Colletotrichum gloeosporioides s.l. associated with Theobroma cacao and other plants in Panama: multilocus phylogenies distinguish host-associated pathogens from asymptomatic endophytes

Colletotrichum interacts with numerous plant species overtly as symptomatic pathogens and cryptically as asymptomatic endophytes. It is not known whether these contrasting ecological modes are optional strategies expressed by individual Colletotrichum species or whether a species' ecology is explicitly pathogenic or endophytic. We explored this question by inferring relationships among 77 C. gloeosporioides s.l. strains isolated from asymptomatic leaves and from anthracnose lesions on leaves and fruits of Theobroma cacao (cacao) and other plants from Panamá. ITS and 5'-tef1 were used to assess diversity and to delineate operational taxonomic units for multilocus phylogenetic analysis. The ITS and 5'-tef1 screens concordantly resolved four strongly supported lineages, clades A-D: Clade A includes the ex type of C. gloeosporioides, clade B includes the ex type ITS sequence of C. boninense, and clades C and D are unidentified. The ITS yielded limited resolution and support within all clades, in particular the C. gloeosporioides clade (A), the focal lineage dealt with in this study. In contrast the 5'-tef1 screen differentiated nine distinctive haplotype subgroups within the C. gloeosporioides clade that were concordant with phylogenetic terminals resolved in a five-locus nuclear phylogeny. Among these were two phylogenetic species associated with symptomatic infections specific to either cacao or mango and five phylogenetic species isolated principally as asymptomatic infections from cacao and other plant hosts. We formally describe two new species, C. tropicale and C. ignotum, that are frequent asymptomatic associates of cacao and other Neotropical plant species, and epitypify C. theobromicola, which is associated with foliar and fruit anthracnose lesions of cacao. Asymptomatic Colletotrichum strains isolated from cacao plants grown in China included six distinct C. gloeosporioides clade taxa, only one of which is known to occur in the Neotropics.     

A Randomised Trial Evaluating the Safety and Immunogenicity of the Novel Single Oral Dose Typhoid Vaccine M01ZH09 in Healthy Vietnamese Children

Background

The emergence of drug resistant typhoid fever is a major public health problem, especially in Asia. An oral single dose typhoid vaccine would have major advantages. M01ZH09 is a live oral single dose candidate typhoid vaccine containing Salmonella enterica serovar Typhi (Ty2 aroC ⁻ ssaV ⁻) ZH9 with two independently attenuating deletions. Studies in healthy adults demonstrated immunogenicity and an acceptable safety profile.

Objectives

We conducted a randomised placebo controlled, single-blind trial to evaluate the safety and immunogenicity of M01ZH09 in healthy Vietnamese children aged 5 to 14 years.

Methods

Subjects were randomly assigned to receive either a nominal dose of 5×10⁹ CFU of M01ZH09 or placebo and were followed up for 28 days. The primary safety outcome was the proportion of subjects with any adverse event attributed to M01ZH09. The primary immunogenicity endpoint was the proportion of subjects who showed a positive immune response to M01ZH09 in the Salmonella Typhi lipopolysaccharide (LPS) specific serum IgA and IgG ELISA.

Principal Findings

One hundred and fifty-one children were enrolled, 101 subjects received M01ZH09 and 50 subjects received placebo. An intention to treat analysis was conducted. There were no serious adverse events and no bacteraemias. In the M01ZH09 group, 26 (26%; 95% CI, 18–5%) of 101 subjects experienced adverse events compared to 11 (22%; 95% CI, 12–36%) of 50 subjects in the placebo group (odds ratio (OR) [95%CI]  = 1.23 [0.550–2.747]; p = 0.691). Faecal shedding of S. Typhi (Ty2 aroC ⁻ ssaV ⁻) ZH9 was detected in 51 (51%; 95% CI, 41–61%) of 100 M01ZH09 subjects. No shedding was detected beyond day 3. A positive immune response, defined as 70% increase (1.7 fold change) in LPS specific serum IgG (day 14 or 28) and/or 50% increase (1.5 fold change) in LPS specific serum IgA (day 7 or 14) from baseline was detected in 98 (97%; 95% CI, 92–99%) of 101 M01ZH09 recipients and 8 (16%; 95% CI, 7–29%) of 50 placebo recipients. Twenty-eight (100%; 95% CI, 88–100%) of 28 vaccine recipients who were evaluated in the LPS specific IgA ELISPOT assay showed a positive response compared to none of the 14 placebo recipients tested.

