Mutations in Cu/Zn superoxide dismutase (SOD1) cause autosomal dominant amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease with no effective treatment. Despite ample evidence indicating involvement of mutation-induced SOD1 protein misfolding and aggregation in ALS pathogenesis, the molecular mechanisms that control cellular management of misfolded, aggregation-prone SOD1 mutant proteins remain unclear. Here, we report that parkin, an E3 ubiquitin-protein ligase which is linked to Parkinson's disease, is a novel regulator of cellular defense against toxicity induced by ALS-associated SOD1 mutant proteins. We find that parkin mediates K63-linked polyubiquitination of SOD1 mutants in cooperation with the UbcH13/Uev1a E2 enzyme and promotes degradation of these misfolded SOD1 proteins by the autophagy-lysosome system. In response to strong proteotoxic stress associated with proteasome impairment, parkin promotes sequestration of misfolded and aggregated SOD1 proteins to form perinuclear aggresomes, regulates positioning of lysosomes around misfolded SOD1 aggresomes, and facilitates aggresome clearance by autophagy. Our findings reveal parkin-mediated cytoprotective mechanisms against misfolded SOD1 toxicity and suggest that enhancing parkin-mediated cytoprotection may provide a novel therapeutic strategy for treating ALS.

Alcohol use disorders (AUDs) are a major public health issue and produce enormous societal and economic burdens. Current Food and Drug Administration (FDA)-approved pharmacotherapies for treating AUDs suffer from deleterious side effects and are only effective in a subset of individuals. It is therefore essential to find improved medications for the management of AUDs. Emerging evidence suggests that anticonvulsants are a promising class of drugs for treating individuals with AUDs. In these studies, we used integrative functional genomics to demonstrate that genes that encode Kv7 channels (i.e. Kcnq2/3) are related to alcohol (ethanol) consumption, preference and acceptance in rodents. We then tested the ability of the FDA-approved anticonvulsant retigabine, a Kv7 channel opener, to reduce voluntary ethanol consumption of Wistar rats in a two-bottle choice intermittent alcohol access paradigm. Systemic administration and microinjections of retigabine into the nucleus accumbens significantly reduced alcohol drinking, and retigabine was more effective at reducing intake in high- versus low-drinking populations of Wistar rats. Prolonged voluntary drinking increased the sensitivity to the proconvulsant effects of pharmacological blockade of Kv7 channels and altered surface trafficking and SUMOylation patterns of Kv7.2 channels in the nucleus accumbens. These data implicate Kcnq2/3 in the regulation of ethanol drinking and demonstrate that long-term drinking produces neuroadaptations in Kv7 channels. In addition, these results have identified retigabine as a potential pharmacotherapy for treating AUDs and Kv7 channels as a novel therapeutic target for reducing heavy drinking.

Food-derived peptides, such as β-casomorphin BCM7, have potential to cross the gastrointestinal tract and blood-brain barrier and are associated with neurological disorders and neurodevelopmental disorders. We previously established a novel mechanism through which BCM7 affects the antioxidant levels in neuronal cells leading to inflammatory consequences. In the current study, we elucidated the effects of casein-derived peptides on neuronal development by using the neurogenesis of neural stem cells (NSCs) as an experimental model. First, the transient changes in intracellular thiol metabolites during NSC differentiation (neurogenesis) were investigated. Next, the neurogenic effects of food-derived opioid peptides were measured, along with changes in intracellular thiol metabolites, redox status and global DNA methylation levels. We observed that the neurogenesis of NSCs was promoted by human BCM7 to a greater extent, followed by A2-derived BCM9 in contrast to bovine BCM7, which induced increased astrocyte formation. The effect was most apparent when human BCM7 was administered for 1day starting on 3days postplating, consistent with immunocytochemistry. Furthermore, neurogenic changes regulated by bovine BCM7 and morphine were associated with an increase in the glutathione/glutathione disulfide ratio and a decrease in the S-adenosylmethionine/S-adenosylhomocysteine ratio, indicative of changes in the redox and the methylation states. Finally, bovine BCM7 and morphine decreased DNA methylation in differentiating NSCs. In conclusion, these results suggest that food-derived opioid peptides and morphine regulated neurogenesis and differentiation of NSCs through changes in the redox state and epigenetic regulation.

