cis-Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNA viruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The downstream of 5′ cyclization sequence (5′CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5′CS, and the presence of DCS-PK facilitates the formation of 5′-3′ RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution.
Angiogenesis occurs during tissue growth, development and wound healing. It is also required for tumor progression and represents a rational target for therapeutic intervention. NBM-T-BMX-OS01 (BMX), derived from the semisynthesis of osthole, an active ingredient isolated from Chinese herb Cnidium monnieri (L.) Cuss., was recently shown to enhance learning and memory in rats. In this study, we characterized the anti-angiogenic activities of NBM-T-BMX-OS01 (BMX) in an effort to develop novel inhibitors to suppress angiogenesis and tumor growth. BMX inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and endothelial tube formation in human umbilical endothelial cells (HUVECs). BMX also attenuated VEGF-induced microvessel sprouting from aortic rings ex vivo and reduced HCT116 colorectal cancer cells-induced angiogenesis in vivo. Moreover, BMX inhibited the phosphorylation of VEGFR2, FAK, Akt and ERK in HUVECs exposed to VEGF. BMX was also shown to inhibit HCT116 cell proliferation and to suppress the growth of subcutaneous xenografts of HCT116 cells in vivo. Taken together, this study provides evidence that BMX modulates vascular endothelial cell remodeling and leads to the inhibition of tumor angiogenesis. These results also support the role of BMX as a potential drug candidate and warrant the clinical development in the treatment of cancer.
Picorna-like viruses in the Picornavirales order are a large group of positive-strand RNA viruses that include numerous important pathogens for plants, insects, and humans. In these viruses, nonstructural protein 2C is one of the most conserved proteins and contains ATPase activity and putative RNA helicase activity. Here we expressed 2C protein of Ectropis obliqua picorna-like virus (EoV; genus Iflavirus, family Iflaviridae, order Picornavirales) in a eukaryotic expression system and determined that EoV 2C displays ATP-independent nucleic acid helix destabilizing and strand annealing acceleration activity in a concentration-dependent manner, indicating that this picornaviral 2C is more like an RNA chaperone than like the previously predicted RNA helicase. Our further characterization of EoV 2C revealed that divalent metal ions, such as Mg2+ and Zn2+, inhibit 2C-mediated helix destabilization to different extents. Moreover, we determined that EoV 2C also contains ATPase activity like that of other picornaviral 2C proteins and further assessed the functional relevance between its RNA chaperone-like and ATPase activities using mutational analysis as well as their responses to Mg2+. Our data show that, when one of the two 2C activities was dramatically inhibited or almost abolished, the other activity could remain intact, showing that the RNA chaperone-like and ATPase activities of EoV 2C can be functionally separated. This report reveals that a picorna-like virus 2C protein displays RNA helix destabilizing and strand annealing acceleration activity, which may be critical for picornaviral replication and pathogenesis, and should foster our understanding of picorna-like viruses and viral RNA chaperones.
Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2′-O cap of their RNA; alternatively, they “snatch” host mRNA cap to form the 5′ end of viral RNA. The function of 2′-O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2′-O methylation is replicative, but its viral RNA lacks 2′-O methylation and is recognized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vaccine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2′-O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2′-O methylation was stable in cell culture after being passaged for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and retained the engineered mutation in vivo. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2′-O methylation-defective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2′-O methyltransferases.
The role of many genes and interactions among genes involved in flowering time have been studied extensively in Arabidopsis, and the purpose of this study was to investigate how effectively results obtained with the model species Arabidopsis can be applied to the Brassicacea with often larger and more complex genomes. Brassica rapa represents a very close relative, with its triplicated genome, with subgenomes having evolved by genome fractionation. The question of whether this genome fractionation is a random process, or whether specific genes are preferentially retained, such as flowering time (Ft) genes that play a role in the extreme morphological variation within the B. rapa species (displayed by the diverse morphotypes), is addressed. Data are presented showing that indeed Ft genes are preferentially retained, so the next intriguing question is whether these different orthologues of Arabidopsis Ft genes play similar roles compared with Arabidopsis, and what is the role of these different orthologues in B. rapa. Using a genetical–genomics approach, co-location of flowering quantitative trait loci (QTLs) and expression QTLs (eQTLs) resulted in identification of candidate genes for flowering QTLs and visualization of co-expression networks of Ft genes and flowering time. A major flowering QTL on A02 at the BrFLC2 locus co-localized with cis eQTLs for BrFLC2, BrSSR1, and BrTCP11, and trans eQTLs for the photoperiod gene BrCO and two paralogues of the floral integrator genes BrSOC1 and BrFT. It is concluded that the BrFLC2 Ft gene is a major regulator of flowering time in the studied doubled haploid population.
