Identification of novel Leishmania major antigens that elicit IgG2a response in resistant and susceptible mice

Experimental murine models with high, intermediate and low levels of genetically based susceptibility to Leishmania major infection reproduce almost entire spectrum of clinical manifestations of the human disease. There are increasing non-comparative studies on immune responses against isolated antigens of L. major in different murine strains. The aim of the present study was to find out whether there is an antigen that can induce protective immune response in resistant and susceptible murine strains. To do that, crude antigenic extract of procyclic and metacyclic promastigotes of L. major was prepared and subjected to SDS-PAGE electrophoresis. Western-blotting was used to search for antigen(s) capable of raising high antibody level of IgG2a versus IgG1 in the sera of both infected resistant and susceptible strains. Two novel antigens from metacyclic promastigotes of L. major (140 and 152 kDa) were potentially able to induce specific dominant IgG2a responses in BALB/c and C57BL/6 mice. The 2 antigens also reacted with IgG antibody of cutaneous leishmaniasis patients. We confirm that 140 and 152 kDa proteins of L. major promastigotes are inducing IgG production in mice and humans.     

Multiple cis-regulatory elements regulate distinct and complex patterns of developmental and wound-induced expression of Arabidopsis thaliana 4CL gene family members

Lignin is an important biopolymer that is deposited in secondary cell walls of plant cells (e.g., tracheary elements) and in response to stresses such as wounding. Biosynthesis of lignin monomers occurs via the phenylpropanoid pathway, in which the enzyme 4-coumarate:CoA ligase (4CL) plays a key role by catalyzing the formation of hydroxycinnamoyl-CoA esters, subsequently reduced to the corresponding monolignols (hydroxycinnamoyl alcohols). 4CL is encoded by a family of four genes in Arabidopsis thaliana (At4CL1-At4CL4), which are developmentally regulated and co-expressed with other phenylpropanoid genes. We investigated in detail the wound-induced expression of At4CL1-At4CL4, and found that At4CL1 and At4CL2 mRNA accumulation follows biphasic kinetics over a period of 72 h, while At4CL4 expression is rapidly activated for a period of at least 12 h before declining. In order to localize cis-regulatory elements involved in the developmental and wound-induced regulation of the At4CL gene family members, At4CL promoter-beta-glucuronidase (GUS) reporter gene fusions were constructed and transferred into Arabidopsis plants. Analysis of these plants revealed that the promoter fragments direct discrete and distinct patterns of expression, some of which did not recapitulate expected patterns of wound-induced expression. The locations of regulatory elements associated with the At4CL2 gene were investigated in detail using a series of transgenic Arabidopsis plants containing promoter fragments and parts of the transcribed region of the gene fused to GUS. Positive and negative regulatory elements effective in modulating developmental expression or wound responsiveness of the gene were located both in the promoter and transcribed regions of the At4CL2 gene.An erratum to this article can be found at     

Imaging features that discriminate between foci induced by high- and low-LET radiation in human fibroblasts

In this study, we investigated the formation of radiation-induced foci in normal human fibroblasts exposed to X rays or 130 keV/mum nitrogen ions using antibodies to phosphorylated protein kinase ataxia telangiectasia mutated (ATMp) and histone H2AX (gamma-H2AX). High-content automatic image analysis was used to quantify the immunofluorescence of radiation-induced foci. The size of radiation-induced foci increased for both proteins over a 2-h period after nitrogen-ion irradiation, while the size of radiation-induced foci did not change after exposure to low-LET radiation. The number of radiation-induced ATMp foci showed a more rapid rise and greater frequency after X-ray exposure and was resolved more rapidly such that the frequency of radiation-induced foci decreased by 90% compared to 60% after exposure to high-LET radiation 2 h after 30 cGy. In contrast, the kinetics of radiation-induced gamma-H2AX focus formation was similar for high- and low-LET radiation in that it reached a plateau early and remained constant for up to 2 h. High-resolution 3D images of radiation-induced gamma-H2AX foci and dosimetry computation suggest that multiple double-strand breaks from nitrogen ions are encompassed within large nuclear domains of 4.4 Mbp. Our work shows that the size and frequency of radiation-induced foci vary as a function of radiation quality, dose, time and protein target. Thus, even though double-strand breaks and radiation-induced foci are correlated, the dynamic nature of both contradicts their accepted equivalence for low doses of different radiation qualities.     

