Ovarian cancer G protein-coupled receptor 1 (OGR1) has been shown to be a proton sensing receptor in vitro. We have shown that OGR1 functions as a tumor metastasis suppressor gene when it is over-expressed in human prostate cancer cells in vivo. To examine the physiological functions of OGR1, we generated conditional OGR1 deficient mice by homologous recombination. OGR1 deficient mice were viable and upon gross-inspection appeared normal. Consistent with in vitro studies showing that OGR1 is involved in osteoclastogenesis, reduced osteoclasts were detected in OGR1 deficient mice. A pH-dependent osteoclasts survival effect was also observed. However, overall abnormality in the bones of these animals was not observed. In addition, melanoma cell tumorigenesis was significantly inhibited in OGR1 deficient mice. OGR1 deficient mice in the mixed background produced significantly less peritoneal macrophages when stimulated with thioglycolate. These macrophages also showed altered extracellular signal-regulated kinases (ERK) activation and nitric oxide (NO) production in response to lipopolysaccharide. OGR1-dependent pH responses assessed by cAMP production and cell survival in macrophages or brown fat cells were not observed, presumably due to the presence of other proton sensing receptors in these cells. Our results indicate that OGR1's role in osteoclastogenesis is not strong enough to affect overall bone development and its role in tumorigenesis warrants further investigation. The mice generated can be potentially used for several disease models, including cancers or osteoclast-related diseases.

Verticillium dahliae Kleb. is a phytopathogenic fungus that causes wilt disease in a wide range of crops, including cotton. The life cycle of V. dahliae includes three vegetative phases: parasitic, saprophytic and dormant. The dormant microsclerotia are the primary infectious propagules, which germinate when they are stimulated by root exudates. In this study, we report the first application of Agrobacterium tumefaciens-mediated transformation (ATMT) for construction of insertional mutants from a virulent defoliating isolate of V. dahliae (V592). Changes in morphology, especially a lack of melanized microsclerotia or pigmentation traits, were observed in mutants. Together with the established laboratory unimpaired root dip-inoculation approach, we found insertional mutants to be affected in their pathogenicities in cotton. One of the genes tagged in a pathogenicity mutant encoded a glutamic acid-rich protein (VdGARP1), which shared no significant similarity to any known annotated gene. The vdgarp1 mutant showed vigorous mycelium growth with a significant delay in melanized microsclerotial formation. The expression of VdGARP1 in the wild type V529 was organ-specific and differentially regulated by different stress agencies and conditions, in addition to being stimulated by cotton root extract in liquid culture medium. Under extreme infertile nutrient conditions, VdGARP1 was not necessary for melanized microsclerotial formation. Taken together, our data suggest that VdGARP1 plays an important role in sensing infertile nutrient conditions in infected cells to promote a transfer from saprophytic to dormant microsclerotia for long-term survival. Overall, our findings indicate that insertional mutagenesis by ATMT is a valuable tool for the genome-wide analysis of gene function and identification of pathogenicity genes in this important cotton pathogen.

High rates of esophageal cancer (EC) are found in people of the Henan Taihang Mountain, Fujian Minnan, and Chaoshan regions of China. Historical records describe great waves of populations migrating from north-central China (the Henan and Shanxi Hans) through coastal Fujian Province to the Chaoshan plain. Although these regions are geographically distant, we hypothesized that EC high-risk populations in these three areas could share a common ancestry. Accordingly, we used 16 East Asian-specific Y-chromosome biallelic markers (single nucleotide polymorphisms; Y-SNPs) and six Y-chromosome short tandem repeat (Y-STR) loci to infer the origin of the EC high-risk Chaoshan population (CSP) and the genetic relationship between the CSP and the EC high-risk Henan Taihang Mountain population (HTMP) and Fujian population (FJP). The predominant haplogroups in these three populations are O3*, O3e*, and O3e1, with no significant difference between the populations in the frequency of these genotypes. Frequency distribution and principal component analysis revealed that the CSP is closely related to the HTMP and FJP, even though the former is geographically nearer to other populations (Guangfu and Hakka clans). The FJP is between the CSP and HTMP in the principal component plot. The CSP, FJP and HTMP are more closely related to Chinese Hans than to minorities, except Manchu Chinese, and are descendants of Sino-Tibetans, not Baiyues. Correlation analysis, hierarchical clustering analysis, and phylogenetic analysis (neighbor-joining tree) all support close genetic relatedness among the CSP, FJP and HTMP. The network for haplogroup O3 (including O3*, O3e* and O3e1) showed that the HTMP have highest STR haplotype diversity, suggesting that the HTMP may be a progenitor population for the CSP and FJP. These findings support the potentially important role of shared ancestry in understanding more about the genetic susceptibility in EC etiology in high-risk populations and have implications for determining the molecular basis of this disease.

