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Steroid receptor coactivator 1 (Src-1) and Twist1 are aberrantly upregulated in a variety of tumors and play an important role in tumor progression. However, the exact role of Src-1 and Twist1 in nasopharyngeal carcinoma (NPC) is uncertain. In this study, we investigated the possible prognostic value and biological effect of Src-1 and Twist1 in NPC. Src-1 and Twist1 expression was detected in a cohort of NPC patients (n = 134) by qRT-PCR. Kaplan-Meier survival analysis was used comparing overall survival (OS) and progression-free survival (PFS). Multivariate analysis was performed using the Cox proportional hazard regression model. Biologic effect of Src-1 and Twist1 in NPC cell lines was evaluated by western blot, colony formation assay, soft agar assay, scratch wound healing assay, transwell invasion assay and tumor xenografts growth. We have found that Src-1 and Twist1 were aberrantly upregulated in human NPC tissues, and associated with advanced tumor stage, distant metastasis and unfavorable prognosis. Knockdown of Src-1 or Twist1 in human NPC cell line CNE-1 suppressed colony formation, anchorage-independent growth, cell migration, invasion and tumor xenografts growth, while enforced expression of Src-1 or Twist1 in human NPC cell line HNE-2 promotes anchorage-independent growth, cell migration and invasion. In addition, Src-1 and Twist1 could suppress E-cadherin expression and increase Vimentin expression, thus suggested that Src-1 and Twist1 enhanced the malignant behaviors of NPC cells via inducing epithelial-mesenchymal transition (EMT). Our data indicated that Src-1 and Twist1 could be possible prognostic biomarkers and potential therapy targets for patients with NPC. [ABSTRACT FROM AUTHOR]

Salvia splendens is a perennial, ornamental herbaceous flower that is widely cultivated as a bedding plant in gardens. The development of novel S. splendens cultivars and investigating the relevant molecular mechanisms are of great significance. In this study, RNA-sequencing and real-time PCR methods were used to analyze the possible molecular mechanism of S. splendens mutant, SX919M. From the wild-type S. splendens 919CK, we firstly selected a natural mutant, SX919M, which displayed multiple branches, clustered spheroids, and radial symmetrical inflorescence with higher numbers of calyces, ovules, stamens, and perianth tubes. Further, the RNA-seq was used to identify the differentially expressed genes (DEGs) in the mutant which included a total of 3568 upregulated and 3290 downregulated unigenes. We further observed that the indole alkaloid biosynthesis pathway showed the highest DEG enrichment, which was supported by a significant increase in the IAA content in mutant SX919M. In addition, we validated three DEGs, namely, CL2200.Contig2_All encoding methyl IAA esterase, CL12462.Contig1_All and CL12462.Contig2_All, which encoded strictosidine synthase, upregulated in mutant SX919M. We selected a novel S. splendens germplasm SX919M with a high ornamental value and determined that the upregulation of IAA biogenesis may be associated with its development. [ABSTRACT FROM AUTHOR]

Angiotensin II (AngII), a vasoactive peptide that elevates arterial blood pressure and results in hypertension, has been reported to directly induce vascular endothelial cell apoptosis. Recent work has demonstrated that propofol pre-treatment attenuates angiotensin II-induced apoptosis in human coronary artery endothelial cells. However, the underlying mechanism remains largely unknown. Here, we investigated human umbilical vein endothelial cells (HUVECs) subjected to angiotensin II-induced apoptosis in the presence or absence of propofol treatment and found that angiotensin II-induced apoptosis was attenuated by propofol in a dose-dependent manner. Furthermore, ELISA assays demonstrated that the ratio of angiotensin (1–7) (Ang (1–7)) to Ang II was increased after propofol treatment. We examined the expression of ACE2, Ang (1–7) and Mas and found that the ACE2-Ang (1–7)-Mas axis was up-regulated by propofol, while ACE2 overexpression increased phosphorylated endothelial nitric oxide synthase (phosphorylated eNOS) expression and siACE2 resulted in the repression of endothelial nitric oxide synthase (eNOS) phosphorylation. In conclusion, our study revealed that propofol can inhibit endothelial cell apoptosis induced by Ang II by activating the ACE2-Ang (1–7)-Mas axis and further up-regulating the expression and phosphorylation of eNOS. [ABSTRACT FROM AUTHOR]

RNA binding protein polypyrimidine tract binding protein 2 (Ptbp2) as a key alternative splicing regulator for male germ cell development is well established. However, its expression levels and role in cryptorchidism testes tissues has not been explored. Additionally, the molecular mechanism of heat stress impacts the correct proliferation and differentiation of germ cells is unclear. To investigate whether changes in Ptbp2 expression are correlated with heat stress-induced germ cell injury in testicular tissue, we used a murine model of intraperitoneal cryptorchidism with surgical operation. Here we present compelling evidence that germ cells are severely damaged in mice with unilateral cryptorchidism, with non-obstructive azoospermia. And the Ptbp2 and Pgk2 mRNA levels were significantly decreased in parallel, leading us to conclude that the negative correlation between Ptbp2 levels and germ cell injury in unilateral cryptorchidism murine model. We hypothesize that Ptbp2 is susceptible to heat stress and its disruption has resulted in stability decline of germ cell transcripts Pgk2 mRNA, which consequently lead to germ cell injury in cryptorchidism testes. Thus, we confirm that Ptbp2 is an essential factor in heat stress-induced sperm cell injury and non-obstructive azoospermia. [ABSTRACT FROM AUTHOR]

