GST, CYP and PON1 polymorphisms in farmers attributing ill health to organophosphate-containing sheep dip

Previously we reported that in sheep dippers exposed to organophosphates the frequency of paraoxonase (PON1) polymorphisms differed between those with or without self-reported ill health. We have now examined whether polymorphisms in other genes involved in xenobiotic metabolism alter disease risk in this population. There were elevated but non-significant risks associated with the CYP2D6 WT genotype (odds ratio (OR) 1.47, 95% CI 0.83-2.60), or a GSTP1*B or *C allele (OR 1.37, 95% CI 0.88-2.01) or being GSTM1*2/GSTT1*2 homozygous (OR 1.61, 95% CI 0.74-3.48). Similar results were generally obtained after the exclusion of subjects to obtain a more homogenous case-referent population: for double null GSTM1 and GSTT1 homozygotes the OR was 2.06 (95% CI 0.85-2.04). In those also likely to have been exposed to diazinon, risks associated with a GSTP1*B or *C allele (OR 1.82, 95% CI 0.92-3.63) or a GSTM1*2/GSTT1*2 homozygous (OR 2.60, 95% CI 0.72-10.42) were elevated but not to a significant extent. Risk associated with PON1 genotype and phenotype varied with CYP2D6 and GSTP1 genotype but not consistently with a priori hypotheses. Further work is necessary to delineate more clearly pathways of organophosphate activation and non-PON1 pathways of detoxification and to confirm whether CYP and GST polymorphisms alter disease risk in populations exposed to organophosphates.     

Hypoxia Induces EMT in Low and Highly Aggressive Pancreatic Tumor Cells but Only Cells with Cancer Stem Cell Characteristics Acquire Pronounced Migratory Potential

Tumor hypoxia induces epithelial-mesenchymal transition (EMT), which induces invasion and metastasis, and is linked to cancer stem cells (CSCs). Whether EMT generates CSCs de novo, enhances migration of existing CSCs or both is unclear. We examined patient tissue of pancreatic ductal adenocarcinoma (PDA) along with carcinomas of breast, lung, kidney, prostate and ovary. For in vitro studies, five established PDA cell lines classified as less (CSC^low) and highly aggressive CSC-like cells (CSC^high) were examined by single and double immunofluorescence microscopy, wound-, transwell-, and time-lapse microscopy. HIF-1α and Slug, as well as HIF-2α and CD133 were co-expressed pointing to a putative co-existence of hypoxia, EMT and CSCs in vivo. CSC^high cells exhibited high basal expression of the mesenchymal Vimentin protein but low or absent expression of the epithelial marker E-cadherin, with the opposite result in CSC^low cells. Hypoxia triggered altering of cell morphology from an epithelial to a mesenchymal phenotype, which was more pronounced in CSC^high cells. Concomitantly, E-cadherin expression was reduced and expression of Vimentin, Slug, Twist2 and Zeb1 enhanced. While hypoxia caused migration in all cell lines, velocity along with the percentage of migrating, polarized and pseudopodia-forming cells was significantly higher in CSC^high cells. These data indicate that hypoxia-induced EMT occurs in PDA and several other tumor entities. However although hypoxia-induced EMT signaling occurs in all tumor cell populations, only the stem-like cells acquire high migratory potential and thus may be responsible for invasion and metastasis.     

Differential Induction of Antimicrobial REGIII by the Intestinal Microbiota and Bifidobacterium breve NCC2950

The intestinal microbiota is a key determinant of gut homeostasis, which is achieved, in part, through regulation of antimicrobial peptide secretion. The aim of this study was to determine the efficiency by which members of the intestinal microbiota induce the antimicrobial peptide REGIII and to elucidate the underlying pathways. We showed that germfree mice have low levels of REGIII-γ in their ileum and colon compared to mice with different intestinal microbiota backgrounds. Colonization with a microbiota of low diversity (altered Schaedler flora) did not induce the expression of REGIII-γ as effectively as a complex community (specific pathogen free). Monocolonization with the probiotic Bifidobacterium breve, but not with the nonprobiotic commensal Escherichia coli JM83, upregulated REGIII-γ expression. Induction of REGIII-γ by B. breve was abrogated in mice lacking MyD88 and Ticam1 signaling. Both live and heat-inactivated B. breve but not spent culture medium from B. breve induced the expression of REGIII-α, the human ortholog and homolog of REGIII-γ, in human colonic epithelial cells (Caco-2). Taken together, the results suggest that REGIII-γ expression in the intestine correlates with the richness of microbiota composition. Also, specific bacteria such as Bifidobacterium breve NCC2950 effectively induce REGIII production in the intestine via the MyD88-Ticam1 pathway. Treatment with this probiotic may enhance the mucosal barrier and protect the host from infection and inflammation.     