Brassica rapa; candidate gene mapping; expression quantitative trait loci (eQTL); FLOWERING LOCUS C (FLC).; flowering time; gene expression networks; genome triplication.
The association between Human Leukocyte Antigen (HLA) class II and rheumatoid arthritis (RA) has been extensively studied, but few reported DR-DQ haplotype. Here we investigated the association of HLA-DRB1, DQA1, DQB1, and DR-DQ haplotypes with RA susceptibility and with anti-CCP antibodies in 281 RA patients and 297 control in Han population. High-resolution genotyping were performed. The HLA-DRB1 shared epitope (SE)-encoding allele *0405 displayed the most significant RA association (P = 1.35×10−6). The grouped DRB1 SE alleles showed great association with RA (P = 3.88×10−13). The DRB1 DRRAA alleles displayed significant protective effects (P = 0.021). The SE-dependent DR-DQ haplotype SE-DQ3/4/5 remained strong association with both anti-CCP -positive (P = 3.71×10−13) and -negative RA (P = 3.89×10−5). Our study revealed that SE alleles and its haplotypes SE-DQ3/4/5 were highly associated with RA susceptibility in Han population. The SE-DQ3/4/5 haplotypes were associated with both anti-CCP positive RA and -negative RA.
Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2′-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2′-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2′-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.
The four serotypes of dengue virus cause severe outbreaks globally in tropical countries with thousands of patients requiring hospitalization. The health care and indirect economic cost of dengue in endemic countries is huge. Despite this, no clinically approved vaccine or antiviral treatment is currently available. Dengue transmission could be stopped with a vaccine that provides full protection to all serotypes. Dengue afflicts many developing countries and a vaccine should therefore be cost-effective and should provide protection with ideally a single injection. Here we present a novel dengue vaccine approach that harbours mutation(s) in the 2′-O-methyltransferase (MTase), a viral enzyme that methylates viral RNA as a strategy to escape the host immune response. Non-methylated RNA is recognized as “foreign” and triggers an interferon response in the cell. The MTase mutant virus is immediately recognized by the host's immune response and hardly has a chance to spread in the organism while an immune response is efficiently triggered by the initially infected cells. Mice and monkeys infected with the mutant virus developed an immune response that fully protected them from a challenge with wild-type virus. Furthermore, we show that MTase mutant dengue virus cannot infect Aedes mosquitoes. Collectively, the results suggest 2′-O-MTase mutant dengue virus as a safe, highly immunogenic vaccine approach.
Human enterovirus type 71 (EV71) is the major pathogen of hand-foot-and-mouth disease (HFMD) and has been associated with severe neurological disease and even death in infants and young children. The pathogenesis of EV71 infection in the human central nervous system remains unclear. In this study, human whole genome microarray was employed to perform transcriptome profiling in SH-SY5Y human neuroblastoma cells infected with EV71. The results indicated that EV71 infection lead to altered expression of 161 human mRNAs, including 74 up-regulated genes and 87 down-regulated genes. Bioinformatics analysis indicated the possible roles of the differentially regulated mRNAs in selected pathways, including cell cycle/proliferation, apoptosis, and cytokine/chemokine responses. Finally, the microarray results were validated using real-time RT-PCR with high identity. Overall, our results provided fundamental information regarding the host response to EV71 infection in human neuroblastoma cells, and this finding will help explain the pathogenesis of EV71 infection and virus-host interaction.
The simultaneous sorption behavior and characteristics of cadmium (Cd) and sulfamethoxazole (SMX) on rice straw biochar were investigated. Isotherms of Cd and SMX were well modeled by the Langmuir equation (R
2>0.95). The calculated maximum adsorption parameter (Q) of Cd was similar in single and binary systems (34 129.69 and 35 919.54 mg/kg, respectively). However, the Q of SMX in a binary system (9 182.74 mg/kg) was much higher than that in a single system (1 827.82 mg/kg). The presence of Cd significantly promoted the sorption of SMX on rice straw biochar. When the pH ranged from 3 to 7.5, the sorption of Cd had the characteristics of a parabola pattern with maximum adsorption at pH 5, while the adsorption quantity of SMX decreased with increasing pH, with maximum adsorption at pH 3. The amount of SMX adsorbed on biochar was positively correlated with the surface area of the biochar, and the maximum adsorption occurred with d 250 biochar (biochar with a diameter of 150–250 μm). Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) showed that the removal of Cd and SMX by rice straw biochar may be attributed to precipitation and the formation of surface complexes between Cd or SMX and carboxyl or hydroxyl groups. The results of this study indicate that rice straw biochar has the potential for simultaneous removal of Cd and SMX from co-contaminated water.
