We developed and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting Japanese encephalitis virus (JEV). The sensitivity of the JEV RT-LAMP assay was in concordance with that of real-time RT-PCR and 10-fold more sensitive than that of conventional RT-PCR, which the detection limit was 24 copies/μl. The JEV RT-LAMP was highly specific, which no cross-reactivity was found with dengue-2 virus, rabies virus, norovirus, astrovirus and human enterovirus 71. The JEV RT-LAMP assay was more simple and less time-consuming compared to the conventional RT-PCR and real-time RT-PCR, which the amplification could be completed in a single tube within 1 h under isothermal conditions at temperature of 63°C. The results suggest that the RT-LAMP assay can be applied as a practical molecular diagnostic tool for JEV infection and surveillance.

Analysis of protein-protein interactions (PPIs) is a valuable approach for the characterization of huge networks of protein complexes or proteins of unknown function. Co-immunoprecipitation (coIP) using affinity resins coupled to protein A/G is the most widely used method for PPI detection. However, this traditional large scale resin-based coIP is too laborious and time consuming. To overcome this problem, we developed a miniaturized sandwich immunoassay platform (MSIP) by combining antibody array technology and coIP methods.

Long-term livestock over-grazing causes nitrogen outputs to exceed inputs in Inner Mongolia, suggesting that low levels of nitrogen fertilization could help restore grasslands degraded by overgrazing. However, the effectiveness of such an approach depends on the response of production and species composition to the interactive drivers of nitrogen and water availability. We conducted a five-year experiment manipulating precipitation (NP: natural precipitation and SWP: simulated wet year precipitation) and nitrogen (0, 25 and 50 kg N ha(-1) yr(-1)) addition in Inner Mongolia. We hypothesized that nitrogen fertilization would increase forage production when water availability was relatively high. However, the extent to which nitrogen would co-limit production under average or below average rainfall in these grasslands was unknown.Aboveground net primary production (ANPP) increased in response to nitrogen when precipitation was similar to or higher than the long-term average, but not when precipitation was below average. This shift in limitation was also reflected by water and nitrogen use efficiency. Belowground live biomass significantly increased with increasing water availability, but was not affected by nitrogen addition. Under natural precipitation (NP treatment), the inter-annual variation of ANPP was 3-fold greater than with stable water availability (CV(ANPP) = 61±6% and 17±3% for NP and SWP treatment, respectively) and nitrogen addition increased CV(ANPP) even more (89±14%). This occurred in part because fertilizer nitrogen left in the soil in dry years remained available for uptake during wet years and because of high production by unpalatable annual species in wet years in the NP treatment. In summary, plant growth by residual fertilizer nitrogen could lead to sufficient yields to offset lack of additional production in dry years. However, the utility of fertilization for restoration may be constrained by shifts in species composition and the lack of response by belowground biomass, which reduces replacement of soil carbon and nitrogen.

ALDH1 has been shown to play a role in the early differentiation of stem cells in some human malignancies. Whether cancer stem cells occur in ALDH1-associated cervical cancer is not known.

Rhizoma Paridis Saponins (RPS), a natural compound purified from Rhizoma Paridis, has been found to inhibit cancer growth in vitro and in animal models of cancer. However, its effects on esophageal cancer remain unexplored. The purpose of this study was to investigate the effects of RPS on tumor growth in a rat model of esophageal cancer and the molecular mechanism underlying the effects. A rat model of esophageal cancer was established by subcutaneous injection of N-nitrosomethylbenzylamine (NMBA, 1 mg/kg) for 10 weeks. RPS (350 mg/kg or 100 mg/kg) was administered by oral gavage once daily for 24 weeks starting at the first NMBA injection. RPS significantly reduced the size and number of tumors in the esophagus of rats exposed to NMBA and inhibited the viability, migration, and invasion of esophageal cancer cells EC9706 and KYSE150 in a dose dependent manner (all P < 0.01). Flow cytometry revealed that RPS induced apoptosis and cell cycle G2/M arrest in the esophageal cancer cells. The expression of cyclooxygenases-2 (COX-2) and Cyclin D1 in rat esophageal tissues and the esophageal cancer cells were also significantly reduced by RPS (all P < 0.01). Consistently, RPS also significantly decreased the release of prostaglandin E2, a downstream molecule of COX-2, in a dose-dependent manner (P < 0.01). Our study suggests that RPS inhibit esophageal cancer development by promoting apoptosis and cell cycle arrest and inhibiting the COX-2 pathway. RPS might be a promising therapeutic agent for esophageal cancer.

