Snakes are widely farmed in China, Southeast Asia, and India, with extensive applications in food and medicine; however, glycosaminoglycans (GAGs) derived from these sources remain largely understudied. In this study, GAGs were extracted and purified from the bone and carcass of the cobra (Naja naja atra) to investigate their structural characteristics and bioactivities. Structural analysis, including 1H NMR spectroscopy and disaccharide composition assays, identified the purified fractions (CBF2 and CCF2) as chondroitin sulfate/dermatan sulfate (CS/DS). These fractions exhibited structural heterogeneity, containing 35.9% and 28.8% iduronic acid, respectively, with disaccharide profiles dominated by A-type units (>80%) and high 4S/6S ratios (10.31 and 18.27, respectively). In vitro assays demonstrated that CBF2 and CCF2 possessed anticoagulant activity, prolonging activated partial thromboplastin time (aPTT) and thrombin time (TT) compared to conventional CS. These findings highlight the critical role of uronic acid epimerization in modulating the biological functions of GAGs.

Amanita exitialis, a deadly mushroom found in eastern Asia, causes the highest death rates among all poisonous mushrooms in China. The aim of the present study was to develop an efficient, accurate, and user-friendly PCRbased method for identifying A. exitialis that could facilitate the prevention, diagnosis, and treatment of associated food poisoning. A. exitialis-specific primers and probes were designed based on the internal transcribed spacer region variations of 27 mushroom species. Specificity was confirmed using conventional and real-time PCR for 23 non-target mushroom species, including morphologically similar and closely related species. Compared to conventional PCR, real-time PCR was more sensitive (detectable DNA concentration: 1.36 x 10-2 ng/mu L vs. 1.36 x 10-3) and efficient (analysis time: 1 h vs. 40 min). Furthermore, the real-time PCR results could be immediately visualized using amplification curve analysis. The results present two robust PCR-based methods for A. exitialis identification that can facilitate food safety.

Zika virus (ZIKV) infection can result in a birth defect of the brain called microcephaly and other severe fetal brain defects. ZIKV enters the susceptible host cells by endocytosis, which is mediated by the interaction of the envelope (E) glycoprotein with cellular surface receptor molecules. However, the cellular factors that used by the ZIKV to gain access to host cells remains elusive. Here, we report that the extracellular domain of integrin beta 4 (ITGB4) is an entry factor of ZIKV. ITGB4 mediates ZIKV infection by directly interacting with the E glycoprotein of ZIKV, and ITGB4 knockout hampers the binding and replication of ZIKV to host cells. A functional monoclonal antibody against ITGB4 or the soluble forms of ITGB4 could decrease the binding and infection of ZIKV to permissive cell lines. Importantly, the ITGB4 antibody blocks the infection of ZIKV to mouse placenta, thus protecting the fetuses from ZIKV infection. Together, our study has demonstrated that ZIKV infection involves ITGB4 dependent binding.

The interactions between plant viruses and insect vectors are very complex. In recent years, RNA sequencing data have been used to elucidate critical genes of Tomato spotted wilt ortho-tospovirus (TSWV) and Frankliniella occidentalis ( F. occidentalis ). However, very little is known about the essential genes involved in thrips acquisition and transmission of TSWV. Based on transcriptome data of F. occidentalis infected with TSWV, we verified the complete sequence of the E3 ubiquitin-protein ligase UBR7 gene ( UBR7 ), which is closely related to virus transmission. Additionally, we found that UBR7 belongs to the E3 ubiquitin–protein ligase family that is highly expressed in adulthood in F. occidentalis . UBR7 could interfere with virus replication and thus affect the transmission efficiency of F. occidentalis . With low URB7 expression, TSWV transmission efficiency decreased, while TSWV acquisition efficiency was unaffected. Moreover, the direct interaction between UBR7 and the nucleocapsid (N) protein of TSWV was investigated through surface plasmon resonance and GST pull-down. In conclusion, we found that UBR7 is a crucial protein for TSWV transmission by F. occidentalis , as it directly interacts with TSWV N. This study provides a new direction for developing green pesticides targeting E3 ubiquitin to control TSWV and F. occidentalis .

