Sequence analysis of the gene encoding H antigen in <Emphasis Type="Italic">Escherichia coli</Emphasis> isolated from food in Morocco

In order to develop other molecular method useful for typing of motile and non motile Escherichia coli strains, a total of 207 strains of E. coli (133 reference strains, 74 food strains) were characterized by analysis of sequences of their amplified flagellin-encoding (fliC) gene products. The collection of reference strains was used for database building of fliC gene sequences. Application of this identification system to 74 E. coli food isolates revealed a reproducible and clear cut classification with very good correlation to results obtained by HhaI restriction of the amplified flagellin gene. The proposed determination of fliC sequences variations should be helpful for epidemiological studies.     

Effect of grain type and processing method on rumen fermentation and milk rumenic acid production

It was hypothesized that differences in starch degradability account for observed differences in rumen vaccenic acid (t11-18:1) and milk rumenic acid (RA) concentrations. To test this hypothesis, starch degradability was varied through grain source and by processing. Eight Holstein cows in mid-lactation were assigned to two 4 × 4 Latin squares with four 21-day periods and four diets: dry rolled barley, ground barley, dry rolled corn and ground corn. Diets contained similar starch content and were supplemented with whole sunflower seed to provide similar total polyunsaturated fatty acid (PUFA) (18:2n-6 + 18:3n-3) contents. Forage/concentrate ratios of all diets were 42 : 58. Rumen, plasma and milk samples were collected in the third week of each period. In situ degradation rates (%/h) for rolled corn, ground corn, rolled barley and ground barley were 5.4, 8.9, 17.0 and 19.4, respectively, for dry matter (DM) and 6.3, 10.8, 25.3 and 43.8, respectively, for starch. DM intakes were greater for corn-based diets (CBD) than for barley-based diets (BBD) with no difference between rolled and ground diets. Daily minimum rumen pH was less (5.2 v. 5.5) and pH duration <5.8 (h/d) was greater (7.4 v. 4.3) for BBD than for CBD. Milk fat content and yield were less for BBD than for CBD with greater values observed for rolling compared with grinding. Variability in milk fat yield was strongly related (R2 = 0.55; P < 0.01) to total starch intake (45%) and milk c9t11-CLA (10%) and none of the t-18:1 isomers or CLA isomers that are typically associated with milk fat depression entered the model. The concentrations (%) of t10-18:1 and t11-18:1 were greater for BBD than for CBD in rumen contents (t10-18:1, 3.5 v. 1.3; t11-18:1, 3.2 v. 1.9), plasma (t10-18:1, 1.2 v. 0.2; t11-18:1, 0.97 v. 0.58) and milk (t10-18:1, 3.8 v. 1.0; t11-18:1, 2.6 v. 1.7) despite greater total PUFA intakes for CBD. Milk RA concentration was greater for BBD than for CBD (1.46 v. 0.89) but was not influenced by the method of grain processing. This study clearly demonstrated that the milk content and profile of t-18:1 and CLA isomers were more strongly influenced by the source of grain starch (barley > corn) than by the method of grain processing indicating that factors inherent in the source of starch were responsible for the observed differences and these factors could not be modified by the processing methods used in this study.     

A molecular mechanism for eflornithine resistance in African trypanosomes

Human African trypanosomiasis, endemic to sub-Saharan Africa, is invariably fatal if untreated. Its causative agent is the protozoan parasite Trypanosoma brucei. Eflornithine is used as a first line treatment for human African trypanosomiasis, but there is a risk that resistance could thwart its use, even when used in combination therapy with nifurtimox. Eflornithine resistant trypanosomes were selected in vitro and subjected to biochemical and genetic analysis. The resistance phenotype was verified in vivo. Here we report the molecular basis of resistance. While the drug's target, ornithine decarboxylase, was unaltered in resistant cells and changes to levels of metabolites in the targeted polyamine pathway were not apparent, the accumulation of eflornithine was shown to be diminished in resistant lines. An amino acid transporter gene, TbAAT6 (Tb927.8.5450), was found to be deleted in two lines independently selected for resistance. Ablating expression of this gene in wildtype cells using RNA interference led to acquisition of resistance while expression of an ectopic copy of the gene introduced into the resistant deletion lines restored sensitivity, confirming the role of TbAAT6 in eflornithine action. Eflornithine resistance is easy to select through loss of a putative amino acid transporter, TbAAT6. The loss of this transporter will be easily identified in the field using a simple PCR test, enabling more appropriate chemotherapy to be administered.     

Development of a cultivation-independent approach for the study of genetic diversity of Sinorhizobium meliloti populations

The development of a species-specific marker for the analysis of the genetic polymorphism of the nitrogen-fixing symbiotic bacterium Sinorhizobium meliloti directly from environmental DNA is reported. The marker is based on terminal-restriction fragment length polymorphism (T-RFLP) methodology targeting specifically the 16S-23S Ribosomal Intergenic Spacer of S. meliloti. Species-specificity and polymorphism of the marker were tested on DNA extracted from soil samples and from a collection of 130 S. meliloti bacterial isolates. These primers and the T-RFLP approach proved useful for the detection and analysis of polymorphism of S. meliloti populations.     