The principal target organ of duck hepatitis A virus type 1 (DHAV-1) is duckling liver, which is an energy-intensive organ and plays important roles in body’s energy metabolism and conversion. As the “power house” of the hepatocytes, mitochondria provide more than 90% of the energy. However, mitochondria are much vulnerable to the oxidative stress for their rich in polyunsaturated fatty acids. Although previous researches have demonstrated that DHAV-1 could induce the oxidative stress in the serum of the infected ducklings, no related study on the mitochondria during the pathological process of DVH has been reported by far. To address this issue, we examined the HE stained tissue pathological slices, detected the hepatic SOD, CAT and GPX activities and MDA contents and analyzed the ATP content, mitochondrial ultrastructure and the mitochondrial SOD, GPX activities and MDA content in the liver tissues. The results showed that the hepatic redox status was significantly disturbed so that causing the mitochondrial dysfunction, ATP depletion and mitochondrial oxidative stress during the process of the DHAV-1 infection, and a prescription formulated with Hypericum japonicum flavone, Radix Rehmanniae Recens polysaccharide and Salvia plebeia flavone (HRS), which had been demonstrated with good anti-oxidative activity in serum, could effectively alleviate the hepatic injury and the oxidative stress in liver tissue induced by DHAV-1 thus alleviating the mitochondrial injury and oxidative stress. In a word, this research discovers the oxidative stress induced mitochondrial dysfunction and oxidative stress during the DVH pathological process and demonstrates HRS exerts good anti-oxidative activity in liver tissue to protect mitochondria against reactive oxygen species (ROS). [ABSTRACT FROM AUTHOR]

Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles. [ABSTRACT FROM AUTHOR]

Posterior capsule opacification (PCO) is the most common complication that causes visual decrease after extracapsular cataract surgery. The primary cause of PCO formation is the proliferation of the residual lens epithelial cells (LECs). The mammalian target of rapamycin (mTOR) plays an important role in the growth and migration of LECs. In the current study, we used small interfering RNA (siRNA) to specifically attenuate mTOR in human lens epithelial B3 cells (HLE B3). We aimed to examine the effect of mTOR-siRNA on the proliferation, migration and epithelial-to-mesenchymal transition (EMT) of HLE B3 cells and explore the underlying mechanisms. The mTOR-siRNA was transfected into HLE B3 cells using lipofectamine 2000. The mRNA and protein levels of mTOR were examined to confirm the efficiency of mTOR-siRNA. The levels of mRNA and protein as well as the activity of mTOR down-stream effectors p70 ribosomal protein S6 kinase (p70S6K) and protein kinase B (PKB, AKT) were examined using real-time PCR or Western blot, respectively. The cell proliferation was determined using cell counting kit (CCK) 8 and cell growth curve assay. The cell migration was examined using Transwell system and Scratch assay. MTOR-siRNA effectively eliminated mTOR mRNA and protein. The proliferation and migration were significantly suppressed by mTOR-siRNA transfection. mTOR-siRNA reduced the mRNA of p70S6K and AKT in a time-dependent manner. Furthermore, the phosphorylation of p70S6K and AKT was decreased by mTOR-siRNA. MTOR-siRNA also eliminated the formation of mTORC1 and mTORC2 protein complex and blocked the transforming growth factor (TGF)-β-induced EMT. Our results suggested that mTOR-siRNA could effectively inhibit the proliferation, migration and EMT of HLE B3 cells through the inhibition of p70S6K and AKT. These results indicated that mTOR-siRNA might be an effective agent inhibiting HLE cells growth and EMT following cataract surgery and provide an alternative therapy for preventing PCO. [ABSTRACT FROM AUTHOR]

Dysfunction of histone acetylation inhibits topoisomerase IIα (Topo IIα), which is implicated in benzene-induced hematotoxicity in patients with chronic benzene exposure. Whether histone deacetylase (HDAC) inhibitors can relieve benzene-induced hematotoxicity remains unclear. Here we showed that hydroquinone, a main metabolite of benzene, increased the HDAC activity, decreased the Topo IIα expression and induced apoptosis in human bone marrow mononuclear cells in vitro, and treatment with two HDAC inhibitors, namely trichostatin A (TSA) or a mixture of ribosome-inactivating proteins MCP30, almost completely reversed these effects. We further established a benzene poisoning murine model by inhaling benzene vapor in a container and found that benzene poisoning decreased the expression and activity of Topo IIα, and impaired acetylation of histone H4 and H3. The analysis of regulatory factors of Topo IIα promoter found that benzene poisoning decreased the mRNA levels of SP1 and C-MYB, and increased the mRNA level of SP3. Both TSA and MCP30 significantly enhanced the acetylation of histone H3 and H4 in Topo IIα promoter and increased the expression and activity of Topo IIα in benzene poisoning mice, which contributed to relieve the symptoms of hematotoxicity. Thus, treatment with HDAC inhibitors represents an attractive approach to reduce benzene-induced hematotoxicity. [ABSTRACT FROM AUTHOR]