Numerous studies have shown that Tanshinones have anti-tumor effects in vitro, but few studies were focusing on the anti-tumor activity of Tanshinones against one special cancer cell line. The aim of this study is to investigate the growth inhibition effect of four Tanshinones on SPC-A-1 cell line and the relationship between their structures and cytotoxicity.

Protein kinase C (PKC) family members such as PKCbetaII may become activated in the hyperglycemic state associated with diabetes. Preclinical and clinical data implicate aberrant PKC activity in the development of diabetic microvasculature abnormalities. Based on this potential etiological role for PKC in diabetic complications, several therapeutic PKC inhibitors have been investigated in clinical trials for the treatment of diabetic patients. In this report, we present the discovery and preclinical evaluation of a novel class of 3-amino-pyrrolo[3,4-c]pyrazole derivatives as inhibitors of PKC that are structurally distinct from the prototypical indolocarbazole and bisindolylmaleimide PKC inhibitors. From this pyrrolo-pyrazole series, several compounds were identified from biochemical assays as potent, ATP-competitive inhibitors of PKC activity with high specificity for PKC over other protein kinases. These compounds were also found to block PKC signaling activity in multiple cellular functional assays. PF-04577806, a representative from this series, inhibited PKC activity in retinal lysates from diabetic rats stimulated with phorbol myristate acetate. When orally administered, PF-04577806 showed good exposure in the retina of diabetic Long-Evans rats and ameliorated retinal vascular leakage in a streptozotocin-induced diabetic rat model. These novel PKC inhibitors represent a promising new class of targeted protein kinase inhibitors with potential as therapeutic agents for the treatment of patients with diabetic microvascular complications.

Tissue-specific iron content is tightly regulated to simultaneously satisfy specialized metabolic needs and avoid cytotoxicity. In the brain, disruption of iron homeostasis may occur in acute as well as progressive injuries associated with neuronal dysfunction and death. We hypothesized that adverse effects of disrupted metal homeostasis on brain function may involve impairment of DNA repair processes. Because in the brain, the base excision repair (BER) pathway is central for handling oxidatively damaged DNA, we investigated effects of elevated iron and zinc on key BER enzymes. In vitro DNA repair assays revealed inhibitory effects of metals on BER activities, including the incision of abasic sites, 5'-flap cleavage, gap filling DNA synthesis and ligation. Using the comet assay, we showed that while metals at concentrations which inhibit BER activities in in vitro assays, did not induce direct genomic damage in cultured primary neurons, they significantly delayed repair of genomic DNA damage induced by sublethal exposure to H(2)O(2). Thus, in the brain even a mild transient metal overload, may adversely affect the DNA repair capacity and thereby compromise genomic integrity and initiate long-term deleterious sequelae including neuronal dysfunction and death.