Patients with inflammatory bowel disease (IBD) or irritable bowel syndrome (IBS) often experience increased sensory responsiveness in the urinary bladder reflecting neurogenic bladder overactivity. Here we demonstrate that colitis-induced up-regulation of the phospholipase C gamma (PLCγ) pathway downstream of brain-derived neurotrophic factor (BDNF) in bladder afferent neurons in the dorsal root ganglia (DRG) plays essential roles in activating these neurons thereby leading to bladder hyperactivity. Upon induction of colitis with 2,4,6-trinitrobenzenesulfonic acid (TNBS) in rats, we found that the phosphorylation (activation) level of cAMP responsive element-binding (p-CREB) protein, a molecular switch of neuronal plasticity, was increased in specifically labeled bladder afferent neurons in the thoracolumbar and lumbosacral DRGs. In rats having reduced levels of BDNF (BDNF+/-), colitis failed to elevate CREB protein activity in bladder afferent neurons. Physiological examination also demonstrated that colitis-induced urinary frequency was not shown in BDNF+/- rats, implicating an essential role of BDNF in mediating colon-to-bladder sensory cross-sensitization. We further implemented in vivo and in vitro studies and demonstrated that BDNF-mediated colon-to-bladder sensory cross-activation involved the TrkB-PLCγ-calcium/calmodulin-dependent protein kinase II (CaMKII) cascade. In contrast, the PI3K/Akt pathway was not activated in bladder afferent neurons during colitis and was not involved in BDNF action in the DRG. Our results suggest that colon-to-bladder sensory cross-sensitization is regulated by specific signal transduction initiated by the up-regulation of BDNF in the DRG.

Transcriptome analysis is important for interpreting the functional elements of the genome and revealing the molecular constituents of cells and tissues. Herein, differentially transcribed genes were identified by deep sequencing after zebrafish (Danio rerio) were exposed to β-diketone antibiotics (DKAs); 23,129 and 23,550 mapped genes were detected in control and treatment groups, a total of 3238 genes were differentially expressed between control and treatment groups. Of these genes, 328 genes (213 up- and 115 down-regulation) had significant differential expression (p < 0.05) and an expression ratio (control/treatment) of >2 or <0.5. Additionally, we performed Gene Ontology (GO) category and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, and found 266 genes in the treatment group with annotation terms linked to the GO category. A total of 77 differentially expressed transcriptional genes were associated with 132 predicted KEGG metabolic pathways. Serious liver tissue damage was reflected and consistent with the differences in genetic classification and function from the transcriptome analysis. These results enhance our understanding of zebrafish developmental processes under exposure to DKA stress. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1357-1371, 2016.

Esophageal adenocarcinoma (EAC) is characterized by rapidly increasing incidence and mortality rates and poor survival. Efficacious preventive and treatment options are urgently needed. An increasing number of pharmacologic agents targeting cancer cell death via autophagy mechanisms are being evaluated in hopes of circumventing apoptotic and therapeutic resistance. We report for the first time, loss of Beclin-1, a key mediator of autophagy, was significantly linked to prognostic factors in EAC. Specifically, Beclin-1 expression loss occurred in 49.0% of EAC patients versus 4.8% of controls. There was a significant inverse correlation between loss of Beclin-1 with histologic grade and tumor stage supporting a tumor suppressive role for Beclin-1. Autophagy modulation linked to cell death was examined in EAC cell lines following treatment with a proanthocyanidin-rich cranberry extract, C-PAC, and the commonly used autophagy inducer, rapamycin. C-PAC induced Beclin-1-independent autophagy in EAC cells characterized by reduced phosphorylation at serine 15 and 93, and significant cell death induction. In contrast, rapamycin-induced autophagy resulted in concomitant, increases in total Beclin-1 levels as well as Beclin-1-phosphorylation in a cell line specific manner, leading to long-term cell survival. Furthermore, autophagic LC3-II was induced by C-PAC following siRNA suppression of Beclin-1 in EAC cells. Together these data support a prognostic role of Beclin-1 in EAC with evidence that Beclin-dependent autophagy induction is agent specific. Future studies are necessary to fully interrogate the role autophagy plays in the progression of normal tissue to EAC and how specific agents targeting autophagic mechanisms can be efficaciously applied for cancer prevention or treatment. © 2015 Wiley Periodicals, Inc.