This study aimed to explore the effects of fucoidan from Fucus vesiculosus on concanavalin A (ConA)-induced acute liver injury in mice. Pretreatment with fucoidan protected liver function indicated by ALT, AST and histopathological changes by suppressing inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). In addition, intrinsic and extrinsic apoptosis mediated by Bax, Bid, Bcl-2, Bcl-xL and Caspase 3, 8, and 9 were inhibited by fucoidan and the action was associated with the TRADD/TRAF2 and JAK2/STAT1 signal pathways. Our results demonstrated that fucoidan from Fucus vesiculosus alleviated ConA-induced acute liver injury via the inhibition of intrinsic and extrinsic apoptosis mediated by the TRADD/TRAF2 and JAK2/STAT1 pathways which were activated by TNF-α and IFN-γ. These findings could provide a potential powerful therapy for T cell-related hepatitis. [ABSTRACT FROM AUTHOR]

Understanding the mechanisms that regulate pluripotency of embryonic stem cells (ESCs) is important to ensure their safe clinical use. CHIR99021 (CHIR)-induced activation of Wnt/β-catenin signaling promotes self-renewal in mouse ESCs (mESCs). β-catenin functions individually or cooperates with transcription factors to activate stemness factors such as c-Myc, Esrrb, Pou5f1, and Nanog. However the relationship between the core pluripotent factor, Kruppel-like factor 4 (also known as GKLF or EZF) and Wnt/β-catenin signaling, remains ambiguous in J1 mESCs. DNA microarray analysis revealed that CHIR-treatment promoted pluripotency-maintaining transcription factors and repressed germ layer specification markers. CHIR also promoted genes related to the development of extracellular regions and the plasma membrane to maintain pluripotency of J1 mESCs. Among the CHIR-regulated genes, Klf4 has not been reported previously. We identified a novel cis element in the Klf4 gene that was activated by β-catenin in J1 mESCs. We determined that β-catenin interacted with this cis element, identifying Klf4 as a β-catenin target gene in this context. Moreover, several microRNAs that targeted the 3′-UTR of Klf4 mRNA were identified, with miR-7a being down-regulated by CHIR in a β-catenin-independent manner in J1 mESCs. These data collectively suggest that CHIR enhances Klf4 expression by repressing miR-7a expression or canonical Wnt pathway activation. [ABSTRACT FROM AUTHOR]

The two major intracellular protein degradation systems, the ubiquitin-proteasome system (UPS) and autophagy, work collaboratively in many biological processes including development, apoptosis, aging, and countering oxidative injuries. We report here that, in human retinal pigment epithelial cells (RPE), ARPE-19 cells, proteasome inhibitors, clasto-lactacystinβ-lactone (LA) or epoxomicin (Epo), at non-lethal doses, increased the protein levels of autophagy-specific genes Atg5 and Atg7 and enhanced the conversion of microtubule-associated protein light chain (LC3) from LC3-I to its lipidative form, LC3-II, which was enhanced by co-addition of the saturated concentration of Bafilomycin A1 (Baf). Detection of co-localization for LC3 staining and labeled-lysosome further confirmed autophagic flux induced by LA or Epo. LA or Epo reduced the phosphorylation of the protein kinase B (Akt), a downstream target of phosphatidylinositol-3-kinases (PI3K), and mammalian target of rapamycin (mTOR) in ARPE-19 cells; by contrast, the induced changes of autophagy substrate, p62, showed biphasic pattern. The autophagy inhibitor, Baf, attenuated the reduction in oxidative injury conferred by treatment with low doses of LA and Epo in ARPE-19 cells exposed to menadione (VK3) or 4-hydroxynonenal (4-HNE). Knockdown of Atg7 with siRNA in ARPE-19 cells reduced the protective effects of LA or Epo against VK3. Overall, our results suggest that treatment with low levels of proteasome inhibitors confers resistance to oxidative injury by a pathway involving inhibition of the PI3K-Akt-mTOR pathway and activation of autophagy. [ABSTRACT FROM AUTHOR]