Biochar; Rice straw; Simultaneous sorption; Cadmium (Cd); Sulfamethoxazole (SMX)
In this study, several nitrilase genes from phylogenetically distinct organisms were expressed and purified in E. coli in order to study their ability to mediate the biotransformation of nitriles. We identified three nitrilases: Acidovorax facilis nitrilase (AcN); Alcaligenes fecalis nitrilase (AkN); and Rhodococcus rhodochrous nitrilase (RkN), which catalyzed iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA). AcN demonstrated 8.8-fold higher activity for IDAN degradation as compared to AkN and RkN. Based on homology modeling and previously described ‘hot spot’ mutations, several AcN mutants were screened for improved activity. One mutant M3 (F168V/L201N/S192F) was identified, which demonstrates a 41% enhancement in the conversion as well as a 2.4-fold higher catalytic efficiency towards IDAN as compared to wild-type AcN.
Here we report the complete genome sequence of a dengue virus serotype 2 (DENV-2) strain, GZ40, isolated in Guangdong, China, in 2010. A phylogenetic analysis classified GZ40 into the Cosmopolitan genotype, while previous Chinese DENV-2 isolates belong to the Asian I genotype. The reemergence of the Cosmopolitan genotype of DENV-2 in China deserves further investigation.
Amur virus was recently identified as the causative agent of hemorrhagic fever with renal syndrome. Here we report the complete genome sequence of an Amur virus isolated from Apodemus peninsulae in Northeastern China. The sequence information provided here is critical for the molecular epidemiology and evolution of Amur virus in China.
We report here the complete genome sequence of a human echovirus type 30 strain ECV30/GX10/05 isolated in Guangxi, China, in 2010. Phylogenetic analysis showed that ECV30/GX10/05 was closely related to a Korean strain isolated in 2008. The sequence information will help in an understanding of the molecular epidemiology and evolution of echovirus.
Seoul virus (SEOV) is responsible for 25% of cases of hemorrhagic fever with renal syndrome in Asia. Here we report the complete genome of strain DPRK08. The sequence information provided here is useful for understanding the molecular character of SEOV in the Democratic People's Republic of Korea (DPRK) and the circulation of SEOV in East Asia.
The emerging human enterovirus 71 (EV71) represents a growing threat to public health, and no vaccine or specific antiviral is currently available. Human intravenous immunoglobulin (IVIG) is clinical used in treating severe EV71 infections. However, the discovery of antibody dependent enhancement (ADE) of EV71 infection illustrates the complex roles of antibody in controlling EV71 infection. In this study, to identify the distinct role of each IgG subclass on neutralization and enhancement of EV71 infection, different lots of pharmaceutical IVIG preparations manufactured from Chinese donors were used for IgG subclass fractionation by pH gradient elution with the protein A-conjugated affinity column. The neutralization and ADE capacities on EV71 infection of each purified IgG subclass were then assayed, respectively. The neutralizing activity of human IVIG is mainly mediated by IgG1 subclass and to less extent by IgG2 subclass. Interestingly, IgG3 fraction did not have neutralizing activity but enhanced EV71 infection in vitro. These results revealed the different roles of human IgG subclasses on EV71 infection, which is of critical importance for the rational design of immunotherapy and vaccines against severe EV71 diseases.
