Artemis was initially discovered as the gene inactivated in human radiosensitive T(-)B(-) severe combined immunodeficiency, a syndrome characterized by the absence of B and T lymphocytes and cellular hypersensitivity to ionizing radiation. Hypomorphic Artemis alleles have also been identified in patients and are associated with combined immunodeficiencies of varying severity. We examine the molecular mechanisms underlying a syndrome of partial immunodeficiency caused by a hypomorphic Artemis allele using the mouse as a model system. This mutation, P70, leads to premature translation termination that deletes a large portion of a nonconserved C terminus. We find that homozygous Artemis-P70 mice exhibit reduced numbers of B and T lymphocytes, thereby recapitulating the patient phenotypes. The hypomorphic mutation results in impaired end processing during the lymphoid-specific DNA rearrangement known as V(D)J recombination, defective double-strand break repair, and increased chromosomal instability. Biochemical analyses reveal that the Artemis-P70 mutant protein interacts with the DNA-dependent protein kinase catalytic subunit and retains significant, albeit reduced, exo- and endonuclease activities but does not undergo phosphorylation. Together, our findings indicate that the Artemis C terminus has critical in vivo functions in ensuring efficient V(D)J rearrangements and maintaining genome integrity.

Disease epizootics in freshwater culture crustaceans (crab, crayfish and shrimp) gained high attention recently in China, due to intensive developments of freshwater aquacultures. Spiroplasma was identified as a lethal pathogen of the above three freshwater crustaceans in previous studies. Further characterization of these freshwater crustacean Spiroplasma strains were analyzed in the current study. Phylogenetic position was investigated by analysis of partial nucleotide sequences of 16S ribosomal RNA (rRNA), gyrB and rpoB genes, together with complete sequencing of 23S rRNA gene and 16S-23S rRNA intergenetic spacer regions (ISRs). Phylogenetic analysis of these sequences showed that the above-mentioned three freshwater crustacean Spiroplasma strains were identical and had a close relationship with Spiroplasma mirum. Furthermore, the genomic size, serological studies and experimental infection characteristics confirmed that three freshwater crustacean Spiroplasma strains are a single species other than traditional S. mirum. Therefore, these data suggest that a single species of Spiroplasma infects all three investigated freshwater crustaceans in China, and is a potential candidate for a new species within the Spiroplasma genus. These results provide critical information for the further investigations in fresh aquaculture epizootics related to tremor diseases, caused by this infectious agent.

Perifosine is a membrane-targeted alkylphospholipid developed to inhibit the PI3K/Akt pathway and has been suggested as a favorable candidate for combined use with radiotherapy. In this study, we investigated the effect of the combined treatment of perifosine and radiation (CTPR) on prostate cancer cells in vitro and on prostate cancer xenografts in vivo.

Gene duplication is supposed to be the major source for genetic innovations. However, how a new duplicate gene acquires functions by integrating into a pathway and results in adaptively important phenotypes has remained largely unknown. Here, we investigated the biological roles and the underlying molecular mechanism of the young kep1 gene family in the Drosophila melanogaster species subgroup to understand the origin and evolution of new genes with new functions. Sequence and expression analysis demonstrates that one of the new duplicates, nsr (novel spermatogenesis regulator), exhibits positive selection signals and novel subcellular localization pattern. Targeted mutagenesis and whole-transcriptome sequencing analysis provide evidence that nsr is required for male reproduction associated with sperm individualization, coiling, and structural integrity of the sperm axoneme via regulation of several Y chromosome fertility genes post-transcriptionally. The absence of nsr-like expression pattern and the presence of the corresponding cis-regulatory elements of the parental gene kep1 in the pre-duplication species Drosophila yakuba indicate that kep1 might not be ancestrally required for male functions and that nsr possibly has experienced the neofunctionalization process, facilitated by changes of trans-regulatory repertories. These findings not only present a comprehensive picture about the evolution of a new duplicate gene but also show that recently originated duplicate genes can acquire multiple biological roles and establish novel functional pathways by regulating essential genes.

A collection of in vitro evidence has demonstrated that Notch signaling plays a key role in the growth of neurites in differentiated neurons. However, the effects of Notch signaling on axon outgrowth in an in vivo condition remain largely unknown.