A simple and reliable HPLC method was developed for quantification of chondroitin sulfate (CS). The procedure is based on precolumn hydrolysis of CS to liberate galactosamine and subsequent derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Hydrolysis and derivatization conditions were optimized. A linear correlation coefficient of 0.9999 was calculated within the range of 10-1500 mu g/mL from the standard curve. The method produces good precision and good accuracy (100.75 % recovery). An advantage over other common methods is its ability to quantify CS of all molecular weights and structures, as evidenced by the determination of CS fractions with narrow molecular weight distributions obtained through depolymerization by different methods, while enzymatic HPLC was proven to be infeasible. Extraction recoveries of CS from monosaccharide mixed samples were > 93 %. The reliability was also validated by a small difference (-1.95 % to 4.12 %) relative to enzymatic HPLC results in analysing representative CS samples of different animal origins and suppliers.

Low molecular weight (LWM) hyaluronic acid (HA) and chondroitin sulfate (CS) have a wide range of applications. To determine their molecular weight (MW), we developed a gel permeation chromatography (GPC) method, which is calibrated based on serrated peaks in the chromatograms. MW calibrants were obtained from the enzymolysis of HA and CS using hyaluronidase. The identical structure of calibrants and samples ensured the soundness of the method. The highest confidence MWs were up to 14,454 and 14,605 for HA and CS, respectively, and the standard curves showed very high correlation coefficients. Thanks to the changeless relationship between MW and its contribution to the GPC integral, the second calibration curves could be derived via one GPC column, also embodied correlation coefficients of >0.9999. The discrepancies of MW values were minuscule, and the measurement of a sample could be conducted in <30 min. The accuracy of the method was verified using LWM heparins, and the measured Mw values showed a 1.2 %-2.0 % error relative to pharmacopeia results. The MW results obtained for LWM-HA and LWM-CS samples were also consistent with the results obtained by multiangle laser light scattering. The method was also verified be able to measure the very low MWs.

Host plants deploy the small RNA (sRNA)-directed RNA silencing pathway to resist invasion by acellular microorganisms (viruses/viroids/satellites), and, in turn, this pathway is exploited by pathogenic agents to create an environment conducive to infection. Previous known sRNA-RNA systems consist of host endogenous microRNAs (miRNAs) mediating the regulation of host mRNAs and virus/viroid/satellite-derived small interfering RNAs (vsiRNAs) targeting their genomic RNAs. However, more in-depth explorations have substantially expanded the understanding of the complexity of sRNA-RNA regulatory networks. Here, we review some recently discovered sRNA-mediated regulatory systems. Specifically, in addition to virus-encoded proteins acting as virulence factors, vsiRNAs can serve as important pathogenic determinants targeting host mRNAs and noncoding RNAs to promote virus/viroid/satellite infection and trigger symptoms that may be side effects of infection. Additionally, virus-activated but host-derived siRNAs (vasiRNAs) regulate endogenous plant gene expression related to virus resistance or pathogenicity. The inhibitory effect of miRNAs on plant endogenous mRNAs and viral RNAs (vRNAs) has also been identified. Furthermore, siRNA-based interregulation occurring between viruses and their parasite satellite RNAs (satRNAs) enables coexisting virus-satRNA-plant homoeostasis. Thus, the underlying mechanisms of plant-virus/viroid/satellite competition and symbiosis are largely obscured by these diverse sRNA-RNA combinations. Guided by the intricate regulatory network-based principle at the RNA level, practically applicable and feasible strategies have been developed for the management of plant viruses/viroids/satellites for which effective control measures are lacking.