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a seven transmembrane receptor known as a potential stem cell marker for intestinal crypts and hair follicles, has recently been found to be overexpressed in some types of human cancers. However, the role of LGR5 in cervical cancer remains unclear. In this study, the expression of LGR5 gradually increases from normal cervix to cervical cancer in situ and to cervical cancers as revealed by immunohistochemistry and western blot analyses. Through knocking down or overexpressing LGR5 in SiHa and HeLa cells, the expression level of LGR5 was found to be positively related to cell proliferation in vitro and to tumor formation in vivo. Further investigation indicated that LGR5 protein could significantly promote the acceleration of cell cycle. Moreover, the TOP-Flash reporter assay and western blot for β-catenin, cyclinD1, and c-myc proteins, target genes of the Wnt/β-catenin pathway, indicated that LGR5 significantly activated Wnt/β-catenin signaling. Additionally, the blockage of Wnt/β-catenin pathway resulted in a significant inhibition of cell proliferation induced by LGR5. Taken together, these results demonstrate that LGR5 can promote proliferation and tumor formation in cervical cancer cells by activating the Wnt/β-catenin pathway.

The transcription factor gene Pax6 is widely considered a master regulator of eye development in bilaterian animals. However, the existence of visual organs that develop without Pax6 input and the considerable pleiotropy of Pax6 outside the visual system dictate further studies into defining ancestral functions of this important regulator. Previous work has shown that the combinatorial knockdown of the insect Pax6 orthologs eyeless (ey) and twin of eyeless (toy) perturbs the development of the visual system but also other areas of the larval head in the red flour beetle Tribolium castaneum. To elucidate the role of Pax6 during Tribolium head development in more detail, we studied head cuticle morphology, brain anatomy, embryonic head morphogenesis, and developmental marker gene expression in combinatorial ey and toy knockdown animals. Our experiments reveal that Pax6 is broadly required for patterning the anterior embryonic head. One of the earliest detectable roles is the formation of the embryonic head lobes, which originate from within the ocular segment and give rise to large parts of the supraesophageal brain including the mushroom body, a part of the posterior head capsule cuticle, and the visual system. We present further evidence that toy continues to be required for the development of the larval eyes after formation of the embryonic head lobes in cooperation with the eye developmental transcription factor dachshund (dac). The sum of our findings suggests that Pax6 functions as a competence factor throughout the development of the insect ocular segment. Comparative evidence identifies this function as an ancestral aspect of bilaterian head development.

Unexplained infertility requires that more sensitive and mechanism-based biomarkers should be developed and used independently of or in addition to conventional semen parameters for an infertility diagnosis. In the present study, semen samples were collected from young men participating in the Male Reproductive Health in Chongqing College students (MARCHS) cohort study in the follow-up stage in 2014. Conventional semen parameters were measured in all 656 participants, whereas sperm mitochondrial membrane potential (MMP), mitochondrial DNA copy number (mtDNAcn), mtDNA integrity and apoptotic parameters were measured among 627, 386, 362, and 628 participants, respectively. We found that sperm MMP was significantly positively correlated with all of conventional semen parameters including semen volume (r = 0.090, p = 0.025), sperm concentration (r = 0.301, p<0.01), total sperm count (r = 0.324, p<0.01), and progressive motility (r = 0.399, p<0.01); sperm MMP was also negatively correlated with Annexin V+ sperm (r = -0.553, p<0.01); mtDNAcn was significantly negatively correlated with sperm concentration (r = -0.214, p<0.01), total sperm count (r = -0.232, p<0.01), and progressive motility (r = -0.164, p = 0.01); mtDNA integrity was also significantly positively correlated with sperm concentration (r = 0.195, p<0.01), total sperm count (r = 0.185, p<0.01), and progressive motility (r = 0.106, p = 0.043). After adjusting for potential confounders, these relationships remained significant. Furthermore, we explored the potential effects of lifestyles on such mitochondrial biomarkers and found that the current drinkers displayed a higher level of sperm MMP; additionally, mt DNAcn was increased with age. The results indicated that certain mitochondrial biomarkers could serve as predictors of semen quality in a general population, and the study provides a baseline for the effects of population characteristics and lifestyles on such mitochondrial markers.

