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Available evidence suggests the involvement of microRNAs (miRNAs) in the pathological process of several diseases. Nonetheless, molecular mechanism underlying biological effects of miRNAs such as pacemaker exosome-derived miR-127-5p in embryonic-like stem cells (ESCs) differentiation into pacemaker cell is yet to be clarified. Through real time quantitative polymerase chain reaction (qPCR) or western blotting (WB) techniques, levels of miRNAs, miR-127-5p, and NKx2.5 expressions were quantitatively measured. Cellular differentiation (CD) was probed with flow cytometric (FC) and WB techniques. Prediction of miR-127-5p association with NKx2.5 was studied through bioinformatics tools before verification with luciferase assays. Promotion of ESCs differentiation to pacemaker through miR-127-5p was measured with qPCR and WB techniques. Through the same assaying methods, up-regulation of pace-making genes (Shox2, HCN4, Cx45, Tbx3 and Tbx18) expression was observed in Nkx2.5 knockdown group. However, Nkx2.5 expression was down-regulated during differentiation of pacemaker-like cells into ESCs. Furthermore, techniques (such as qPCR, WB, immunofluorescent staining and FC) were employed to demonstrate that overexpressed miR-127-5p could suppress NKx2.5 expression. Through NKx2.5 targeting, ESCs could be differentiated into pacemaker-like cells with miR-127-5p possibly serving as a crucial positive regulator. On the account of our findings, further researches are needed to unearth the possible underlying mechanism and role of exosome-derived miRNAs in cell signaling. • MiR-127-5p could act as a significant positive regulator in ESCs differentiated into pacemaker-like cells by targeting NKx2.5. • NKx2.5 expression was down-regulated during differentiation of pacemaker-like cells into ESCs. • This work is the first to describe the role of miR-127-5p in the regulation of NKx2.5 expression in cardiac development. [ABSTRACT FROM AUTHOR]

Highlights: [•] miR-612 suppresses tumorsphere and clone formation of HCC cells. [•] miR-612 reduces drug resistance of HCC cells. [•] miR-612 suppresses tumorigenesis of HCC in NOD/SCID mice. [•] miR-612 inhibits an invasive frontier of HCC xenografts. [•] miR-612 suppresses Wnt/β-catenin signaling. [Copyright &y& Elsevier]

Abstract: Daxx is a multifunctional protein, regulating a wide range of important functions including apoptosis and transcription. However, the way Daxx is regulated is poorly understood. In our previous studies, we have found that Daxx forms a complex with the E3 ubiquitin ligase Mdm2 and the de-ubiquitinase Hausp. In the present work, we show that Daxx is ubiquitinated by Mdm2 in both in vitro and in vivo systems and Mdm2 reduces Daxx expression upon over-expression. We further demonstrate that Hausp critically controls the cellular level of Daxx most likely by inducing Daxx de-ubiquitination. These results reveal Mdm2 and Hausp as important regulators for Daxx functions by controlling Daxx ubiquitination and stability. [Copyright &y& Elsevier]

Abstract: In addition to the subventricular zone, the dentate gyrus of the hippocampus is one of the few brain regions in which neurogenesis continues into adulthood. Perturbation of neurogenesis can alter hippocampal function, and previous studies have shown that neurogenesis is dysregulated in Alzheimer disease (AD) brain. Bone morphogenetic protein-4 (BMP4) and its antagonist Noggin have been shown to play important roles both in embryonic development and in the adult nervous system, and may regulate hippocampal neurogenesis. Previous data indicated that increased expression of BMP4 mRNA within the dentate gyrus might contribute to decreased hippocampal cell proliferation in the APPswe/PS1ΔE9 mouse AD model. However, it is not known whether the BMP antagonist Noggin contributes to the regulation of neurogenesis. We therefore studied the relative expression levels and localization of BMP4 and its antagonist Noggin in the dentate gyrus and whether these correlated with changes in neurogenesis in 6–12mo old APPswe/PS1ΔE9 transgenic mice. Bromodeoxyuridine (BrdU) was used to label proliferative cells. We report that decreased neurogenesis in the APP/PS1 transgenic mice was accompanied by increased expression of BMP4 and decreased expression of Noggin at both the mRNA and protein levels; statistical analysis showed that the number of proliferative cells at different ages correlated positively with Noggin expression and negatively with BMP4 expression. Intraventricular administration of a chimeric Noggin/Fc protein was used to block the action of endogenous BMP4; this resulted in a significant increase in the number of BrdU-labeled cells in dentate gyrus subgranular zone and hilus in APP/PS1 mice. These results suggest that BMP4 and Noggin co-modulate neurogenesis. [Copyright &y& Elsevier]

The epidermal growth factor receptor (EGFR) is known to bind to the F-actin cytoskeleton in intact cells, and this interaction has been suggested to sequester the EGFR signal transduction system to specific loci within the cell. In this study, the interaction of the EGFR with actin is examined in a reconstituted cell free system. Soluble protein components of the cytosol from A431 cells are shown to dramatically enhance binding of the EGFR to F-actin in a saturable, concentration-dependent fashion. Most of the intracellular C-terminal portion of the EGFR is found to be required for this interaction. Binding of the EGFR to F-actin strongly deactivates the receptor by diminishing EGFR autophosphorylation activity and enhancing tyrosine phosphatase activity toward the EGFR. These results suggest that a ternary or larger protein signaling complex forms on an F-actin cellular scaffold, providing a spatially restricted signal modulation site that can be regulated by cytoskeletal remodeling. [Copyright &y& Elsevier]

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) are dynamic and multiprotein complexes implicated in a variety of important biochemical events. Due to alternative mRNA splicing, PML has at least six nuclear isoforms that share a common N-terminus but differ in their C-terminal regions. However, the unique role of each PML isoform is not clear. Here, we report the characterization of the deubiquitinase ataxin-3 as a specific binding partner of PML isoform II (PML-II). Ataxin-3 was identified as a potential binding protein of PML-II in a yeast-hybrid screen employing the unique C-terminal region of PML-II as bait. Ataxin-3 only binds to the C-terminal region of PML-II and not that of other PML isoforms. The interaction between ataxin-3 and PML-II was confirmed by co-immunoprecipition assays, and immunofluorescent microscopy revealed that PML-II and ataxin-3 were co-localized in PML-NBs. In addition, PML-II not only interacts with ataxin-3 with a normal range of poly-Q repeats (13Q), but also with a pathological form of ataxin-3 with extended poly-Q repeats (79Q). Importantly, the deubiquitinase activity of ataxin-3 was inhibited by PML-II. Our results suggest that PML-II may be a negative regulator of ataxin-3. • The C-terminus of PML-II interacts with ataxin-3. • PML-II interacts with the full-length ataxin-3 independent on the expansion of PolyQ. • PML-II recruits ataxin-3 to PML-NBs in PML KO cells. • PML-II inhibits the DUB activity of ataxin-3. [ABSTRACT FROM AUTHOR]

Abstract: Apoptin, a protein from chicken anemia virus, selectively induces apoptosis of transformed or tumor cells, but not in normal cells. However, the mechanism of action of Apoptin is still not well understood. Using yeast two-hybrid and immunoprecipitation approaches, we found that Apoptin interacted with Heat shock cognate protein 70 (Hsc70). In vivo, Apoptin induced the translocation of endogenous Hsc70 from the cytoplasm to the nucleus, and both were co-localized in the nucleus. In addition, Apoptin induced Akt phosphorylation, which was markedly inhibited by Hsc70 knockdown, suggesting that Hsc70 may play a critical role in Apoptin-induced Akt phosphorylation. These findings help to further understand the molecular mechanism of Apoptin. [Copyright &y& Elsevier]

Abstract: Schwann cells derived from peripheral nerve sheath tumors from individuals with Neurofibromatosis Type 1 (NF1) are deficient for the protein neurofibromin, which contains a GAP-related domain (NF1-GRD). Neurofibromin-deficient Schwann cells have increased Ras activation, increased proliferation in response to certain growth stimuli, increased angiogenic potential, and altered cell morphology. This study examined whether expression of functional NF1-GRD can reverse the transformed phenotype of neurofibromin-deficient Schwann cells from both benign and malignant peripheral nerve sheath tumors. We reconstituted the NF1-GRD using retroviral transduction and examined the effects on cell morphology, growth potential, and angiogenic potential. NF1-GRD reconstitution resulted in morphologic changes, a 16–33% reduction in Ras activation, and a 53% decrease in proliferation in neurofibromin-deficient Schwann cells. However, NF1-GRD reconstitution was not sufficient to decrease the in vitro angiogenic potential of the cells. This study demonstrates that reconstitution of the NF1-GRD can at least partially reverse the transformation of human NF1 tumor-derived Schwann cells. [Copyright &y& Elsevier]

Abstract Background Aberrant expression of interferon regulatory factor 4 (IRF4) has been reported in several hematologic malignancies. However, the prognostic significance of IRF4 expression in esophageal squamous cell carcinoma (ESCC) remains unknown. Methods IRF4 protein expression in ESCC tumor specimens was determined immunohistochemically. The correlation of IRF4 expression with clinico-pathological features was assessed from a cohort of 100 patients with primary ESCC. Kaplan-Meier and Cox proportional regression analyses were used to evaluate the association between IRF4 expression and patient survival. Results A Kaplan-Meier analysis indicated that patients with high IRF4 expression had a significantly longer overall survival rate than those with low IRF4 expression (p = 0.0006). Furthermore, multi-variate analyses revealed that IRF4 protein expression is an independent prognostic indicator for ESCC patients. Conclusion Our results suggest that increased IRF4 protein expression correlates with improved outcome in ESCC. IRF4 may therefore represent a promising prognostic biomarker and potential immuno-therapeutic target for patients with ESCC. Highlights • Increased expression of IRF4 is closely related to improved ESCC outcome. • IRF4 serve as an independent predictor of a positive prognosis in ESCC patients. • IRF4 may represent a promising prognostic biomarker and potential immuno-therapeutic target for ESCC patients. [ABSTRACT FROM AUTHOR]

