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Hepatocyte apoptosis is a crucial factor affecting liver quality in brain-dead donors. The identification of key molecular proteins involved in brain-death (BD)-induced hepatocyte apoptosis may help determine an effective method for improving the quality of livers from brain-dead donors. In this study, we used in vivo and in vitro models to investigate the role of chitinase-3-like protein 1 (CHI3L1) in promoting liver cell apoptosis after BD. Chitin was used to inhibit CHI3L1 in a rat model of BD. Macrophage polarization of THP-1 cells and hypoxia/reoxygenation (H/R) of LO-2 cells were used to mimic BD-induced cell stress in liver. We found that CHI3L1 played a vital role in promoting liver cell apoptosis. Six hours after BD, CHI3L1 expression was significantly upregulated in liver macrophages and was associated with BD-induced M1 polarization of these cells. In liver cells cultured under H/R conditions, recombinant CHI3L1 activated the protease-activated receptor 2 (PAR2)/c-June N-terminal kinase (JNK) apoptotic pathway and aggravated apoptosis. Compared with the control group, chitin particles inhibited the expression of CHI3L1 in the liver of brain dead rats, thereby reducing activation of the hepatocyte surface receptor, PAR2, and its downstream JNK/caspase-3 signaling pathway, ultimately reducing hepatocyte apoptosis. In conclusion, our results indicate that CHI3L1 relies on a PAR2/JNK-mediated mechanism to promote BD-induced hepatocyte apoptosis. • CHI3L1 upregulated in liver macrophages after Brain death (BD). • CHI3L1 upregulation associated with BD-induced M1 polarization. • CHI3L1 activated PAR2/JNK pathway, promotes BD-induced hepatocyte apoptosis. • Chitin inhibits CHI3L1 expression and PAR2/JNK/caspase-3 signaling pathway. • Chitin reduces hepatocyte apoptosis. [ABSTRACT FROM AUTHOR]

Bacterial resistance has become a serious threat to human health. In particular, the gradual development of resistance to polymyxins, the last line of defense for human infections, is a major issue. Secreted proteins contribute to the interactions between bacteria and the environment. In this study, we compared the secretomes of polymyxin B-sensitive and -resistant Escherichia coli strains by data-independent acquisition mass spectrometry. In total, 87 differentially expressed secreted proteins were identified in polymyxin B-resistant E. coli compared to the sensitive strain. A GO enrichment analysis indicated that the differentially expressed proteins were involved in biological processes, including bacterial-type flagellum-dependent cell motility, ion transport, carbohydrate derivative biosynthetic process, cellular response to stimulus, organelle organization, and cell wall organization or biogenesis. The differentially expressed secreted proteins in polymyxin B-resistant bacteria were enriched for multiple pathways, suggesting that the resistance phenotype depends on complex regulatory mechanisms. A potential biomarker or drug target (YebV) was found in polymyxin B-resistant E. coli. This work clarifies the secretome changes associated with the acquisition of polymyxin resistance and may contribute to drug development. • Quantitative secretome analysis of polymyxin B-resistant E. coli was firstly reported. • Polymyxins resistance is closely related to the improvement of antioxidant capacity. • Polymyxin B-resistant E. coli highly express YebV outside the cell. [ABSTRACT FROM AUTHOR]

Ginsenoside Rh2 (G-Rh2), a component of ginseng extraction, exerted anti-tumor property in the occurrence and progress of human tumors. Vestibular schwannoma (VS) is a kind of benign tumor. Extraction of traditional Chinese herb has been applied to treat VS as adjuvant therapy. Nevertheless, G-Rh2-related molecular mechanisms in VS progress are not yet clear. The purpose of current study is to unveil the function and potential molecular mechanism of Rh2 in VS cellular functions. At first, the viability and apoptosis of VS cells treated with different concentrations of Rh2 were assessed. Autophagy and DNA damage response can be induced by multiple drugs. Here, we observed the changes of autophagy and DNA damage in Rh2-induced VS cells. Based on the experimental data, treatment with Rh2 contributed to cell apoptosis by inducing DNA damage and suppressing DNA damage. LAMP2 (lysosomal associated membrane protein 2), an autophagy inducer, was downregulated in Rh2-treated VS cells. Through mechanism study, we determined that Rh2 led to the transcriptional inactivation of LAMP2 by downregulating its transcription activator NR2F2 (nuclear receptor subfamily 2 group F member 2). In addition, NR2F2 overexpression recovered the role of Rh2 in cell functions, which was further rescued by the silence of LAMP2. Collectively, our study unveiled a novel NR2F2/LAMP2 axis in Rh2-mediated VS cells, which potentially contributes to the therapy for VS. Image 1 • Rh2 induced apoptosis of primary VS cells by regulating DNA damage and autophagy. • LAMP2 was downregulated in Rh2-induced primary VS cells and its recovery attenuated Rh2-mediated cellular functions. • NR2F2 positively regulated LAMP2 expression through transcriptional activation. • NR2F2/LAMP3 axis regulated Rh2-mediated functions of primary VS cells. [ABSTRACT FROM AUTHOR]

