Selective and efficient immobilization of cadmium in soil by layered double hydroxides intercalated with the mercaptosuccinic acid.

Cadmium (Cd) contamination in cropland poses a significant threat to the quality of agricultural products, but even though in-situ remediation has been extensively applied, non-selective immobilization remains an issue. In order to develop a material that specifically immobilizes Cd in soil, a layered double hydroxide, intercalated with mercaptosuccinic acid (MSA-CFA), was synthesized through co-precipitation. In this case, the MSA-CFA's maximum adsorption capacity was increased from the 513.8 mg·g-1 for unintercalated hydrotalcite CFA to 692.6 mg·g-1. Besides, MSA-CFA efficiently removed 99.25 % of Cd from soil water-extract solution and immobilized up to 70.03 % of bio-available Cd. However, interestingly, its immobilization effects on beneficial metal elements Fe, Mn and Zn were milder, being equivalent to 2/7, 5/7 and 1/2 that of lime, respectively. Moreover, XRD and XPS techniques revealed isomorphous substitution with calcium and sulfhydryl complexation during the Cd adsorption by MSA-CFA. Compared with CFA, the increased adsorption capacity of MSA-CFA for Cd was due to intercalated MSA acting as a new adsorption site, while the enhanced selectivity was contributed by sulfhydryl's affinity for Cd. Altogether, MSA-CFA showed great promise as a competitive and highly efficient candidate amendment in Cd-contaminated soil remediation.

A vicinal oxygen chelate protein facilitates viral infection by triggering the unfolded protein response in Nicotiana benthamiana.

Vicinal oxygen chelate (VOC) proteins are members of an enzyme superfamily with dioxygenase or non-dioxygenase activities. However, the biological functions of VOC proteins in plants are poorly understood. Here, we show that a VOC in Nicotiana benthamiana (NbVOC1) facilitates viral infection. NbVOC1 was significantly induced by infection by beet necrotic yellow vein virus (BNYVV). Transient overexpression of NbVOC1 or its homolog from Beta vulgaris (BvVOC1) enhanced BNYVV infection in N. benthamiana, which required the nuclear localization of VOC1. Consistent with this result, overexpressing NbVOC1 facilitated BNYVV infection, whereas, knockdown and knockout of NbVOC1 inhibited BNYVV infection in transgenic N. benthamiana plants. NbVOC1 interacts with the basic leucine zipper transcription factors bZIP17/28, which enhances their self-interaction and DNA binding to the promoters of unfolded protein response (UPR)-related genes. We propose that bZIP17/28 directly binds to the NbVOC1 promoter and induces its transcription, forming a positive feedback loop to induce the UPR and facilitating BNYVV infection. Collectively, our results demonstrate that NbVOC1 positively regulates the UPR that enhances viral infection in plants.

Correlates of condom use among male university students from eastern China who engage in casual sex.

Consistent condom use with casual partners is critical for preventing the transmission of human immunodeficiency virus (HIV) among male university students. This study aimed to determine the level of consistent condom use and explore the correlates of condom use consistency in male university students in eastern China. A descriptive cross-sectional survey was conducted in 13 universities in Zhejiang Province, which involved the recruitment of 31,674 students by stratified random sampling. Among them, 545 male students who engaged in casual sex in the year prior to this study were included. Adjusted and unadjusted logistic regression models were used to examine the correlates associated with consistent condom use. Among the 545 male university students, only 205 (37.6%) consistently used condoms in the previous year. The following correlates were associated with higher rates of consistent condom use: 1) Knowledge, specifically, the number of correct answers to "HIV infection can be determined by appearance" (AOR: 2.06, 95% CI: 1.21-3.49); 2) never finding casual partners on the internet during the past over the prior year (AOR: 0.63; 95% CI: 0.40-0.99); 3) never drinking alcohol before casual sex during the last over the prior year (AOR: 0.30; 95% CI: 0.20-0.46); 4) never engaging in commercial sex (AOR: 0.57; 95% CI: 0.34-0.96); and 5) high condom self-efficacy score (AOR: 2.55; 95% CI: 1.44-4.49). The study found a low level of consistent condom use among male university students. Promoting condom self-efficacy, reducing web-based casual sex, drinking before sex, and commercial sex are essential to improving the level of consistent condom use among male university students to reduce the transmission of HIV.

Fast Delivery of Multifunctional NIR-II Theranostic Nanoaggregates Enabled by the Photoinduced Thermoacoustic Process.