植物检疫是一种生物安全措施,通过立法手段防止植物及其产品在流通过程中传播有害生物.检测鉴定是植物检疫措施实施的核心.该文概述植物检疫的作用及其面临的挑战、植物检疫检测技术的应用现状及发展趋势,并对多组学时代下植物检疫检测技术进行展望.

Quarantine insects are economically important pests that frequently invade new habitats. A rapid and accurate monitoring method to trace the geographical sources of invaders is required for their prevention, detection, and eradication. Current methods based on genetics are typically time-consuming. Here, we developed a novel tracing method based on insect gut microbiota. The source location of the insect gut microbiota can be used to rapidly determine the geographical origin of the insect. We analyzed 179 gut microbiota samples from 591 individuals of 22 quarantine insect species collected from 36 regions in China. The gut microbiota of these insects primarily included Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, Proteobacteria, and Tenericutes. The diversity of the insect gut microbiota was closely associated with geographical and environmental factors. Different insect species could be distinguished based on the composition of gut microbiota at the phylum level. Populations of individual insect species from different regions could be distinguished based on the composition of gut microbiota at the phylum, class, and order levels. A method for determining the geographical origins of invasive insect species has been established; however, its practical application requires further investigations before implementation.

对于多数植物病毒而言,其在田间的自然扩散主要依赖昆虫等介体生物,而媒介昆虫的垂直传播是植物病毒长期存在并发生的重要原因.对媒介昆虫垂直传播病毒机制的研究不仅可以为未来开发高效低毒农药奠定基础,更可为植物病毒与昆虫的互作和病毒病的预测预报提供新的视野及角度.媒介昆虫在植物病毒传播过程中的具体作用在近几年被广泛研究.该文综述了近年来植物病毒在昆虫体内垂直传播的研究进展,包括昆虫传播植物病毒的方式、植物病毒在昆虫体内的垂直传播方式以及虫媒病毒垂直传播的可能机制等.在整个垂直传播的过程中,植物病毒的衣壳蛋白、磷蛋白和媒介昆虫唐氏综合症细胞黏附分子、硫酸乙酰肝素糖蛋白、热激蛋白以及卵黄原蛋白,甚至共生菌都有参与.最后,基于媒介昆虫和植物病毒的关系对未来植物病毒病的绿色防控和生物防控进行了展望.

The use of omics techniques to analyze the differences between genetic engineering organisms and their parents can identify unintended effects and explore whether such unintended effects will have negative consequences. In order to evaluate whether genetic engineering will cause changes in crops beyond the changes introduced by conventional plant breeding, we compared the extent of transcriptome and metabolome modification in the leaves of three lines developed by RNA interference (RNAi)-based genetic engineering and three lines developed by conventional breeding. The results showed that both types of plant breeding methods can manifest changes at the short interfering RNA (siRNA), transcriptomic, and metabolic levels. Relative expression analysis of potential off-target gene revealed that there was no broad gene decline in the three RNAi-based genetic engineering lines. We found that the number of DEGs and DAMs between RNAi-based genetic engineering lines and the parental line was less than that between conventional breeding lines. These unique DEGs and DAMs between RNAi-based genetic engineering lines and the parental lines were not enriched in detrimental metabolic pathways. The results suggest that RNAi-based genetic engineering do not cause unintended effects beyond those found in conventional breeding in maize.

Conserved and multifunctional Geminivirus Replication-associated Protein (Rep) specifically recognizes the replication origin and initiates viral DNA replication. We report the X-ray crystallography-based structures of two complexes containing the N-terminal domain (5-117aa) of Tomato yellow leaf curl virus (TYLCV) Rep: the catalytically-dead Rep in complex with nonanucleotide ssDNA (Rep5-117 Y101F-ssDNA) as well as the catalytically-active phosphotyrosine covalent adduct (Rep5-117-ssDNA). These structures provide functional insight into the role of Rep in viral replication. Metal ions stabilize the DNA conformation by interacting with the phosphate group of adenine and thus promote formation of the catalytic center. Furthermore, we identified a compound that inhibits the binding of Rep to ssDNA and dsDNA and found that the addition of metal ions compromises the inhibitory effectiveness of this compound. This study demonstrates the mechanism of DNA recognition and cleavage process of viral Rep, emphasizing the role of metal ions.