Diversity of mRNAs from mytilin B, one of the five mytilins identified in the Mediterranean mussel, Mytilus galloprovincialis, has been investigated from circulating hemocytes. One mussel expressed simultaneously two to ten different mytilin B mRNAs as observed in denaturing gradient gel electrophoresis (DGGE), defining 10 individual DGGE patterns (named A to J) within the mussels from Messina, Sicily (Italy). Three patterns accounted for 79% of the individuals whereas other patterns were found in only 2-7% of the 57 analyzed mussels. Base mutations were observed at specific locations, mainly within COOH-terminus and 3'UTR, leading to 36 nucleotide sequence variants and 21 different coding sequences (cds) segregating in two different clusters. Most of the base mutations were silent, and the number of pro-peptide variants was restricted to four. Finally, as the two amino acid replacements occurred within COOH-terminus, mature peptide from mytilin B appeared unique. Multiple sequencing of partial mytilin B gene from one mussel revealed that one to four randomly distributed mutation points occurred within intron-3. Only one sequence out of the 91 analyzed contained 16 mutation points. In addition, this sequence was the only one containing four out of the six mutation points occurring within exon-4, that code for most of the COOH-terminus domain, including the unique amino acid replacement. Statistical tests for neutrality indicated negative selection pressure on signal and mature peptide domains, but possible positive selection pressure for COOH-terminus domain.

Homing endonucleases (HEs) cut long DNA target sites with high specificity to initiate and target the lateral transfer of mobile introns or inteins. This high site specificity of HEs makes them attractive reagents for gene targeting to promote DNA modification or repair. We have generated several hundred catalytically active, monomerized versions of the well-characterized homodimeric I-CreI and I-MsoI LAGLIDADG family homing endonuclease (LHE) proteins. Representative monomerized I-CreI and I-MsoI proteins (collectively termed mCreIs or mMsoIs) were characterized in detail by using a combination of biochemical, biophysical and structural approaches. We also demonstrated that both mCreI and mMsoI proteins can promote cleavage-dependent recombination in human cells. The use of single chain LHEs should simplify gene modification and targeting by requiring the expression of a single small protein in cells, rather than the coordinate expression of two separate protein coding genes as is required when using engineered heterodimeric zinc finger or homing endonuclease proteins.

Hepatitis C virus (HCV) infection is characterized by persistent replication of a complex mixture of viruses termed a "quasispecies." Transmission is generally associated with a stringent population bottleneck characterized by infection by limited numbers of "transmitted/founder" (T/F) viruses. Characterization of T/F genomes of human immunodeficiency virus type 1 (HIV-1) has been integral to studies of transmission, immunopathogenesis, and vaccine development. Here, we describe the identification of complete T/F genomes of HCV by single-genome sequencing of plasma viral RNA from acutely infected subjects. A total of 2,739 single-genome-derived amplicons comprising 10,966,507 bp from 18 acute-phase and 11 chronically infected subjects were analyzed. Acute-phase sequences diversified essentially randomly, except for the poly(U/UC) tract, which was subject to polymerase slippage. Fourteen acute-phase subjects were productively infected by more than one genetically distinct virus, permitting assessment of recombination between replicating genomes. No evidence of recombination was found among 1,589 sequences analyzed. Envelope sequences of T/F genomes lacked transmission signatures that could distinguish them from chronic infection viruses. Among chronically infected subjects, higher nucleotide substitution rates were observed in the poly(U/UC) tract than in envelope hypervariable region 1. Fourteen full-length molecular clones with variable poly(U/UC) sequences corresponding to seven genotype 1a, 1b, 3a, and 4a T/F viruses were generated. Like most unadapted HCV clones, T/F genomes did not replicate efficiently in Huh 7.5 cells, indicating that additional cellular factors or viral adaptations are necessary for in vitro replication. Full-length T/F HCV genomes and their progeny provide unique insights into virus transmission, virus evolution, and virus-host interactions associated with immunopathogenesis.

The ASK1-JNK3 signaling pathway plays a pivotal role in the pathogenesis of Parkinson's disease (PD). The specific binding of β-arrestin2 to JNK3 is essential for activation of the ASK1-JNK3 cascade, representing a potential therapeutic target for preventing dopaminergic neuronal death in PD. The aim of this study was to identify a novel strategy for the prevention of dopaminergic neuronal death in PD.

