Factors associated with typical enteropathogenic Escherichia coli infection among children <5 years old with moderate-to-severe diarrhoea in rural western Kenya, 2008-2012.

Typical enteropathogenic Escherichia coli (tEPEC) infection is a major cause of diarrhoea and contributor to mortality in children <5 years old in developing countries. Data were analysed from the Global Enteric Multicenter Study examining children <5 years old seeking care for moderate-to-severe diarrhoea (MSD) in Kenya. Stool specimens were tested for enteric pathogens, including by multiplex polymerase chain reaction for gene targets of tEPEC. Demographic, clinical and anthropometric data were collected at enrolment and ~60-days later; multivariable logistic regressions were constructed. Of 1778 MSD cases enrolled from 2008 to 2012, 135 (7.6%) children tested positive for tEPEC. In a case-to-case comparison among MSD cases, tEPEC was independently associated with presentation at enrolment with a loss of skin turgor (adjusted odds ratio (aOR) 2.08, 95% confidence interval (CI) 1.37-3.17), and convulsions (aOR 2.83, 95% CI 1.12-7.14). At follow-up, infants with tEPEC compared to those without were associated with being underweight (OR 2.2, 95% CI 1.3-3.6) and wasted (OR 2.5, 95% CI 1.3-4.6). Among MSD cases, tEPEC was associated with mortality (aOR 2.85, 95% CI 1.47-5.55). This study suggests that tEPEC contributes to morbidity and mortality in children. Interventions aimed at defining and reducing the burden of tEPEC and its sequelae should be urgently investigated, prioritised and implemented.

Diarrhoea, enteric pathogen detection and nutritional indicators among controls in the Global Enteric Multicenter Study, Kenya site: an opportunity to understand reference populations in case-control studies of diarrhoea.

Given the challenges in accurately identifying unexposed controls in case-control studies of diarrhoea, we examined diarrhoea incidence, subclinical enteric infections and growth stunting within a reference population in the Global Enteric Multicenter Study, Kenya site. Within 'control' children (0-59 months old without diarrhoea in the 7 days before enrolment, n = 2384), we examined surveys at enrolment and 60-day follow-up, stool at enrolment and a 14-day post-enrolment memory aid for diarrhoea incidence. At enrolment, 19% of controls had ⩾1 enteric pathogen associated with moderate-to-severe diarrhoea ('MSD pathogens') in stool; following enrolment, many reported diarrhoea (27% in 7 days, 39% in 14 days). Controls with and without reported diarrhoea had similar carriage of MSD pathogens at enrolment; however, controls reporting diarrhoea were more likely to report visiting a health facility for diarrhoea (27% vs. 7%) or fever (23% vs. 16%) at follow-up than controls without diarrhoea. Odds of stunting differed by both MSD and 'any' (including non-MSD pathogens) enteric pathogen carriage, but not diarrhoea, suggesting control classification may warrant modification when assessing long-term outcomes. High diarrhoea incidence following enrolment and prevalent carriage of enteric pathogens have implications for sequelae associated with subclinical enteric infections and for design and interpretation of case-control studies examining diarrhoea.

Overnight improvements in two REM sleep-sensitive tasks are associated with both REM and NREM sleep changes, sleep spindle features, and awakenings for dream recall.

Memory consolidation is associated with sleep physiology but the contribution of specific sleep stages remains controversial. To clarify the contribution of REM sleep, participants were administered two REM sleep-sensitive tasks to determine if associated changes occurred only in REM sleep. Twenty-two participants (7 men) were administered the Corsi Block Tapping and Tower of Hanoi tasks prior to and again after a night of sleep. Task improvers and non-improvers were compared for sleep structure, sleep spindles, and dream recall. Control participants (N = 15) completed the tasks twice during the day without intervening sleep. Overnight Corsi Block improvement was associated with more REM sleep whereas Tower of Hanoi improvement was associated with more N2 sleep. Corsi Block improvement correlated positively with %REM sleep and Tower of Hanoi improvement with %N2 sleep. Post-hoc analyses suggest Tower of Hanoi effects-but not Corsi Block effects-are due to trait differences. Sleep spindle density was associated with Tower of Hanoi improvement whereas spindle amplitude correlated with Corsi Block improvement. Number of REM awakenings for dream reporting (but not dream recall per se) was associated with Corsi Block, but not Tower of Hanoi, improvement but was confounded with REM sleep time. This non-replication of one of 2 REM-sensitive task effects challenges both 'dual-process' and 'sequential' or 'sleep organization' models of sleep-dependent learning and points rather to capacity limitations on REM sleep. Experimental awakenings for sampling dream mentation may not perturb sleep-dependent learning effects; they may even enhance them.