Bactrocera dorsalis is an invasive polyphagous pest causing considerable ecological and economic damage worldwide. We report a high-quality chromosome-level genome assembly and combine various transcriptome data to explore the molecular mechanisms of its rapid adaptation to new environments. The expansions of the DDE transposase superfamily and key gene families related to environmental adaptation and enrichment of the expanded and unique gene families in metabolism and defence response pathways explain its environmental adaptability. The relatively high but not significantly different expression of heat-shock proteins, regardless of the environmental conditions, suggests an intrinsic mechanism underlying its adaptation to high temperatures. The mitogen-activated protein kinase pathway plays a key role in adaptation to new environments. The prevalence of duplicated genes in its genome explains the diversity in the B. dorsalis complex. These findings provide insights into the genetic basis of the invasiveness and diversity of B. dorsalis, explaining its rapid adaptation and expansion.

Phytolacca is the largest genus of Phytolaccaceae. Owing to interspecific hybridization, infraspecific variation, and apparent weak genetic control of many qualitative characters, which have obscured boundaries between species, the classification and phylogenetic relationships of this genus are unclear. Native Phytolacca is disjunctly distributed in America, eastern Asia, and Africa, and the biogeographic history of the genus remained unresolved. In this study, we used the whole chloroplast genome and three markers (nrDNA, rbcL, and matK) to reconstruct phylogenetic relationships within Phytolacca, analyze divergence times, and infer biogeographic histories. The phylogenetic results indicate that Phytolacca is monophyletic, which is inconsistent with the infrageneric classification based on morphology. According to the divergence time estimation, Phytolacca began to diversify at approximately 20.30 Ma during the early Miocene. Central America, including Mexico, Costa Rica, and Colombia, is the center of species diversity. Biogeographical analysis indicated five main dispersal events and Phytolacca originated from Central and South America. Birds may be the primary agents of dispersal because of the fleshy fruiting of Phytolacca. This study extended sampling and added more genetic characteristics to infer the evolutionary history of Phytolacca, providing new insights for resolving the classification and elucidating the dispersal events of Phytolacca.

对水稻中多种病原细菌的检测,使用常规方法往往耗时耗力,而多重PCR可以更加高效地进行多种细菌的检测.根据水稻细菌性谷枯病菌gyrB基因,水稻细菌性叶鞘褐腐病菌PfsI/R quorum sensing位点以及水稻细菌性条斑病菌和水稻白叶枯病菌含铁细胞接受因子基因设计引物,建立4种水稻病菌的多重PCR检测方法,对方法进行特异性和灵敏度测试,并对采自不同地区的水稻样本进行检测.结果 显示,多重PCR方法能同步地快速检测出水稻细菌性谷枯病菌、水稻细菌性叶鞘褐腐病菌、水稻细菌性条斑病菌或水稻白叶枯病菌,检测灵敏度达到103 cfu/mL的菌液浓度,利用该方法对我国不同地区的58份水稻种子进行检测,其中17个样本检测出水稻细菌性条斑病菌或水稻白叶枯病菌,未检测到水稻细菌性谷枯病菌和水稻细菌性叶鞘褐腐病菌.

