Shotgun identification of the structural proteome of shrimp white spot syndrome virus and iTRAQ differentiation of envelope and nucleocapsid subproteomes

White spot syndrome virus (WSSV) is a major pathogen that causes severe mortality and economic losses to shrimp cultivation worldwide. The genome of WSSV contains a 305-kb double-stranded circular DNA, which encodes 181 predicted ORFs. Previous gel-based proteomics studies on WSSV have identified 38 structural proteins. In this study, we applied shotgun proteomics using off-line coupling of an LC system with MALDI-TOF/TOF MS/MS as a complementary and comprehensive approach to investigate the WSSV proteome. This approach led to the identification of 45 viral proteins; 13 of them are reported for the first time. Seven viral proteins were found to have acetylated N termini. RT-PCR confirmed the mRNA expression of these 13 newly identified viral proteins. Furthermore iTRAQ (isobaric tags for relative and absolute quantification), a quantitative proteomics strategy, was used to distinguish envelope proteins and nucleocapsid proteins of WSSV. Based on iTRAQ ratios, we successfully identified 23 envelope proteins and six nucleocapsid proteins. Our results validated 15 structural proteins with previously known localization in the virion. Furthermore the localization of an additional 12 envelope proteins and two nucleocapsid proteins was determined. We demonstrated that iTRAQ is an effective approach for high throughput viral protein localization determination. Altogether WSSV is assembled by at least 58 structural proteins, including 13 proteins newly identified by shotgun proteomics and one identified by iTRAQ. The localization of 42 structural proteins was determined; 33 are envelope proteins, and nine are nucleocapsid proteins. A comprehensive identification of WSSV structural proteins and their localization should facilitate the studies of its assembly and mechanism of infection.     

气候变化情景下少花蒺藜草在中国的分布区变化

少花蒺藜草(Cenchrus spinifex)是我国的入侵种植物之一,严重影响我国的畜牧养殖业和生态环境。为了预测未来气候变化情景下,少花蒺藜草的适生分布区变化,该研究基于MaxEnt模型,利用103个少花蒺藜草的地理分布数据和19个气候环境因子,分析预测在RCP 4.5、RCP 8.5两种未来气候变化情景下,2050s和2070s时段在我国范围内少花蒺藜草的适生分布区。结果表明：(1)少花蒺藜草的当前适生分布区占研究区域面积的4.00%,主要分布于内蒙古自治区、吉林省、辽宁省三省(区)接壤的东北地区。(2)未来少花蒺藜草的适生分布区面积有所增加,其中中等适生区所占面积扩张程度最大,达到38.26%。(3)年平均气温、温度季节性变化标准差、最湿季降水量是影响少花蒺藜草分布的主要气候因子。(4)未来少花蒺藜草的分布质心总体向西移动。综上认为,目前在中国范围内,少花蒺藜草的已入侵区域还远小于潜在可入侵区域,未来还可能向我国干旱半干旱区进一步扩散,为防止少花蒺藜草在我国北方地区大面积扩散带来的危害,未来需要重点关注对其的预防措施和入侵态势。该研究结果为我国防治入侵种植物提供重要的理论依据和...     

A Quantitative Chemical Proteomics Approach to Profile the Specific Cellular Targets of Andrographolide,a Promising Anticancer Agent That Suppresses Tumor Metastasis