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a transcription factor which regulates the expression of many cytoprotective genes. In the present study, we found that the expression of Nrf2 was suppressed in prostate tumor of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. Similarly, the expression of Nrf2 and the induction of NQO1 were also substantially suppressed in tumorigenic TRAMP C1 cells but not in non-tumorigenic TRAMP C3 cells. Examination of the promoter region of the mouse Nrf2 gene identified a CpG island, which was methylated at specific CpG sites in prostate TRAMP tumor and in TRAMP C1 cells but not in normal prostate or TRAMP C3 cells, as shown by bisulfite genomic sequencing. Reporter assays indicated that methylation of these CpG sites dramatically inhibited the transcriptional activity of the Nrf2 promoter. Chromatin immunopreceipitation (ChIP) assays revealed increased binding of the methyl-CpG-binding protein 2 (MBD2) and trimethyl-histone H3 (Lys9) proteins to these CpG sites in the TRAMP C1 cells as compared to TRAMP C3 cells. In contrast, the binding of RNA Pol II and acetylated histone H3 to the Nrf2 promoter was decreased. Furthermore, treatment of TRAMP C1 cells with DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-aza) and histone deacetylase (HDAC) inhibitor trichostatin A (TSA) restored the expression of Nrf2 as well as the induction of NQO1 in TRAMP C1 cells. Taken together, these results indicate that the expression of Nrf2 is suppressed epigenetically by promoter methylation associated with MBD2 and histone modifications in the prostate tumor of TRAMP mice. Our present findings reveal a novel mechanism by which Nrf2 expression is suppressed in TRAMP prostate tumor, shed new light on the role of Nrf2 in carcinogenesis and provide potential new directions for the detection and prevention of prostate cancer.

Yersinia enterocolitica is an enteric pathogen that invades the intestinal mucosa and proliferates within the lymphoid follicles (Peyer's patches). The attachment invasion locus (ail) mediates invasion by Y. enterocolitica and confers an invasive phenotype upon non-invasive E. coli; ail is the primary virulence factor of Y. enterocolitica. The ferrioxamine receptor (foxA) located on the Y. enterocolitica chromosome, together with its transport protein, transports a siderophore specific for ferric ion. Currently, ail is the primary target gene for nucleic acid detection of pathogenic Y. enterocolitica.

The prevalence of obesity and diabetes is increasing dramatically throughout the world. Studies have shown that excess adiposity is a critical predictor of new onset T2DM. This meta-analysis is aimed to assess the metabolic effects of fluoxetine in T2DM.

The peach tree, Prunus persica (L.) Batsch, is widely cultivated in China, and its flowers have been used for centuries in traditional Chinese medicine to treat gut motility disorders. But few studies have explored the pharmacological effect of Prunus persica (L.) Batsch flowers on gastrointestinal motility. In this study, the activities of different extracts from Prunus persica (L.) Batsch flowers on the smooth muscle contractions were evaluated using isolated colon model, and the ethyl acetate extract (EAE) showed the strongest effects in vitro. EAE (10(-8)-10(-5) g/mL) caused a concentration-dependent stimulatory effect in rat colonic tissue. Additionally, ketotifen (100 µM), cimetidine (10 µM), and pyrilamine (1 µM) produced a significant inhibition of contractions caused by EAE. Furthermore, immunofluorescence and toluidine blue staining revealed increased numbers of mast cells in the EAE group, and EAE increased histamine release from the colonic tissues. These data indicate that EAE has significant prokinetic activity and acts by a mechanism that mainly involves mast cell degranulation. Our study provides a pharmacological basis for the use of an extract of Prunus persica (L.) Batsch flowers in the treatment of gut motility disorders.

Calcium phosphate- (CaP-) based composite scaffolds have been used extensively for the bone regeneration in bone tissue engineering. Previously, we developed a biomimetic composite nanofibrous membrane of gelatin/β-tricalcium phosphate (TCP) and confirmed their biological activity in vitro and bone regeneration in vivo. However, how these composite nanofibers promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unknown. Here, gelatin/β-TCP composite nanofibers were fabricated by incorporating 20 wt% β-TCP nanoparticles into electrospun gelatin nanofibers. Electron microscopy showed that the composite β-TCP nanofibers had a nonwoven structure with a porous network and a rough surface. Spectral analyses confirmed the presence and chemical stability of the β-TCP and gelatin components. Compared with pure gelatin nanofibers, gelatin/β-TCP composite nanofibers caused increased cell attachment, proliferation, alkaline phosphatase activity, and osteogenic gene expression in rat BMSCs. Interestingly, the expression level of the calcium-sensing receptor (CaSR) was significantly higher on the composite nanofibrous scaffolds than on pure gelatin. For rat calvarial critical sized defects, more extensive osteogenesis and neovascularization occurred in the composite scaffolds group compared with the gelatin group. Thus, gelatin/β-TCP composite scaffolds promote osteogenic differentiation of BMSCs in vitro and bone regeneration in vivo by activating Ca(2+)-sensing receptor signaling.

Reduced neutrophil apoptosis plays an important role in the pathogenesis of acute exacerbation chronic obstructive pulmonary disease (AECOPD). The p38 mitogen-activated protein kinase (MAPK) signaling pathway is involved in neutrophil apoptosis. 1α,25-Dihydroxyvitamin D3 (1α,25VitD3) can induce tumor cell apoptosis. The aim of this study was to assess the effects of 1α,25VitD3 on peripheral blood neutrophil apoptosis in AECOPD and examine the role of the p38 MAPK signaling pathway.

Few gene markers selectively identify mesenchymal progenitor cells inside the bone marrow. We have investigated a cell population located in the mouse bone marrow labeled by Connective Tissue Growth Factor reporter expression (CTGF-EGFP). Bone marrow flushed from CTGF reporter mice yielded an EGFP+ stromal cell population. Interestingly, the percentage of stromal cells retaining CTGF reporter expression decreased with age in vivo and was half the frequency in females compared to males. In culture, CTGF reporter expression and endogenous CTGF expression marked the same cell types as those labeled using Twist2-Cre and Osterix-Cre fate mapping approaches, which previously had been shown to identify mesenchymal progenitors in vitro. Consistent with this past work, sorted CTGF+ cells displayed an ability to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and into osteoblast, adipocyte, and stromal cell lineages after transplantation into a parietal bone defect. In vivo examination of CTGF reporter expression in bone tissue sections revealed that it marked cells highly localized to the trabecular bone region and was not expressed in the perichondrium or periosteum. Mesenchymal cells retaining high CTGF reporter expression were adjacent to, but distinct from mature osteoblasts lining bone surfaces and endothelial cells forming the vascular sinuses. Comparison of CTGF and Osterix reporter expression in bone tissue sections indicated an inverse correlation between the strength of CTGF expression and osteoblast maturation. Down-regulation of CTGF reporter expression also occurred during in vitro osteogenic differentiation. Collectively, our studies indicate that CTGF reporter mice selectively identify a subpopulation of bone marrow mesenchymal progenitor cells that reside in the trabecular bone region.

The development of an effective vaccine is critical for prevention of a Middle East respiratory syndrome coronavirus (MERS-CoV) pandemic. Some studies have indicated the receptor-binding domain (RBD) protein of MERS-CoV spike (S) is a good candidate antigen for a MERS-CoV subunit vaccine. However, highly purified proteins are typically not inherently immunogenic. We hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. Therefore, the immunogenicity of a novel MERS-CoV RBD-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. Different vaccination regimens were compared in BALB/c mice immunized 3 times intramuscularly (i.m.) with a vaccine containing 10 µg of recombinant MERS-CoV RBD in combination with either aluminium hydroxide (alum) alone, alum and polyriboinosinic acid (poly I:C) or alum and cysteine-phosphate-guanine (CpG) oligodeoxynucleotides (ODN). The immune responses of mice vaccinated with RBD, incomplete Freund's adjuvant (IFA) and CpG ODN by a subcutaneous (s.c.) route were also investigated. We evaluated the induction of RBD-specific humoral immunity (total IgG and neutralizing antibodies) and cellular immunity (ELISpot assay for IFN-γ spot-forming cells and splenocyte cytokine production). Our findings indicated that the combination of alum and CpG ODN optimized the development of RBD-specific humoral and cellular immunity following subunit vaccination. Interestingly, robust RBD-specific antibody and T-cell responses were induced in mice immunized with the rRBD protein in combination with IFA and CpG ODN, but low level of neutralizing antibodies were elicited. Our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. The vaccination regimen used in this study is promising and could improve the protection offered by the MERS-CoV subunit vaccine by eliciting effective humoral and cellular immune responses.