β2-Syntrophin is a Cdk5 substrate that restrains the motility of insulin secretory granules

The molecular basis for the interaction of insulin granules with the cortical cytoskeleton of pancreatic β-cells remains unknown. We have proposed that binding of the granule protein ICA512 to the PDZ domain of β2-syntrophin anchors granules to actin filaments and that the phosphorylation/dephosphorylation of β2-syntrophin regulates this association. Here we tested this hypothesis by analyzing INS-1 cells expressing GFP-β2-syntrophin through the combined use of biochemical approaches, imaging studies by confocal and total internal reflection fluorescence microscopy as well as electron microscopy. Our results support the notion that β2-syntrophin restrains the mobility of cortical granules in insulinoma INS-1 cells, thereby reducing insulin secretion and increasing insulin stores in resting cells, while increasing insulin release upon stimulation. Using mass spectrometry, in vitro phosphorylation assays and β2-syntrophin phosphomutants we found that phosphorylation of β2-syntrophin on S75 near the PDZ domain decreases its binding to ICA512 and correlates with increased granule motility, while phosphorylation of S90 has opposite effects. We further show that Cdk5, which regulates insulin secretion, phosphorylates S75. These findings provide mechanistic insight into how stimulation displaces insulin granules from cortical actin, thus promoting their motility and exocytosis.     

Molecular Characterization of Leishmania Species Isolated from Cutaneous Leishmaniasis in Yemen

Background

Cutaneous leishmaniasis (CL) is a neglected tropical disease endemic in the tropics and subtropics with a global yearly incidence of 1.5 million. Although CL is the most common form of leishmaniasis, which is responsible for 60% of DALYs lost due to tropical-cluster diseases prevalent in Yemen, available information is very limited.

Methodology/Principal Findings

This study was conducted to determine the molecular characterization of Leishmania species isolated from human cutaneous lesions in Yemen. Dermal scrapes were collected and examined for Leishmania amastigotes using the Giemsa staining technique. Amplification of the ribosomal internal transcribed spacer 1(ITS-1) gene was carried out using nested PCR and subsequent sequencing. The sequences from Leishmania isolates were subjected to phylogenetic analysis using the neighbor-joining and maximum parsimony methods. The trees identified Leishmania tropica from 16 isolates which were represented by two sequence types.

Conclusions/Significance

The predominance of the anthroponotic species (i.e. L. tropica) indicates the probability of anthroponotic transmission of cutaneous leishmaniasis in Yemen. These findings will help public health authorities to build an effective control strategy taking into consideration person–to-person transmission as the main dynamic of transmission of CL.     

FGFR3 mutational status and protein expression in patients with bladder cancer in a Jordanian population

Bladder cancer accounts for nearly 5% of all newly diagnosed cancers in Jordan, with a much higher frequency in males. Recent studies have shown that activating mutations in FGFR3 are the most common findings in non-invasive low grade bladder tumors. In this study, we, retrospectively, investigated a cohort of 121 bladder cancer patients with various grades and stages of the tumor for molecular changes in FGFR3. Overexpression of FGFR3 was observed in 49%, 34%, 15%, and 2% of pTa, pT1, pT2, and pT3 cases, respectively. Further, FGFR3 expression was positive in 45%, 26%, and 30% of G1, G2 and G3 cases, respectively. Mutational analysis of exons 7, 10 and 15 of FGFR3 identified four previously reported mutations, namely R248C (n = 4; 10%), S249C (n = 23; 59%), Y375C (n = 7; 18%), G382R (n = 4; 10%), and one novel mutation, G382E (n = 1; 3%). Our results indicate that both mutations and overexpression of FGFR3 are correlated together, and are more prevalent in early stage (pTa and pT1) and low grade (G1 and G2) bladder tumors. Survival analysis showed no contribution of changes in FGFR3 on the patient's survival. Multivariate Cox proportional hazards model analysis of overall survival for the following variables: age, gender, stage and grade of tumor, and FGFR3 (expression and mutation) revealed that age, stage and grade of tumor are independent predictors of overall survival in patients with bladder cancer. Our work is the first to address the molecular status of FGFR3 in Jordanian patients with bladder cancer, and provides further support for FGFR3 as a key player in the initiation of bladder tumors.     

Production of tyrosol by Candida albicans biofilms and its role in quorum sensing and biofilm development

Tyrosol and farnesol are quorum-sensing molecules produced by Candida albicans which accelerate and block, respectively, the morphological transition from yeasts to hyphae. In this study, we have investigated the secretion of tyrosol by C. albicans and explored its likely role in biofilm development. Both planktonic (suspended) cells and biofilms of four C. albicans strains, including three mutants with defined defects in the Efg 1 and Cph 1 morphogenetic signaling pathways, synthesized extracellular tyrosol during growth at 37°C. There was a correlation between tyrosol production and biomass for both cell types. However, biofilm cells secreted at least 50% more tyrosol than did planktonic cells when tyrosol production was related to cell dry weight. The addition of exogenous farnesol to a wild-type strain inhibited biofilm formation by up to 33% after 48 h. Exogenous tyrosol appeared to have no effect, but scanning electron microscopy revealed that tyrosol stimulated hypha production during the early stages (1 to 6 h) of biofilm development. Experiments involving the simultaneous addition of tyrosol and farnesol at different concentrations suggested that the action of farnesol was dominant, and 48-h biofilms formed in the presence of both compounds consisted almost entirely of yeast cells. When biofilm supernatants were tested for their abilities to inhibit or enhance germ tube formation by planktonic cells, the results indicated that tyrosol activity exceeds that of farnesol after 14 h, but not after 24 h, and that farnesol activity increases significantly during the later stages (48 to 72 h) of biofilm development. Overall, our results support the conclusion that tyrosol acts as a quorum-sensing molecule for biofilms as well as for planktonic cells and that its action is most significant during the early and intermediate stages of biofilm formation.