The characterization of Listeria spp. isolated from food products and the food-processing environment.

AIM: To enhance the information pertaining to the epidemiology of a collection of 378 Listeria spp. isolates obtained from several food-processing plants in Ireland over a 3-year period (2004-2007). METHODS AND RESULTS: The collection was characterized by pulsed-field gel electrophoresis (PFGE). The most prevalent pulse-type was PFGE profile I (n=14·5%) that consisted mainly of environmental Listeria spp. samples. Serotyping of 145 Listeria monocytogenes isolates was performed. The most common serovar was 1/2a and comprised 57·4% (n=77) of the L. monocytogenes collection. The other serovars were as follows: 4b (14·1%, n=19), 1/2b (9·7%, n=13), 4c (4·4%, n=6) and 1/2c (6·7%, n=9), respectively. Eleven isolates were identified as non-Listeria spp., the remaining ten L. monocytogenes isolates were nontypeable. The antimicrobial susceptibility testing revealed the antibiotic that isolates displayed the most resistance to was gentamicin (5%) followed by sulfamethoxazole-trimethoprim (2%), tetracycline and ciprofloxacin (1·5%). CONCLUSIONS: The subtyping has indicated the diversity of the Listeria spp. The presence of serotype 1/2a, 1/2b and 4b in both raw and cooked ready-to-eat food products is a public health concern, as these serotypes are frequently associated with foodborne outbreaks and sporadic cases of human listeriosis. In addition, the emergence of antimicrobial-resistant L. monocytogenes isolates could have serious therapeutic consequences. SIGNIFICANCE AND IMPACT OF STUDY: The molecular subtyping and the further characterization of these isolates may be valuable particularly in the context of a suspected common source outbreak in the future.

Forecasting extreme stratospheric polar vortex events.

Extreme polar vortex events known as sudden stratospheric warmings can influence surface winter weather conditions, but their timing is difficult to predict. Here, we examine factors that influence their occurrence, with a focus on their timing and vertical extent. We consider the roles of the troposphere and equatorial stratosphere separately, using a split vortex event in January 2009 as the primary case study. This event cannot be reproduced by constraining wind and temperatures in the troposphere alone, even when the equatorial lower stratosphere is in the correct phase of the quasi biennial oscillation. When the flow in the equatorial upper stratosphere is also constrained, the timing and spatial evolution of the vortex event is captured remarkably well. This highlights an influence from this region previously unrecognised by the seasonal forecast community. We suggest that better representation of the flow in this region is likely to improve predictability of extreme polar vortex events and hence their associated impacts at the surface.

Application of high rate, high temperature anaerobic digestion to fungal thermozyme hydrolysates from carbohydrate wastes.