Drug target identification is a critical step toward understanding the mechanism of action of a drug, which can help one improve the drug''s current therapeutic regime and expand the drug''s therapeutic potential. However, current in vitro affinity-chromatography-based and in vivo activity-based protein profiling approaches generally face difficulties in discriminating specific drug targets from nonspecific ones. Here we describe a novel approach combining isobaric tags for relative and absolute quantitation with clickable activity-based protein profiling to specifically and comprehensively identify the protein targets of andrographolide (Andro), a natural product with known anti-inflammation and anti-cancer effects, in live cancer cells. We identified a spectrum of specific targets of Andro, which furthered our understanding of the mechanism of action of the drug. Our findings, validated through cell migration and invasion assays, showed that Andro has a potential novel application as a tumor metastasis inhibitor. Moreover, we have unveiled the target binding mechanism of Andro with a combination of drug analog synthesis, protein engineering, and mass-spectrometry-based approaches and determined the drug-binding sites of two protein targets, NF-κB and actin.As most drugs exert pharmacological effects by interacting with their target proteins, the identification of these target proteins is a critical step in unraveling the mechanisms of drug action. It is also imperative for our understanding of the pharmacodynamics of a known drug, suggesting potentially unrevealed actions and thus refining future clinical applications of the substance. Traditional approaches used to identify protein targets of a drug typically utilize immobilized drug affinity chromatography coupled with mass spectrometry (MS)¹ (1, 2). These methods can be applied to cell lysates, but not in an in vivo setting, because of the requirement of a solid support. In vitro target profiling might not accurately reflect the drug''s actions in the in vivo physiological environment. To overcome this limitation, several groups have used activity-based protein profiling (ABPP) combined with bio-orthogonal click chemistry to identify drug targets both in vitro and in vivo (supplemental Fig. S1) (3–15). ABPP probes exert their functions via covalent reactions with the target proteins or photoaffinity-based labeling via the incorporation of photoreactive groups. With the increasing sensitivity of modern MS platforms, low-abundance protein targets can be successfully identified. Although both conventional affinity chromatography and recent ABPP-based methods allow us to detect a set of candidate protein targets for a drug, it remains difficult to discriminate specific interactions from nonspecific ones. Thus, more time and effort are needed for subsequent validation because of the presence of a large number of nonspecific binders. Therefore, there is an urgent need to develop comprehensive unbiased methods for specific target identification. Quantitative proteomics has been used to profile enriched kinases using cell-lysate-based kinobead pull-down. However, these types of experiments are mainly suitable for studying kinase inhibitors (16). Recently, proteomics methods based on stable isotope labeling of amino acids in cell culture (SILAC) have been applied to determine the specific binders of small molecules or proteins with certain post-translational modifications (17–19). These studies have shed light on how quantitative proteomics can improve the specificity of target protein identification. Nevertheless, because of the inherent limitations of SILAC, the complete incorporation of isotopic amino acids via such an approach takes a long time. Furthermore, it is also extremely difficult to apply the SILAC approach to tissue and body fluid samples, which are of particular relevance to biomedical research.Here we introduce a clickable activity-based protein profiling (ICABPP) approach based on the use of isobaric tags for relative and absolute quantitation (iTRAQ) for the unbiased specific and comprehensive identification of target proteins in live cells. iTRAQ is a stable isotope labeling approach for multiplexed quantitative proteome profiling (20). An overview of the technique is illustrated in Fig. 1A. In this assay, cells are first incubated with a clickable probe or with DMSO, which serves as a negative control. After the probe permeates the cell, and covalently binds to its dedicated in situ targets, the washed cells are lysed, clicked with biotin-N₃ tag, and enriched through avidin pull-down in parallel. The beads are washed thoroughly, and the bond proteins are directly digested on the beads with trypsin. The resulting peptides are labeled with their respective iTRAQ reagents, pooled together for further identification and quantification via LC-MS/MS. This technique enabled us to discriminate specific protein targets from nonspecific, and endogenously biotinylated proteins. Biological replicates of probe- or DMSO-treated samples are included to overcome experimental variations. As shown in Fig. 1B, nonspecific binding proteins'' iTRAQ reporters have equal or similar intensities, whereas specific target proteins enriched by the probe show highly differential intensities relative to the DMSO-treated control samples (as illustrated by the significantly higher reporter intensities of 116 and 117 versus 113 and 114 shown in Fig. 1B). The multiplexing nature of the iTRAQ-based chemical proteomics method allows replicated enrichments to be compared within a single LC-MS/MS analysis, thereby increasing the accuracy of specific target identifications, and minimizing experimental errors.Open in a separate windowFig. 1.Identifying specific drug targets using ICABPP approach in live cells. A, live cells were treated with DMSO, and clickable probe in duplicate. Cells were lysed and tagged with biotin-N3 using click chemistry in parallel. The biotin-bearing target proteins were enriched through avidin pull-down, and directly digested on beads. The resulting peptides of the two biological replicates of control pulled-down samples were labeled with 113 and 114, respectively, and two probe pulled-down samples were labeled with 116 and 117, respectively. The labeled peptides were combined in order to be identified, and quantified via LC-MS/MS. B, for the nonspecific targets, the iTRAQ reporters showed similar intensities, whereas for the specific targets, the reporters showed highly differential intensities.In this context, the ICABPP approach was applied to identify protein targets of andrographolide (Andro) (Fig. 2), a natural product with known anti-inflammation, and anti-cancer effects (21–25), in live cancer cells. A spectrum of 75 potential Andro targets was identified with high confidence, which suggested that Andro may exert anti-cancer effects by acting on multiple targets to interfere with several cellular signaling pathways. Two targets, NF-κB and β-actin, were validated by in vitro binding assay, and direct binding site mapping. Furthermore, our data revealed a novel mechanism of Andro in suppressing tumor metastasis.Open in a separate windowFig. 2.Chemical structures of Andro, reduced Andro analog RA, and Andro-based clickable ABPP probes P1 and P2.     