Conclusions

This was the first phase II trial of a novel oral candidate typhoid vaccine in children in an endemic country. M01ZH09 had an appropriate safety profile and was immunogenic in children.

Trial Registration

Controlled-trials.comISRCTN91111837     

A genetic map of potato (Solanum tuberosum) integrating molecular markers,including transposons,and classical markers

A genetic map of potato (Solanum tuberosum L.) integrating molecular markers with morphological and isozyme markers was constructed using a backcross population of 67 diploid potato plants. A general method for map construction is described that differs from previous methods employed in potato and other outbreeding plants. First, separate maps for the female and male parents were constructed. The female map contained 132 markers, whereas the male map contained 138 markers. Second, on the basis of the markers in common the two integrated parental maps were combined into one with the computer programme JoinMap. This combined map consisted of 175 molecular markers, 10 morphological markers and 8 isozyme markers. Ninety-two of the molecular markers were derived from DNA sequences flanking either T-DNA inserts in potato or reintegrated maize transposable elements originating from these T-DNA constructs. Clusters of distorted segregation were found on chromosomes 1,2,8 and 11 for the male parent and chromosome 5 for both parents. The total length of the combined map is 1120 cM.     

Rescue of DeltaF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in cftr, a gene encoding a PKA-regulated Cl(-) channel. The most common mutation results in a deletion of phenylalanine at position 508 (DeltaF508-CFTR) that impairs protein folding, trafficking, and channel gating in epithelial cells. In the airway, these defects alter salt and fluid transport, leading to chronic infection, inflammation, and loss of lung function. There are no drugs that specifically target mutant CFTR, and optimal treatment of CF may require repair of both the folding and gating defects. Here, we describe two classes of novel, potent small molecules identified from screening compound libraries that restore the function of DeltaF508-CFTR in both recombinant cells and cultures of human bronchial epithelia isolated from CF patients. The first class partially corrects the trafficking defect by facilitating exit from the endoplasmic reticulum and restores DeltaF508-CFTR-mediated Cl(-) transport to more than 10% of that observed in non-CF human bronchial epithelial cultures, a level expected to result in a clinical benefit in CF patients. The second class of compounds potentiates cAMP-mediated gating of DeltaF508-CFTR and achieves single-channel activity similar to wild-type CFTR. The CFTR-activating effects of the two mechanisms are additive and support the rationale of a drug discovery strategy based on rescue of the basic genetic defect responsible for CF.     

Endogenous biotin‐binding proteins: an overlooked factor causing false positives in streptavidin‐based protein detection

Biotinylation is widely used in DNA, RNA and protein probing assays as this molecule has generally no impact on the biological activity of its substrate. During the streptavidin‐based detection of glycoproteins in Lactobacillus rhamnosus GG with biotinylated lectin probes, a strong positive band of approximately 125 kDa was observed, present in different cellular fractions. This potential glycoprotein reacted heavily with concanavalin A (ConA), a lectin that specifically binds glucose and mannose residues. Surprisingly, this protein of 125 kDa could not be purified using a ConA affinity column. Edman degradation of the protein, isolated via cation and anion exchange chromatography, lead to the identification of the band as pyruvate carboxylase, an enzyme of 125 kDa that binds biotin as a cofactor. Detection using only the streptavidin conjugate resulted in more false positive signals of proteins, also in extracellular fractions, indicating biotin‐associated proteins. Indeed, biotin is a known cofactor of numerous carboxylases. The potential occurence of false positive bands with biotinylated protein probes should thus be considered when using streptavidin‐based detection, e.g. by developing a blot using only the streptavidin conjugate. To circumvent these false positives, alternative approaches like detection based on digoxigenin labelling can also be used.     

