An Xiang

Hepatitis B viral core protein disrupts human host gene expression by binding to promoter regions

BackgroundThe core protein (HBc) of hepatitis B virus (HBV) has been implicated in the malignant transformation of chronically-infected hepatocytes and displays pleiotropic functions, including RNA- and DNA-binding activities. However, the mechanism by which HBc interacts with the human genome to exert effects on hepatocyte function remains unknown. This study investigated the distribution of HBc binding to promoters in the human genome and evaluated its effects on the related genes’ expression.ResultsWhole-genome chromatin immunoprecipitation microarray (ChIP-on-chip) analysis was used to identify HBc-bound human gene promoters. Gene Ontology and pathway analyses were performed on related genes. The quantitative polymerase chain reaction assay was used to verify ChIP-on-chip results. Five novel genes were selected for luciferase reporter assay evaluation to assess the influence of HBc promoter binding. The HBc antibody immunoprecipitated approximately 3100 human gene promoters. Among these, 1993 are associated with known biological processes, and 2208 regulate genes with defined molecular functions. In total, 1286 of the related genes mediate primary metabolic processes, and 1398 encode proteins with binding activity. Sixty-four of the promoters regulate genes related to the mitogen-activated protein kinase (MAPK) pathways, and 41 regulate Wnt/beta-catenin pathway genes. The reporter gene assay indicated that HBc binding up-regulates proto-oncogene tyrosine-protein kinase (SRC), type 1 insulin-like growth factor receptor (IGF1R), and neurotrophic tyrosine kinase receptor 2 (NTRK2), and down-regulates v-Ha-ras Harvey rat sarcoma viral oncogene (HRAS).ConclusionHBc has the ability to bind a large number of human gene promoters, and can disrupt normal host gene expression. Manipulation of the transcriptional profile in HBV-infected hepatocytes may represent a key pathogenic mechanism of HBV infection.

An aptamer-based immunoassay in microchannels of a portable analyzer for detection of microcystin-leucine-arginine

The rapid detection of microcystin-leucine-arginine (MC-LR), the most highly toxic among MCs, is significantly important to environmental and human health protection and prevention of MC-LR from being used as a bioweapon. Although aptamers offer higher affinity, specificity, and stability with MC-LR than antibodies in the immunodetection of MC-LR due to steric hindrance between two antibodies and limited epitopes of MC-LR for use in a sandwich immunoassay, no sandwich immunoassay using an aptmer has been developed for MC-LR detection. This study is aimed at developing an aptamer-antibody immunoassay (AAIA) to detect MC-LR using a portable analyzer. The aptamers were immobilized onto the glass surface of a microchamber to capture MC-LR. MC-LR and horseradish peroxidase (HRP)-labeled antibody were pulled into the microchamber to react with the immobilized aptamer. The chemiluminescence (CL) catalyzed by HRP was tested by a photodiode-based portable analyzer. MC-LR at 0.5-4.0 μg/L was detected quantitatively by the AAIA, with a CL signal sensitivity of 0.3 μg/L. The assay took less than 35 min for a single sample and demonstrated a high specificity, detecting only MC-LR, but not MC-LA, MC-YR, or nodularin-R. The recovery of two spiked real environmental samples calculated as 94.5-112.7%. Therefore, this AAIA was proved to be a rapid and simple method to detect MC-LR in the field by a single analyst.

The hepatitis B virus (HBV) core protein enhances the transcription activation of CRE via the CRE/CREB/CBP pathway.

We previously reported that hepatitis B virus core protein (HBc) can bind to the Enhancer I (Enh I) domain and can accumulate with transcription coactivator cAMP response element (CRE). This raises the possibility that HBc may interact with CRE/CREB and regulate CRE transcription activation. In this study, we investigated the function and mechanisms of HBc in regulating CRE transcriptional activation using the HepG2 cell line. Our results showed the following: (1) HBc expression significantly increases HBV CRE transcriptional activation; (2) phosphorylation of the serine residues in the arginine-rich domain (ARD) of HBc protein impacts the function of transcriptional activation by the CRE; (3) HBc protein significantly increases HBV CRE transcriptional activation following forskolin treatment; (4) HBc nonspecifically binds to CRE and enhances the binding of the cAMP response element-binding protein (CREB) to CRE; and (5) HBc increases the concurrent accumulation of CREB and CBP at the CRE region. HBc activates Enh I through its binding to CRE, increasing the concurrent accumulation of CREB/CBP on CRE, and thus increases CRE transcriptional activation.