Ursolic acid (UA), a well-known natural triterpenoid found in abundance in blueberries, cranberries and apple peels, has been reported to possess many beneficial health effects. These effects include anticancer activity in various cancers, such as skin cancer. Skin cancer is the most common cancer in the world. Nuclear factor E2-related factor 2 (Nrf2) is a master regulator of antioxidative stress response with anticarcinogenic activity against UV- and chemical-induced tumor formation in the skin. Recent studies show that epigenetic modifications of Nrf2 play an important role in cancer prevention. However, the epigenetic impact of UA on Nrf2 signaling remains poorly understood in skin cancer. In this study, we investigated the epigenetic effects of UA on mouse epidermal JB6 P+ cells. UA inhibited cellular transformation by 12-O-tetradecanoylphorbol-13-acetate at a concentration at which the cytotoxicity was no more than 25%. Under this condition, UA induced the expression of the Nrf2-mediated detoxifying/antioxidant enzymes heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferase 1A1. DNA methylation analysis revealed that UA demethylated the first 15 CpG sites of the Nrf2 promoter region, which correlated with the reexpression of Nrf2. Furthermore, UA reduced the expression of epigenetic modifying enzymes, including the DNA methyltransferases DNMT1 and DNMT3a and the histone deacetylases (HDACs) HDAC1, HDAC2, HDAC3 and HDAC8 (Class I) and HDAC6 and HDAC7 (Class II), and HDAC activity. Taken together, these results suggest that the epigenetic effects of the triterpenoid UA could potentially contribute to its beneficial effects, including the prevention of skin cancer.

Thiamine (vitamin B1, VB1) can act as a plant defence trigger, or priming agent, leading to a rapid counterattack on pathogen invasion. In this study, the priming effect of thiamine on rice (Oryza sativa cv. Nipponbare) and its activity against root-knot nematode (Meloidogyne graminicola) infection were evaluated. Thiamine treatment and subsequent nematode inoculation activated hydrogen peroxide (H2O2) accumulation and lignin deposition in plant roots, and this correlated with enhanced transcription of OsPAL1 and OsC4H, two genes involved in the phenylpropanoid pathway. The number of nematodes in rice roots was slightly but significantly reduced, and the development of the nematodes was delayed, whereas no direct toxic effects of VB1 on nematode viability and infectivity were observed. The combined application of thiamine with l-2-aminooxy-3-phenylpropionic acid (AOPP), an inhibitor of phenylalanine ammonia-lyase (PAL), significantly hampered the VB1-priming capacity. These findings indicate that thiamine-induced priming in rice involves H2O2 and phenylpropanoid-mediated lignin production, which hampers nematode infection. Further cellular and molecular studies on the mechanism of thiamine-induced defence will be useful for the development of novel nematode control strategies.

To assess whether changes in antiretroviral drugs other than thymidine nucleoside reverse transcriptase inhibitors (NRTI) may have a body fat impact in HIV-infected patients with lipoatrophy.

Some evidence indicated that chemoresistance associates with the acquisition of cancer stem-like properties. Recent studies suggested that chemokines can promote the chemoresistance and stem cell properties in various cancer cells, while the underling mechanism is still not completely illustrated. In our study, we found that CCL21 can upregulate the expression of P-glycoprotein (P-gp) and stem cell property markers such as Bmi-1, Nanog, and OCT-4 in colorectal cancer (CRC) HCT116 cells and then improve the cell survival rate and mammosphere formation. Our results suggested that Snail was crucial for CCL21-mediated chemoresistance and cancer stem cell property in CRC cells. Further, we observed that CCL21 treatment increased the protein but not mRNA levels of Snail, which suggested that CCL21 upregulates Snail via posttranscriptional ways. The downstream signals AKT/GSK-3β mediated CCL21 induced the upregulation of Snail due to the fact that CCL21 treatment can obviously phosphorylate both AKT and GSK-3β. The inhibitor of PI3K/Akt, LY294002 significantly abolished CCL21 induced chemoresistance and mammosphere formation of HCT116 cells. Collectively, our results in the present study revealed that CCL21 can facilitate chemoresistance and stem cell property of CRC cells via the upregulation of P-gp, Bmi-1, Nanog, and OCT-4 through AKT/GSK-3β/Snail signals, which suggested a potential therapeutic approach to CRC patients.

It is important to know the tumor resistance against cisplatin before the treatment of non-small cell lung cancer (NSCLC). The purpose of this study was to evaluate the response to treatment and survival in patients with NSCLC treated with cisplatin-based chemotherapy according to excision repair cross-complementation 1 (ERCC1) expression.

Hepatocellular carcinoma, a lethal malignant neoplasm with poor prognosis, has dismal results of surgical resection and chemoradiotherapy. Norcantharidin (NCTD), the demethylated analog of cantharidin derived from a traditional Chinese medicine, Mylabris, has been used in the treatment of cancer. However, the detailed mechanisms underlying this process are generally unclear.