Schizophrenia (SCZ), bipolar disorder (BD) and depressive disorder (DD) are common psychiatric disorders, which show common genetic vulnerability. Previous gene-disease association studies have reported correlations between d-amino acid oxidase activator (DAOA) gene polymorphisms and the three psychiatric disorders. However, the findings were contradictory. A meta-analysis was therefore conducted to provide more robust investigations into DAOA polymorphisms and the risk of SCZ, BD and DD.

Pseudomonas sp. strain 2-92, isolated from a Canadian field plot under long-term mineral fertilization, strongly inhibits the growth of Fusarium graminearum, Rhizoctonia solani, and Gaeumannomyces graminis. Here, we report the draft genome sequence of Pseudomonas sp. strain 2-92.

Diabetes mellitus (DM) is one of the primary causes of premature death and disability worldwide. We performed a systematic review and meta-analysis of the published literature regarding the trends in prevalence, awareness, treatment, and control of diabetes mellitus in mainland China. PUBMED, EMBASE, Chinese Biomedical Database, China National Infrastructure database, Chinese Wan Fang database, and Chongqing VIP database were searched. Fifty-six eligible studies were included. Increasing trends in the prevalence, treatment, and control of diabetes in mainland China from 1979 to 2012 were observed. The pooled prevalence, awareness, treatment, and control of diabetes mellitus were 6.41%, 45.81%, 42.54%, and 20.87%, respectively. A higher prevalence of diabetes mellitus was found in urban (7.48%, 95%CI = 5.45~9.50) than rural (6.53%, 95%CI = 4.30~8.76) areas. Furthermore, an increasing chronological tendency was shown in different subgroups of age with regard to the prevalence of diabetes. A higher awareness of DM was found in urban (44.25%, 95%CI = 32.60~55.90) than rural (34.27%, 95%CI = 21.00~47.54) populations, and no significant differences were found in the treatment, and control of diabetes among the subgroups stratified by gender and location. From 1979 to 2012, the prevalence, treatment, and control of diabetes mellitus increased; nevertheless, there was no obvious improvement in the awareness of diabetes.

In the present study, the wastewater sample collected from the Dongming discharging river in Shijiazhuang city was analysed using both chemical analysis and biological assays including the Salmonella mutagenicity test, micronucleus test and single-cell gel electrophoresis. Chemical analysis of the sample was performed using gas chromatography mass spectrometry and inductively coupled plasma mass spectrometry. The Salmonella mutagenicity test was performed on Salmonella typhimurium TA97, TA98, TA100 and TA102 strains with and without S9 mixture. The mice received the wastewater in natura through drinking water at concentrations of 25%, 50%, and 100%. One group of mice was exposed for 2 consecutive days, and the other group of mice was exposed for 15 consecutive days. To establish the levels of primary DNA damage, single-cell gel electrophoresis was performed on treated mouse liver cell. The concentrations of chromium and lead in the sample exceeded the national standard (GB20922-2007) by 0.78 and 0.43-fold, respectively. More than 30 organic compounds were detected, and some of the detected compounds were mutagens, carcinogens and environmental endocrine disrupters. A positive response for Salmonella typhimurium TA98 strain was observed. Mouse exposure via drinking water containing 50% and 100% of wastewater for 15 consecutive days caused a significant increase of MN frequencies in a dose-response manner. Mouse exposure via drinking water containing 50% and 100% of wastewater for 15 consecutive days caused a significant increase of the Olive tail moments in a dose-response manner. All the results indicated that the sample from the Dongming discharging river in Shijiazhuang city exhibited genotoxicity and might pose harmful effects on the local residents.

Mitogen-activated protein kinase kinase 4 (MAP2K4) gene acts as the direct upstream activator of c-Jun NH2-terminal kinase pathway, which plays an important role in regulating neuron survival and apoptosis in response to cerebral ischemia. However, the association between MAP2K4 gene polymorphisms and ischemic stroke (IS) has not yet been published. Therefore, this study investigates the association between MAP2K4 gene polymorphism rs3826392 and IS susceptibility, as well as its quantitative traits in Southern Chinese Han population.

