Field evaluation of a novel,rapid diagnostic assay,and molecular epidemiology of enterotoxigenic E. coli among Zambian children presenting with diarrhea

BackgroundEnterotoxigenic Escherichia coli (ETEC) is one of the top aetiologic agents of diarrhea in children under the age of 5 in low-middle income countries (LMICs). The lack of point of care diagnostic tools for routine ETEC diagnosis results in limited data regarding the actual burden and epidemiology in the endemic areas. We evaluated performance of the novel Rapid LAMP based Diagnostic Test (RLDT) for detection of ETEC in stool as a point of care diagnostic assay in a resource-limited setting.MethodsWe conducted a cross-sectional study of 324 randomly selected stool samples from children under 5 presenting with moderate to severe diarrhea (MSD). The samples were collected between November 2012 to September 2013 at selected health facilities in Zambia. The RLDT was evaluated by targeting three ETEC toxin genes [heat labile toxin (LT) and heat stable toxins (STh and STp)]. Quantitative PCR was used as the “gold standard” to evaluate the diagnostic sensitivity and specificity of RLDT for detection of ETEC. We additionally described the prevalence and seasonality of ETEC.ResultsThe study included 324 participants, 50.6% of which were female. The overall prevalence of ETEC was 19.8% by qPCR and 19.4% by RLDT. The children between 12 to 59 months had the highest prevalence of 22%. The study determined ETEC toxin distribution was LT 28/321(9%), ST 18/321(6%) and LT/ST 16/321(5%). The sensitivity and specificity of the RLDT compared to qPCR using a Ct 35 as the cut-off, were 90.7% and 97.5% for LT, 85.2% and 99.3% for STh and 100% and 99.7% for STp, respectively.ConclusionThe results of this study suggest that RLDT is sufficiently sensitive and specific and easy to implement in the endemic countries. Being rapid and simple, the RLDT also presents as an attractive tool for point-of-care testing at the health facilities and laboratories in the resource-limited settings.     

Linearizing ecological models with time-varying parameters

An ecological model with time-varying parameters can be linearized about a point which is either a local equilibrium or fixed in time, but not in general both. Each choice has its advantages, but neither choice is anywhere near as useful as the linearization of a model with constant parameters. Linearizations of a model of annual plankton cycles can capture some qualitative behavior of the original nonlinear model.     

Lack of response to all-trans retinoic acid supplementation in adult dogs following left pneumonectomy.

We showed previously that removing 55-58% of the lung by right pneumonectomy (R-PNX) in adult dogs triggers compensatory growth of the remaining lung, but removing 42-45% of the lung by left PNX (L-PNX) does not. We also showed that, following R-PNX, supplemental all-trans retinoic acid (RA) selectively enhances alveolar capillary endothelial cell volume (Yan X, Bellotto DJ, Foster DJ, Johnson RL, Jr., Hagler HH, Estrera AS, and Hsia CC. J Appl Physiol 96: 1080-1089, 2004). We hypothesized that RA supplementation might enhance compensation following L-PNX and tested this hypothesis by administering RA (2 mg.kg(-1).day(-1), 4 days/wk) or placebo orally to litter-matched adult foxhounds for 4 mo following L-PNX. Resting lung function was measured under anesthesia. Air and tissue volumes of the remaining lung were assessed by high-resolution computed tomography scan and by detailed postmortem morphometric analysis of the fixed lung. There was no significant difference in resting lung function, lung volume, alveolar structure, or septal ultrastructure between RA and placebo treatment groups. We conclude that RA supplementation does not induce post-PNX compensatory lung growth in the absence of existing cellular growth activities initiated by other primary signals.     