Anti-inflammatory polyphenols, such as epigallocatechin-3-gallate (EGCG), have been shown to protect against the toxicity of environmental pollutants. It is well known that bioactive food compounds such as polyphenols may exert their protection by modulating inflammatory pathways regulated through nuclear factor-kappa B (NF-κB) signaling. EGCG has been reported to inhibit NF-κB activation. We hypothesize that EGCG can protect against polychlorinated biphenyl (PCB)-induced endothelial inflammation in part through epigenetic regulation of NF-κB-regulated inflammatory genes. In order to test this hypothesis, human endothelial cells (EA.hy926) were exposed to physiologically relevant levels of coplanar PCB 126 and/or 15 or 30 μM of EGCG, followed by quantification of NF-κB subunit p65, histone acetyltransferase p300 and histone deacetylases (HDACs) accumulation through chromatin immunoprecipitation assay in the promoter region of inflammatory genes. In addition, the enrichment of the acetylated H3 was also quantified. PCB 126 exposure increased the expression of vascular inflammatory mediators, including interleukin (IL)-6, C-reactive protein, intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and IL-1α/β, which were prevented by pretreatment with EGCG. This inhibitory effect by EGCG correlated with abolished nuclear import of p65, decreased chromatin binding of p65 and p300, as well as increased chromatin binding of HDAC 1/2. Furthermore, EGCG induced hypoacetylation of H3, which accounts for deactivation of downstream genes. These data suggest that EGCG-induced epigenetic modifications can decrease PCB-induced vascular toxicity.

Short chain fatty acids (SCFAs) are produced in the colonic lumen mainly by bacterial fermentation of dietary fiber. Emerging evidence shows that SCFA has important physiological and pathophysiological effects on colonic and systemic events. Recently, propionate, known as a kind of SCFA, has been shown to lower fatty acid contents in plasma and reduce food intake. However, the detailed mechanism underlying the propionate-mediated lipid metabolism action remains poorly understood. The intestinal lipid metabolism process is critical for systemic energy homeostasis. Therefore, we investigate here the effects of propionate on intestinal lipid metabolism. Results show that propionate induced peroxisome proliferator-activated receptor α (PPARα) expression time-dependently and concentration-dependently in YAMC (a mouse intestinal epithelial cell line) cells. The expression levels of PPARα-responsive genes such as carnitine palmitoyl transferase II (CPTII) and trifunctional protein α (TFPα) were up-regulated in the presence of propionate, thereby suppressing triglyceride (TG) accumulation. Furthermore, propionate-mediated PPARα induction required phosphorylation of extracellular signal-regulated kinase. Collectively, these data indicate that propionate regulates intestinal lipid metabolism through the induction of PPARα expression. Results suggest that the inhibitory effect of propionate on TG accumulation partly contributes to the propionate-mediated fatty acid-lowering effect.

Persistence of latently infected cells in presence of Anti-Retroviral Therapy presents the main obstacle to HIV-1 eradication. Much effort is thus placed on identification of compounds capable of HIV-1 latency reversal in order to render infected cells susceptible to viral cytopathic effects and immune clearance. We identified the BAF chromatin remodeling complex as a key player required for maintenance of HIV-1 latency, highlighting its potential as a molecular target for inhibition in latency reversal. Here, we screened a recently identified panel of small molecule inhibitors of BAF (BAFi's) for potential to activate latent HIV-1. Latency reversal was strongly induced by BAFi's Caffeic Acid Phenethyl Ester and Pyrimethamine, two molecules previously characterized for clinical application. BAFi's reversed HIV-1 latency in cell line based latency models, in two ex vivo infected primary cell models of latency, as well as in HIV-1 infected patient's CD4 + T cells, without inducing T cell proliferation or activation. BAFi-induced HIV-1 latency reversal was synergistically enhanced upon PKC pathway activation and HDAC-inhibition. Therefore BAFi's constitute a promising family of molecules for inclusion in therapeutic combinatorial HIV-1 latency reversal.