This network meta-analysis aimed to investigate the effectiveness and safety of 100 mg BID and 200 mg QD oral celecoxib in the treatment of OA of the knee or hip. PubMed, Embase and Cochrane Library were searched through from inception to August 2014. Bayesian network meta-analysis was used to combine direct and indirect evidences on treatment effectiveness and safety. A total of 24 RCTs covering 11696 patients were included. For the comparison in between the two dosage regimens, 100 mg BID oral celecoxib exhibited a greater probability to be the preferred one either in terms of pain intensity or function at the last follow-up time point. For total gastrointestinal (GI) adverse effects (AEs), both of the two dosage regimens demonstrated a higher incidence compared to the placebo group. Further analyses of GI AEs revealed that only 200 mg QD was associated with a significantly higher risk of abdominal pain when compared with placebo. Furthermore, 100 mg BID showed a significantly lower incidence of skin AEs when compared with 200 mg QD and placebo. Maybe 100 mg BID should be considered as the preferred dosage regimen in the treatment of knee or hip OA.

To evaluate the efficacy and safety of single-dose intra-articular bupivacaine plus morphine after knee arthroscopic surgery.

Antibiotic-resistant mechanisms are associated with fitness costs. However, why antibiotic-resistant bacteria usually show increasing adaptation to hosts is largely unknown, especially from the host's perspective. The present study reveals the host's varied response to balofloxacin-resistant Escherichia coli (BLFX-R) using an integrated proteome and metabolome approach and identifies myo-inositol and phagocytosis-related proteins as crucial biomarkers. Originally, macrophages have an optimal attractive preference to BLFX-S due to more polarization of BLFX-S than BLFX-R, which renders faster elimination to BLFX-S than BLFX-R. The slower elimination to BLFX-R may be reversed by exogenous myo-inositol. Primarily, myo-inositol depolarizes macrophages, elevating adherence to both BLFX-S and BLFX-R. Since the altered adherence is equal to both strains, the myo-inositol-treated macrophages are free of the barrier to BLFX-R and thereby promote phagocytosis of BLFX-R. This work provides a novel strategy based on metabolic modulation for eliminating antibiotic-resistant bacteria with a high degree of host adaptation.

Heart failure (HF) is accompanied by complex alterations in myocardial energy metabolism. Up to 40% of HF patients have dyssynchronous ventricular contraction, which is an independent indicator of mortality. We hypothesized that electromechanical dyssynchrony significantly affects metabolic remodeling in the course of HF. We used a canine model of tachypacing-induced HF. Animals were paced at 200 bpm for 6 weeks either in the right atrium (synchronous HF, SHF) or in the right ventricle (dyssynchronous HF, DHF). We collected biopsies from left ventricular apex and performed comprehensive metabolic pathway analysis using multi-platform metabolomics (GC/MS; MS/MS; HPLC) and LC-MS/MS label-free proteomics. We found important differences in metabolic remodeling between SHF and DHF. As compared to Control, ATP, phosphocreatine (PCr), creatine, and PCr/ATP (prognostic indicator of mortality in HF patients) were all significantly reduced in DHF, but not SHF. In addition, the myocardial levels of carnitine (mitochondrial fatty acid carrier) and fatty acids (12:0, 14:0) were significantly reduced in DHF, but not SHF. Carnitine parmitoyltransferase I, a key regulatory enzyme of fatty acid ß-oxidation, was significantly upregulated in SHF but was not different in DHF, as compared to Control. Both SHF and DHF exhibited a reduction, but to a different degree, in creatine and the intermediates of glycolysis and the TCA cycle. In contrast to this, the enzymes of creatine kinase shuttle were upregulated, and the enzymes of glycolysis and the TCA cycle were predominantly upregulated or unchanged in both SHF and DHF. These data suggest a systemic mismatch between substrate supply and demand in pacing-induced HF. The energy deficit observed in DHF, but not in SHF, may be associated with a critical decrease in fatty acid delivery to the ß-oxidation pipeline, primarily due to a reduction in myocardial carnitine content.

Mesenchymal stem cells (MSCs) are well known having beneficial effects on intracerebral hemorrhage (ICH) in previous studies. The therapeutic mechanisms are mainly to investigate proliferation, differentiation, and immunomodulation. However, few studies have used MSCs to treat blood-brain barrier (BBB) leakage after ICH. The influence of MSCs on the BBB and its related mechanisms were investigated when MSCs were transplanted into rat ICH model in this study.