There are no sensitive and specific biomarkers that aid in the clinical diagnosis and prognosis of hepatocellular carcinoma (HCC). The aim of the present study was to determine the mRNA and protein expression levels of beclin 1 (BECN1) and nuclear factor-κB (NF-κB)p65 in patients with HCC, to evaluate their value as potential diagnostic and prognostic biomarkers. Immunohistochemistry and in situ hybridization were used to detect the expression of hepatic BECN1 and NF-kBp65 in patients with HCC, hepatitis B virus (HBV) or cirrhosis, as compared with the expression levels in healthy subjects. The expression level of the BECN1 protein in the HCC tissue was significantly high compared with that in the cirrhotic, hepatitis and normal tissues. The expression of the BECN1 protein in the hepatitis tissue was significantly high compared with that of the cirrhotic and normal tissues. The expression of the BECN1 mRNA in the cancer tissue was significantly high compared with that of the cirrhotic and normal tissues, and the expression of the BECN1 mRNA in the hepatitis tissue was significantly higher than that of the cirrhotic and normal tissues. The expression of the NF-κBp65 protein in the cancer tissue was significantly high compared with that of the cirrhotic, hepatitis and normal tissues. The expression of the NF-κBp65 mRNA in-the cancer tissue was significantly high compared with that of the cirrhotic, hepatitis and normal tissues. BECN1 expression was positively correlated with NF-κBp65 expression in HCC. The abnormal expression of BECN1 and NF-κBp65 was closely associated with the development of HCC. Finally, a search in GeneGo pathway database observed a link between BECN1 and NF-κBp65 through multiple proteins. These results indicate that BECN1 and NF-κBp65 are upregulated in HCC, and that they may serve as useful biomarkers for HCC.
beclin 1; nuclear factor-κB; immunohistochemistry; in situ hybridization; hepatocellular carcinoma
Puerarin has multiple pharmacological effects and is widely prescribed for patients with cardiovascular diseases including hypertension, cerebral ischemia, myocardial ischemia, diabetes mellitus, and arteriosclerosis. We have successfully prepared puerarin-loaded solid lipid nanoparticles (Pue-SLNs) for oral administration. Pue-SLNs are prepared using monostearin, soya lecithin, and poloxamer 188. SLNs may alter the course of puerarin absorption predominantly to and through lymphatic routes and regions, presumably following a transcellular path of lipid absorption, especially by enterocytes and polar epithelial cells of the intestine. The alteration of absorption might influence the metabolic profile of puerarin when incorporated into SLNs. In the present study, we investigated the metabolic profile of puerarin in rat plasma and urine using rapid resolution liquid chromatography–tandem mass spectrometry after a single-dose intragastric administration of Pue-SLNs in comparison with puerarin suspension. Two glucuronidated metabolites of puerarin, puerarin-4′-O-glucuronide and puerarin-7-O-glucuronide, were detected in rat plasma and urine after intragastric administration of Pue-SLNs, with the latter acting as the major metabolite. Similar results were found in rat plasma and urine after intragastric administration of puerarin suspension. The results suggest that incorporation of puerarin into SLNs does not change either the position of glucuronidation or the metabolic pathway of puerarin in rats.
puerarin; solid lipid nanoparticles; metabolic profiling
Viral self-replicating sub-genomic replicons represent a powerful tool for studying viral genome replication, antiviral screening and chimeric vaccine development. Many kinds of flavivirus replicons have been developed with broad applications.
The replicon system of JEV live vaccine strain SA14-14-2 was successfully developed in this study. Two kinds of replicons that express enhanced green fluorescent protein (EGFP) and Renilla luciferase (R.luc) were constructed under the control of SP6 promoter, respectively. Robust EGFP and R.luc signals could be detected in the replicon-transfected BHK-21 cells. Furthermore, the potential effects of selected amino acids in the C-terminal of envelope protein on replication were characterized using the replicon system.
Our results provide a useful platform not only for the study of JEV replication, but also for antiviral screening and chimeric vaccine development.
Japanese encephalitis virus (JEV); SA14-14-2; Replicon
To further probe into whether swamp buffaloes were domesticated once or multiple times in China, this survey examined the mitochondrial DNA (mtDNA) Control Region (D-loop) diversity of 471 individuals representing 22 populations of 455 Chinese swamp buffaloes and 16 river buffaloes. Phylogenetic analysis revealed that Chinese swamp buffaloes could be divided into two distinct lineages, A and B, which were defined previously. Of the two lineages, lineage A was predominant across all populations. For predominant lineage A, Southwestern buffalo populations possess the highest genetic diversity among the three hypothesized domestication centers (Southeastern, Central, and Southwestern China), suggesting Southwestern China as the most likely location for the domestication of lineage A. However, a complex pattern of diversity is detected for the lineage B, preventing the unambiguous pinpointing of the exact place of domestication center and suggesting the presence of a long-term, strong gene flow among swamp buffalo populations caused by extensive migrations of buffaloes and frequent human movements along the Yangtze River throughout history. Our current study suggests that Southwestern China is the most likely domestication center for lineage A, and may have been a primary center of swamp buffalo domestication. More archaeological and genetic evidence is needed to show the process of domestication.