Clinical trials have provided conflicting results regarding whether epidermal growth factor receptor (EGFR) overexpression predicts poor survival in cervical cancer patients. In this study, we perform a meta-analysis of the association between EGFR expression and survival in cervical cancer patients. We searched clinical studies in the Medline, PubMed, Embase, and Web of Science databases. A total of 22 studies with 2,505 patients were included, and pooled hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated for each study. Heterogeneity was assessed using Higgins I2 to select a Mantel-Haenszel fixed effects model (I2 ≤50%) or a DerSimonian-Laird random effects model (I2 ≥50%). High EGFR levels predicted poor overall survival (OS) (HR: 1.40, 95% CI: 1.10-1.78) and disease-free survival (DFS) (HR: 1.84, 95% CI: 1.51-2.24). Stratified analyses showed that EGFR overexpression was significantly related to poor DFS in patients treated with chemoradiation or surgery. Moreover, the pooled odds ratios (ORs) revealed associations between EGFR expression and clinicopathological features, such as lymph node metastasis (OR: 1.72, 95% CI: 1.23-2.40) and tumor size ≥4 cm (OR: 1.64, 95% CI: 1.20-2.23). This meta-analysis demonstrates that EGFR overexpression is closely associated with reduced survival in patients with cervical cancer. These results may facilitate the individualized management of clinical decisions for anti-EGFR therapies in cervical cancer patients.

Inhibition of the Wnt antagonist sclerostin increases bone mass in patients with osteoporosis and in preclinical animal models. Here we show increased levels of the Wnt antagonist Dickkopf-1 (DKK-1) in animals treated with sclerostin antibody, suggesting a negative feedback mechanism that limits Wnt-driven bone formation. To test our hypothesis that co-inhibition of both factors further increases bone mass, we engineer a first-in-class bispecific antibody with single residue pair mutations in the Fab region to promote efficient and stable cognate light-heavy chain pairing. We demonstrate that dual inhibition of sclerostin and DKK-1 leads to synergistic bone formation in rodents and non-human primates. Furthermore, by targeting distinct facets of fracture healing, the bispecific antibody shows superior bone repair activity compared with monotherapies. This work supports the potential of this agent both for treatment and prevention of fractures and offers a promising therapeutic approach to reduce the burden of low bone mass disorders.

Rubus L. is a large and taxonomically complex genus, species of which exhibit apomixis, polyploidy, and frequent hybridization. Most of Chinese Rubus are assigned in two major sections, Idaeobatus and Malachobatus. To explore the phylogenetic relationships within Chinese Rubus, inferences upon three chloroplast DNA (rbcL, rpl20-rps12, and trnG-trnS), nuclear ribosomal ITS, and two low-copy nuclear markers (GBSSI-2 and PEPC) were deduced in 142 Rubus taxa from 17 subsections in 6 sections. nrITS and GBSSI-2 were the most informative among the six DNA regions assessed. Phylogenetic relationships within Rubus were well-resolved by combined nuclear datasets rather than chloroplast markers. The phylogenetic inferences strongly supported that section Idaeobatus was a polyphyletic group with four distant clades. All samples of sect. Malachobatus formed a monophyletic clade, in which R. tsangorum and R. amphidasys of sect. Dalibardastrum, and R. peltatus from subsection Peltati of sect. Idaeobatus were always nested. Rubus pentagonus (2n = 2x = 14) from subsect. Alpestres of sect. Idaeobatus was a sister group to the polyploid sect. Malachobatus, as well as the polytomy of three sect. Cyalctis members. This suggests that some polyploids of Malachobatus might originate from common ancestors, via polyploidization of hybrids between R. pentagonus and sect. Cylactis species. They had experienced species explosion in a short time. Section Dalibardastrum species have potential parental lineages from subsects. Moluccani and Stipulosi of sect. Malachobatus. Based on molecular phylogenies, we also provided recommendations for the taxonomic treatments of four taxa. In addition, our results showed certain incongruence between chloroplast and nuclear markers, which might be due to hybridization and introgression.

