Neurexin-1 is required for synapse formation and larvae associative learning in Drosophila

Neurexins are highly polymorphic cell-surface adhesive molecules in neurons. In cultured mammalian cell system, they were found to be involved in synaptogenesis. Here, we report for the first time that Drosophila neurexin is required for synapse formation and associative learning in larvae. Drosophila genome encodes a single functional neurexin (CG7050; Neurexin-1 or Nrx-1), which is a homolog of vertebrate alpha-neurexin. Neurexin-1 is expressed in central nervous system and highly enriched in synaptic regions of the ventral ganglion and brain. Neurexin-1 null mutants are viable and fertile, but have shortened lifespan. The synapse number is decreased in central nervous system in Neurexin-1 null mutants. In addition, Neurexin-1 null mutants exhibit associative learning defect in larvae.     

Baculiferins A–O,O-sulfated pyrrole alkaloids with anti-HIV-1 activity,from the Chinese marine sponge Iotrochota baculifera

Fifteen new DOPA-derived pyrrole alkaloids, named baculiferins A–O (2–16), were isolated from the Chinese marine sponge Iotrochota baculifera, together with the known alkaloids purpurone (1) and ningalin A (17). Most of the new compounds contain one to three O-sulfate units. Their structures were determined by extensive spectroscopic analysis including ¹H and ¹³C NMR (COSY, HMQC, HMBC) and ESIMS data. A possible pathway for the biosynthetic origin of the isolated alkaloids is proposed, in which DOPA is assumed to be a joint biogenetic precursor. Baculiferins C, E–H, and K–N (4, 6–9, 12–15) were found to be potent inhibitors against the HIV-1 IIIB virus in both, MT4 and MAGI cells. Additional bioassay revealed that baculiferins could dramatically bind to the HIV-1 target proteins Vif, APOBEC3G, and gp41, for which structure–activity relationships are discussed.     

Development of a quantitative, high-throughput cell-based enzyme-linked immunosorbent assay for detection of colony-stimulating factor-1 receptor tyrosine kinase inhibitors

Inhibitors of receptor tyrosine kinases are implicated as therapeutic agents for the treatment of many human diseases including cancer, inflammation and diabetes. Cell-based assays to examine inhibition of receptor tyrosine kinase mediated intracellular signaling are often laborious and not amenable to high-throughput cell-based screening of compound libraries. Here we describe the development of a nonradioactive, sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the activation and inhibition of ligand-induced phosphorylation of the colony-stimulating factor-1 receptor (CSF-1R) in 96-well microtiter plate format. The assay involves the capture of the Triton X-100 solubilized human CSF-1R, from HEK293E cells overexpressing histidine epitope-tagged CSF-1R (CSF-1R/HEK293E), with immobilized CSF-1R antibody and detection of phosphosphorylation of the activated receptor with a phosphotyrosine specific antibody. The assay exhibited a 5-fold increase in phosphorylated CSF-1R signal from CSF-1R/HEK293E cells treated with colony-stimulating factor (CSF-1) relative to treated vector control cells. Additionally, using a histidine epitope-specific capture antibody, this method can also be adapted to quantify the phosphorylation state of any recombinantly expressed, histidine-tagged receptor tyrosine kinase. This method is a substantial improvement in throughput and quantitation of CSF-1R phosphorylation over conventional immunoblotting techniques.     

Synthesis of NaYF(4):Yb,Er/single-walled carbon nanohorns nanocomposite and its application as cells label

A novel nanocomposite synthesis method of amino-modified NaYF(4):Yb,Er upconversion luminescent nanoparticles and single-walled carbon nanohorns was developed via covalent linkage for the first time. The nanocomposite was covalently coupled with rabbit anti-CEA8 antibody and then used successfully as a cell labeling agent for the immunolabeling and imaging of HeLa cells.     

Genetic analysis of two phosphodiesterases reveals cyclic diguanylate regulation of virulence factors in Dickeya dadantii

Cyclic diguanylate (c‐di‐GMP) is a second messenger implicated in the regulation of various cellular properties in several bacterial species. However, its function in phytopathogenic bacteria is not yet understood. In this study we investigated a panel of GGDEF/EAL domain proteins which have the potential to regulate c‐di‐GMP levels in the phytopathogen Dickeya dadantii 3937. Two proteins, EcpB (contains GGDEF and EAL domains) and EcpC (contains an EAL domain) were shown to regulate multiple cellular behaviours and virulence gene expression. Deletion of ecpB and/or ecpC enhanced biofilm formation but repressed swimming/swarming motility. In addition, the ecpB and ecpC mutants displayed a significant reduction in pectate lyase production, a virulence factor of this bacterium. Gene expression analysis showed that deletion of ecpB and ecpC significantly reduced expression of the type III secretion system (T3SS) and its virulence effector proteins. Expression of the T3SS genes is regulated by HrpL and possibly RpoN, two alternative sigma factors. In vitro biochemical assays showed that EcpC has phosphodiesterase activity to hydrolyse c‐di‐GMP into linear pGpG. Most of the enterobacterial pathogens encode at least one T3SS, a major virulence factor which functions to subvert host defences. The current study broadens our understanding of the interplay between c‐di‐GMP, RpoN and T3SS and the potential role of c‐di‐GMP in T3SS regulation among a wide range of bacterial pathogens.     