Receptor-like protein tyrosine phosphatases (RPTPs) are type I transmembrane glycoproteins with N-glycans whose catalytic activities are regulated by dimerization. However, the intrinsic mechanism involved in dimerizing processes remains obscure. In this study, receptor protein tyrosine phosphatase rho (PTPRT) is identified as a novel substrate of N-Acetylglucosaminyltransferase V (GnT-V). We show that addition of β1,6 GlcNAc branches on PTPRT prolongs PTPRT's cell-surface retention time. GnT-V overexpression enhances galectin-3's cell-surface retention and promotes PTPRT's dimerization mediated by galectin-3. Increased dimerization subsequently reduces PTPRT's catalytic activity on the dephosphorylation of signal transducer and activator of transcription 3 (STAT3) at tyrosine residue 705 (pY705 STAT3), then the accumulated pY705 STAT3 translocates into the nucleus. Collectively, these findings provide an insight into the molecular mechanism by which GnT-V promotes cell migration, suggesting that accumulation of β1,6 GlcNAc branched N-glycans promotes PTPRT's dimerization and decreases its catalytic activity, resulting in enhanced cell migratory capacity. [ABSTRACT FROM AUTHOR]

Recently, there is ample evidence suggesting the important role of microRNAs (miRNAs) in autoimmune diseases via modulating B cells function. Primary biliary cholangitis (PBC) is a progressive immune-mediated liver disease with unclear pathogenic mechanism. Whether the miRNA in peripheral B cells of PBC involve the mechanisms of pathology and progression is not known. The present study explores miRNA deregulation in peripheral B-cell of PBC from stage I to IV and healthy controls. Peripheral B cells were obtained from 72 PBC patients (stage I, n = 17; stage II, n = 23; stage III, n = 16; stage IV, n = 16) and 15 healthy controls. Initially, in a discovery study, miRNA array analysis was performed, subsequently, in a validation study, quantitative PCR was used to investigate miRNA expression profile in B cells of PBS patients compared to healthy controls. Based on bioinformatics analysis, we identified the potential biological processes and significant signaling pathways affected by these microRNAs, and generated the microRNA–gene network. The discovery study identified 558 miRNAs differentially expressed in B cells of PBC patients compared to controls. Interestingly, among all differentially expressed miRNAs, hsa-miR-223-3p and hsa-miR-21-5p were the only miRNAs that showed consistent and significant down-regulation from stage I to stage III of PBC. Bioinformatics showed that potential target genes of both miRNAs involved in migration, cell differentiation, apoptosis, and signal transduction pathways. In conclusion, our results suggest that the expression profiles of miRNA in peripheral B cells of PBC patients are closely associated with the disease progression, especially the down-regulation of hsa-miR-223-3p and hsa-miR-21-5p. Taken together, our study offers novel perspectives on the role of miRNAs in PBC pathogenesis. [ABSTRACT FROM AUTHOR]

Leucine-rich-alpha-2-glycoprotein 1 (LRG1) has been shown to be involved in various human malignancies. Whether it plays a role in colorectal cancer (CRC) development remains unclear. Here, we investigated whether and through what mechanism LRG1 functions in human CRC cells. The plasma level of LRG1 was significantly increased in CRC patients, but it was remarkably decreased in patients with resected colorectal cancers. Meanwhile, both mRNA and protein levels of LRG1 were remarkable overexpressed in CRC tissues than normal tissues. The knockdown of LRG1 significantly inhibited cell proliferation, induced cell cycle arrest at the G0/G1 phase, and promoted apoptosis in SW480 and HCT116 cells in vitro. In addition, LRG1 silencing led to the downregulation of the levels of key cell cycle factors, such as cyclin D1, B, and E and anti-apoptotic B-cell lymphoma-2(Bcl-2). However, it up-regulated the expression of pro-apoptotic Bax and cleaved caspase-3. Furthermore, RUNX1 could be induced by LRG1 in a concentration-dependent manner, while the knockdown of RUNX1 blocked the promotion of the proliferation and inhibition of apoptosis induced by LRG1. Collectively, these findings indicate that LRG1 plays a crucial role in the proliferation and apoptosis of CRC by regulating RUNX1 expression. Thus, LRG1 may be a potential detection biomarker as well as a marker for monitoring recurrence and therapeutic target for CRC. [ABSTRACT FROM AUTHOR]

The cockroach, Periplaneta americana, is an obnoxious and notorious pest of the world, with a strong ability to adapt to a variety of complex environments. However, the molecular mechanism of this adaptability is mostly unknown. In this study, the genes and microbiota composition associated with the adaptation mechanism were studied by analyzing the transcriptome and 16S rDNA pyrosequencing of the P. americana midgut, respectively. Midgut transcriptome analysis identified 82,905 unigenes, among which 64 genes putatively involved in digestion (11 genes), detoxification (37 genes) and oxidative stress response (16 genes) were found. Evaluation of gene expression following treatment with cycloxaprid further revealed that the selected genes (CYP6J1, CYP4C1, CYP6K1, Delta GST, alpha-amylase, beta-glucosidase and aminopeptidase) were upregulated at least 2.0-fold at the transcriptional level, and four genes were upregulated more than 10.0-fold. An interesting finding was that three digestive enzymes positively responded to cycloxaprid application. Tissue expression profiles further showed that most of the selected genes were midgut-biased, with the exception of CYP6K1. The midgut microbiota composition was obtained via 16S rDNA pyrosequencing and was found to be mainly dominated by organisms from the Firmicutes phylum, among which Clostridiales, Lactobacillales and Burkholderiales were the main orders which might assist the host in the food digestion or detoxification of noxious compounds. The preponderant species, Clostridium cellulovorans, was previously reported to degrade lignocellulose efficiently in insects. The abundance of genes involved in digestion, detoxification and response to oxidative stress, and the diversity of microbiota in the midgut might provide P. americana high capacity to adapt to complex environments. [ABSTRACT FROM AUTHOR]