Higher Prevalence of Sexual Transmitted Diseases and Correlates of Genital Warts among Heterosexual Males Attending Sexually Transmitted Infection Clinics (MSCs) in Jiangmen,China: Implication for the Up-Taking of STD Related Service

Background

Increasing burden of STDs is one of China’s major public health concerns. However, only a limited number of studies have ever investigated the prevalence of these STDs, particular for genital warts and its correlates among heterosexual males attending STD clinics in China. In order to fill this gap, we conducted a cross-sectional study among MSCs in Jiangmen, China, between the years of 2009 and 2010.

Method

The eligible participants were recruited from several STD-clinics in public hospitals. We collected demographic information and behaviors of the participants. After HIV and syphilis testing, we further checked whether the participants had genital warts and genital herpes. In addition, urine samples were collected from part of the participants for CT and NG testing.

Results

Of the 533 eligible participants, over three-fifths were aged 35 or below, nearly three quarters had no college degree, over three-fifths were residence of Jiangmen. The prevalence of HIV, syphilis, genital warts, genital herpes, CT and NG were 0.19%, 7.50%, 7.32%, 5.25%, 9.73% and 6.19%, respectively. Living with family members (versus living alone), no STD-related service in past year, experiencing STDs related symptoms in past year, and sex with FSWs in last three months were positively associated with genital warts, with adjusted ORs of 5.54 (95% CI 1.94–15.81), 2.26 (95% CI 1.08–4.74), 1.99 (95% CI 1.00–3.99) and 2.01 (95% CI 1.00–4.04), respectively.

Conclusion

Our study indicates that the prevalence of STDs among MSCs in Jiangmen was high, which may further spread HIV among MSCs. Targeted interventions that focused on STDs related services uptake should be implemented urgently.     

一株具有抑制单胺氧化酶作用的干酪乳杆菌筛选

【目的】通过体外模型从健康人体粪便内分离筛选出具有抑制单胺氧化酶(MAO)活性的乳酸菌,为今后乳酸菌体内抗衰老的研究提供参考。【方法】采用单胺氧化酶体外抑制模型对乳酸菌的发酵上清及无细胞提取物进行了筛选,并对筛选出的样品进行了两种指标的测定,即样品的剂量效应,以及样品与酶的预保温时间对酶活抑制率的影响;同时利用膜分离技术对不同分子量范围的样品进行了MAO的抑制测定。以筛选出的菌株JH-23为目的菌,通过16S rDNA序列分析及API细菌鉴定系统对菌株进行鉴定。【结果】筛选出的菌株JH-23无细胞提取物对MAO的抑制率达到33.7%。样品经冻干后,在反应浓度为16 mg/mL时抑制率达到53.2%,且MAO抑制率随预保温时间的增加而上升,在30 min之后抑制效果趋于平稳;粗样品经48 h透析后,透析液中的MAO抑制率较透析前明显升高。菌株JH-23的鉴定结果显示其属于干酪乳杆菌。【结论】开发了一种以单胺氧化酶作为靶位酶的新式体外筛选模型,该模型方便快捷且灵敏性高,对之后的抗衰老体内研究有所帮助。筛选出的干酪乳杆菌JH-23细胞裂解物对MAO有抑制作用,其中起到MAO抑制作用的主要是细胞内的小分子类物质。     

A genome-wide association study identified new variants associated with mathematical abilities in Chinese children