Controlled expression and structural organization of a Lactococcus lactis bacteriophage lysin encoded by two overlapping genes.

The phi vML3 bacteriophage lysin is specific for lactococci and could be used to promote enzyme release during cheese manufacture. The level of lysin expression from the cloned gene using its own upstream sequences is very low. Expression in Escherichia coli by using a synthetic hybrid lysin gene and a series of BAL 31 deletions of the original cloned DNA fragment suggested that the start of the gene had previously been incorrectly assigned. Reevaluation of homology between the lysin and Bacillus subtilis PZA protein 15 led to the identification of a new potential ribosome binding site (RBS). A 0.72-kb PCR-generated fragment including this RBS and the complete lysin gene was expressed and inducibly controlled. The translational start of the lysin gene was identified as an isoleucine codon, and this may lead to a low translation rate. During the analysis of the BAL 31 deletion fragments, two proteins of 20 and 8 kDa were shown to be expressed from the originally defined lysin gene. The DNA sequence has a second open reading frame with a good RBS and two potential start methionines. The smaller lysin protein was isolated, and the N terminus was sequenced, confirming that one methionine codon acted as the start of a second gene. The larger lysin protein has homology with lysozymes. The smaller lysin protein has some features resembling those of a holin. The possible roles of these two proteins in lysis of lactococci are discussed.     

Lipid rafts in T cell signalling and disease

Lipid rafts is a blanket term used to describe distinct areas in the plasma membrane rich in certain lipids and proteins and which are thought to perform diverse functions. A large number of studies report on lipid rafts having a key role in receptor signalling and activation of lymphocytes. In T cells, lipid raft involvement was demonstrated in the early steps during T cell receptor (TCR) stimulation. Interestingly, recent evidence has shown that signalling in these domains differs in T cells isolated from patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Here, we discuss these findings and explore the potential of lipid rafts as targets for the development of a new class of agents to downmodulate immune responses and for the treatment of autoimmune diseases.     

Enhancing cell survival of atrazine degrading Rhodococcus erythropolis NI86/21 cells encapsulated in alginate beads

AIMS: To develop a method to produce beads with encapsulated Rhodococcus erythropolis NI86/21 with high cell density, extended shelf life, ease of handling and good atrazine degradation capabilities in both liquid and in agricultural soil. METHODS AND RESULTS: Our findings show that the supplementary recovery step in nutrient broth media shortly after cell encapsulation facilitates cell survival in both wet and dry beads upon extended storage at 4 degrees C. Air drying has little or no impact on encapsulated R. erythropolis cell's ability to degrade atrazine in liquid or soil. Bead storage for periods extending up to 12 months at 4 degrees C did not affect the capacity of R. erythropolis encapsulated cells to degrade atrazine in either BMN or nonsterile soil extracts. Bentonite-amended beads formulated with 1% skim milk and exposed to the supplementary growth step, outperformed all other bead formats. These beads provided adequate numbers of vigorous R. erythropolis cells in either liquid or soil media to degrade atrazine. CONCLUSIONS: Supplementary growth in nutrient broth media immediately following cell encapsulation greatly enhances R. erythropolis cells survival in both wet and dry beads upon extended storage at 4 degrees C. Wet and dried beads have similar capacity for atrazine degradation, and their usefulness and appeal in agronomic practise will only be known after bioassay evaluation and successful demonstration at field scale using incurred residues. SIGNIFICANCE AND IMPACT OF THE STUDY: R. erythropolis NI86/21 encapsulated cells have the potential to reduce residual atrazine in soil, thereby minimizing the likelihood of off-site transport to ground or river water and reduce the loss of crops because of phytotoxicity of residual herbicide. Owing to their ease of handling, storage and possible compatibilities with pre-existing mechanical equipment, dried bead formats are ideally suited for agricultural and remediational applications.     