Multifunctional nanoaggregates are widely used in cancer phototheranostics. However, it is challenging to construct their multifunctionality with a single component, and deliver them rapidly and efficiently without complex modifications. Herein, a NIR-absorbing small molecule named TBT-2(TP-DPA) is designed and certify its theranostic potentials. Then, their nanoaggregates, which are simply encapsulated by DSPE-PEG, demonstrate a photothermal efficiency of 51% while keeping a high photoluminescence quantum yield in the NIR region. Moreover, the nanoaggregates can be excited and delivered by an 808 nm pulse laser to solid tumors within only 40 min. The delivery efficiency and theranostic efficacy are better than that of the traditional enhanced permeability and retention (EPR) effect (generally longer than 24 hours). This platform is first termed as the photoinduced thermoacoustic (PTA) process, and confirm its application requires both NIR-responsive materials and pulse laser irradiation. This study not only inspires the design of multifunctional nanoaggregates, but also offers a feasible approach to their fast delivery. The platform reported here provides a promising prospect to boost the development of multifunctional theranostic drugs and maximize the efficacy of used medicines for their clinical applications.

Texture and Magnetic Property of Fe-6.5 wt % Si Steel Strip with Cu-Rich Particles Modification.

Based on the ordered phase effectively suppressed by rapid solidification technology, the grain refinement concept using Cu is incorporated into the soft magnetic materials. Cu dosage not only could refine the grain size with an average grain size of 8.7 µm but also improve the continuity and consistency of Fe-6.5 wt % Si steel strip. It mainly attributes to the Cu-rich particles precipitating at the grain boundary, nailing the grain boundaries movement and inhibiting the grain growth, and then improving the magnetic properties and mechanical properties. The 1.5 wt % Cu sample exhibits an excellent magnetic property with the saturation magnetization of 236.54 emu/g, which mainly attributes to the strong η, λ, Goss texture formation and the band structure optimization of Si-Cu comodification. Furthermore, the mechanical properties of the steel strip are effectively improved, and the failure plastic deformation of 1.5 wt % Cu steel strip is about 11%. The rapid solidification with Cu-dosage refinement technology also has a remarkable reference on the mechanical properties and magnetic properties modification of other metal materials.

Optimization of single-tube nested PCR for the detection of Echinococcus spp.

Echinococcosis is a serious zoonotic life-threatening parasitic disease caused by metacestodes of Echinococcus spp., and appropriate sensitive diagnosis and genotyping techniques are required to detect infections and study the genetic characterization of Echinococcus spp. isolates. In this study, a single-tube nested PCR (STNPCR) method was developed and evaluated for the detection of Echinococcus spp. DNA based on the COI gene. STNPCR was 100 times more sensitive than conventional PCR and showed the same sensitivity to common nested PCR (NPCR); but with a lower risk of cross-contamination. The limit of detection of the developed STNPCR method was estimated to be 10 copies/µL of the recombinant standard plasmids of Echinococcus spp. COI gene. In clinical application, 8 cyst tissue samples and 12 calcification tissue samples were analysed by conventional PCR with outer and inner primers and resulted in 100.00% (8/8) and 8.33% (1/12), 100.00% (8/8) and 16.67% (2/12) positive reactions, respectively, while STNPCR and NPCR were all able to identify the presence of genomic DNA in 100.00% (8/8) and 83.33% (10/12) of the same samples. Due to its high sensitivity combined with the potential for the elimination of cross-contamination, the STNPCR method was suitable for epidemiological investigations and characteristic genetic studies of Echinococcus spp. tissue samples. The STNPCR method can effectively amplify low concentrations of genomic DNA from calcification samples and cyst residues infected with Echinococcus spp. Subsequently, the sequences of positive PCR products were obtained, which were useful for haplotype analysis, genetic diversity, and evolution studies of Echinococcus spp., and understanding of Echinococcus spp. dissemination and transmission among the hosts.

Theileria annulata SVSP455 interacts with host HSP60.