A novel IgM–H-Ficolin complement pathway to attack allogenic cancer cells in vitro

The pentameric serum IgMs are critical to immune defense and surveillance through cytotoxicity against microbes and nascent cancer cells. Ficolins, a group of oligomeric lectins with an overall structure similar to C1q and mannose-binding lectin (MBL) participate in microbe infection and apoptotic cell clearance by activating the complement lectin pathway or a primitive opsonophagocytosis. It remains unknown whether serum IgMs interplay with ficolins in cancer immunosurveillance. Here we report a natural cancer killing of different types of cancer cells by sera from a healthy human population mediated by a novel IgM–H-ficolin complement activation pathway. We demonstrate for the first time that H-ficolin bound to a subset of IgMs in positive human sera and IgM–H-ficolin deposited on cancer cells to activate complement attack in cancer cells. Our data suggest that the IgM–H-ficolin -mediated complement activation pathway may be another defensive strategy for human cancer immunosurveillance.

Co-delivery nanocarriers targeting folate receptor and encapsulating 2-deoxyglucose and α-tocopheryl succinate enhance anti-tumor effect in vivo

A combination administration of chemical agents was highlighted to treat tumors. Recently, tumor cell has been found to be different from normal cell in metabolic manner. Most of cancer cells prefer aerobic glycolysis to mitochondrial oxidative phosphorylation (OXPHOS) to satisfy energy and biomass synthesis requirement to survive, grow and proliferate, which provides novel and potential therapeutic targets for chemotherapy. Here, 2-deoxy-d-glucose (2-DG), a potent inhibitor of glucose metabolism, was used to inhibit glycolysis of tumor cells; α-tocopheryl succinate (α-TOS), a water-insoluble vitamin E derivative, was chosen to suppress OXPHOS. Our data demonstrated that the combination treatment of 2-DG and α-TOS could significantly promote the anti-tumor efficiency in vitro compared with administration of the single drug. In order to maximize therapeutic activity and minimize negative side effects, a co-delivery nanocarrier targeting folate receptor (FR) was developed to encapsulate 2-DG and α-TOS simultaneously based on our previous work. Transmission electron microscope, dynamic light scattering method and UV-visible spectrophotometers were used to investigate morphology, size distribution and loading efficiency of the α-TOS-2-DG-loaded and FR-targeted nanoparticles (TDF NPs). The TDF NPs were found to possess a layer-by-layer shape, and the dynamic size was <100 nm. The final encapsulation efficiencies of α-TOS and 2-DG in TDF NPs were 94.3%±1.3% and 61.7%±7.7% with respect to drug-loading capacities of 8.9%±0.8% and 13.2%±2.6%, respectively. Almost no α-TOS release was found within 80 h, and release of 2-DG was sustained and slow within 72 h. The results of FR binding assay and fluorescence biodistribution revealed that TDF NPs could target FR highly expressed on tumor cell in vitro and in vivo. Further, in vivo anti-tumor experiments showed that TDF NPs had an improved biological function with less toxicity. Thus, our work indicates that the co-delivery TDF NPs have a great potential in tumor therapy.

MOTS-c peptide increases survival and decreases bacterial load in mice infected with MRSA

ABSTRACT Sepsis is a life‐threatening disease characterized by uncontrolled inflammatory responses upon pathogen infections, especially for the antibiotic‐resistant strains, such as Methicillin‐resistant S. aureus (MRSA). Here we demonstrated that a Mitochondria‐derived peptide (MOTS‐c) could significantly improve the survival rate and decrease bacteria loads in MRSA‐challenged mice, accompanied with declined levels of pro‐inflammatory cytokines, such as TNF‐&agr;, IL‐6 and IL‐1&bgr;, but with increased level of anti‐inflammatory cytokine IL‐10. Moreover this peptide enhanced bactericidal capacity of macrophages. Meanwhile, MOTS‐c inhibited the phosphorylation mitogen‐activated protein kinases (MAPK), and enhanced the expression of aryl hydrocarbon receptor (AhR) and signal transducer and activator of transcriptional 3 (STAT3) in macrophages. Overall, MOTS‐c plays a beneficial role in curbing the overwhelming inflammatory bursts in the fight against MRSA infection. It may serve as a potential therapeutic agent in sepsis treatment. HighlightMOTS‐c improved survival status in mice during MRSA infection.MOTS‐c strongly enhanced bactericidal capacity of macrophages.MOTS‐c exerted an anti‐inflammatory effect via suppressing MAPKs and increasing Ahr/STAT3 signaling pathways.

