G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

The bovine uterus is susceptible to infection, and the elevated cortisol level due to stress are common in cows after delivery. The essential trace element selenium plays a pivotal role in the antioxidant and anti-inflammatory defence system of body. This study investigated whether selenium supplementation protected endometrial cells from inflammation in the presence of high-level cortisol. The primary bovine endometrial epithelial cells were subjected to Escherichia coli lipopolysaccharide to establish cellular inflammation model. The gene expression of inflammatory mediators and proinflammatory cytokines was measured by quantitative PCR. The key proteins of NF-κB and MAPK signalling pathways were detected by Western blot and immunofluorescence. The result showed that pre-treatment of Na2 SeO3 (1, 2 and 4 μΜ) decreased the mRNA expression of proinflammatory genes, inhibited the activation of NF-κB and suppressed the phosphorylation of extracellular signal-regulated kinase, P38MAPK and c-Jun N-terminal kinase. This inhibition of inflammation was more apparent in the presence of high-level cortisol (30 ng/mL). These results indicated that selenium has an anti-inflammatory effect, which is mediated via NF-κB and MAPK signalling pathways and is augmented by cortisol in bovine endometrial epithelial cells.

The Cauliflower mosaic virus (CaMV) 35S promoter (P35S) is a commonly used target for detection of genetically modified organisms (GMOs). There are currently 24 reported detection methods, targeting different regions of the P35S promoter. Initial assessment revealed that due to the absence of primer binding sites in the P35S sequence, 19 of the 24 reported methods failed to detect P35S in MON88913 cotton, and the other two methods could only be applied to certain GMOs. The rest three reported methods were not suitable for measurement of P35S in some testing events, because SNPs in binding sites of the primer/probe would result in abnormal amplification plots and poor linear regression parameters. In this study, we discovered a conserved region in the P35S sequence through sequencing of P35S promoters from multiple transgenic events, and developed new qualitative and quantitative detection systems targeting this conserved region. The qualitative PCR could detect the P35S promoter in 23 unique GMO events with high specificity and sensitivity. The quantitative method was suitable for measurement of P35S promoter, exhibiting good agreement between the amount of template and Ct values for each testing event. This study provides a general P35S screening method, with greater coverage than existing methods.

Cardiomyocyte progenitor cells play essential roles in early heart development, which requires highly controlled cellular organization. microRNAs (miRs) are involved in various cell behaviors by post-transcriptional regulation of target genes. However, the roles of miRNAs in human cardiomyocyte progenitor cells (hCMPCs) remain to be elucidated. Our previous study showed that miR-134 was significantly downregulated in heart tissue suffering from congenital heart disease, underlying the potential role of miR-134 in cardiogenesis. In the present work, we showed that the upregulation of miR-134 reduced the proliferation of hCMPCs, as determined by EdU assay and Ki-67 immunostaining, while the inhibition of miR-134 exhibited an opposite effect. Both up- and downregulation of miR-134 expression altered the transcriptional level of cell-cycle genes. We identified Meis2 as the target of miR-134 in the regulation of hCMPC proliferation through bioinformatic prediction, luciferase reporter assay and western blot. The over-expression of Meis2 mitigated the effect of miR-134 on hCMPC proliferation. Moreover, miR-134 did not change the degree of hCMPC differentiation into cardiomyocytes in our model, suggesting that miR-134 is not required in this process. These findings reveal an essential role for miR-134 in cardiomyocyte progenitor cell biology and provide new insights into the physiology and pathology of cardiogenesis.