Glycine has been shown to protect against ischemic stroke through various mechanisms. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) which antagonize Akt-dependent cell survival has been linked to neuronal damage. However, whether glycine has a neuroprotective property in intracerebral hemorrhage (ICH) was unknown. This study aimed to determine the protective effect of glycine in rats ICH. Adult male Sprague-Dawley (SD) rats were subjected to left striatum infusion of autologous blood. ICH animals received glycine (0.2–3 mg/kg, icv) at 1 h after ICH with or without pre-injection of Akt Inhibitor IV (100 μM, 2 μl, icv) 0.5 h prior to glycine treatment. Our results showed that in the perihematomal area PTEN was up-regulated in the early stage after ICH. However, glycine treatment decreased PTEN protein level and increased the phosphorylation level of AKT (p-AKT) in the perihematomal area. With the administration of glycine, neuronal death was significantly reduced and Evans blue leakage was alleviated as well as the brain edema after ICH. Moreover, hematoma volume was decreased and neurobehavioral outcome was improved. Nevertheless, Akt Inhibitor IV abolished the neuroprotective effects of glycine after ICH. Together, our findings demonstrate, for the first time, the protective role of glycine on ICH rats, and suggest that the neuroprotective effect of glycine was mediated through PTEN/Akt signal pathway. [ABSTRACT FROM AUTHOR]

Retroviral integrase catalyzes the integration of retroviral genome into host chromosomal DNA, which is a prerequisite of effective viral replication and infection. The human immunodeficiency virus type 1 (HIV-1) integrase has previously been reported to be regulated by the ubiquitination, but the molecular characterization of integrase ubiquitination is still unclear. In this study, we analyzed the ubiquitination of avian leukosis virus (ALV) integrase in detail. The ubiquitination assay showed that, like HIV-1, ALV integrase could also be modified by ubiquitination when expressed in 293 T and DF-1 cells. Domain mapping analysis revealed that the ubiquitination of ALV integrase might mainly occurred in the catalytic core and the N-terminal zinc-binding domains. Both lysine and non-lysine residues within integrase of ALV and HIV-1 were responsible for the ubiquitin conjugation, and the N-terminal HHCC zinc-binding motif might play an important role in mediating integrase ubiquitination. Interestingly, mass spectrometry analysis identified the Thr10 and Cys37 residues in the HHCC zinc-binding motif as the ubiquitination sites, indicating that ubiquitin may be conjugated to ALV integrase through direct interaction with the non-lysine residues. These findings revealed the detailed features of retroviral integrase ubiquitination and found a novel mechanism of ubiquitination mediated by the non-lysine residues within the N-terminal zinc-binding domain of integrase. [ABSTRACT FROM AUTHOR]

The retroviral integrase plays an essential role in the integration of reverse-transcribed retroviral cDNA into the host cell genome, and serves as an important target for anti-viral therapeutics. In this study, we identified the COP9 signalosome subunit 6 (CSN6) as a novel avian leukosis virus (ALV) integrase binding protein. Co-immunoprecipitation and GST pull-down assays showed that CSN6 bound to ALV integrase likely through direct interaction of CSN6 to the catalytic core of the integrase. We further demonstrated CSN6 inhibited integrase activity in vitro ; knockdown of CSN6 in DF-1 promoted ALV production. These results indicated that CSN6 may be a negative regulator of ALV replication by binding to and inhibiting integrase. Our findings provided the insight into the integrase-based host defense system and may have implications in the development of integrase-based anti-viral strategies. [ABSTRACT FROM AUTHOR]

Abstract: Pluripotent embryonic stem (ES) cells are the most versatile cells, with the potential to differentiate into all types of cell lineages including neural precursor cells (NPCs), which can be expanded in large numbers for significant periods of time to provide a reliable cell source for transplantation in neurodegenerative disorders such as Parkinson’s disease (PD). In the present study, we used the MESPU35 mouse ES cell line, which expresses enhanced green fluorescent protein that enables one to distinguish between transplanted cells and cells of host origin. Embryoid bodies (EBs) were formed and were induced to NPCs in N2 selection medium plus fibronectin. Praxiology and immunohistochemistry methods were used to observe the survival, differentiation, and therapeutic effect of NPCs after grafted into the striatum of PD rats. We found that mouse ESc were differentiated into nestin-positive NPCs 6 days after the EBs formed and cultured in the N2 selection medium. The number of survival NPCs was increased significantly by fibronectin. About 23.76±2.29% of remaining cells were tyrosine hydroxylase (TH)-positive 12 days after NPCs were cultured in N2 selective medium. The survival rates of NPCs were 2.10±0.41% and about 90.90±3.00% of the engrafted NPCs were TH-positive 6 weeks after transplantation into the striatum of PD rats. The rotation of PD rats was relieved 3 weeks after the NPCs transplantation and this effect was kept for at least 6 weeks. It suggests that most of the survival NPCs derived from ES cells differentiated into TH-positive neurons after grafted into the striatum of PD rats, which produces therapeutic effect on PD. [Copyright &y& Elsevier]