Seroprevalence of HBV and HCV markers among intravenous drug abusers and subjects affected by chronic active hepatitis

In order to investigate the seroprevalence of HBV and HCV in neapolitan area, we studied blood specimens of 180 IVDAs, 115 CAH patients and 72 healthy subjects using hepatitis B core (anti-HBs) and hepatitis C antibody (anti-HCV). High frequency of anti-HBs (80.9%) and strong association with an unexpected seroprevalence of anti-HCV (67%) was found in CAH patients. Our study does not explain these results, but suggest either a possible inference between both viruses or that serum from CAH subjects contains a component that gives false positivity.     

Quick and accurate detection of Sclerotinia sclerotiorum and Phomopsis spp. in soybean seeds using qPCR and seed‐soaking method

Seed‐borne pathogenic fungi can cause serious damage to soybean crops by reducing the germination, vigour and emergence of the seeds. Special attention should be paid to pathogen detection in seeds to prevent its introduction in disease‐free areas. Considering the importance of rapid and successful diagnosis of seed‐borne pathogenic fungi in soybeans, this study evaluated a method to detect Sclerotinia sclerotiorum and Phomopsis spp. in seeds using quantitative polymerase chain reaction (qPCR). Naturally infested samples were subjected to detection using qPCR and blotter test, and the findings were compared. Using soybean seeds soaked in water, both pathogens were detected at an infestation level up a 0.0625% (one infected seed out of 1,599 healthy seeds) by qPCR. This technique allowed the detection of 300 fg of S. sclerotiorum and 30 fg of Phomopsis spp. DNA in the seed samples. Phomopsis spp. was detected in 40.7% of the evaluated seed batches (81 batches) and S. sclerotiorum was detected in 32.1% of the evaluated batches, although most of the seeds had low infestation levels. It was up to 28.5 times more efficient to use qPCR rather than blotter test to detect pathogens with a low incidence of occurrence in soybean seeds. If routinely used to test healthy seeds, qPCR would contribute to reducing soybean losses due to diseases as well as decreasing the costs required to control those diseases.     

Distinctively variable sequence-based nuclear DNA markers for multilocus phylogeography of the soybean- and rice-infecting fungal pathogen Rhizoctonia solani AG-1 IA

A series of multilocus sequence-based nuclear DNA markers was developed to infer the phylogeographical history of the Basidiomycetous fungal pathogen Rhizoctonia solani AG-1 IA infecting rice and soybean worldwide. The strategy was based on sequencing of cloned genomic DNA fragments (previously used as RFLP probes) and subsequent screening of fungal isolates to detect single nucleotide polymorphisms (SNPs). Ten primer pairs were designed based on these sequences, which resulted in PCR amplification of 200-320 bp size products and polymorphic sequences in all markers analyzed. By direct sequencing we identified both homokaryon and heterokaryon (i.e. dikaryon) isolates at each marker. Cloning the PCR products effectively estimated the allelic phase from heterokaryotic isolates. Information content varied among markers from 0.5 to 5.9 mutations per 100 bp. Thus, the former RFLP codominant probes were successfully converted into six distinctively variable sequence-based nuclear DNA markers. Rather than discarding low polymorphism loci, the combination of these distinctively variable anonymous nuclear markers would constitute an asset for the unbiased estimate of the phylogeographical parameters such as population sizes and divergent times, providing a more reliable species history that shaped the current population structure of R. solani AG-1 IA.     

Investigation of alginate gel inhomogeneity in simulated gastro-intestinal conditions using magnetic resonance imaging and transmission electron microscopy

The inhomogeneity of alginate gel beads prepared by an external diffusion method has been characterised using spatially resolved nuclear magnetic resonance or “magnetic resonance imaging” (MRI) and transmission electron microscopy (TEM). The beads exhibited various degrees of inhomogeneity although reducing the length of exposure to the gelling bath and the presence of non-gelling ions decreased gel inhomogeneity. In order to gain information regarding the gastro-intestinal functionality of these beads for in vivo applications, they were exposed to simulated gastro-intestinal conditions. The increased polymer concentration at the edge of the beads was shown to persist throughout our gastro-intestinal model despite the centre of the bead becoming progressively more porous in nature. The porosity of the alginate gels has been quantified by image analysis of transmission electron micrographs and shown to depend on both location within the bead and gastro-intestinal conditions. We suggest that such changes in porosity of these alginate beads during simulated gastro-intestinal conditions may make these an attractive option for controlled delivery applications in vivo.     