Here we report the generation and analysis of genome-wide exon-level transcriptome data from 16 brain regions comprising the cerebellar cortex, mediodorsal nucleus of the thalamus, striatum, amygdala, hippocampus, and 11 areas of the neocortex. The dataset was generated from 1,340 tissue samples collected from one or both hemispheres of 57 postmortem human brains, spanning from embryonic development to late adulthood and representing males and females of multiple ethnicities. We also performed genotyping of 2.5 million SNPs and assessed copy number variations for all donors. Approximately 86% of protein-coding genes were found to be expressed using stringent criteria, and over 90% of these were differentially regulated at the whole transcript or exon level across regions and/or time. The majority of these spatiotemporal differences occurred before birth, followed by an increase in the similarity among regional transcriptomes during postnatal lifespan. Genes were organized into functionally distinct co-expression networks, and sex differences were present in gene expression and exon usage. Finally, we demonstrate how these results can be used to profile trajectories of genes associated with neurodevelopmental processes, cell types, neurotransmitter systems, autism, and schizophrenia, as well as to discover associations between SNPs and spatiotemporal gene expression. This study provides a comprehensive, publicly available dataset on the spatiotemporal human brain transcriptome and new insights into the transcriptional foundations of human neurodevelopment.
Chikungunya virus belongs to the genus Alphavirus in the family Togaviridae. Here we report the complete genome sequence of a chikungunya virus strain, GD05/2010, isolated in 2010 from a patient with chikungunya fever in Guangdong, China. The sequence information is important for surveillance of this emerging arboviral infection in China.
Liver toxicity (hepatotoxicity) is a critical issue in drug discovery and development. Standard preclinical evaluation of drug hepatotoxicity is generally performed using in vivo animal systems. However, only a small number of preselected compounds can be examined in vivo due to high experimental costs. A more efficient yet accurate screening technique which can identify potentially hepatotoxic compounds in the early stages of drug development would thus be valuable. Here, we develop and apply a novel genomic prediction technique for screening hepatotoxic compounds based on in vitro human liver cell tests. Using a training set of in vivo rodent experiments for drug hepatotoxicity evaluation, we discovered common biomarkers of drug-induced liver toxicity among six heterogeneous compounds. This gene set was further triaged to a subset of 32 genes that can be used as a multi-gene expression signature to predict hepatotoxicity. This multi-gene predictor was independently validated and showed consistently high prediction performance on five test sets of in vitro human liver cell and in vivo animal toxicity experiments. The predictor also demonstrated utility in evaluating different degrees of toxicity in response to drug concentrations which may be useful not only for discerning a compound’s general hepatotoxicity but also for determining its toxic concentration.
Drug hepatocellular toxicity; Co-expression Extrapolation; Multi-gene Expression-based Predictor
Here we report the first complete genome sequence of a dengue virus serotype 4 genotype II strain, GZ30, isolated in Guangzhou, Guangdong Province, China, in 2010. The sequence information provided herein will help us to understand the molecular epidemiology of dengue virus and predict the risk of severe diseases in mainland China.
RNA interference (RNAi) is a eukaryotic gene-silencing mechanism that functions in antiviral immunity in diverse organisms. To combat RNAi-mediated immunity, viruses encode viral suppressors of RNA silencing (VSRs) that target RNA and protein components in the RNAi machinery. Although the endonuclease Dicer plays key roles in RNAi immunity, little is known about how VSRs target Dicer. Here, we show that the B2 protein from Wuhan nodavirus (WhNV), the counterpart of Flock House virus (FHV), suppresses Drosophila melanogaster RNAi by directly interacting with Dicer-2 (Dcr-2) and sequestering double-stranded RNA (dsRNA) and small interfering RNA (siRNA). Further investigations reveal that WhNV B2 binds to the RNase III and Piwi-Argonaut-Zwille (PAZ) domains of Dcr-2 via its C-terminal region, thereby blocking the activities of Dcr-2 in processing dsRNA and incorporating siRNA into the RNA-induced silencing complex (RISC). Moreover, we uncover an interrelationship among diverse activities of WhNV B2, showing that RNA binding enhances the B2–Dcr-2 interaction by promoting B2 homodimerization. Taken together, our findings establish a model of suppression of Drosophila RNAi by WhNV B2 targeting both Dcr-2 and RNA and provide evidence that an interrelationship exists among diverse activities of VSRs to antagonize RNAi.