secretory capacity, contributing to a decline in functional islet mass and a deregulated glucose homeostasis. This is linked to several features of aging, such as induction of cellular senescence or the formation of modified proteins, such as advanced glycation end products (AGEs) - the latter mainly examined in relation to hyperglycemia and in disease models. However, age-related changes of endocrine islets under normoglycemic and non-pathologic conditions are poorly investigated. Therefore, a characterization of pancreatic tissue sections as wells as plasma samples of wild-type mice (C57BL/6J) at various age groups (2.5, 5, 10, 15, 21 months) was performed. Our findings reveal that mice at older age are able to secret sufficient amounts of insulin to maintain normoglycemia. During aging the pancreatic islet area increased and the islet size doubled in 21 months old mice when compared to 2.5 months old mice, whereas the islet number was unchanged. This was accompanied by an age-dependent decrease in Ki-67 levels and pancreatic duodenal homeobox-1 (PDX-1), indicating a decline in proliferative and regenerative capacity of pancreatic islets with advancing age. In contrast, the number of p16Ink4a-positive nuclei within the islets was elevated starting from 10 months of age. Interestingly, AGEs accumulated exclusively in the islet blood vessels of old mice associated with increased amounts of inflammatory markers, such as the inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine (3-NT). In summary, the age-related increase in islet size and area was associated with the induction of senescence, accompanied by an accumulation of non-enzymatically modified proteins in the islet vascular system., Graphical abstract fx1, Highlights • Aging induces an increase in pancreatic islet size and area, but not number. • Age-related endocrine islet expansion is associated with senescence induction. • Aged islets accumulate AGEs and nitrated proteins exclusively in the vessels.

NLRP3/IL-1β activation via thioredoxin (TRX)/thioredoxin-interacting protein (TXNIP) following mitochondria ROS (mtROS) overproduction plays a key role in inflammation. However, the involvement of this process in tubular damage in the kidneys of patients with diabetic nephropathy (DN) is unclear. Here, we demonstrated that mtROS overproduction is accompanied by decreases in TRX expression and TXNIP up-regulation. In addition, we discovered that mtROS overproduction is also associated with increases in NLRP3/IL-1β and TGF-β expression in the kidneys of patients with DN and db/db mice. We reversed these changes in db/db mice by administering a peritoneal injection of MitoQ, an antioxidant targeting mtROS. Similar results were observed in human tubular HK-2 cells subjected to high-glucose (HG) conditions and treated with MitoQ. Treating HK-2 cells with MitoQ suppressed the dissociation of TRX from TXNIP and subsequently blocked the interaction between TXNIP and NLRP3, leading to the inhibition of NLRP3 inflammasome activation and IL-1β maturation. The effects of MitoQ were enhanced by pretreatment with TXNIP siRNA and abolished by pretreatment with monosodium urate (MSU) and TRX siRNA in vitro. These results suggest that mitochondrial ROS-TXNIP/NLRP3/IL-1β axis activation is responsible for tubular oxidative injury, which can be ameliorated by MitoQ via the inhibition of mtROS overproduction., Graphical abstract fx1, Highlights • Reactive oxygen species promotes renal damage in diabetic nephropathy. • Mitochondrial ROS- TXNIP-NLRP3 biological axis involved in tubular injury of DN. • Inhibition of mitochondrial ROS by MitoQ ameliorated the renal tubular injury.

Graphical abstract, Netrin-1, an axon guidance protein, is difficult to detect using immunohistochemistry. We performed a multi-step, blinded, and controlled protocol optimization procedure to establish an efficient and effective fluorescent immunohistochemistry protocol for characterizing Netrin-1 expression. Coronal mouse brain sections were used to test numerous antigen retrieval methods and combinations thereof in order to optimize the stain quality of a commercially available Netrin-1 antibody. Stain quality was evaluated by experienced neuroanatomists for two criteria: signal intensity and signal-to-noise ratio. After five rounds of testing protocol variants, we established a modified immunohistochemistry protocol that produced a Netrin-1 signal with good signal intensity and a high signal-to-noise ratio. The key protocol modifications are as follows: • Use phosphate buffer (PB) as the blocking solution solvent. • Use 1% sodium dodecyl sulfate (SDS) treatment for antigen retrieval. The original protocol was optimized for use with the Netrin-1 antibody produced by Novus Biologicals. However, we subsequently further modified the protocol to work with the antibody produced by Abcam. The Abcam protocol uses PBS as the blocking solution solvent and adds a citrate buffer antigen retrieval step.