Abstract Recent clinical researches demonstrated "obesity paradox" in chronic obstructive pulmonary disease (COPD) patients. However, why obesity is beneficial to COPD development remains unclear. Obesity is distinguished by hyperinsulinemia, and cellular senescence of airway epithelial cells (AEC) is involved in COPD progression. In this study, we aim to investigate the roles of insulin in high concentration in AEC cellular senescence. Human bronchial epithelial cells (BEAS-2B) and small airway epithelial cells (HSAEpiC) were used for experiments and treated with insulin in indicated time period and concentration with or without 1% cigarette smoke extract (CSE) exposure, and autophagy activator (rapamycin) or inhibitor (chloroquine) after exposed to 1% CSE. The expression of senescence protein (p21), cell cycle related protein (cyclinE), and autophagy protein (LC3B, Beclin-1 and p62) were determined by western blot. Cellular senescence was detected by Senescence-β-Galactosidase staining. Autophagy level was detected by GFP-LC3B fluorescent light. Results showed that insulin receded cellular senescence in different time and concentration, as indicated by decreased expression of p21, increased expression of cyclinE and down-regulated ratio of SA-β-Gal stained cells in BEAS-2B and HSAEpiC under physiological or CSE exposure condition. 5 μg/ml insulin treated for 48 h up-regulated the ratio of LC3B II/I, Beclin-1 and number of spot dots of GFP-LC3B, and down-regulated the level of p62 in AEC cells after 1% CSE exposure, while 1% CSE showed an opposite effect. Activating autophagy receded cellular senescence, and vice versa. Meanwhile, the effect of 5 μg/ml insulin on receding cellular senescence could be alleviated by autophagy inhibitor chloroquine or reinforced by autophagy activator rapamycin. In conclusion, insulin in high concentration could reduce the CSE induced cellular senescence of human airway epithelial cells through autophagy pathway. Highlights • Cellular senescence in human epithelial cells wasreducedby high concentration of insulinwith or without CSE exposure. • Autophagy was activated by high concentration of insulinin human epithelial cellsafter CSE exposure. • Cellular senescence was modulatedby autophagyin human epithelial cells. • Insulin could reduce cellular senescence in human epithelial cells though autophagy pathway. [ABSTRACT FROM AUTHOR]

Abstract: The epithelial Ca2+ channel TRPV6 constitutes the apical Ca2+ entry mechanism in active Ca2+ transport in kidney and intestine, but little is known about regulation mechanism of TRPV6. We performed yeast two-hybrid screen with TRPV6 C-terminus since TRPV6 has PDZ (Post-synaptic density-95, Drosophila discs-large protein, Zonula occludens protein 1) binding motif at its C terminal end. As a result, we found that 4 PDZ domain-containing protein, PDZK2, interacts with TRPV6 through its fourth PDZ domain. Glutathione S-transferase pull-down assay shows that TRPV6 and PDZK2 directly interact and that TRPV6 C-terminal PDZ binding motif is primarily responsible for this interaction. Mutant Δ4 lacking last 4 amino acid EYQI did not interact with PDZK2. Heterologous overexpression of both TRPV6 and PDZK2 did not show any effect on the activation of TRPV6. On the other hand, the peak current amplitude of mutant Δ4 decreased compared with that of WT TRPV6. When introduced into HEK293 cells expressing TRPV6, PDZ binding motif peptide (EYQI) markedly reduced the peak current amplitude in divalent free (DVF) solution. Knocking down the endogenous PDZK2 of HEK293 cells by RNAi significantly decreased DVF current density. Taken together, we propose that PDZK2 is an essential TRPV6 interacting protein as a physiological modulator of TRPV6. [Copyright &y& Elsevier]

Abstract: Dizocilpine maleate (MK-801) causes the blockage of the glutamic acid (Glu) receptors in the central nervous system that are involved in pain transmission. However, the mechanism of action of MK-801 in pain-related neurons is not clear, and it is still unknown whether Glu is involved in the modulation of this processing. This study examines the effect of MK-801, Glu on the pain-evoked response of pain-excitation neurons (PENs) and pain-inhibition neurons (PINs) in the nucleus accumbens (NAc) of rats. The trains of electric impulses applied to the sciatic nerve were used as noxious stimulation. The electrical activities of PENs or PINs in NAc were recorded by a glass microelectrode. Our results revealed that the lateral ventricle injection of Glu increased the discharged frequency and shortened the discharged latency of PEN, and decreased the discharged frequency and prolonged the discharged inhibitory duration (ID) of PIN in NAc of rats evoked by the noxious stimulation, while intra-NAc administration of MK-801 produced the opposite response. On the basis of above findings we can deduce that Glu, MK-801 and N-methyl-d-aspartate (NMDA) receptor are involved in the modulation of nociceptive information transmission in NAc. [Copyright &y& Elsevier]