Dosage Regulation of the Active X Chromosome in Human Triploid Cells

In mammals, dosage compensation is achieved by doubling expression of X-linked genes in both sexes, together with X inactivation in females. Up-regulation of the active X chromosome may be controlled by DNA sequence–based and/or epigenetic mechanisms that double the X output potentially in response to autosomal factor(s). To determine whether X expression is adjusted depending on ploidy, we used expression arrays to compare X-linked and autosomal gene expression in human triploid cells. While the average X:autosome expression ratio was about 1 in normal diploid cells, this ratio was lower (0.81–0.84) in triploid cells with one active X and higher (1.32–1.4) in triploid cells with two active X''s. Thus, overall X-linked gene expression in triploid cells does not strictly respond to an autosomal factor, nor is it adjusted to achieve a perfect balance. The unbalanced X:autosome expression ratios that we observed could contribute to the abnormal phenotypes associated with triploidy. Absolute autosomal expression levels per gene copy were similar in triploid versus diploid cells, indicating no apparent global effect on autosomal expression. In triploid cells with two active X''s our data support a basic doubling of X-linked gene expression. However, in triploid cells with a single active X, X-linked gene expression is adjusted upward presumably by an epigenetic mechanism that senses the ratio between the number of active X chromosomes and autosomal sets. Such a mechanism may act on a subset of genes whose expression dosage in relation to autosomal expression may be critical. Indeed, we found that there was a range of individual X-linked gene expression in relation to ploidy and that a small subset (∼7%) of genes had expression levels apparently proportional to the number of autosomal sets.     

Evaluation of a mathematical model structure describing the effect of (gel) structure on the growth of Listeria innocua, Lactococcus lactis and Salmonella Typhimurium

Aims:  To evaluate a novel secondary model structure ( Int J Food Microbiol 2008; 128: 67) that describes the effect of medium structure on the maximum specific growth rate ( μ _max) of Salmonella Typhimurium on the growth of S. Typhimurium, Listeria innocua , Lactococcus lactis and Listeria monocytogenes .
Methods and Results:  In the present study, the novel secondary model is validated for S . Typhimurium in more realistic media, namely, pasteurized milk and a cheese mimicking medium. The predictions were accurate. Next, the secondary model structure was evaluated in a two step and a global regression procedure on literature data. On the one hand, the growth of two other micro-organisms, namely L. innocua and L. lactis , in monoculture for varying gelatine concentrations was tested and on the other hand the growth rate of L. monocytogenes was fitted in a broth of which the viscosity was altered with polyvinylpyrrolidone. The model was able to describe the effect of increasing gelatine concentration or viscosity accurately.
Conclusions:  The proposed secondary model structure is able to describe the effect of gelatine concentration on the μ _max of the micro-organisms tested in this study.
Significance and Impact of the Study:  In predictive microbiology, much attention has been paid to the effect of food structure on the μ _max of bacteria. However, to the authors' knowledge, a lack of secondary models still exists to describe this effect. Although the proposed model is empirical, the model parameters have clear biological meaning. The predictive power of the model to describe the effect of food structure is clearly illustrated.     

Comprehensive proteomic analysis of human cervical-vaginal fluid using colposcopy samples

Background  

Cervical-vaginal fluid (CVF) plays an important role in the prevention of gynecological infections, although little is known about the contribution of CVF proteins to the immunity of the lower female genital tract. In order to analyze the protein composition of human CVF, we used CVF samples that are routinely collected during colposcopy, but are usually discarded. Since these samples are available in large quantities we aimed to analyze their usefulness for proteomics experiments. The samples were analyzed using different prefractionation techniques (ultrafiltration and C₄(RP)-LC protein separation) followed by C₁₈(RP)-LC peptide separation and identification by MALDI-TOF-TOF mass spectrometry. To determine the reproducibility of this proteomics platform we analyzed three technical replicates. Using spectral counting, protein abundances were estimated in a semiquantitative way. We also compared the results obtained in this study with those from previous studies derived from patients with different physiological conditions in order to determine an overlapping protein set.