BACKGROUND: Theileria annulata, a transforming parasite, invades bovine B cells, dendritic cells and macrophages, promoting the uncontrolled proliferation of these cells. This protozoan evolved intricate strategies to subvert host cell signaling pathways related to antiapoptotic signaling to enable survival and proliferation within the host cells. However, the molecular mechanisms of the cell transformation induced by T. annulata remain largely unclear. Although some studies have predicted that the subtelomere-encoded variable secreted protein (SVSP) family plays roles in host-parasite interactions, the evidence for this is limited. METHODS: In the present study, the SVSP455 (TA05545) gene, a member of the SVSP gene family, was used as the target molecule. The expression pattern of SVSP455 in different life-cycle stages of T. annulata infection was explored using a quantitative real-time PCR assay, and the subcellular distribution of SVSP455 was observed using confocal microscopy. The host cell proteins interacting with SVSP455 were screened using the Y2H system, and their interactions were verified in vivo and in vitro using both bimolecular fluorescence complementation and confocal microscopy, and co-immunoprecipitation assays. The role played by SVSP455 in cell transformation was further explored by using overexpression, RNA interference and drug treatment experiments. RESULTS: The highest level of the SVSP455 transcript was detected in the schizont stage of T. annulata, and the protein was located both on the surface of schizonts and in the host cell cytoplasm. In addition, the interaction between SVSP455 and heat shock protein 60 was shown in vitro, and their link may regulate host cell apoptosis in T. annulata-infected cells. CONCLUSION: Our findings are the first to reveal that T. annulata-secreted SVSP455 molecule directly interacts with both exogenous and endogenous bovine HSP60 protein, and that the interaction of SVSP455-HSP60 may manipulate the host cell apoptosis signaling pathway. These results provide insights into cancer-like phenotypes underlying Theilera transformation and therapeutics for protection against other pathogens.

DEVELOPMENT OF A TRIPLEX REAL-TIME PCR ASSAY TO DETECT ECHINOCOCCUS SPECIES IN CANID FECAL SAMPLES.

Echinococcosis is a zoonotic disease with great significance to public health, and appropriate detection and control strategies should be adopted to mitigate its impact. Most cases of echinococcosis are believed to be transmitted by the consumption of food and/or water contaminated with canid stool containing Echinococcus spp. eggs. Studies assessing Echinococcus multilocularis, Echinococcus granulosus sensu stricto, and Echinococcus shiquicus coinfection from contaminated water-derived, soil-derived, and food-borne samples are scarce, which may be due to the lack of optimized laboratory detection methods. The present study aimed to develop and evaluate a novel triplex TaqMan-minor groove binder probe for real-time polymerase chain reaction (rtPCR) to simultaneously detect the 3 Echinococcus spp. mentioned above from canid fecal samples in the Qinghai-Tibetan Plateau area (QTPA). The efficiency and linearity of each signal channel in the triplex rtPCR assay were within acceptable limits for the range of concentrations tested. Furthermore, the method was shown to have good repeatability (standard deviation ≤0.32 cycle threshold), and the limit of detection was estimated to be 10 copies plasmid/µl reaction. In summary, the evaluation of the present method shows that the newly developed triplex rtPCR assay is a highly specific, precise, consistent, and stable method that could be used in epidemiological investigations of echinococcosis.

Performance of HIV detection in Zhejiang province in China: The Pareto principle at work.

BACKGROUND: Timely detection of HIV infection is critical for curbing the AIDS epidemic, and building an extensive and effective HIV laboratory network is of great importance. Therefore, improving quality management of the laboratory network and optimizing detection strategies are desirable research issues. METHODS: We assessed the applicability of the Pareto principle to HIV detection performance. We conducted a retrospective review of basic information and numbers of screening tests among an HIV laboratory network (1,452 laboratories) in Zhejiang province in 2014 and statistically analyzed HIV testing data for different population categories. RESULTS: Approximately, 80% of the cumulative HIV screening tests and positive screening tests originated from 17.3% (251/1,452) and 11.7% (170/1,452) of the laboratories in the whole province, respectively, and similar patterns were observed at the prefectural level. We found that the top five population screening categories (25%, 5/20) had the highest contribution (approximately 80%) to not only the number of screening tests (77.2%) but also the numbers of positive (76.4%) and confirmed positive tests (81.5%). CONCLUSIONS: The Pareto principle provides a method for identifying noteworthy laboratories to deliver prior quality supervision and developing highly efficient screening strategies that best suit local needs.

Listeria monocytogenes MenI Encodes a DHNA-CoA Thioesterase Necessary for Menaquinone Biosynthesis, Cytosolic Survival, and Virulence.