Background: Mitochondrial dysfunction would ultimately lead to myocardial cell apoptosis and death during ischemia-reperfusion injuries. Autophagy could ameliorate mitochondrial dysfunction by autophagosome forming, which is a catabolic process to preserve the mitochondrial’s structural and functional integrity. HO-1 induction and expression are important protective mechanisms. This study in order to investigate the role of HO-1 during mitochondrial damage and its mechanism. Methods and Results: The H9c2 cardiomyocyte cell line were incubated by hypoxic and then reoxygenated for the indicated time (2, 6, 12, 18, and 24 h). Cell viability was tested with CCK-8 kit. The expression of endogenous HO-1(RT-PCR and Western blot) increased with the duration of reoxygenation and reached maximum levels after 2 hours of H/R; thereafter, the expression gradually decreased to a stable level. Mitochondrial dysfunction (Flow cytometry quantified the ROS generation and JC-1 staining) and autophagy (The Confocal microscopy measured the autophagy. RFP-GFP-LC3 double-labeled adenovirus was used for testing.) were induced after 6 hours of H/R. Then, genetic engineering technology was employed to construct an Lv-HO1-H9c2 cell line. When HO-1 was overexpressed, the LC3II levels were significantly increased after reoxygenation, p62 protein expression was significantly decreased, the level of autophagy was unchanged, the mitochondrial membrane potential was significantly increased, and the mitochondrial ROS level was significantly decreased. Furthermore, when the HO-1 inhibitor ZnPP was applied the level of autophagy after reoxygenation was significantly inhibited, and no significant improvement in mitochondrial dysfunction was observed. Conclusions: During myocardial hypoxia-reoxygenation injury, HO-1 overexpression induces autophagy to protect the stability of the mitochondrial membrane and reduce the amount of mitochondrial oxidation products, thereby exerting a protective effect. [ABSTRACT FROM AUTHOR]

The S-phase checkpoint plays an essential role in regulation of the ribonucleotide reductase (RNR) activity to maintain the dNTP pools. How eukaryotic cells respond appropriately to different levels of replication threats remains elusive. Here, we have identified that a conserved GSK-3 kinase Mck1 cooperates with Dun1 in regulating this process. Deleting MCK1 sensitizes dun1Δ to hydroxyurea (HU) reminiscent of mec1Δ or rad53Δ. While Mck1 is downstream of Rad53, it does not participate in the post-translational regulation of RNR as Dun1 does. Mck1 phosphorylates and releases the Crt1 repressor from the promoters of DNA damage-inducible genes as RNR2-4 and HUG1. Hug1, an Rnr2 inhibitor normally silenced, is induced as a counterweight to excessive RNR. When cells suffer a more severe threat, Mck1 inhibits HUG1 transcription. Consistently, only a combined deletion of HUG1 and CRT1, confers a dramatic boost of dNTP levels and the survival of mck1Δdun1Δ or mec1Δ cells assaulted by a lethal dose of HU. These findings reveal the division-of-labor between Mck1 and Dun1 at the S-phase checkpoint pathway to fine-tune dNTP homeostasis. [ABSTRACT FROM AUTHOR]

Cohesin acetyltransferases ESCO1 and ESCO2 play a vital role in establishing sister chromatid cohesion. How ESCO1 and ESCO2 are controlled in a DNA replication-coupled manner remains unclear in higher eukaryotes. Here we show a critical role of UL4-ING igases (CRL4s) in cohesion establishment via regulating ESCO2 in human cells. Depletion of CUL4A, CUL4B or DDB1 subunits substantially reduces the normal cohesion efficiency. We also show that MMS22L, a vertebrate ortholog of yeast Mms22, is one of DB1 and UL4-ssociated actors (DCAFs) involved in cohesion. Several lines of evidence show selective interaction of CRL4s with ESCO2 through LxG motif, which is lost in ESCO1. Depletion of either CRL4s or ESCO2 causes a defect in SMC3 acetylation, which can be rescued by HDAC8 inhibition. More importantly, both CRL4s and PCNA act as mediators for efficiently stabilizing ESCO2 on chromatin and catalyzing SMC3 acetylation. Taken together, we propose an evolutionarily conserved mechanism in which CRL4s and PCNA promote ESCO2-dependent establishment of sister chromatid cohesion. [ABSTRACT FROM AUTHOR]