A kinetic and clonal analysis of heterogeneity in K562 cells

Expression of markers of differentiation was measured in a clone of the continuous cell line K562, derived originally from the cells of a patient with leukemia. Three of the markers were lineage specific, R18 for erythropoiesis and 80H.5 and My-1 for granulopoiesis. The fourth marker was the self-renewal capacity of clonogenic cells. The markers were measured as a function of time in pooled colonies from day 2 to day 12, and at a point of time in individual colonies. Evidence of an orderly pattern of marker appearance and disappearance was not seen. Rather, their expression appeared to occur at random during growth.     

SeSaMe:Metagenome Sequence Classification of Arbuscular Mycorrhizal Fungi-associated Microorganisms

Arbuscular mycorrhizal fungi (AMF) are plant root symbionts that play key roles in plant growth and soil fertility. They are obligate biotrophic fungi that form coenocytic multinucleated hyphae and spores. Numerous studies have shown that diverse microorganisms live on the surface of and inside their mycelia, resulting in a metagenome when whole-genome sequencing (WGS) data are obtained from sequencing AMF cultivated in vivo. The metagenome contains not only the AMF sequences, but also those from associated microorganisms. In this study, we introduce a novel bioinformatics program, Spore-associated Symbiotic Microbes (SeSaMe), designed for taxonomic classification of short sequences obtained by next-generation DNA sequencing. A genus-specific usage bias database was created based on amino acid usage and codon usage of a three consecutive codon DNA 9-mer encoding an amino acid trimer in a protein secondary structure. The program distinguishes between coding sequence (CDS) and non-CDS, and classifies a query sequence into a genus group out of 54 genera used as reference. The mean percentages of correct predictions of the CDS and the non-CDS test sets at the genus level were 71% and 50% for bacteria, 68% and 73% for fungi (excluding AMF), and 49% and 72% for AMF (Rhizophagus irregularis), respectively. SeSaMe provides not only a means for estimating taxonomic diversity and abundance but also the gene reservoir of the reference taxonomic groups associated with AMF. Therefore, it enables users to study the symbiotic roles of associated microorganisms. It can also be applicable to other microorganisms as well as soil metagenomes. SeSaMe is freely available at www.fungalsesame.org.     

Eight variable microsatellite loci for a Neotropical tree, Jacaranda copaia (Aubl.) D.Don (Bignoniaceae)

The Dendrogene Project (Genetic Conservation within Managed Forests in Amazonia) aims to understand the genetic and ecological processes that underpin tree species survival and in particular their response to forest management regimes. As part of the project, we developed eight microsatellite markers for Jacaranda copaia to be used for genetic structure, gene flow and reproductive biology studies. Polymorphism was evaluated using 96 adult trees from the Tapajos National Forest in the Eastern Brazilian Amazon. An average of 22 alleles per locus were detected, with expected heterozygosity ranging from 0.731 to 0.94.     

Resistance to Trypanosoma cruzi infection in relation to the timing of IgG humoral response

The Biozzi "high" (BH) and "low" (BL) responder mice (Selection III) differed in their susceptibility to Trypanosoma cruzi. The BH strain responded quickly to the infection, similar to the reaction of (CBA X C57B1/10)F1 mice but in contrast to the susceptible BL strain. We suggest that the IgG response mounted by the host during the prepatent period of the infection is crucial to the outcome of the infection.     

Polarity Effects in the Hisg Gene of Salmonella Require a Site within the Coding Sequence

A single site in the middle of the coding sequence of the hisG gene of Salmonella is required for most of the polar effect of mutations in this gene. Nonsense and insertion mutations mapping upstream of this point in the hisG gene all have strong polar effects on expression of downstream genes in the operon; mutations mapping promotor distal to this site have little or no polar effect. Two previously known hisG mutations, mapping in the region of the polarity site, abolish the polarity effect of insertion mutations mapping upstream of this region. New polarity site mutations have been selected which have lost the polar effect of upstream nonsense mutations. All mutations abolishing the function of the site are small deletions; three are identical, 28-bp deletions which have arisen independently. A fourth mutation is a deletion of 16 base pairs internal to the larger deletion. Several point mutations within this 16-bp region have no effect on the function of the polarity site. We believe that a small number of polarity sites of this type are responsible for polarity in all genes. The site in the hisG gene is more easily detected than most because it appears to be the only such site in the hisG gene and because it maps in the center of the coding sequence.