OsSHOC1 and OsPTD1 are essential for crossover formation during rice meiosis

Meiosis is essential for eukaryotic sexual reproduction and plant fertility, and crossovers (COs) are essential for meiosis and the formation of new allelic combinations in gametes. In this study, we report the isolation of a meiotic gene, OsSHOC1, and the identification of its partner, OsPTD1. Osshoc1 was sterile both in male and female gametophytes, and it showed a striking reduction in the number of meiotic COs, indicating that OsSHOC1 was required for normal CO formation. Further investigations showed that OsSHOC1 physically interacted with OsPTD1 and that the latter was also required for normal CO formation and plant fertility. Additionally, the expression profiles of both genes were consistent with their functions. Our results suggest that OsSHOC1 and OsPTD1 are essential for rice fertility and CO formation, possibly by stabilizing the recombinant intermediates during meiosis.     

Rapid identification of an adult plant stripe rust resistance gene in hexaploid wheat by high-throughput SNP array genotyping of pooled extremes

Key message

High-throughput SNP array analysis of pooled extreme phenotypes in a segregating population by KASP marker genotyping permitted rapid, cost-effective location of a stripe rust resistance QTL in wheat.

Abstract

German wheat cultivar “Friedrichswerther” has exhibited high levels of adult plant resistance (APR) to stripe rust in field environments for many years. F_2:3 lines and F₆ recombinant inbred line (RILs) populations derived from a cross between Friedrichswerther and susceptible landrace Mingxian 169 were evaluated in the field in 2013, 2016 and 2017. Illumina 90K iSelect SNP arrays were used to genotype bulked extreme pools and parents; 286 of 1135 polymorphic SNPs were identified on chromosome 6B. Kompetitive Allele-Specific PCR (KASP) markers were used to verify the chromosome region associated with the resistance locus. A linkage map was constructed with 18 KASP-SNP markers, and a major effect QTL was identified within a 1.4 cM interval flanked by KASP markers IWB71602 and IWB55937 in the region 6BL3-0-0.36. The QTL, named QYr.nwafu-6BL, was stable across environments, and explained average 54.4 and 47.8% of the total phenotypic variation in F_2:3 lines and F₆ RILs, respectively. On the basis of marker genotypes, pedigree analysis and relative genetic distance QYr.nwafu-6BL is likely to be a new APR QTL. Combined high-throughput SNP array genotyping of pooled extremes and validation by KASP assays lowers sequencing costs compared to genome-wide association studies with SNP arrays, and more importantly, permits rapid isolation of major effect QTL in hexaploid wheat as well as improving accuracy of mapping in the QTL region. QYr.nwafu-6BL with flanking KASP markers developed and verified in a subset of 236 diverse lines can be used in marker-assisted selection to improve stripe rust resistance in breeding programs.

     

The Saccharomyces cerevisiae PDS1 and RAD9 checkpoint genes control different DNA double-strand break repair pathways

In response to DNA damage, the Saccharomyces cerevisiae securin Pds1 blocks anaphase promotion by inhibiting ESP1-dependent degradation of cohesins. PDS1 is positioned downstream of the MEC1- and RAD9-mediated DNA damage-induced signal transduction pathways. Because cohesins participate in postreplicative repair and the pds1 mutant is radiation sensitive, we identified DNA repair pathways that are PDS1-dependent. We compared the radiation sensitivities and recombination phenotypes of pds1, rad9, rad51 single and double mutants, and found that whereas pds1 rad9 double mutants were synergistically more radiation sensitive than single mutants, pds1 rad51 mutants were not. To determine the role of PDS1 in recombinational repair pathways, we measured spontaneous and DNA damage-associated sister chromatid exchanges (SCEs) after exposure to X rays, UV and methyl methanesulfonate (MMS) and after the initiation of an HO endonuclease-generated double-strand break (DSB). The rates of spontaneous SCE and frequencies of DNA damage-associated SCE were similar in wild type and pds1 strains, but the latter exhibited reduced viability after exposure to DNA damaging agents. To determine whether pds1 mutants were defective in other pathways for DSB repair, we measured both single-strand annealing (SSA) and non-homologous end joining (NHEJ) in pds1 mutants. We found that the pds1 mutant was defective in SSA but efficient at ligating cohesive ends present on a linear plasmid. We therefore suggest that checkpoint genes control different pathways for DSB repair, and PDS1 and RAD9 have different roles in recombinational repair.     

簇毛麦端体6VS的显微切割及其专化DNA序列的克隆和分析

从簇毛麦(Haynaldia villosa (L.) Schur.)组合CA9211/RW15(6D/6V异代换系)幼胚培养SC2后代中,用原位杂交方法鉴定出T240-6为6VS端体异代换系. 以此为材料,采用微细玻璃针切割法及"单管反应"技术体系,对6VS进行切割分离及LA (Linker adaptor)-PCR扩增.扩增带在100～3 000 bp 之间,大部分集中在600～1 500 bp.利用32P标记的簇毛麦基因组为探针进行Southern杂交,证实扩增产物来源于簇毛麦.扩增产物纯化后,连接到pGEM-T载体上,构建了6VS DNA质粒文库.对文库的分析表明,文库大约有17 000个白色克隆;插入片段分布在100～1 500 bp,平均600 bp.点杂交结果表明,37%克隆有中度到强烈的杂交信号,证明含有中度或高度重复序列;63%克隆有较弱的信号或没有信号,证明为单/低拷贝序列克隆.从文库中获得8个簇毛麦特异克隆,对其中两个克隆pHVMK22和 pHVMK134进行了RFLP分析和序列分析,并利用该探针对小麦抗白粉病基因Pm21进行了检测.RFLP 结果表明,两个克隆一个为低拷贝序列克隆(pHVMK22),另一个为高度重复序列克隆,均为簇毛麦专化DNA序列.以pHVMK22为探针对抗、感病小麦(Triticum aestivum L.)品系的Southern杂交发现抗病品系有一条2 kb的特征带, 该探针可能作为检测抗病基因Pm21的探针.