Mathematical ability is moderately heritable, and it is a complex trait which can be evaluated in several different categories. A few genetic studies have been published on general mathematical ability. However, no genetic study focused on specific mathematical ability categories. In this study, we separately performed genome-wide association studies on 11 mathematical ability categories in 1146 students from Chinese elementary schools. We identified seven genome-wide significant single nucleotide polymorphisms (SNPs) with strong linkage disequilibrium among each other (all r² > 0.8) associated with mathematical reasoning ability (top SNP: rs34034296, p = 2.01 × 10⁻⁸, nearest gene: CUB and Sushi multiple domains 3, CSMD3). We replicated one SNP (rs133885) from 585 SNPs previously reported to be associated with general mathematical ability associated with division ability in our data (p = 1.053 × 10⁻⁵). In the gene- and gene-set enrichment analysis by MAGMA, we found three significant enrichments of associations with three mathematical ability categories for three genes (LINGO2, OAS1 and HECTD1). We also observed four significant enrichments of associations with four mathematical ability categories for three gene sets. Our results suggest new candidate genetic loci for the genetics of mathematical ability.     

Insular shifts in body size of rice frogs in the Zhoushan Archipelago, China

1. Differences in body size between mainland and island populations have been reported for reptiles, birds and mammals. Despite widespread recognition of insular shifts in body size in these taxa, there have been no reports of such body size shifts in amphibians. 2. We provide the first evidence of an insular shift in body size for an amphibian species, the rice frog Rana limnocharis. We found significant increases in body size of rice frogs on most sampled islands in the Zhoushan archipelago when compared with neighbouring mainland China. 3. Large body size in rice frogs on islands was significantly related to increased population density, in both breeding and non-breeding seasons. Increases in rice frog density were significantly related to higher resource availability on islands. Increased resource availability on islands has led to higher carrying capacities, which has subsequently facilitated higher densities and individual growth rates, resulting in larger body size in rice frogs. We also suggest that large body size has evolved on islands, as larger individuals are competitively superior under conditions of harsh intraspecific competition at high densities. 4. Increases in body size in rice frogs were not related to several factors that have been implicated previously in insular shifts in body size in other taxa. We found no significant relationships between body size of rice frogs and prey size, number of larger or smaller frog species, island area or distance of islands from the mainland. 5. Our findings contribute to the formation of a broad, repeatable ecological generality for insular shifts in body size across a range of terrestrial vertebrate taxa, and provide support for recent theoretical work concerning the importance of resource availability for insular shifts in body size.     

A novel manual pooling system for preparing three-dimensional pools of a deep coverage soybean bacterial artificial chromosome library

Compared with hybridization‐based techniques, polymerase chain reaction‐based screening of large insert libraries has been used widely as it is fast, easy and sensitive. However, various pooling strategies are needed to ensure efficient screening. It is time‐consuming and labourious to prepare three‐dimensional pools for a deep coverage bacterial artificial chromosome (BAC) library of soybean (1.12 × 10⁹ bp) in the absence of robotic facility. In the present study, we describe a novel manual pooling system for preparing three‐dimensional pools of a soybean BAC library. This simple technique enables a single researcher to construct three‐dimensional pools for a deep‐coverage (12 haploid genome equivalents) BAC library of soybean in less than 2 months without any robotic manipulation. When the prepared three‐dimensional pools were screened with 29 polymerase chain reaction‐based markers, an average of 9.2 clones per marker were identified. These identified clones will be useful either in quantitative trait loci gene isolation or in synteny study between soybean and other legumes including Lotus japonicus. This efficient pooling system could be applied to any other BAC libraries without the need for robotic manipulation.     

Unmarked gene deletion and host–vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus

Sulfolobus islandicus is being used as a model for studying archaeal biology, geo-biology and evolution. However, no genetic system is available for this organism. To produce an S. islandicus mutant suitable for genetic analyses, we screened for colonies with a spontaneous pyrEF mutation. One mutant was obtained containing only 233 bp of the original pyrE sequence in the mutant allele and it was used as a host to delete the β-glycosidase (lacS) gene. Two unmarked gene deletion methods were employed, namely plasmid integration and segregation, and marker replacement and looping out, and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus–E. coli plasmid shuttle vectors were constructed, which genetically complemented ΔpyrEFΔlacS mutation after transformation. Thus, a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers.