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.

It has been demonstrated that certain NK-1 antagonists could reduce proliferation of several cancer cell lines, however, it is unknown whether SR140333 exerts proliferation inhibition in breast cancer cell line.

Cancer stem cells (CSCs) are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs.

A very small proportion of people remain negative for HIV infection after repeated HIV-1 viral exposure, which is called HIV-1 resistance. Understanding the mechanism of HIV-1 resistance is important for the development of HIV-1 vaccines and Acquired Immune Deficiency Syndrome (AIDS) therapies. In this study, we analyzed the gene expression profiles of CD4+ T cells from HIV-1-resistant individuals and HIV-susceptible individuals. One hundred eighty-five discriminative HIV-1 resistance genes were identified using the Minimum Redundancy-Maximum Relevance (mRMR) and Incremental Feature Selection (IFS) methods. The virus protein target enrichment analysis of the 185 HIV-1 resistance genes suggested that the HIV-1 protein nef might play an important role in HIV-1 infection. Moreover, we identified 29 infection information exchanger genes from the 185 HIV-1 resistance genes based on a virus-host interaction network analysis. The infection information exchanger genes are located on the shortest paths between virus-targeted proteins and are important for the coordination of virus infection. These proteins may be useful targets for AIDS prevention or therapy, as intervention in these pathways could disrupt communication with virus-targeted proteins and HIV-1 infection.

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.

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.

Recent findings have revealed that gut microbiota plays a substantial role in modulating diseases such as autism, rheumatoid arthritis, allergies, and cancer that occur at sites distant to the gut. Athymic nude mice have been employed for tumorigenic research for decades; however, the relationships between the gut microbiome and host's response in drug treatment to the grafted tumors have not been explored. In this study, we analyzed the fecal microbiome of nonxenograft and xenograft nude mice treated with phytosaponins from a popular medicinal plant, Gynostemma pentaphyllum (Gp). Analysis of enterobacterial repetitive intergenic consensus (ERIC)-PCR data showed that the microbiota profile of xenograft mice departed from that of the nonxenograft mice. After ten days of treatment with Gp saponins (GpS), the microbiota of the treated mice was closer to the microbiota at Day 0 before the implantation of the tumor. Data obtained from 16S pyrosequencing of fecal samples reiterates the differences in microbiome between the nonxenograft and xenograft mice. GpS markedly increased the relative abundance of Clostridium cocleatum and Bacteroides acidifaciens, for which the beneficial effects on the host have been well documented. This study, for the first time, characterizes the properties of gut microbiome in nude mice responding to tumor implant and drug treatment. We also demonstrate that dietary saponins such as GpS can potentially regulate the gut microbial ecosystem by increasing the number of symbionts. Interestingly, this regulation of the gut ecosystem might, at least in part, be responsible for or contribute to the anticancer effect of GpS.

Genomic in situ hybridization (GISH) has been widely used to detect rye (Secale cereale L.) chromosomes in wheat (Triticum aestivum L.) introgression lines. The routine procedure of GISH using genomic DNA of rye as a probe is time-consuming and labor-intensive because of the preparation and labeling of genomic DNA of rye and denaturing of chromosomes and probes. In this study, new oligonucleotide probes Oligo-1162, Oligo-pSc200 and Oligo-pSc250 were developed. The three new probes can be used for non-denaturing fluorescence in situ hybridization (ND-FISH) assays and replace genomic DNA of rye as a probe to discriminate rye chromosomes in wheat backgrounds. In addition, previously developed oligonucleotide probes Oligo-pSc119.2-1, Oligo-pSc119.2-2, Oligo-pTa535-1, Oligo-pTa535-2, Oligo-pTa71-2, Oligo-pAWRC.1 and Oligo-CCS1 can also be used for ND-FISH of wheat and rye. These probes have provided an easier, faster and more cost-effective method for the FISH analysis of wheat and hybrids derived from wheat × rye.

Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by "DS-Cav1" mutations and by an appended C-terminal trimerization motif or "foldon" from T4-bacteriophage fibritin. Here we investigate the creation of a cysteine zipper to allow for the removal of the phage foldon, while maintaining the immunogenicity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide "rings", with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cysteine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen.

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.

Prediction of disease-specific survival (DSS) for individual patient with gastric cancer after R0 resection remains a clinical concern. Since the clinicopathologic characteristics of gastric cancer vary widely between China and western countries, this study is to evaluate a nomogram from Memorial Sloan-Kettering Cancer Center (MSKCC) for predicting the probability of DSS in patients with gastric cancer from a Chinese cohort.

Many stem cells grow into three-dimensional (3D) spheres or colonies, such as neural progenitor cells (NPCs) and embryonic stem cells (ESCs). Sphere morphology helps maintaining the stemness of stem cells. Our previous study demonstrated that forced growth of RT4 and HEK293 cells into 3D sphere on low attachment surface could induce stem cell properties. The close relationship between 3D sphere morphology and stem cell stemness drives us to hypothesize that 3D sphere formation induces fibroblasts reprogramming. The key gene Sox2 for reprogramming fibroblasts into NPCs was found to be overexpressed in 3D sphere cultured mouse fibroblasts. These cells exhibited similar morphological and molecular features to NPCs in vitro, were capable of differentiating into neurons, astrocytes and oligodendrocytes, and could generate long-term expandable neurospheres while maintaining differentiation capability. When engrafted into hippocampus of adult rat brain, the 3D sphere cells differentiated into neural cells. Thus, NPCs can be generated from fibroblasts directly through a physical approach without introducing exogenous reprogramming factors.

Drug combinatorial therapy could be more effective in treating some complex diseases than single agents due to better efficacy and reduced side effects. Although some drug combinations are being used, their underlying molecular mechanisms are still poorly understood. Therefore, it is of great interest to deduce a novel drug combination by their molecular mechanisms in a robust and rigorous way. This paper attempts to predict effective drug combinations by a combined consideration of: (1) chemical interaction between drugs, (2) protein interactions between drugs' targets, and (3) target enrichment of KEGG pathways. A benchmark dataset was constructed, consisting of 121 confirmed effective combinations and 605 random combinations. Each drug combination was represented by 465 features derived from the aforementioned three properties. Some feature selection techniques, including Minimum Redundancy Maximum Relevance and Incremental Feature Selection, were adopted to extract the key features. Random forest model was built with its performance evaluated by 5-fold cross-validation. As a result, 55 key features providing the best prediction result were selected. These important features may help to gain insights into the mechanisms of drug combinations, and the proposed prediction model could become a useful tool for screening possible drug combinations.

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.

Cultivated rice consists of two important ecotypes, upland and irrigated, that have respectively adapted to either dry land or irrigated cultivation. Upland rice, widely adopted in rainfed upland areas in virtue of its little water requirement, contains abundant untapped genetic resources, such as genes for drought adaptation. With water shortage exacerbated and population expanding, the need for breeding crop varieties with drought adaptation becomes more and more urgent. However, a previous oversight in upland rice research reveals little information regarding its genetic mechanisms for upland adaption, greatly hindering progress in harnessing its genetic resources for breeding and cultivation.

The human sulfatase 1 (hSulf-1) gene encodes an endosulfatase that functions to inhibit the heparin-binding growth factor signaling, including the basic fibroblast growth factor (bFGF)-mediated pathway, by desulfating the cell surface heparan sulfate proteoglycans (HSPGs). bFGF could stimulate cell cycle progression and inhibit cell apoptosis, this biological effect can be reversed by hSulf-1. However, molecular mechanisms have not been fully reported. In the current study, by reactivation of hSulf-1 expression and function in the hSulf-1-negative hepatocellular carcinoma (HCC) cell lines and HCC xenograft tumors, we found that hSulf-1 blocked the bFGF effect on the promotion of cell cycle and inhibition of apoptosis. The bFGF-stimulated activation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) pathways was suppressed by hSulf-1, which led to a decreased expression of the target genes Cyclin D1 and Survivin, then finally induced cell cycle arrest and apoptosis in HCC cells. Our data suggested that hSulf-1 may be a suitable target for cancer therapy.