Graphical abstract, Highlights • IMI toxication was evaluated with three different methods. • Pathological lesions were observed after IMI exposure in gills, liver and brain. • IMI exposure induced iNOS, 8-OHdG and TNF-α activation in gills, liver and brain. • IMI exposure caused upregulation iNOS, caspase 3 and MT1 expressions in brain., Imidacloprid (IMI) is a neonicotinoid that is widely used for the protection of crops and carnivores from insects and parasites, respectively. It is well known that imidacloprid exposure has a harmful effect on several organisms. However, there is little information about imidacloprid toxicity in aquatic animals, particularly fish. Thus, in the current study, we assessed the histopathological changes; activation of iNOS, 8-OHdG and TNF-α; and expression levels of caspase 3, iNOS, CYP1A and MT1 genes in the common carp exposed to imidacloprid. For this purpose, fish were exposed to either a low dose (140 mg/L) or a high dose (280 mg/L) of imidacloprid for 24 h, 48 h, 72 h and 96 h. After IMI exposure, we detected hyperplasia of secondary lamellar cells and mucous cell hyperplasia in the gills, as well as hydropic degeneration in hepatocytes and necrosis in the liver. Moreover, 8-OHdG, iNOS and TNF-α activation was found particularly in the gills and liver but also moderately in the brain. Transcriptional analysis showed that caspase 3 expression was altered low dose and high doses of IMI for 72 h and 96 h exposure (p 0.05) except with low and high doses of IMI for 96 h (p

Erosive pustular dermatosis of the scalp (EPDS) is a rare inflammatory disease with predominance in the elderly population. Clinically, the condition is characterized by sterile pustules and chronic crusted erosions. Bacteriologic and mycologic workups tend to have negative findings, and histopathologic evaluation findings are nonspecific. Affected areas undergo a continuous cycle of healing and recurrence, resulting in atrophic skin with new areas of pustules and erosions. If left untreated, these areas may progress and result in scarring alopecia or cutaneous malignancies. Although the exact etiology remains unknown, EPDS tends to occur in areas of cutaneous atrophy because of numerous etiologies such as long-standing solar damage, surgical/medical/physical trauma, or, in the case of our patient, a burn.1 Although EPDS has been documented after skin grafts in elderly patients, to the best of our knowledge, it has yet to be described in a pediatric patient after skin graft for a burn.

Highlights • The association between ORMDL1 expression and clinipathological characteristic in colorectal cancer (CRC) has not been reported before, which is addressed in this study. • Although ORMDL1 is generally upregulated in CRC, high expression of ORMDL1 is associated with longer survival; the combination of ORMDL1 expression and other biomarkers to stratify and predict prognosis in CRC is worth to explore. • ORMDL1 seems to influence cell cytoskeleton dynamics through Rho GTPase, other than canonical EMT related transcription factors., Background The ORMDL1 gene is known as a crucial negative regulator of sphingolipid biogenesis. However, the ORMDL1 gene has rarely been studied in a tumor-related context. Therefore, its prognostic value and functional significance in colorectal cancer (CRC) remain to be explored. Methods TCGA CRC cohort analysis, qRT-PCR, and immunohistochemistry (IHC) were used to examine the ORMDL1 expression level. The association between ORMDL1 expression and various clinical characteristics was analyzed by chi-square tests. The overall survival (OS) of CRC patients was analyzed by Kaplan-Meier analysis. In vitro and in vivo cell-based assays were performed to explore the role of ORMDL1 in cell proliferation, invasion and migration. Transcriptional changes in cells with either ORMDL1 knockdown or overexpression were compared and analyzed. Results ORMDL1 was upregulated in CRC tissues in both the TCGA and our cohort. Interestingly, its expression was significantly lower in patients with metastasis than in patients without metastasis, and the high expression group had longer OS than the low expression group. Knockdown of ORMDL1 expression can promote proliferation, colony formation and invasion, while attenuating migration in CRC cell lines. In contrast, forced overexpression of ORMDL1 reduced cell proliferation, colony formation and invasion, while enhancing cell migration. Stable knockdown of ORMDL1 can promote cancer cell proliferation in vivo to some extent. Finally, Rho GTPase activity was influenced by ORMDL1, and the expression of ORMDL1 was enhanced by DTT treatment. Conclusion ORMDL1 is upregulated and may serve as a biomarker to predict favourable outcomes in colorectal cancer.

The hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote carriers of ASGR1 deletions exhibit ∼34% lower risk of coronary artery disease and ∼10% to 14% reduction of non-HDL cholesterol. Since the proprotein convertase PCSK9 is a major degrader of the low-density lipoprotein receptor (LDLR), we investigated the degradation and functionality of LDLR and/or PCSK9 by endogenous/overexpressed ASGR1 using Western blot and immunofluorescence in HepG2-naïve and HepG2-PCSK9-knockout cells. ASGR1, like PCSK9, targets LDLR, and both independently interact with/enhance the degradation of the receptor. This lack of cooperativity between PCSK9 and ASGR1 was confirmed in livers of wildtype (WT) and Pcsk9−/− mice. ASGR1 knockdown in HepG2-naïve cells significantly increased total (∼1.2-fold) and cell-surface (∼4-fold) LDLR protein. In HepG2-PCSK9-knockout cells, ASGR1 silencing led to ∼2-fold higher levels of LDLR protein and DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)-LDL uptake associated with ∼9-fold increased cell-surface LDLR. Overexpression of WT-ASGR1/2 primarily reduced levels of immature non-O-glycosylated LDLR (∼110 kDa), whereas the triple Ala-mutant of Gln240/Trp244/Glu253 (characterized by loss of carbohydrate binding) reduced expression of the mature form of LDLR (∼150 kDa), suggesting that ASGR1 binds the LDLR in both a sugar-dependent and -independent fashion. The protease furin cleaves ASGR1 at the RKMK103↓ motif into a secreted form, likely resulting in a loss of function on LDLR. Altogether, we demonstrate that LDLR is the first example of a liver-receptor ligand of ASGR1. We conclude that silencing of ASGR1 and PCSK9 may lead to higher LDL uptake by hepatocytes, thereby providing a novel approach to further reduce LDL cholesterol levels.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is a common pathological condition that causes intractable myelopathy and radiculopathy, mainly the result of an endochondral ossification-like process. Our previous genome-wide association study identified six susceptibility loci for OPLL, including the cell division cycle 5-like (CDC5L) gene region. Here, we found CDC5L to be expressed in type II collagen-producing chondrocyte-like fibroblasts in human OPLL specimens, as well as in differentiating ATDC5 chondrocytes. Cdc5l siRNA transfection in murine chondrocytes decreased the expression of the early chondrogenic genes Sox9 and Col2a1, diminished the cartilage matrix production, and enhanced the expression of parathyroid-hormone-related protein (a resting chondrocyte marker). We also showed that Cdc5l shRNA suppressed the growth of cultured murine embryonal metatarsal cartilage rudiments and that Cdc5l knockdown suppressed the growth of ATDC5 cells. Fluorescence-activated cell sorting analysis revealed that the G2/M cell cycle transition was blocked; our data showed that Cdc5l siRNA transfection enhanced expression of Wee1, an inhibitor of the G2/M transition. Cdc5l siRNA also decreased the pre-mRNA splicing efficiency of Sox9 and Col2a1 genes in both ATDC5 cells and primary chondrocytes; conversely, loss of Cdc5l resulted in enhanced splicing of Wee1 pre-mRNA. Finally, an RNA-binding protein immunoprecipitation assay revealed that Cdc5l bound directly to these target gene transcripts. Overall, we conclude that Cdc5l promotes both early chondrogenesis and cartilage growth and may play a role in the etiology of OPLL, at least in part by fine-tuning the pre-mRNA splicing of chondrogenic genes and Wee1, thus initiating the endochondral ossification process.

Macroautophagy dysregulation is implicated in multiple neurological disorders, such as Parkinson's disease. While autophagy pathways are heavily researched in heterologous cells and neurons, regulation of autophagy in the astrocyte, the most abundant cell type in the mammalian brain, is less well understood. Missense mutations in the Synj1 gene encoding Synaptojanin1 (Synj1), a neuron-enriched lipid phosphatase, have been linked to Parkinsonism with seizures. Our previous study showed that the Synj1 haploinsufficient (Synj1+/−) mouse exhibits age-dependent autophagy impairment in multiple brain regions. Here, we used cultured astrocytes from Synj1-deficient mice to investigate its role in astrocyte autophagy. We report that Synj1 is expressed in low levels in astrocytes and represses basal autophagosome formation. We demonstrate using cellular imaging that Synj1-deficient astrocytes exhibit hyperactive autophagosome formation, represented by an increase in the size and number of GFP-microtubule-associated protein 1A/1B-light chain 3 structures. Interestingly, Synj1 deficiency is also associated with an impairment in stress-induced autophagy clearance. We show, for the first time, that the Parkinsonism-associated R839C mutation impacts autophagy in astrocytes. The impact of this mutation on the phosphatase function of Synj1 resulted in elevated basal autophagosome formation that mimics Synj1 deletion. We found that the membrane expression of the astrocyte-specific glucose transporter GluT-1 was reduced in Synj1-deficient astrocytes. Consistently, AMP-activated protein kinase activity was elevated, suggesting altered glucose sensing in Synj1-deficient astrocytes. Expressing exogenous GluT-1 in Synj1-deficient astrocytes reversed the autophagy impairment, supporting a role for Synj1 in regulating astrocyte autophagy via disrupting glucose-sensing pathways. Thus, our work suggests a novel mechanism for Synj1-related Parkinsonism involving astrocyte dysfunction.