Listeria monocytogenes is a Gram-positive, intracellular pathogen that is highly adapted to invade and replicate in the cytosol of eukaryotic cells. Intermediate metabolites in the menaquinone biosynthesis pathway are essential for the cytosolic survival and virulence of L. monocytogenes, independent of the production of menaquinone (MK) and aerobic respiration. Determining which specific intermediate metabolite(s) are essential for cytosolic survival and virulence has been hindered by the lack of an identified 1,4-dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) thioesterase essential for converting DHNA-CoA to DHNA in the MK synthesis pathway. Using the recently identified Escherichia coli DHNA-CoA thioesterase as a query, homology sequence analysis revealed a single homolog in L. monocytogenes, LMRG_02730 Genetic deletion of LMRG_02730 resulted in an ablated membrane potential, indicative of a nonfunctional electron transport chain (ETC) and an inability to aerobically respire. Biochemical kinetic analysis of LMRG_02730 revealed strong activity toward DHNA-CoA, similar to its E. coli homolog, further demonstrating that LMRG_02730 is a DHNA-CoA thioesterase. Functional analyses in vitro, ex vivo, and in vivo using mutants directly downstream and upstream of LMRG_02730 revealed that DHNA-CoA is sufficient to facilitate in vitro growth in minimal medium, intracellular replication, and plaque formation in fibroblasts. In contrast, protection against bacteriolysis in the cytosol of macrophages and tissue-specific virulence in vivo requires the production of 1,4-dihydroxy-2-naphthoate (DHNA). Taken together, these data implicate LMRG_02730 (renamed MenI) as a DHNA-CoA thioesterase and suggest that while DHNA, or an unknown downstream product of DHNA, protects the bacteria from killing in the macrophage cytosol, DHNA-CoA is necessary for intracellular bacterial replication.

Retinoblastoma cell-derived Twist protein promotes regulatory T cell development.

BACKGROUND: The development of tumor tissue-infiltrating regulatory T cell (Treg) is incompletely understood. This study investigates the role of retinoblastoma cell (Rbc)-derived Twist­related protein 1 (Twist) in the Treg development. METHODS: The surgically removed Rb tissues were collected. Rbcs were cultured with CD4+ T cells to assess the role of Rbc-derived Twist in the Treg generation. RESULTS: We found that more than 90% Rbcs expressed Twist. Foxp3+ Tregs were detected in the Rb tissues that were positively correlated with the Twist expression in Rbcs, negatively associated with Rb patient survival and sight survival. Treating Rbcs with hypoxia promoted the Twist expression that could be detected in the cytoplasm, nuclei and on the cell surface. Twist activated CD4+ T cells by binding the TLR4/myeloid differentiation factor 2 complex and promoted the transforming growth factor-ß-inducible early gene 1 product and Foxp3 expression. These Rbc-induced Foxp3+ Tregs showed immune-suppressive function on CD8+ T cell proliferation. CONCLUSIONS: Rbcs express Twist, that induces IL-4+ Foxp3+ Tregs; the latter can inhibit CD8+ cytotoxic T cell activities. Therefore, Twist may play an important role in the pathogenesis of Rb.

Structural characterization of Lanzhou lily (Lilium davidii var. unicolor) polysaccharides and determination of their associated antioxidant activity.

BACKGROUD: The Lanzhou lily (Lilium davidii var. unicolor) is the only Lilium species that is used for both culinary and medicinal purposes in China. Its bulbs contain various bioactive substances, such as polysaccharides, saponins and colchicine. Lanzhou lily polysaccharides are known to have anti-immunity, anti-tumor and anti-oxidation functions. RESULTS: The present study used a Box-Behnken design to optimize the ultrasound-assisted extraction of Lanzhou lily polysaccharides. Compared to other enzymes, trypsin significantly increased the polysaccharide yields, whereas the protein content of polysaccharides extracted with trypsin was the lowest. Monosaccharide mainly includes glucose (> 50%) and mannose (> 10%). 1,1-Diphenyl-2-picrylhydrazyl radical scavenging activity, chelating activity, total antioxidant capacity and hydroxyl radical scavenging activity of Lanzhou lily polysaccharides extracted with trypsin were stronger than those extracted without enzymes (control). Structural characteristics of Lanzhou lily polysaccharides extracted with trypsin and extracted without enzymes were characterized by scanning electron microscopy and nuclear magnetic resonance spectroscopy. When water extracted polysaccharide and trypsin extracted polysaccharide concentrations were 200 µg mL-1 , Raw264.7 proliferation rates were 101.69% and 159.41%, respectively. CONCLUSION: The Lanzhou lily polysaccharide was identified as α-(1 → 6)-d-glucan. Consequently, the effects of both potential antioxidant and proliferative activity of trypsin are significant. © 2020 Society of Chemical Industry.