Genetic engineering (GM) has great potential to improve maize productivity, but rises some concerns on unintended effects, and equivalent as their comparators. There are some limitations through targeted analysis to detect the UE in genetically modified organisms in many previous studies. We here reported a case-study on the effects of introducing herbicides and insect resistance (HIR) gene cassette on molecular profiling (transcripts and metabolites) in a popular maize variety Zhengdan958 (ZD958) in China. We found that introducing HIR gene cassette bring a limited numbers of differential abundant genes (DAGs) or differential abundant metabolites (DAMs) between transgenic events and non-transgenic control. In contrast, averaged 10 times more DAGs and DAMs were observed when performed comparison under different growing environments in three different ecological regions of China than the numbers induced by gene effects. Major biological pathways relating to stress response or signaling transduction could explain somehow the effects of growing environments. We further compared two transgenic events mediated ZD958 (GM-ZD958) with either transgenic parent GM-Z58, and other genetic background nonGM-Z58, nonGM-ZD958, and Chang7-2. We found that the numbers of DAGs and DAMs between GM-ZD958 and its one parent maize variety, Z58 or GM-Z58 is equivalent, but not Chang7-2. These findings suggest that greater effects due to different genetic background on altered molecular profiling than gene modification itself. This study provides a case evidence indicating marginal effects of gene pleiotropic effects, and environmental effects should be emphasized.

The taxonomically challenging genus Amaranthus (Family Amaranthaceae) includes important agricultural weed species that are being spread globally as grain contaminants. We hypothesized that the ALS gene will help resolve these taxonomic challenges and identify potentially harmful resistant biotypes. We obtained 153 samples representing 26 species from three Amaranthus subgenera and included in that incorporated ITS, ALS (domains C, A and D) and ALS (domains B and E) sequences. Subgen. Albersia was well supported, but subgen. Amaranthus and subgen. Acnida were not. Amaranthus tuberculatus, A. palmeri and A. spinosus all showed different genetic structuring. Unique SNPs in ALS offered reliable diagnostics for most of the sampled Amaranthus species. Resistant ALS alleles were detected in sixteen A. tuberculatus samples (55.2%), eight A. palmeri (27.6%) and one A. arenicola (100%). These involved Ala122Asn, Pro197Ser/Thr/Ile, Trp574Leu, and Ser653Thr/Asn/Lys substitutions, with Ala122Asn, Pro197Thr/Ile and Ser653Lys being reported in Amaranthus for the first time. Moreover, different resistant mutations were present in different A. tuberculatus populations. In conclusion, the ALS gene is important for species identification, investigating population genetic diversity and understanding resistant evolution within the genus Amaranthus.

Release of various genetically modified organisms (GMOs) into market during the past decade contributes to concerns regarding the traceability of food and feed products. In this study, a comprehensive list of inserted elements of commercialized GMOs has been collected, which contained information of 191 singular genetically modified (GM) varieties. Several elements were selected to develop a universal analytical approach for screening GM presence in food and feed products. This approach achieved coverage of those singular GM events as well as their hybrid crosses. The developed screening approach showed great specificity and sensitivity of less than 25 copies that meet the labeling demands for all countries. Moreover, microfluidic chip and digital PCR were combined in this approach to detect GM varieties and estimate GM content, both of which achieved high-throughput and accurate identification of unauthorized GMOs. Overall, this new analytical approach will serve as a functional tool for accurate control of authorized and unauthorized GMOs.

The fungus Leptosphaeria maculans leading to Phoma stem canker (blackleg) of Brassica napus (oilseed rape, canola) produces the phytotoxin sirodesmin PL, which is responsible for major yield losses of oilseed rape worldwide. Polymerase chain reaction (PCR) remains the gold standard diagnostic tool for L. maculans, but the required expensive equipment and long time make it inappropriate for fast field test. Herein, a portable system for rapid assaying L. maculans and L. biglobosa is designed around recombinase polymerase amplification (RPA) with fluorescent probe as the signal indicator, which allowed the real-time assay of amplification performed on a portable device between 37 and 42 degrees C. The time needed to observe the positive reaction results is controlled within 30 min. The proposed assay system is a good choice for on-site disease screening of oilseed rape plant where rapid detection is valuable, including port quarantine, agriculture quality testing, and pathogen spreading control.