Chronic alcoholism often causes liver injuries characterized by hepatic steatosis, inflammation as well as oxidative stress and finally leads to advanced cirrhosis and liver cancer. Fas-activated serine/threonine kinase (FASTK) and its homologs are gradually known as multifunctional proteins involved in various biological processes; however, the role of FASTK and its family members in alcoholic liver disease (ALD) is still unexplored. Here we found that, among FASTK family members, the expression of FASTK was specifically induced both in livers of mice received chronic ethanol ingestion and in ethanol-stimulated hepatocytes. Animal studies showed that genetic deletion of FASTK attenuated chronic ethanol ingestion-induced liver damage, steatosis, and inflammation. Moreover, FASTK deficiency was associated with improved oxidative/anti-oxidative system homeostasis and reduced reactive oxygen species (ROS) generation in livers upon chronic ethanol stimulation. Importantly, FASTK ablation preserved hepatic sirtuin-1 (SIRT1) expression/activity upon chronic ethanol ingestion and SIRT1 silencing via adenovirus-mediated small interfering RNA transfer diminished FASTK deletion-elicited beneficial effects on alcohol-associated hepatic steatosis, inflammation, and oxidative stress. Mechanistically, ethanol increased the phosphorylation of human antigen R (HuR, a RNA binding protein that stabilizes SIRT1 mRNA) and triggered the dissociation of HuR-SIRT1 mRNA complex, in turn promoting SIRT1 mRNA decay. Genetic deletion of FASTK diminished ethanol-induced HuR phosphorylation and HuR-SIRT1 mRNA complex dissociation, thereby enhancing SIRT1 mRNA stability. Collectively, these findings for the first time highlight a critical role of FASTK in the pathogenesis of ALD and implicate HuR-SIRT1 mRNA complex involves in this process.

ZNF32 is a recently identified zinc finger protein and its functions remain largely unknown. Autophagy has been shown to affect cell proliferation and survival. Here, we innovatively show the effect of ZNF32 on cell autophagy and autophagy-associated cell death in breast carcinoma cells and also elucidate its underlying mechanisms. We examined the autophagic activity and LC3 II expression in human carcinoma cell lines with increased or decreased ZNF32 expression. Pharmacological inhibition (rapamycin) or activation (EGF) assays were used to investigate the function of the AKT/mTOR pathway during this process. H2O2- and diamide-induced MCF-7 cell death models were used to elucidate the role of ZNF32-associated autophagy in breast carcinoma cell death. Our results show that increasing ZNF32 expression in MCF-7 cells inhibits autophagy initiation by activating the AKT/mTOR pathway, and further reduced autophagy-associated cell death and maintained MCF-7 cell survival. Conversely, impairing ZNF32 expression by transfecting ZNF32 siRNA strongly promoted autophagy, further augmenting autophagy-associated cell death. Furthermore, correlations between ZNF32 and autophagy were observed in both MCF-7 xenograft tumors and in breast cancer patients. In conclusion, ZNF32 acts as an effective autophagy inhibitor to protect breast cancer cells from excessive stimulus-autophagy-induced cell death.

Fluoride and arsenic are two common inorganic contaminants in drinking water that are associated with impairment in child development and retarded intelligence. The present study was conducted to explore the effects on spatial learning, memory, glutamate levels, and group I metabotropic glutamate receptors (mGluRs) expression in the hippocampus and cortex after subchronic exposure to fluoride, arsenic, and a fluoride and arsenic combination in rats. Weaned male Sprague-Dawley rats were assigned to four groups. The control rats drank tap water. Rats in the three exposure groups drank water with sodium fluoride (120 mg/L), sodium arsenite (70 mg/L), and a sodium fluoride (120 mg/L) and sodium arsenite (70 mg/L) combination for 3 months. Spatial learning and memory was measured in Morris water maze. mGluR1 and mGluR5 mRNA and protein expression in the hippocampus and cortex was detected using RT-PCR and Western blot, respectively. Compared with controls, learning and memory ability declined in rats that were exposed to fluoride and arsenic both alone and combined. Combined fluoride and arsenic exposure did not have a more pronounced effect on spatial learning and memory compared with arsenic and fluoride exposure alone. Compared with controls, glutamate levels decreased in the hippocampus and cortex of rats exposed to fluoride and combined fluoride and arsenic, and in cortex of arsenic-exposed rats. mGluR5 mRNA and protein expressions in the hippocampus and mGluR5 protein expression in the cortex decreased in rats exposed to arsenic alone. Interestingly, compared with fluoride and arsenic exposure alone, fluoride and arsenic combination decreased mGluR5 mRNA expression in the cortex and protein expression in the hippocampus, suggesting a synergistic effect of fluoride and arsenic. These data indicate that fluoride and arsenic, either alone or combined, can decrease learning and memory ability in rats. The mechanism may be associated with changes of glutamate level and mGluR5 expression in cortex and hippocampus.