A novel combined capillary chip for rapid identification of gene mutation

To develop a simple micro-platform for gene mutation detection, we used a DNA hybridization approach in a combined microchannel. A glass groove immobilized with oligonucleotide probes by UV-crosslinking, and encased in a transparent heat-shrinkable polythene tube. After heat treatment, the polythene tube stretched tightly over the glass groove and was designated as a combined capillary chip (CCC). The CCC assay was optimized with 10 μL reaction solutions shuttling back and forth at 50 μL min−1 for 10 min, give a detection minimum of 0.1 nM target DNA sequences. In hepatitis B virus (HBV) YMDD mutations detection, 61.3% (92/150) was of a single genotype and it was authenticated with sequence analysis. The other 38.7% (58/150) was mixed genotype detected, but 18.9% (11/58) has a missed diagnosis in sequence analysis. It proved a higher sensitivity than sequence analysis. The CCC assay has a simple fabricating process, simple structure and higher specificity in gene mutation detection. These features are promising for clinical gene mutation analysis.

Azoramide, a novel regulator, favors adipogenesis against osteogenesis through inhibiting the GLP-1 receptor-PKA-β-catenin pathway

BackgroundThe reciprocal fate decision of mesenchymal stem cells (MSCs) to either bone or adipocytes is determined by Wnt-related signaling and the glucagon-like peptide-1 receptor (GLP-1R). Azoramide, an ER stress alleviator, was reported to have an antidiabetic effect. In this study, we investigated the function of azoramide in regulating the lineage determination of MSCs for either adipogenic or osteogenic differentiation.MethodsIn this study, microcomputed tomography and histological analysis on bone morphogenetic protein (BMP)2-induced parietal periosteum bone formation assays, C3H10T1/2 and mouse bone marrow MSC-derived bone formation and adipogenesis assays, and specific staining for bone tissue and lipid droplets were used to evaluate the role of azoramide on the lineage determination of MSC differentiation. Cells were harvested for Western blot and quantitative real-time polymerase chain reaction (PCR), and immunofluorescence staining was used to explore the potential mechanism of azoramide for regulating MSC differentiation.ResultsBased on MSC-derived bone formation assays both in vivo and in vitro, azoramide treatment displayed a cell fate determining ability in favor of adipogenesis over osteogenesis. Further mechanistic characterizations disclosed that both the GLP-1R agonist peptide exendin-4 (Ex-4) and GLP-1R small interfering (si)RNA abrogated azoramide dual effects. Moreover, cAMP-protein kinase A (PKA)-mediated nuclear β-catenin activity was responsible for the negative function of azoramide on bone formation in favor of adipogenesis.ConclusionsThese data provide the first evidence to show that azoramide may serve as an antagonist against GLP-1R in MSC lineage determination.

Androgen receptor‑mediated upregulation of quaking affects androgen receptor‑related prostate cancer development and anti‑androgen receptor therapy

The androgen receptor (AR) has a crucial role in prostate cancer. RNA-binding protein-mediated post-transcriptional regulation is important in the initiation and development of cancer. The present study attempted to elucidate the mutual association of AR and RNA-binding protein quaking (QKI) in the development of prostate cancer. Dual-luciferase reporter demonstrated that AR can positively regulate the expression of QKI in prostate cancer cell lines due to its effective transcription regulating function. In addition, QKI may increase expression of AR by heat shock protein 90, which is a coactivator of AR, and silencing QKI can increase the sensitive of Casodex, which is an antagonist of AR in castration-resistant prostate cancer. This may be a new strategy for advanced prostate cancer.

H-Ficolin-IgM initiates complement-dependent attack against cancer cells

The pentameric serum IgMs are critical to immune defense and surveillance through cytotoxicity against microbes and nascent cancer cells. Ficolins, a group of oligomeric lectins with an overall structure similar to C1q and mannose-binding lectin (MBL) participate in microbe infection and apoptotic cell clearance by activating the complement lectin pathway or a primitive opsonophagocytosis. It remains unknown whether serum IgMs interplay with ficolins in cancer immunosurveillance. Here we report a natural cancer killing of different types of cancer cells by sera from a healthy human population mediated by a novel IgM–H-ficolin complement activation pathway. We demonstrate for the first time that H-ficolin bound to a subset of IgMs in positive human sera and IgM–H-ficolin deposited on cancer cells to activate complement attack in cancer cells. Our data suggest that the IgM–H-ficolin -mediated complement activation pathway may be another defensive strategy for human cancer immunosurveillance.