Herbal extract of Artemisia maritima induces selective anti-tumor effects in three human lung carcinoma cells (H1299, NCI-H1437, PC-14) through activating apoptosis-related proteins, G2/M cell cycle arrest and suppression of cell migration.

PURPOSE: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths in the United States, accounting for 85% of all diagnosed lung cancers and resulting in over 100,000 deaths per year. The main purpose of the current study was to investigate the antiproliferative effects of ethanolic extract of Artemisia maritima in three human lung cancer cell lines along with studying the effects of the herbal extract on cellular apoptosis, G2/M cell cycle arrest and cell migration. METHODS: The CCK-8 assay was used to determine cell viability. Apoptosis was detected by using acridine orange (AO)/ethidium bromide (EB) staining, annexin V/propidium iodide (PI) assay and western blot. The cell migration was determined by wound healing assay while the effects on cell cycle were evaluated by flow cytometry. RESULTS: The results showed that herbal extract of Artemisia maritima decreased the viability of all three cell lines H1299, NCI-H1437, PC-14 dose-dependently with maximum effect on NCI-H1437 cell line. The antiproliferative effects were due to the activation of mitochondrial-dependent apoptotic pathway as seen by fluorescence microscopy which showed chromatin condensation and nuclear fragmentation. This was also associated with increase in Bax and decrease in Bcl-2 levels. Artemisia maritima extract treatment also led to G2/M phase cell cycle arrest along with strong inhibition of cell migration. CONCLUSIONS: In conclusion, the results of the current study clearly indicate that Artemisia maritima extract exhibits antiproliferative effects in Nonsmall cell lung cancer cells by triggering apoptosis, cell cycle arrest and inhibition of cell migration.

Coenurosis of Yak, Bos grunniens, caused by Taenia multiceps: A Case Report with Molecular Identification in Qinghai Tibetan Plateau Area, China.

Coenurosis is an important zoonotic helminthic disease caused by the larval stage of the tapeworm Taenia multiceps. This parasite typically infects the brain of the intermediate hosts, including sheep, goat, cattle and even humans. We report a case of T. multiceps infection in a yak confirmed by clinical symptoms, morphological characteristics, and molecular and phylogenetic analyses. The coenurus was thin-walled, whitish, and spherical in shape with a diameter of 10 cm. The parasite species was identified as T. multiceps by PCR amplification and sequencing of the 18S rRNA, cox1 and nad1 genes. Three gene sequences all showed high homology (all above 97%) with the reference sequences from different hosts. Moreover, phylogenetic reconstructions with the 3 published Taenia gene sequences confirmed that the Qinghai yak isolate was closely related to T. multiceps. Although there are advanced diagnosis and treatment methods for coenurosis, early infection is difficult to diagnose. Importantly, the findings of yak infection case should not be ignored due to its zoonotic potential.

Identification of an amphipathic peptide sensor of the Bacillus subtilis fluid membrane microdomains.

Regions of increased fluidity are newly found bacterial membrane microdomains that are composed of short, unsaturated and branched fatty acyl chains in a fluid and disordered state. Currently, little is known about how proteins are recruited and localized to these membrane domains. Here, we identify a short amphipathic α-peptide in a previously unreported crystal structure and show that it is responsible for peripheral localization of the phosphate acyltransferase PlsX to the fluid microdomains in Bacillus subtilis. Mutations disrupting the amphipathic interaction or increasing the nonpolar interaction are found to redistribute the protein to the cytosol or other part of the plasma membrane, causing growth defects. These results reveal a mechanism of peripheral membrane sensing through optimizing nonpolar interaction with the special lipids in the microdomains. This finding shows that the fluid membrane microdomains may take advantage of their unique lipid environment as a means of recruiting and organizing proteins.

Development of Beet necrotic yellow vein virus-based vectors for multiple-gene expression and guide RNA delivery in plant genome editing.

Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus (BNYVV) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive-stranded RNAs. Here, we have established a BNYVV full-length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV-based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co-localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV-based vectors were used to deliver NbPDS guide RNAs for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV-based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants.

EZH1 is an antipsychotic-sensitive epigenetic modulator of social and motivational behavior that is dysregulated in schizophrenia.