The aim of the present study was to examine the effect of the traditional Chinese medicine Qili qiangxin on cardiomyocyte apoptosis following myocardial infarction (MI) in a rat model. MI was induced in rats by ligation of the anterior descending coronary artery. Survivors were randomly divided into the sham operation, MI, and Qili qiangxin groups (4 g/kg per day). After 28 days, infarction size was measured. In the non-infarcted zones (NIZ), the apoptotic index (AI) was measured by terminal deoxynucleotidyl transferase (TdT)-mediated digoxigenin-conjugated dUTP nick-end labeling (TUNEL). Expression of Fas was detected by immunohistochemistry, and the expression of xanthine oxidase (XO) and caspase-3 by western blot analysis. In addition, the XO and ·O2-, ·OH-scavenging activity of myocardial tissue in NIZ was measured by colorimetry. Compared to the MI group, AI and the expression of Fas and caspase-3 were significantly decreased in NIZ. The activity of XO was also considerably reduced while ·O2- and ·OH-scavenging activity was significantly increased in the Qili qiangxin group. Ventricular remodeling was attenuated but there were no significant differences in infarct size (IS) or XO expression levels between the Qili qiangxin and MI groups. In conclusion, the results suggest that Qili qiangxin may inhibit cardiomyocyte apoptosis in NIZ in rats. The potential mechanism involved may be associated with its ability to reduce reactive oxygen species (ROS) and to depress the expression of Fas and caspase-3.

Alcoholic fatty liver disease (AFLD) is characterized by hepatic steatosis and carries an elevated risk of cirrhosis and hepatocellular carcinoma. However, the mechanism of AFLD has not been elucidated thoroughly, and there are still no efficient therapies in clinic. Notably, butyrate, one kind of short-chain fatty acids produced by gut microbiota, has been shown to improve methionine-choline-deficient diet-induced non-alcoholic steatohepatitis. And our previous study found that butyrate ameliorated endotoxemia in db/db mice. In this study, we aimed to explore the role of butyrate in the development of AFLD.

The aim of this study is to investigate the role and potential mechanism of p75NTR in mineralization in vivo using p75NTR-knockout mice and in vitro using ectomesenchymal stem cells (EMSCs).

The current study aims to investigate the effect of κ-opioid receptor (κ-OR) activation on sodium palmitate (SP)-induced human umbilical vein endothelial cells (HUVECs) inflammatory response and elucidate the underlying mechanisms.

To study the effects of tetramethylpyrazine (TMP) on the proliferation of hepatic stellate cells-T6 (HSC-T6), and the expression of connective tissue growth factor (CTGF) and Smad2/3 in these cells, HSC-T6 cells were cultured with TMP at different concentrations after transforming growth factor-β1 (TGF-β1) stimulation. MTT assay was used to assess the cell proliferation. Cells were divided into the control group, TGF-β1-treated group and TMP-treated groups, which were treated with different concentrations of TMP. Immunocytochemistry and western blot were performed to detect the expression levels of CTGF and Smad2/3 in HSC-T6 cells. MTT analysis indicated that TMP significantly inhibited the proliferation of HSC-T6 cells, in dose-dependent and time-dependent manners. Immunocytochemistry detection and western blot showed that TMP could diminish TGF-β1-induced CTGF over-expression in HSC-T6 cells. Similarly, the enhancing effects of TGF-β1 on Smad2/3 expressions in HSC-T6 cells could also be counteracted by TMP treatment. Nuclear translocation of Smad2/3 was blocked by TMP treatment. Correlation analysis suggested a positive correlation between CTGF and Smad2/3 expression levels in HSC-T6 cells. TMP exerts anti-hepatic fibrosis effect through decreasing the expression of CTGF and Smad2/3, as well as inhibiting the proliferation of HSC-T6 cells. Our study provides cellular and molecular bases for further application of TMP in the clinical treatment for hepatic fibrosis.