Background: Vascular endothelial growth factor (VEGF) is commonly overexpressed in a variety of tumor types including lung cancer. As a key regulator of angiogenesis, it promotes tumor survival, growth, and metastasis through the activation of the downstream protein kinase B (AKT) and extracellular signal-regulated kinase (ERK 1/2) activation. The VEGF promoter contains a 36 bp guanine-rich sequence (VEGFq) which is capable of forming quadruplex (four-stranded) DNA. This sequence has been implicated in the down-regulation of both basal and inducible VEGF expression and represents an ideal target for inhibition of VEGF expression. Results: Our experiments demonstrate sequence-specific interaction between a G-rich quadruplex-forming oligonucleotide encoding a portion of the VEGFq sequence and its double stranded target sequence, suggesting that this G-rich oligonucleotide binds specifically to its complementary C-rich sequence in the genomic VEGF promoter by strand invasion. We show that treatment of A549 non-small lung cancer cells (NSCLC) with this oligonucleotide results in decreased VEGF expression and growth inhibition. The VEGFq oligonucleotide inhibits proliferation and invasion by decreasing VEGF mRNA/protein expression and subsequent ERK 1/2 and AKT activation. Furthermore, the VEGFq oligonucleotide is abundantly taken into cells, localized in the cytoplasm/nucleus, inherently stable in serum and intracellularly, and has no effect on non-transformed cells. Suppression of VEGF expression induces cytoplasmic accumulation of autophagic vacuoles and increased expression of LC3B, suggesting that VEGFq may induce autophagic cell death. Conclusion: Our data strongly suggest that the G-rich VEGFq oligonucleotide binds specifically to the C-rich strand of the genomic VEGF promoter, via strand invasion, stabilizing the quadruplex structure formed by the genomic G-rich sequence, resulting in transcriptional inhibition. Strand invading oligonucleotides represent a new approach to specifically inhibit VEGF expression that avoids many of the problems which have plagued the therapeutic use of oligonucleotides. This is a novel approach to specific inhibition of gene expression. [ABSTRACT FROM AUTHOR]

Resveratrol, a non-flavonoid polyphenolic compound, is structurally and functionally similar to estrogen and has drawn great attention for its potentially beneficial effects on diabetes. However, it is not known whether it shares the same protective effect against diabetes as estrogen and the underlying mechanisms. The aim of the present study was to investigate the protective effects of phytoestrogen resveratrol and exogenous 17β-estradiol against streptozotocin (STZ)-induced type 1 diabetes. Female mice were ovariectomized (OVX) and chronically injected with different concentrations of resveratrol (0.1, 1 or 10 mg/kg) and 17β-estradiol (0.01, 0.1 or 1 mg/kg) subcutaneously for 4 weeks, and the levels of blood glucose, plasma insulin, plasma antioxidant capacity, the changes of pancreatic islet cells and the expressions of glucose transporter 4 (GLUT4), insulin receptor substrate 1 (IRS-1) and phosphorylation of extracellular signal-regulated kinase (p-ERK) were detected. Resveratrol and 17β-estradiol significantly inhibited the increase of the blood glucose level and the rise of plasma malondialdehyde in STZ-induced diabetic mice, improved the levels of plasma antioxidant capacity and plasma insulin, protected the pancreatic islet cells, and increased the expressions of GLUT4 and IRS-1, but decreased p-ERK expression in skeletal muscle and myocardial tissue. The results suggest that resveratrol or 17β-estradiol shows obvious protection against STZ-induced diabetes in OVX mice, the mechanisms probably involve their ameliorating antioxidant activities and islet function, promoting muscle glucose uptake and inhibiting the expression of p-ERK. [ABSTRACT FROM AUTHOR]

Background: Trichinellosis is a serious food-borne parasitic zoonosis worldwide. In the effort to develop vaccine against Trichinella infection, we have identified Trichinella spiralis Heat shock protein 70 (Ts-Hsp70) elicits partial protective immunity against T. spiralis infection via activating dendritic cells (DCs) in our previous study. This study aims to investigate whether DCs were activated by Ts-Hsp70 through TLR2 and/or TLR4 pathways. Methods and findings: After blocking with anti-TLR2 and TLR4 antibodies, the binding of Ts-Hsp70 to DCs was significantly reduced. The reduced binding effects were also found in TLR2 and TLR4 knockout (TLR2-/- and TLR4-/-) DCs. The expression of TLR2 and TLR4 on DCs was upregulated after treatment with Ts-Hsp70 in vitro. These results suggest that Ts-Hsp70 is able to directly bind to TLR2 and TLR4 on the surface of mouse bone morrow-derived DCs. In addition, the expression of the co-stimulatory molecules (CD80, CD83) on Ts-Hsp70-induced DCs was reduced in TLR2-/- and TLR4-/- mice. More evidence showed that Ts-Hsp70 reduced its activation on TLR2/4 knockout DCs to subsequently activate the naïve T-cells. Furthermore, Ts-Hsp70 elicited protective immunity against T. spiralis infection was reduced in TLR2-/- and TLR4-/- mice correlating with the reduced humoral and cellular immune responses. Conclusion: This study demonstrates that Ts-Hsp70 activates DCs through TLR2 and TLR4, and TLR2 and TLR4 play important roles in Ts-Hsp70-induced DCs activation and immune responses. [ABSTRACT FROM AUTHOR]