Peritrophin was first isolated from insect peritrophic membrane (PM) and was thought to protect insects from invasion of microorganisms and to stimulate digestion of food. In this study, a peritrophin-like gene (EsPT) was obtained from Eriocheir sinensis. The full length cDNA of EsPT was 1232 bp, which contained 1005 bp ORF encoding a protein of 334 amino acids, including a 22 amino acid signal peptide, and 3 conserved chitin binding type 2 domains (ChtBD2) characterized by having a 6-cysteine motif. Phylogenetic analysis showed that EsPT was clustered together with 2 insect peritrophin-44-like proteins (MdP44L from Musca domestica and CcP44L from Ceratitis capitata), an insect chitin binding peritrophin-A domain containing protein (CfPT from Coptotermes formosanus) and a crustacean peritrophin (MnPT from Macrobrachium nipponense). Tissue distribution analysis revealed that EsPT was mainly expressed in hepatopancreas, intestine and hemocytes. The expression of EsPT is regulated by lipopolysaccharide, peptidoglycan, Staphylococcus aureus, Vibrio parahaemolyticus and Aeromonas hydrophila challenge. The recombinant EsPT could bind to different microbes, and enhanced the clearance of V. parahaemolyticus in vivo. In crabs, silencing of EsPT by siRNA suppressed the elimination of V. parahaemolyticus and increasing number of bacteria, finally upregulated the expression of anti-lipopolysaccharide factor (ALF) and clip domain serine proteases (cSP). The results might indicate that EsPT was involved in the anti-bacterial innate immunity of crabs.

C-type lectins (CTLs) have crucial functions in recognizing and eliminating pathogens in innate immunity. This study identified a novel low-density lipoprotein receptor class A (LDLa) domain-containing CTL, designated as EsCTLDcp, from the Chinese mitten crab Eriocheir sinensis. The EsCTLDcp cDNA is 1258 bp long, with a 975 bp open reading frame that encodes a 324-amino acid protein. EsCTLDcp contains a signal peptide, an LDLa, and a single C-type lectin-like domain. EsCTLDcp was only expressed in the hepatopancreas of normal crabs, and its expression was regulated following crab challenge with pathogen-associated molecular patterns and with bacteria. The recombinant EsCTLDcp agglutinates Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Vibrio parahaemolyticus and Aeromonas hydrophila) in the presence of calcium. rEsCTLDcp also binds to various bacteria including S. aureus, Bacillus thuringiensis, Bacillus subtilis, Escherichia coli, Vibrio natriegens, V. parahaemolyticus, and A. hydrophila. The rEsCTLDcp protein helped the crabs clear the virulent Gram-negative bacterium V. parahaemolyticus in vivo, as well as interacted with VP24, an envelope protein of white spot syndrome virus (WSSV). These data suggest that EsCTLDcp functions as a pattern-recognition receptor involved in the innate immunity of E. sinensis.

The Tibetan antelope (Pantholops hodgsonii) is endemic to the extremely inhospitable high-altitude environment of the Qinghai-Tibetan Plateau, a region that has a low partial pressure of oxygen and high ultraviolet radiation. Here we generate a draft genome of this artiodactyl and use it to detect the potential genetic bases of highland adaptation. Compared with other plain-dwelling mammals, the genome of the Tibetan antelope shows signals of adaptive evolution and gene-family expansion in genes associated with energy metabolism and oxygen transmission. Both the highland American pika, and the Tibetan antelope have signals of positive selection for genes involved in DNA repair and the production of ATPase. Genes associated with hypoxia seem to have experienced convergent evolution. Thus, our study suggests that common genetic mechanisms might have been utilized to enable high-altitude adaptation.