Polymorphisms in genes of the steroid hormone biosynthesis and metabolism pathways and endometrial cancer risk

Objectives: The incidence of endometrial cancer has recently increased substantially and studies have shown that altered levels of exogenous and endogenous hormones are associated with individual variation in endometrial cancer risk. The environmental and reproductive risk factors that influence these hormones are well known, however, genetic variants involved in hormone biosynthesis and estrogen metabolism have not been well established in endometrial cancer. Methods: To determine whether polymorphisms in genes of the steroid hormone biosynthesis and metabolism pathways are associated with endometrial cancer risk, 28 polymorphisms in 18 genes were genotyped in 191 endometrial cancer cases and 291 healthy controls. Results: The GSTM1 deletion and the variant (GG) genotype of the CYP1B1 rs1800440 polymorphism were associated with a decreased risk of developing endometrial cancer. Furthermore, combinations of haplotypes in CYP1A1, CYP1B1 and GSTs were associated with a decreased risk. The analysis of the repeat polymorphisms revealed that women with the long repeat allele length of the ESR1 (GT)n repeat polymorphism were at an increased risk of developing endometrial cancer. Conversely, women with two long repeat length alleles of the (CAG)n repeat polymorphism in the AR correlated with a decrease in endometrial cancer risk compared to women with one or two alleles with the short repeat length. Conclusions: The findings are consistent with our hypothesis that variability in genes involved in steroidogenesis and estrogen metabolism may alter the risk of developing endometrial cancer, suggesting that they may be useful as biomarkers for genetic susceptibility to endometrial cancer.     

Destabilization of the Homotetrameric Assembly of 3-Deoxy-d-Arabino-Heptulosonate-7-Phosphate Synthase from the Hyperthermophile Pyrococcus furiosus Enhances Enzymatic Activity

Many proteins adopt homomeric quaternary structures to support their biological function, including the first enzyme of the shikimate pathway that is ultimately responsible for the biosynthesis of the aromatic amino acids in plants and microorganisms. This enzyme, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAH7PS), adopts a variety of different quaternary structures depending on the organism in which it is found. The DAH7PS from the hyperthermophilic archaebacterium Pyrococcus furiosus was previously shown to be tetrameric in its crystalline form, and this quaternary association is confirmed in an improved structure in a different crystal system. This tetramer is also present in solution as revealed by small-angle X-ray scattering and analytical ultracentrifugation. This homotetrameric form has two distinct interfaces, both of which bury over 10% each of the surface area of a single monomer. Substitution of Ile for Asp in the hydrophobic region of one interface gives a protein with a remarkable 4-fold higher maximum catalytic rate than the wild-type enzyme. Analytical ultracentrifugation at pH 7.5 reveals that the tetrameric form is destabilized; although the protein crystallizes as a tetramer, equilibrium exists between tetrameric and dimeric forms with a dissociation constant of 22 μM. Thus, under the conditions of kinetic assay, the enzyme is primarily dimeric, revealing that the dimeric form is a fully functional catalyst. However, in comparison to the wild-type protein, the thermal stability of the dimeric protein is significantly compromised. Thus, an unusual compromise of enzymatic activity versus stability is observed for this DAH7PS from an organism that favors a hyperthermophilic environment.     

Aspergillus nidulans hypB encodes a Sec7-domain protein important for hyphal morphogenesis.

Aspergillus nidulans strains containing the hypB5 temperature sensitive allele have a restrictive phenotype of wide, highly-branched hyphae. The hypB locus was cloned by phenotype complementation using a genomic plasmid library. hypB5 is predicted gene AN6709 in the A. nidulans genome database, which encodes a putative Sec7 domain protein, likely to act early in COPI-mediated vesicle formation for retrograde Golgi to ER transport. The A. nidulans hypB5 allele has a single mutation, cytosine to guanine predicted to cause a nonconservative amino acid change, alanine to proline, in a conserved helix adjacent to the Sec7p nucleotide binding site. This would likely reduce the stability of a highly conserved loop important for nucleotide binding, and is consistent with temperature sensitivity of hypB5 strains. Deletion of AN6709 showed that hypB(Sec7) was not essential. AN6709Delta hyphae resembled the hypB5 restrictive phenotype. As has been shown previously for hypA1 mutants, shifting established hypB5 mutant hyphae from a growth temperature of 28-42 degrees C caused septation in and death of tip cells and growth activation of basal cells.