p38 has long been known as a central mediator of protein kinase A (PKA) signaling in brown adipocytes, which positively regulate the transcription of uncoupling protein 1 (UCP-1). However, the physiological role of p38 in adipose tissues, especially the white adipose tissue (WAT), is largely unknown. Here, we show that mice lacking p38α in adipose tissues display a lean phenotype, improved metabolism, and resistance to diet-induced obesity. Surprisingly, ablation of p38α causes minimal effects on brown adipose tissue (BAT) in adult mice, as evident from undetectable changes in UCP-1 expression, mitochondrial function, body temperature (BT), and energy expenditure. In contrast, genetic ablation of p38α in adipose tissues not only markedly facilitates the browning in WAT upon cold stress but also prevents diet-induced obesity. Consistently, pharmaceutical inhibition of p38α remarkably enhances the browning of WAT and has metabolic benefits. Furthermore, our data suggest that p38α deficiency promotes white-to-beige adipocyte reprogramming in a cell-autonomous manner. Mechanistically, inhibition of p38α stimulates the UCP-1 transcription through PKA and its downstream cAMP-response element binding protein (CREB), which form a positive feedback loop that functions to reinforce the white-to-beige phenotypic switch during cold exposure. Together, our study reveals that inhibition of p38α is able to promote WAT browning and confer metabolic benefits. Our study also indicates that p38α in WAT represents an exciting pharmacological target to combat obesity and metabolic diseases. [ABSTRACT FROM AUTHOR]

Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) remain the predominant pathogens in hand, foot, and mouth disease (HFMD), but the factors underlying the pathogenesis of EV71 and CA16 infections have not been elucidated. Recently, the functions of microRNAs (miRNAs) in pathogen-host interactions have been highlighted. In the present study, we performed comprehensive miRNA profiling in EV71- and CA16-infected human umbilical vein endothelial cells (HUVECs) at multiple time points using high-throughput sequencing. The results showed that 135 known miRNAs exhibited remarkable differences in expression. Of these, 30 differentially expressed miRNAs presented opposite trends in EV71- and CA16-infected samples. Subsequently, we mainly focused on the 30 key differentially expressed miRNAs through further screening to predict targets. Gene ontology (GO) and pathway analysis of the predicted targets showed the enrichment of 14 biological processes, 9 molecular functions, 8 cellular components, and 85 pathways. The regulatory networks of these miRNAs with predicted targets, GOs, pathways, and co-expression genes were determined, suggesting that miRNAs display intricate regulatory mechanisms during the infection phase. Consequently, we specifically analyzed the hierarchical GO categories of the predicted targets involved in biological adhesion. The results indicated that the distinct changes induced by EV71 and CA16 infection may be partly linked to the function of the blood-brain barrier. Taken together, this is the first report describing miRNA expression profiles in HUVECs with EV71 and CA16 infections using high-throughput sequencing. Our data provide useful insights that may help to elucidate the different host-pathogen interactions following EV71 and CA16 infection and offer novel therapeutic targets for these infections. [ABSTRACT FROM AUTHOR]

Imprinted genes display biased expression of paternal and maternal alleles- and are only found in mammals and flowering plants. Compared to several hundred imprinted genes that are functionally characterized in mammals, very few imprinted genes were confirmed in plants and even fewer of them have been functionally investigated. Here, we report a new imprinted gene, NUWA, in plants. NUWA is an essential gene, because loss of its function resulted in reduced transmission through the female gametophyte and defective cell/nuclear proliferation in early Arabidopsis embryo and endosperm. NUWA is a maternally expressed imprinted gene, as only the maternal allele of NUWA is transcribed and translated from gametogenesis to the 16-cell globular embryo stage after fertilization, and the de novo transcription of the maternal allele of NUWA starts from the zygote stage. Different from other identified plant imprinted genes whose encoded proteins are mostly localized to the nucleus, the NUWA protein was localized to the mitochondria and was essential for mitochondria function. Our work uncovers a novel imprinted gene of a previously unidentified type, namely, a maternal-specific expressed nuclear gene with its encoded protein localizing to and controlling the function of the maternally inherited mitochondria. This reveals a unique mechanism of maternal control of the mitochondria and adds an extra layer of complexity to the regulation of nucleus-organelle coordination during early plant development. [ABSTRACT FROM AUTHOR]