Studies on the effect of system retention time on bacterial populations colonizing a three-stage continuous culture model of the human large gut using FISH techniques

Fluorescence in situ hybridization was used to quantitate bacteria growing in a three-stage continuous culture system inoculated with human faeces, operated at two system retention times (60 and 20 h). Twenty-three different 16S rRNA gene oligonucleotide probes of varying specificities were used to detect bacteria. Organisms belonging to genera Bacteroides and Bifidobacterium, together with the Eubacterium rectale/Clostridium coccoides group, the Atopobium, Faecalibacterium prausnitzii and Eubacterium cylindroides groups, as well as the segmented filamentous bacteria, the Roseburia intestinalis group and lactic acid bacteria, were all present in high numbers in the continuous culture system. Other groups and species such as Ruminococci and Enterobacteria also persisted in the model, though not always at levels that allowed reliable quantitation. Some organisms such as Streptococci and Corynebacteria, present in the faecal inoculum, did not colonize the system. Other probes specific for Eubacterium lentum and for members of the genus Desulfovibrio did not detect these organisms at any time. Short chain fatty acid production was always highest in vessel I of the continuous culture system, however, a marked increase in acetate formation and a reduction in butyrate production occurred when system retention time was reduced to 20 h, which correlated with reductions in the numbers of butyrate-producing Roseburia.     

PCR Analysis of the Tri13 gene to Determine the Genetic Potential of Fusarium graminearum Isolates from Iran to Produce Nivalenol and Deoxynivalenol

Fusarium graminearum trichothecene producing isolates can be broadly divided into two chemotypes based on the production of the 8- ketotrichothecenes deoxynivalenol (DON) and nivalenol (NIV). Functional Tri13 gene required for the production of NIV and 4- acetyl NIV, whereas in the isolates producing DON and its acetylated derivates, this gene is nonfunctional. In this study, a total of 57 isolates from different fields of Mazandaran province, Iran were identified as F. graminearum using classical methods and species specific primers. In order to assess the potential of isolates to produce NIV or DON, we used PCR to determine whether isolates carried a functional or nonfunctional Tri13 gene. Out of the 57 tested F. graminearum isolates with Tri13 PCR assays, 46 yielded an amplicon similar to the size predicted for nivalenol production, while 11 yielded an amplicon similar to the size predicted for deoxynivalenol production. From regions where more than one F. graminearum isolate was obtained, isolates were not exclusively of a single chemotype. It seems that genetic diversity among the isolates has relation with geographical region and wheat cultivar. The assay can provide information about the distribution of Tri13 haplotype that can be used in tracing of trichothecene contaminated samples.     

Distribution and diversity of Dipteran-specific cry and cyt genes in native Bacillus thuringiensis strains obtained from different ecosystems of Iran