BACKGROUND: With the capacity to modulate gene networks in an environmentally-sensitive manner, the role of epigenetic systems in mental disorders has come under intense investigation. Dysregulation of epigenetic effectors, including microRNAs and histone-modifying enzymes, may better explain the role of environmental risk factors and the observed heritability rate that cannot be fully attributed to known genetic risk alleles. Here, we aimed to identify novel epigenetic targets of the schizophrenia-associated microRNA 132 (miR-132). METHODS: Histone modifications were quantified by immunodetection in response to viral-mediated overexpression of miR-132 while a luminescent reporter system was used to validate targets of miR-132 in vitro. Genome-wide profiling, quantitative PCR and NanoSting were used to quantify gene expression in post-mortem human brains, neuronal cultures and prefrontal cortex (PFC) of mice chronically exposed to antipsychotics. Following viral-mediated depletion of Enhancer of Zeste 1 (EZH1) in the murine PFC, behaviors including sociability and motivation were assessed using a 3-chambered apparatus and forced-swim test, respectively. RESULTS: Overexpression of miR-132 decreased global histone 3 lysine 27 tri-methylation (H3K27me3), a repressive epigenetic mark. Moreover, the polycomb-associated H3K27 methyltransferase, EZH1, is regulated by miR-132 and upregulated in the PFC of schizophrenics. Unlike its homolog EZH2, expression of EZH1 in the murine PFC decreased following chronic exposure to antipsychotics. Viral-mediated depletion of EZH1 in the mouse PFC attenuated sociability, enhanced motivational behaviors, and affected gene expression pathways related to neurotransmission and behavioral phenotypes. CONCLUSIONS: EZH1 is dysregulated in schizophrenia, sensitive to antipsychotic medications, and a brain-enriched miR-132 target that controls neurobehavioral phenotypes.

Development of LAMP assays for the molecular detection of taeniid infection in canine in Tibetan rural area.

For field-identification of taeniid cestodes in canine animals in Tibetan area, loop-mediated isothermal amplification (LAMP) assays for Echinococcus multilocularis, E. shiquicus, Taenia hydatigena, T. multiceps, T. pisiformis and T. crassiceps were developed and evaluated along with the reported assay for E. granulosus. The LAMP assays showed specific reaction with their corresponding target species DNA with the detection limit of 1 to 10 pg. Moreover, the assays for E. granulosus, E. multilocularis, T. hydatigena and T. multiceps could detect DNA extracted from 3 or more eggs of their corresponding target species. Then, the LAMP assays were applied on samples containing 3 to 35 taeniid eggs obtained from 61 field-collected canine feces in Qinghai, and the result was compared with a reported multiplex PCR and sequence analysis. The LAMP assays and the PCR detected single species DNA of E. granulosus, E. shiquicus, T. hydatigena and T. multiceps in 5, 2, 44 and 2 samples, respectively. In the rest 8 samples, DNA of both E. granulosus and T. hydatigena were detected by the PCR but the LAMP assays detected those DNAs in 2 samples and only T. hydatigena DNA in 6 samples. It was assumed that less than 3 E. granulosus eggs were mixed in the samples although the samples contained 21 to 27 eggs in total. In conclusion, the LAMP assays were less sensitive than the multiplex PCR, but would have adequate sensitivity for field use in Tibetan area.

Crystal structure of the thioesterification conformation of Bacillus subtilis o-succinylbenzoyl-CoA synthetase reveals a distinct substrate-binding mode.

o-Succinylbenzoyl-CoA (OSB-CoA) synthetase (MenE) is an essential enzyme in bacterial vitamin K biosynthesis and an important target in the development of new antibiotics. It is a member of the adenylating enzymes (ANL) family, which reconfigure their active site in two different active conformations, one for the adenylation half-reaction and the other for a thioesterification half-reaction, in a domain-alternation catalytic mechanism. Although several aspects of the adenylating mechanism in MenE have recently been uncovered, its thioesterification conformation remains elusive. Here, using a catalytically competent Bacillus subtilis mutant protein complexed with an OSB-CoA analogue, we determined MenE high-resolution structures to 1.76 and 1.90 Å resolution in a thioester-forming conformation. By comparison with the adenylation conformation, we found that MenE's C-domain rotates around the Ser-384 hinge by 139.5° during domain-alternation catalysis. The structures also revealed a thioesterification active site specifically conserved among MenE orthologues and a substrate-binding mode distinct from those of many other acyl/aryl-CoA synthetases. Of note, using site-directed mutagenesis, we identified several residues that specifically contribute to the thioesterification half-reaction without affecting the adenylation half-reaction. Moreover, we observed a substantial movement of the activated succinyl group in the thioesterification half-reaction. These findings provide new insights into the domain-alternation catalysis of a bacterial enzyme essential for vitamin K biosynthesis and of its adenylating homologues in the ANL enzyme family.