The study of the folding of single domains, in the context of their multidomain environment, is important because more than 70% of eukaryotic proteins are composed of multiple domains. The structures of the tandem immunoglobulin (Ig) domain pairs A164-A165 and A168-A169, from the A-band of the giant muscle protein titin, reveal that they form tightly associated domain arrangements, connected by a continuous β-strand. We investigate the thermodynamic and kinetic properties of these tandem domain pairs. While A164-A165 apparently behaves as a single cooperative unit at equilibrium, unfolding without the accumulation of a large population of intermediates, domains in A168-A169 behave independently. Although A169 appears to be stabilized in the tandem protein, we show that this is due to nonspecific stabilization by extension. We elucidate the folding and unfolding pathways of both tandem pairs and show that cooperativity in A164-A165 is a manifestation of the relative refolding and unfolding rate constants of each individual domain. We infer that the differences between the two tandem pairs result from a different pattern of interactions at the domain/domain interface.

'Hongmian miyou' (Citrus grandis L. Osbeck) is mutated from 'Guanxi miyou', with a different spongy layer coloration. Trifoliate orange (Poncirus trifoliata) is widely used as rootstocks in 'Guanxi miyou' grafting, whereas 'Hongmian miyou' is incompatible with available trifoliate orange rootstocks. To explore the reasons for the etiolation of leaves of 'Hongmian miyou'/trifoliate orange, anatomical differences among different graft unions, gene expression profiles, and auxin levels of scion were investigated in this study. A histological assay indicated that there was no significant difference in anatomical structure between the compatible and incompatible combinations. A total of 1950 significant differentially-expressed genes (DEGs) were identified and analyzed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that genes involved in carbohydrate metabolism, energy metabolism, amino acid metabolism, and plant hormone signal transduction were significantly enriched. Moreover, the expression of nine genes in the auxin pathway were upregulated and three were downregulated in compatible combinations compared with those in the incompatible group. Further experiments verified that indole-3-acetic acid (IAA) content increases in the compatible graft combination, which suggests that IAA might promote graft compatibility.

Cassava (Manihot esculenta Crantz) is an important tropical starchy root crop that is adapted to drought but extremely cold sensitive. A cold-tolerant, high-quality, and robust supply of cassava is urgently needed. Here, we clarify genome-wide distribution and classification of CCGG hemi-methylation and full-methylation, and detected 77 much candidate QTLsepi for cold stress and 103 much candidate QTLsepi for storage root quality and yield in 186 cassava population, generated by crossing two non-inbred lines with female parent KU50 and male parent SC124 (KS population). We developed amplified-fragment single nucleotide polymorphism and methylation (AFSM) genetic map in this population. We also constructed the AFSM QTL map, identified 260 much candidate QTL genes for cold stress and 301 much candidate QTL genes for storage root quality and yield, based on the years greenhouse and field trials. This may accounted for a significant amount of the variation in the key traits controlling cold tolerance and the high quality and yield of cassava.

The aim of the present study was to explore the effect of aloe-emodin (AE)-induced photodynamic activity in human gastric cancer cells. AE was used as a photosensitizer to explore the effect of photodynamic therapy (PDT) in human gastric cancer cells (SGC-7901). An MTT assay was used to detect the effect of AE-induced PDT in optimal concentrations and illumination energy densities in human gastric cancer cells. Following AE-induced PDT, morphological changes of the cells and the rate of cell death were evaluated by TUNEL assay and flow cytometry, respectively. The expression levels of caspase-9 and caspase-3 were determined by western blot analysis. The AE and AE-induced PDT demonstrated a significant inhibitive effect on the proliferation of human gastric cancer cells in dose-dependent and energy-dependent manners. For subsequent experiments, 10 µM AE and 12.8 J/cm2 illumination energy density were used. Typical morphological changes of apoptosis were observed in the cells using a TUNEL assay 12 h subsequent to AE-induced PDT. The percentage of apoptotic cells treated with AE-induced PDT significantly increased when compared with the control group, the 10 µM AE group and the illumination group (P<0.05). Upregulation of caspase-9 and caspase-3 protein levels was also observed following AE-induced PDT. The present study revealed that 10 µM AE-induced PDT had an inhibitory effect on human gastric cancer cells, and it may induce cell apoptosis by upregulating caspase-9 and caspase-3, which indicated that the mitochondrial pathway may be involved. AE-induced PDT has the potential to be a novel therapy for the treatment of human gastric cancer. However, further investigations are required.