Systemic siRNA administration to target and treat glioblastoma, one of the most deadly cancers, requires robust and efficient delivery platform without immunogenicity. Here we report newly emerged multivalent naked RNA nanoparticle (RNP) based on pRNA 3-way-junction (3WJ) from bacteriophage phi29 to target glioblastoma cells with folate (FA) ligand and deliver siRNA for gene silencing. Systemically injected FA-pRNA-3WJ RNPs successfully targeted and delivered siRNA into brain tumor cells in mice, and efficiently reduced luciferase reporter gene expression (4-fold lower than control). The FA-pRNA-3WJ RNP also can target human patient-derived glioblastoma stem cells, thought to be responsible for tumor initiation and deadly recurrence, without accumulation in adjacent normal brain cells, nor other major internal organs. This study provides possible application of pRNA-3WJ RNP for specific delivery of therapeutics such as siRNA, microRNA and/or chemotherapeutic drugs into glioblastoma cells without inflicting collateral damage to healthy tissues.

Recently, we have found that a number of microRNAs (miRNAs) and proteins are involved in the response to acupuncture therapy in hypertensive rats. Our bioinformatics study suggests an association between these miRNAs and proteins, which include miR-339 and sirtuin 2 (Sirt2). In this paper, we aimed to investigate whether Sirt2 was a direct target of miR-339 in neurons. In human SH-SY5Y cells, the luciferase assay implied that Sirt2 was likely a target of miRNA-339. Overexpression of miR-339 downregulated Sirt2 expression, while knockdown of miR-339 upregulated Sirt2 expression in human SH-SY5Y cells and rat PC12 cells. In addition, overexpression of miR-399 increased the acetylation of nuclear factor-κB (NF-κB) and forkhead box protein O1 (FOXO1) in SH-SY5Y cells, which are known targets of Sirt2. Our findings demonstrate that miR-339 regulates Sirt2 in human and rat neurons. Since Sirt2 plays a critical role in multiple important cellular functions, our data imply that acupuncture may act through epigenetic changes and subsequent action on their targets, such as miRNA-339/Sirt2/NF-κB/FOXO1 axis. Some physiological level changes of neurons after altering the miR-339 levels are needed to validate the suggested therapeutic role of miR-339/Sirt2/NF-κB/FOXO1 axis in response to acupuncture therapy in the future work.

A single GAG codon deletion in the gene encoding torsinA is linked to most cases of early-onset torsion dystonia. TorsinA is an ER-localized membrane-associated ATPase from the AAA+ superfamily with an unknown biological function. We investigated the formation of oligomeric complexes of torsinA in cultured mammalian cells and found that wild type torsinA associates into a complex with a molecular weight consistent with that of a homohexamer. Interestingly, the dystonia-linked variant torsinAΔE displayed a reduced propensity to form the oligomers compared to the wild type protein. We also discovered that the deletion of the N-terminal membrane-associating region of torsinA abolished oligomer formation. Our results demonstrate that the dystonia-linked mutation in the torsinA gene produces a protein variant that is deficient in maintaining its oligomeric state and suggest that ER membrane association is required to stabilize the torsinA complex.

Normal interfollicular epidermis (IFE) homeostasis is maintained throughout the entire life by its own stem cells that self-renew and generate progeny that undergo terminal differentiation. However, the fine markers of the stem cells in interfollicular epidermis are not well defined yet. Here we found that TLR7 identified the existence of progenitors and interfollicular epidermal stem cells in murine skin. In vitro, TLR7-expressing cells comprised of two subpopulations that were competent to proliferate and exhibited distinct differentiation potentials. Three-dimensional (3D) organotypic culture and skin reconstitution assays showed that TLR7-expressing cells were able to reconstruct the interfollicular epidermis. Finally, TLR7-expressing cells maintained the intact interfollicular epidermal structures revealed in serial transplantation assays in vivo in mice. Taken together, our results suggest that TLR7-expressing cells comprise an interfollicular epidermal stem cell population.

The aim of this study was to investigate whether 6 candidate serum miRNAs and their interactions with serum folate level were associated with the risk for pancreatic cancer (PC).