The transcription factor, runt-related transcription factor 2 (Runx2), plays a pivotal role in the differentiation of the mesenchymal stem cells to the osteochondroblast lineages. We found by the drug repositioning strategy that a proton pump inhibitor, lansoprazole, enhances nuclear accumulation of Runx2 and induces osteoblastogenesis of human mesenchymal stromal cells. Systemic administration of lansoprazole to a rat femoral fracture model increased osteoblastogenesis. Dissection of signaling pathways revealed that lansoprazole activates a noncanonical bone morphogenic protein (BMP)-transforming growth factor-beta (TGF-β) activated kinase-1 (TAK1)-p38 mitogen-activated protein kinase (MAPK) pathway. We found by in cellulo ubiquitination studies that lansoprazole enhances polyubiquitination of the TNF receptor-associated factor 6 (TRAF6) and by in vitro ubiquitination studies that the enhanced polyubiquitination of TRAF6 is attributed to the blocking of a deubiquitination enzyme, cylindromatosis (CYLD). Structural modeling and site-directed mutagenesis of CYLD demonstrated that lansoprazole tightly fits in a pocket of CYLD where the C-terminal tail of ubiquitin lies. Lansoprazole is a potential therapeutic agent for enhancing osteoblastic differentiation.

Human engineered heart tissues have potential to revolutionize cardiac development research, drug-testing, and treatment of heart disease; however, implementation is limited by the need to use pre-differentiated cardiomyocytes (CMs). Here we show that by providing a 3D poly(ethylene glycol)-fibrinogen hydrogel microenvironment, we can directly differentiate human pluripotent stem cells (hPSCs) into contracting heart tissues. Our straight-forward, ontomimetic approach, imitating the process of development, requires only a single cell-handling step, provides reproducible results for a range of tested geometries and size scales, and overcomes inherent limitations in cell maintenance and maturation, while achieving high yields of CMs with developmentally appropriate temporal changes in gene expression. We demonstrate that hPSCs encapsulated within this biomimetic 3D hydrogel microenvironment develop into functional cardiac tissues composed of self-aligned CMs with evidence of ultrastructural maturation, mimicking heart development, and enabling investigation of disease mechanisms and screening of compounds on developing human heart tissue.

Objectives. The relationship among matriptase function, cellular redox status, and maintenance of intestinal barrier integrity has not been established yet. The aim of this study is to reveal if the crosstalk between matriptase activators and intestinal epithelial monolayers can lead to perturbations in physiological redox regulation in vitro. Methods. The effects of suramin and sphingosine-1-phosphate (S1P) were tested on viability of intestinal porcine epithelial IPEC-J2 cells using MTS assay. Measurements of transepithelial electrical resistance (TER) were performed to determine changes in barrier integrity of cell monolayers. Amplex Red assay was used to monitor extracellular hydrogen peroxide production. Occludin distribution pattern was detected prior to and after matriptase activation using immunofluorescent staining technique. Results. TER reduction was observed in suramin-treated IPEC-J2 cell monolayers, which could be attributed to cell cytotoxic properties of 48 hr 50 μM suramin administration. In contrast, S1P treatment increased TER significantly and elevated occludin accumulation in tight junctions. It was also found that extracellular hydrogen peroxide levels were maintained in IPEC-J2 cells exposed to matriptase activators. Discussion. S1P administration not accompanied by redox imbalance might be one of the key strategies in the improvement of barrier function and consequently in the therapy of intestinal inflammations.

Depression is a major mood disorder affecting people worldwide. The posttranscriptional gene regulation mediated by microRNAs (miRNAs) which may have critical roles in the pathogenesis of depression. However, to date, little is known about the effects of the antidepressant drug duloxetine on miRNA expression profile in chronic unpredictable mild stress (CUMS)-induced depression model in mice. Healthy adult male Kunming mice were randomly divided into three groups: control group, model group and duloxetine group. Sucrose preference test and open field test were used to represent the behavioral change. MiRNAs levels in frontal lobe and hippocampus of mice were analyzed using miRNA microarrays assay. We observed that long-term treatment with duloxetine significantly ameliorated the CUMS procedure-induced sucrose preference decreases and mice treated with duloxetine demonstrated a reversal of the number of crossings, and rearings reduced by CUMS. A significant upregulation of miR-132 and miR-18a in hippocampus in the duloxetine treatment group compared with model group, whereas the levels of miR-134 and miR-124a were significantly downregulated. Furthermore, miR-18a showed significant upregulation in frontal lobe in the duloxetine treatment group relative to model group. Our data showed that miRNA expression profile in frontal lobe and hippocampus was affected by duloxetine in mice model of depression. The effect was especially pronounced in the hippocampus, suggesting that hippocampus might be the action site of duloxetine, which presumably worked by regulating the expression of miRNA levels.

In this study, we screened the different human osteosarcoma cell line MG-63 miRNAs after the treatment of curcumin and explored the effects of curcumin on MG-63 cells and its mechanism.