PTEN-induced putative kinase 1 (PINK1) is a Ser/Thr kinase that specifically localizes on the mitochondrial membrane. It cooperates with Parkin to regulate mitochondrial quality control. Mutations in PINK1 protein which account for 8-15% of Parkinson's disease (PD), are the second most common cause of early-onset Autosomal Recessive Parkinson's disease (AR-PD). The lack of methods for PINK1 heterologous expression and purification has slowed progress in the AR-PD research field. To pave the way for direct structural study of this important protein, in this study, we developed an efficient expression system of recombinant PINK1 kinase domain (rPINK1) using Pichia pastoris (P. pastoris). Our results showed that rPINK1 is best expressed in P. pastoris at 25 °C induction. Additionally, we determined that the optimal induction time was 72 h and the optimal induction methanol concentration was 1% for the expression of rPINK1 in P. pastoris. Subsequent purification by Ni affinity chromatography (Ni-NTA) and cation-exchange chromatography (Mono S) produced the protein with purity higher than 95%. The pure rPINK1 was active to phosphorylate ubiquitin in a substrate phosphorylation assay. Overall, these studies provide the first effective method for heterologous expression and purification of the rPINK1 with a high purity. These findings can help contribute to further researches on the interactions study and biochemical characterization of PINK1.

Glucocorticoids have been widely used as anti-inflammatory therapies. The mechanisms of cortisol action in goat does with endometritis, however, have not been reported. The aim of this study was to investigate the mechanism of cortisol in modulation of effects of E. coli-induced endometritis in the does. Does (n = 24) were assigned to four groups (n = 6): control, E. coli, cortisol, and E. coli + cortisol groups. Does in the cortisol and E. coli + cortisol group were treated with cortisol from 3 days before E. coli inoculations occurred to 36 days post-partum. Does in the E. coli and inoculation groups were administered via intrauterine infusion E. coli O55 (109 CFU/mL) at 0 h. Physical indicators, macroscopic and microscopic changes in the endometrium, uterine secretion cytology and bacteriology were evaluated before (0 h) and at 6, 12, 24, 48, and 72 h after E. coli inoculation. The TLR4 and pro-inflammatory cytokine mRNA transcripts were detected using qPCR. The activations of NF-κB and MAPK signaling pathways were detected using Western blot procedures. As a result, cortisol inhibited the inflammatory response of does by reducing the clinical symptoms, morphological endometrial damage, % PMN in uterine secretions, relative abundance of inflammatory gene mRNA transcripts in the endometrium of does. Cortisol inhibited NF-κB activity by reducing MyD88 and IκB phosphorylation. Treatment with cortisol suppressed the phosphorylation of ERK1/2, p38MAPK, and JNK. These results indicate the anti-inflammatory effect of cortisol in the endometrium of does may be regulated by NF-κB and MAPK pathways.

Postpartum uterine infections are common reproductive diseases in postpartum cows. Evidence has shown that plasma β-endorphins increase during bovine uterine inflammation. However, the effect of β-endorphins on the inflammatory response in bovine endometrium has not been clarified. The aim of this study was to investigate the effect of β-endorphins on the inflammatory response of bovine endometrial epithelial and stromal cells, and to explore the possible mechanism. The cells were treated with E. coli lipopolysaccharide (LPS) to simulate inflammation, which was characterized by the significant activation of NF-κB signaling pathway and the increased gene expression of the downstream proinflammatory cytokines (approximately 1.2- to 15-fold increase, P < 0.05). By using Western blot and qPCR techniques, we found that β-endorphins inhibited the key protein expression of NF-κB pathway, and the gene expressions of TNF, IL1B, IL6, CXCL8, nitric oxide synthase 2, and prostaglandin-endoperoxide synthase 2 (P < 0.05). The co-treatment of β-endorphins and opioid antagonists showed that the anti-inflammatory effect of β-endorphins could be blocked (P < 0.05) by non-selective opioid antagonist naloxone or δ opioid receptor antagonist ICI 154129, but not the μ opioid receptor antagonist CTAP (P > 0.05). In conclusion, β-endorphins may inhibit the inflammatory response of bovine endometrial epithelial and stromal cells through δ opioid receptor.