One hundred and twenty-eight Bacillus thuringiensis isolates from fields of different ecological regions of Iran were collected to study the distribution and diversity of Dipteran-specific cry and cyt genes. The percentage of samples with Bt showed significant differences between different regions and also between different fields. The most Bt frequency was observed in the soil samples collected from Caspianic zone (7%) and soils of cotton (17%). Characterization of isolates was based on morphological characteristics of crystals, plasmid profiles and protein band patterns as well as PCR analysis using general and specific primers for 22 different cry and cyt genes encoding proteins active against mosquitoes. Thirty-eight different cry gene profiles were detected in this collection. Several of them were found to be different from all previously published profiles and none of the previous researches reported these numbers of profiles. Strains containing cry2-type genes were the most abundant and represent 57.1% of the isolates. Strains harboring cry24 and cry10 genes were also highly abundant (38.7 and 32.8%, respectively). cry11, cry4, cry17, cry19, cry21, cry29, cyt1, and cry9 genes were less abundant, found in 25.7, 14.3, 11.4, 1.4, 4.3, 1.4, and 10% of the strains, respectively. Among the cry2 gene containing isolates, 37.5% strains harbored cry2Aa, 55% cry2Ab, 2.5% cry2Ac, and 5% other or novel cry2-type genes. Among the cry4 gene containing isolates, 0% strains harbored cry4A, 60% cry4B, and 40% cry4C, cry4D or novel cry4 type genes. Finally, based on crystal morphology, protein patterns and PCR, 21 strains were selected as potentially high Dipteran-active for bioassays. Also our results showed that some of the isolates may harbor minimum a putative novel cry gene.     

Mapping and identification of a potential candidate gene for a novel maturity locus, <Emphasis Type="Italic">E10</Emphasis>, in soybean

Key message

E10 is a new maturity locus in soybean and FT4 is the predicted/potential functional gene underlying the locus.

Abstract

Flowering and maturity time traits play crucial roles in economic soybean production. Early maturity is critical for north and west expansion of soybean in Canada. To date, 11 genes/loci have been identified which control time to flowering and maturity; however, the molecular bases of almost half of them are not yet clear. We have identified a new maturity locus called “E10” located at the end of chromosome Gm08. The gene symbol E10e10 has been approved by the Soybean Genetics Committee. The e10e10 genotype results in 5–10 days earlier maturity than E10E10. A set of presumed E10E10 and e10e10 genotypes was used to identify contrasting SSR and SNP haplotypes. These haplotypes, and their association with maturity, were maintained through five backcross generations. A functional genomics approach using a predicted protein–protein interaction (PPI) approach (Protein–protein Interaction Prediction Engine, PIPE) was used to investigate approximately 75 genes located in the genomic region that SSR and SNP analyses identified as the location of the E10 locus. The PPI analysis identified FT4 as the most likely candidate gene underlying the E10 locus. Sequence analysis of the two FT4 alleles identified three SNPs, in the 5′UTR, 3′UTR and fourth exon in the coding region, which result in differential mRNA structures. Allele-specific markers were developed for this locus and are available for soybean breeders to efficiently develop earlier maturing cultivars using molecular marker assisted breeding.

     

Towards a unified paradigm for sequence‐based identification of fungi

The nuclear ribosomal internal transcribed spacer (ITS) region is the formal fungal barcode and in most cases the marker of choice for the exploration of fungal diversity in environmental samples. Two problems are particularly acute in the pursuit of satisfactory taxonomic assignment of newly generated ITS sequences: (i) the lack of an inclusive, reliable public reference data set and (ii) the lack of means to refer to fungal species, for which no Latin name is available in a standardized stable way. Here, we report on progress in these regards through further development of the UNITE database ( http://unite.ut.ee ) for molecular identification of fungi. All fungal species represented by at least two ITS sequences in the international nucleotide sequence databases are now given a unique, stable name of the accession number type (e.g. Hymenoscyphus pseudoalbidus|GU586904|SH133781.05FU), and their taxonomic and ecological annotations were corrected as far as possible through a distributed, third‐party annotation effort. We introduce the term ‘species hypothesis’ (SH) for the taxa discovered in clustering on different similarity thresholds (97–99%). An automatically or manually designated sequence is chosen to represent each such SH. These reference sequences are released ( http://unite.ut.ee/repository.php ) for use by the scientific community in, for example, local sequence similarity searches and in the QIIME pipeline. The system and the data will be updated automatically as the number of public fungal ITS sequences grows. We invite everybody in the position to improve the annotation or metadata associated with their particular fungal lineages of expertise to do so through the new Web‐based sequence management system in UNITE.