Affinity is an important property of therapeutic antibodies, so improving affinity is critical to the biological activity and clinical efficacy. An anti–HIF–1α nanobody, VHH212, was screened via a native ribosome display library with a 26.6 nM of K D value was used as the parent. In this paper, a Venn-intersection of multi-algorithms screening (VIMAS) strategy for computer-aided binding affinity prediction was designed. Homology modeling and protein docking methods were used to substitute the need for a crystal structure. Finally, a mutant with a 17.5-fold enhancement in binding affinity (1.52 nM) was obtained by using the VIMAS strategy. Furthermore, the biological activity of mutants was verified at the cellular level. Targeting HIF-1α can sensitize PDAC (pancreatic ductal adenocarcinoma) tumors to gemcitabine, which is a potential co-treatment method for pancreatic cancer patients. Our results showed that the cytotoxicity of gemcitabine on pancreatic cancer cell lines increased with the enhanced-affinity of an intrabody under combined treatment. • Provide a strategy for in silico affinity maturation of nanobodies. • Affinity of mutants increased via computer-aided design. • Nanobody-based intrabodies provide a specific strategy for combined therapy. • Targeting HIF-1α with nanobody increased the cytotoxicity of gemcitabine to MIA PaCa-2 and BxPC-3 cell lines. [ABSTRACT FROM AUTHOR]

Aberrant DNA methylation of various genes has been identified to be associated with disease progression in chronic myeloid leukemia (CML). Our study was intended to investigate DLX4 methylation pattern in different clinical stages of CML and further determine its role in regulating DLX4 expression. Real-time quantitative methylation-specific PCR and bisulfite sequencing PCR were applied to detect DLX4 methylation. 5-aza-2′-deoxycytidine (5-aza-dC) was used for demethylation studies. DLX4 was significantly hypermethylated in CML patients ( P = 0.002) especially in blastic phase (BC) stage ( P < 0.001) as compared with controls. Moreover, DLX4 methylation level in BC stage was significantly higher than in chronic phase (CP) stage ( P < 0.001). DLX4 methylation density was significantly increased during the progression of CML among the tested two patients ( P < 0.001). DLX4 hypermethylation occurred with the highest incidence in BC stage (83%), lower incidence in acute phase (AP) stage (43%), and the lowest incidence in CP stage (26%) ( P = 0.001). Moreover, t(9; 22) with additional alteration cases had significantly higher frequency of DLX4 hypermethylation compared with the other cytogenetics ( P = 0.010). Significantly negative correlation was observed between DLX4 methylation and DLX4-TV2 (the shorter DLX4 isoform) expression (R = −0.382, P = 0.001, n = 78) but not between DLX4 methylation and BP1 (the longer DLX4 isoform) expression (R = 0.134, P = 0.244, n = 78) in CML patients. Both DLX4-TV2 and BP1 mRNA were significantly increased after 5-aza-dC treatment in K562 cell line ( P < 0.001). Our study indicated that hypermethylation of DLX4 correlated with disease progression of CML. Moreover, DLX4 expression was regulated by its methylation in CML. [ABSTRACT FROM AUTHOR]

Highlights: [•] PARP1 siRNA enhances docetaxel’s activity against PC3 cells. [•] PARP1 siRNA enhances docetaxel’s activity against EGFR/Akt/FOXO1 pathway. [•] PARP1 siRNA and PARP1 inhibitor differently affect the phosphorylation and expression of FOXO1. [ABSTRACT FROM AUTHOR]

Abstract: MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules that suppress gene expression via degradation or translational inhibition of their target genes. Many miRNAs are associated with cardiac hypertrophy and heart failure. In this study, we pursued to identify miRNAs that negatively regulate cardiac hypertrophy by utilizing a surgical model for regression of cardiac hypertrophy. Microarray analysis revealed that 15 miRNAs out of the 696 miRNAs tested were specifically up-regulated during the regression period. Among these regression-specific miRNAs, nine microRNAs, which have not been previously reported, were further tested for their effects on phenylephrine (PE)-treated neonatal cardiomyocytes. Consequently, five miRNAs (miR-101b, 142-3p, 181d, 24-2∗, and 450a) completely abrogated PE-induced hypertrophy as determined by measurements of cell size and fetal gene expression. Conversely, antagomers of these miRNAs exacerbated the PE-induced hypertrophy. Collectively, these findings suggest that the five miRNAs newly identified by using our cardiac hypertrophy-regression surgical model negatively regulate cardiac hypertrophy and could be used as potential therapeutic targets for the treatment of heart diseases. [Copyright &y& Elsevier]