Progesterone suppresses the innate immune function of bovine endometrium, making the uterus susceptible to bacterial infection. The bovine endometrial epithelial cells (BEEC) are the first line of defense against bacteria, such as Escherichia coli (E.coli) that causes inflammation of endometrium through the recognition of lipopolysaccharide (LPS). The aim of this study was to investigate the effect of progesterone on the inflammatory response and its potential mechanism using E.coli- or LPS-induced BEEC. Concentrations of 1, 3 and 5 ng/mL progesterone were selected. The mRNA expressions of proinflammatory cytokines were determined using qPCR. The activations of NF-κB and MAPK pathways were detected by Western blot and immunofluorescence. An increase in the mRNA expression of IL-1β, IL-6, IL-8 and TNF-α was observed in BEEC treated with progesterone, LPS or E.coli alone. Progesterone inhibited the E.coli- or LPS-induced gene expression of these cytokines. Progesterone treatment alone showed little influence on NF-κB or MAPK pathway. In BEEC stimulated with E.coli or LPS, progesterone inhibited the phosphorylations of IκB, p65, ERK1/2, p38MAPK and JNK, and the translocation of p65 into the nucleus. These results suggested that progesterone has anti-inflammatory effect, which may be mediated by inhibiting NF-κB activation and MAPK phosphorylation in BEEC.

Human zinc finger protein 32 (ZNF32) is a Cys2-His2 zinc-finger transcription factor that plays an important role in cell fate, yet much of its function remains unknown. Here, we reveal that the zebrafish ZNF32 homologue zfZNF32 is expressed in the nervous system, particularly in the lateral line system. ZfZNF32 knock-out zebrafish (zfZNF-/-) were generated using the CRISPR-associated protein 9 system. We found that the regenerative capacity of the lateral line system was increased in zfZNF-/- upon hair cell damage compared with the wild type. Moreover, SOX2 was essential for the zfZNF32-dependent modulation of lateral line system regeneration. Mechanistic studies showed that ZNF32 suppressed SOX2 transcription by directly binding to a consensus sequence (5'-gcattt-3') in the SOX2 promoter. In addition, ZNF32 localizes to the nucleus, and we have identified that amino acids 1-169 (Aa 1-169) and each of three independent nuclear localization signals (NLSs) in ZNF32 are indispensable for ZNF32 nuclear trafficking. Mutating the NLSs disrupted the inhibitory effect of ZNF32 in SOX2 expression, highlighting the critical role of the NLSs in ZNF32 function. Our findings reveal a pivotal role for ZNF32 function in SOX2 expression and regeneration regulation.

Dexamethasone (DXM) has an anti-immunoinflammatory effect, and is often used in acute kidney injury (AKI). However, the effects of DXM on albumin (ALB) have not been fully studied.

Let-7 family microRNAs have been reported to be downregulated in human hepatocellular carcinoma in comparison with normal hepatic tissues. Among them, let-7g was identified as the lowest expression using real-time RT-PCR. However, the mechanism by which let-7g works in hepatocellular carcinoma remains unknown. Here, in our present study, we have had let-7g reexpressed in vitro in hepatocellular carcinoma cell lines MHCC97-H and HCCLM3 via transfection. The proliferation after reexpression of let-7g was assayed using MTT method; the migration and invasion after restoration were detected by wound-healing and Transwell assay, respectively. We found using Western-blotting that let-7g can regulate epithelial-mesenchymal transition (EMT) by downregulating K-Ras and HMGA2A after reexpresssion. Xenografted nude mice were used to observe whether or not reexpression of let-7g could have potential therapeutic ability. In vivo, to observe the association with let-7g expression and overall prognosis, 40 paired cases of hepatocellular carcinoma were analyzed using in situ hybridization (ISH). It was found that reexpression of let-7g can inhibit the proliferation, migration, and invasion significantly, and that low expression of let-7g was significantly associated with poorer overall survival. Taken together, let-7g could be used as a promising therapeutic agent in vivo in the treatment of hepatocellular carcinoma at the earlier stage.

This study was designed to test the hypothesis that κ-opioid receptor (κ-OR) stimulation reduces palmitate-induced HUVECs apoptosis and to investigate its mechanisms.