Myeloid differentiation factor 88 (MyD88) is a universal and essential adapter protein that participates in the activation of the Toll-like receptor (TLR)/interleukin-1 receptor-mediated signaling pathway. In this study, two MyD88 genes (HcMyD88-1 and HcMyD88-2) were identified from triangle-shell pearl mussel (Hyriopsis cumingii). Both HcMyD88-1 and HcMyD88-2 proteins were determined to have a death domain at the N-terminal and a TIR domain at the C-terminal. Both HcMyD88-1 and HcMyD88-2 genes were mainly expressed in the hepatopancreas of healthy mussels. HcMyD88-1 and HcMyD88-2 slightly responded to Gram-negative bacterial challenge. Upon bacterial challenge with Gram-positive Staphyloccocus aureus, HcMyD88-1 and HcMyD88-2 transcription levels remarkably increased at 2 and 6h, respectively. Overexpression of HcMyD88-1 and HcMyD88-2 proteins in Drosophila Schneider 2 cells led to the activation of antimicrobial peptide genes. These results indicated that HcMyD88-2 had higher activity than HcMyD88-1 during the activation of attacin A, drosomycin, and metchnikowin genes, suggesting that HcMyD88 genes may play a role in antibacterial innate immune defense.

To determine the prevalence, epidemiology and genetic diversity of human coronavirus OC43 (HCoV-OC43) among adult patients with acute respiratory infections (ARI) in Beijing,five hundred and fifty-nine nasopharyngeal swab samples were collected from adult patients with ARI in Beijing. The prevalence of HCoV-OC43 infection among these patients was assessed using two different OneStep reverse transcription polymerase chain reaction (RT-PCR) assays. The epidemiological profiles of the patients with HCoV-OC43 infection were described. Partial S and N genes of HCoV-OC43 circulating strains were sequenced followed by phylogenetic analysis and amino acid alignment. Our results showed that the prevalence of HCoV-OC43 infection was 12.52% (95% CI: 9.78-15.26%), and the epidemic peak occurred in autumn. Fifty partial S and 40 partial N fragments were obtained from these patients. Phylogenetic analysis based on neighbour-joining method showed that at least three distinct clusters (A, B, C/D) of HCoV-OC43 strains were circulating among adult patients with ARI in Beijing. In addition, some novel unique clusters (UNT) of HCoV-OC43 were found in the S- and N-based phylogenetic trees. Furthermore, consensus amino acids substitutes for each cluster were also found after alignment of partial S or N sequence coding region in this study. In conclusion, we herein describe the prevalence of HCoV-OC43 among adult patients and provide substantial evidence for the genetic diversity of HCoV-OC43 circulating in Beijing.

Hemorrhagic fever with renal syndrome (HFRS) was first recognized in far eastern Asia in the 1930s, and has been highly prevalent in this region ever since. To reveal the molecular epidemiology of hantaviruses in this region, a total of 374 small mammals (eight species of rodents and one species of shrew) were captured in the Chinese part of the Bolshoy Ussuriysky Island (Heilongjiang Province). Hantavirus sequences were recovered from three striped field mice (Apodemus agrarius), 11 Maximowicz's voles (Microtus maximowiczii), and one flat-skulled shrew (Sorex roboratus). Genetic and phylogenetic analysis revealed the presence of three viruses: Hantaan virus (HTNV), Khabarovsk virus (KHAV), and Kenkeme virus (KKMV). HTNV sequences recovered from A. agrarius were closely related to those identified in Apodemus mice from the surrounding areas, while a new lineage of KHAV was present in M. maximowiczii. Additionally, while the viral sequences recovered from one flat-skulled shrew were most closely related to KKMV, their divergence to the prototype strain suggests that they represent a new viral subtype. Overall, these results suggest that Bolshoy Ussuriysky Island harbors considerable hantavirus diversity.

Ultraviolet irradiation and carcinogens have been reported to induce epigenetic alterations, which potentially contribute to the development of skin cancer. We aimed to study the genome-wide DNA methylation profiles of skin cancers induced by ultraviolet B (UVB) irradiation and 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-1,3-acetate (TPA).