A new syndrome with noncompaction cardiomyopathy, bradycardia, pulmonary stenosis, atrial septal defect and heterotaxy with suggestive linkage to chromosome 6p

We report a three-generation family with nine patients affected by a combination of cardiac abnormalities and left isomerism which, to our knowledge, has not been described before. The cardiac anomalies include non-compaction of the ventricular myocardium, bradycardia, pulmonary valve stenosis, and secundum atrial septal defect. The laterality sequence anomalies include left bronchial isomerism, azygous continuation of the inferior vena cava, polysplenia and intestinal malrotation, all compatible with left isomerism. This new syndrome is inherited in an autosomal dominant pattern. A genome-wide linkage analysis suggested linkage to chromosome 6p24.3–21.2 with a maximum LOD score of 2.7 at marker D6S276. The linkage interval is located between markers D6S470 (telomeric side) and D6S1610 (centromeric side), and overlaps with the linkage interval in another family with heterotaxy reported previously. Taken together, the genomic region could be reduced to 9.4 cM (12 Mb) containing several functional candidate genes for this complex heterotaxy phenotype. M. W. Wessels, B. M. De Graaf: Both authors contributed equally.     

Influence of environmental conditions on the distribution of Central Asian green toads with three ploidy levels

We studied the distribution of Palearctic green toads (Bufo viridis subgroup), an anuran species group with three ploidy levels, inhabiting the Central Asian Amudarya River drainage. Various approaches (one‐way, multivariate, components variance analyses and maximum entropy modelling) were used to estimate the effect of altitude, precipitation, temperature and land vegetation covers on the distribution of toads. It is usually assumed that polyploid species occur in regions with harsher climatic conditions (higher latitudes, elevations, etc.), but for the green toads complex, we revealed a more intricate situation. The diploid species (Bufo shaartusiensis and Bufo turanensis) inhabit the arid lowlands (from 44 to 789 m a.s.l.), while tetraploid Bufo pewzowi were recorded in mountainous regions (340–3492 m a.s.l.) with usually lower temperatures and higher precipitation rates than in the region inhabited by diploid species. The triploid species Bufo baturae was found in the Pamirs (Tajikistan) at the highest altitudes (2503–3859 m a.s.l.) under the harshest climatic conditions.     

DELAYED URIC ACID ACCUMULATION IN PLASMA PROVIDES ADDITIONAL ANTI-OXIDANT PROTECTION AGAINST IRON-TRIGGERED OXIDATIVE STRESS AFTER A WINGATE TEST

Reactive oxygen species are produced during anaerobic exercise mostly by Fe ions released into plasma and endothelial/muscle xanthine oxidase activation that generates uric acid (UA) as the endpoint metabolite. Paradoxically, UA is considered a major antioxidant by virtue of being able to chelate pro-oxidative iron ions. This work aimed to evaluate the relationship between UA and plasma markers of oxidative stress following the exhaustive Wingate test. Plasma samples of 17 male undergraduate students were collected before, 5 and 60 min after maximal anaerobic effort for the measurement of total iron, haem iron, UA, ferric-reducing antioxidant activity in plasma (FRAP), and malondialdehyde (MDA, biomarker of lipoperoxidation). Iron and FRAP showed similar kinetics in plasma, demonstrating an adequate pro-/antioxidant balance immediately after exercise and during the recovery period (5–60 min). Slight variations of haem iron concentrations did not support a relevant contribution of rhabdomyolysis or haemolysis for iron overload following exercise. UA concentration did not vary immediately after exercise but rather increased 29% during the recovery period. Unaltered MDA levels were concomitantly measured. We propose that delayed UA accumulation in plasma is an auxiliary antioxidant response to post-exercise (iron-mediated) oxidative stress, and the high correlation between total UA and FRAP in plasma (R-Square = 0.636; p = 0.00582) supports this hypothesis.     

Defining the Vulnerable Period for Re-Establishment of Clostridium difficile Colonization after Treatment of C. difficile Infection with Oral Vancomycin or Metronidazole

Background

Clostridium difficile is an anaerobic, spore-forming bacterium that is the most common cause of healthcare-associated diarrhea in developed countries. A significant proportion of patients receiving oral vancomycin or metronidazole for treatment of Clostridium difficile infection (CDI) develop recurrences. However, the period of vulnerability to re-establishment of colonization by C. difficile after therapy is not well defined.

Principal Findings

In a prospective study of CDI patients, we demonstrated that most vancomycin-treated patients maintained inhibitory concentrations of vancomycin in stool for 4 to 5 days after therapy, whereas metronidazole was only detectable during therapy. From the time of elimination of the antibiotics to 14 to 21 days after therapy, a majority of stool suspensions supported growth of C. difficile and deep 16S rRNA sequencing demonstrated persistent marked alteration of the indigenous microbiota. By 21 to 28 days after completion of CDI treatment, a majority of stool suspensions inhibited growth of C. difficile and there was evidence of some recovery of the microbiota.

Conclusions

These data demonstrate that there is a vulnerable period for re-establishment of C. difficile colonization after CDI treatment that begins within a few days after discontinuation of treatment and extends for about 3 weeks in most patients.