RNF12/RLIM is a RING domain-containing E3 ubiquitin ligase whose function has only begun to be elucidated recently. Although RLIM was reported to play important roles in some biological processes such as imprinted X-chromosome inactivation and regulation of TGF-β pathway etc., other functions of RLIM are largely unknown. Here, we identified RLIM as a novel E3 ubiquitin ligase for c-Myc, one of the most frequently deregulated oncoproteins in human cancers. RLIM associates with c-Myc in vivo and in vitro independently of the E3 ligase activity of RLIM. Moreover, RLIM promotes the polyubiquitination of c-Myc protein independently of Ser62 and Thr58 phosphorylation of c-Myc. However, RLIM-mediated ubiquitination does not affect c-Myc stability. Instead, RLIM inhibits the transcriptional activity of c-Myc through which RLIM restrains cell proliferation. Our results suggest that RLIM may function as a tumor suppressor by controlling the activity of c-Myc oncoprotein. [ABSTRACT FROM AUTHOR]

Background and Purpose: It has been accepted that AMPK (Adenosine monophosphate–activated protein kinase) activation exhibits many beneficial effects on glucolipid metabolism. Lysophosphatidylcholine (LPC) is an important lysophospholipid which can improve blood glucose levels in diabetic mice and attenuate inflammation by activating AMPK signal pathway in macrophages. Synthetic alkylphospholipids (ALPs), such as miltefosine, is used as an alternate of LPC for the clinical application. Here, we investigated whether miltefosine could have an impact on hepatic steatosis and related metabolic disorders. Experimental Approach: Mice were fed with high fat diet (HFD) for 16 weeks to generate an obese model. Next, the obese mice were randomly divided into three groups: saline-treated and miltefosine-treated (2.5 or 5 mg/kg/d) groups. Miltefosine was intraperitoneally administrated into mice for additional 4 weeks plus HFD treatment. Key Results: It was shown that miltefosine treatment could substantially improve glucose metabolism, prevented hepatic lipid accumulation, and inhibited liver inflammation in HFD-fed mice by activating AMPK signal pathway. In vitro, miltefosine stimulated AMPKα phosphorylation both in time and dose dependent manner and decreased lipid accumulation in liver cells. When a specific AMPK inhibitor compound C was used to treat mice, the antagonistic effects of miltefosine on HFD-induced mouse hyperlipidaemia and liver steatosis were abolished. Treatment with miltefosine also dramatically inhibited the HFD-induced liver inflammation in mice. Conclusions and Implications: Here we demonstrated that miltefosine might be a new activator of AMPK signal pathway in vivo and in vitro and be useful for treatment of hepatic steatosis and related metabolic disorders. [ABSTRACT FROM AUTHOR]

Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small, non-coding RNAs that affect the post-transcriptional regulation of various biological pathways. To date, it is not fully understood how miRNAs regulate mitochondrial biogenesis. This study aimed at the identification of the role of miRNA-27b in mitochondria biogenesis. The mitochondria content in C2C12 cells was significantly increased during myogenic differentiation and accompanied by a marked decrease of miRNA-27b expression. Furthermore, the expression of the predicted target gene of miRNA-27b, forkhead box j3 (Foxj3), was also increased during myogenic differentiation. Luciferase activity assays confirmed that miRNA-27b directly targets the 3’-untranslated region (3’-UTR) of Foxj3. Overexpression of miRNA-27b provoked a decrease of mitochondria content and diminished expression of related mitochondrial genes and Foxj3 both at mRNA and protein levels. The expression levels of downstream genes of Foxj3, such as Mef2c, PGC1α, NRF1 and mtTFA, were also decreased in C2C12 cells upon overexpression of miRNA-27b. These results suggested that miRNA-27b may affect mitochondria biogenesis by down-regulation of Foxj3 during myocyte differentiation. [ABSTRACT FROM AUTHOR]

Six1 is one of the transcription factors that act as master regulators of development and are frequently dysregulated in cancers. However, the role of Six1 in pancreatic cancer is not clear. Here we show that the relative expression of Six1 mRNA is increased in pancreatic cancer and correlated with advanced tumor stage. In vitro functional assays demonstrate that forced overexpression of Six1 significantly enhances the growth rate and proliferation ability of pancreatic cancer cells. Knockdown of endogenous Six1 decreases the proliferation of these cells dramatically. Furthermore, Six1 promotes the growth of pancreatic cancer cells in a xenograft assay. We also show that the gene encoding cyclin D1 is a direct transcriptional target of Six1 in pancreatic cancer cells. Overexpression of Six1 upregulates cyclin D1 mRNA and protein, and significantly enhances the activity of the cyclin D1 promoter in PANC-1 cells. We demonstrate that Six1 promotes cell cycle progression and proliferation by upregulation of cyclin D1. These data suggest that Six1 is overexpressed in pancreatic cancer and may contribute to the increased cell proliferation through upregulation of cyclin D1. [ABSTRACT FROM AUTHOR]