The objective of this study was to examine the feasibility of using a two-step, fully biological and sustainable strategy for the treatment of carbohydrate rich wastes. The primary step in this strategy involves the application of thermostable enzymes produced by the thermophilic, aerobic fungus, Talaromyces emersonii, to carbohydrate wastes producing a liquid hydrolysate discharged at elevated temperatures. To assess the potential of thermophilic treatment of this hydrolysate, a comparative study of thermophilic and mesophilic digestion of four sugar rich thermozyme hydrolysate waste streams was conducted by operating two high rate upflow anaerobic hybrid reactors (UAHR) at 37 degrees C (R1) and 55 degrees C (R2). The operational performance of both reactors was monitored from start-up by assessing COD removal efficiencies, volatile fatty acid (VFA) discharge and % methane of the biogas produced. Rapid start-up of both R1 and R2 was achieved on an influent composed of the typical sugar components of the organic fraction of municipal solid waste (OFMSW). Both reactors were subsequently challenged in terms of volumetric loading rate (VLR) and it was found that a VLR of 9 gCOD l(-1)d(-1) at a hydraulic retention time (HRT) of 1 day severely affected the thermophilic reactor with instability characterised by a build up of volatile fatty acid (VFA) intermediates in the effluent. The influent to both reactors was changed to a simple glucose and sucrose-based influent supplied at a VLR of 4.5 gCOD l(-1)d(-1) and HRT of 2 days prior to the introduction of thermozyme hydrolysates. Four unique thermozyme hydrolysates were subsequently supplied to the reactors, each for a period of 10 HRTs. The applied hydrolysates were derived from apple pulp, bread, carob powder and cardboard, all of which were successfully and comparably converted by both reactors. The % total carbohydrate removal by both reactors was monitored during the application of the sugar rich thermozyme hydrolysates. This approach offers a sustainable technology for the treatment of carbohydrate rich wastes and highlights the potential of these wastes as substrates for the generation of second-generation biofuels.

Efficacy of a solar concentrator to Inactivate E. coli and C. perfringens spores in latrine waste in Kenya.

Alternative sanitation options are needed for effective waste management in low-income countries where centralized, large-scale waste treatment is not easily achievable. A newly designed solar concentrator technology utilizes solar thermal energy to treat feces contained in drums. This pilot study assessed the efficacy of the new design to inactivate microbes in 13 treatment drums under field conditions in Kenya. Three-quarters of the drums contained <1000 E. coli/g of total solids following 6 h of solar thermal treatment and inactivation of thermotolerant C. perfringens spores ranged from <1.8 to >5.0 log10. Nearly all (94%) samples collected from treatment drums achieved thermophilic temperatures (>50 °C) during the treatment period, however this alone did not ensure samples met the WHO E. coli guideline; higher, sustained thermophilic temperatures tended to be more effective in reaching this guideline. The newly designed solar concentrator was capable of inactivating thermotolerant, environmentally-stable microorganisms as, or possibly more, efficiently than a previous design. Additional data are needed to better characterize how temperature, time, and other parameters affect the ability of the solar concentrator to inactivate microbes in feces.

Toll-like receptor 3 L412F polymorphism promotes a persistent clinical phenotype in pulmonary sarcoidosis.

BACKGROUND/INTRODUCTION: Sarcoidosis is a multi-systemic disorder of unknown etiology, characterized by the presence of non-caseating granulomas in target organs. In 90% of cases, there is thoracic involvement. Fifty to seventy percent of pulmonary sarcoidosis patients will experience acute, self-limiting disease. For the subgroup of patients who develop persistent disease, no targeted therapy is currently available. AIM: To investigate the potential of the single nucleotide polymorphism (SNP), Toll-like receptor 3 Leu412Phe (TLR3 L412F; rs3775291), as a causative factor in the development of and in disease persistence in pulmonary sarcoidosis. To investigate the functionality of TLR3 L412F in vitro in primary human lung fibroblasts from pulmonary sarcoidosis patients. DESIGN: SNP-genotyping and cellular assays, respectively, were used to investigate the role of TLR3 L412F in the development of persistent pulmonary sarcoidosis. METHODS: Cohorts of Irish sarcoidosis patients (n = 228), healthy Irish controls (n = 263) and a secondary cohort of American sarcoidosis patients (n = 123) were genotyped for TLR3 L412F. Additionally, the effect of TLR3 L412F in primary lung fibroblasts from pulmonary sarcoidosis patients was quantitated following TLR3 activation in the context of cytokine and type I interferon production, TLR3 expression and apoptotic- and fibroproliferative-responses. RESULTS: We report a significant association between TLR3 L412F and persistent clinical disease in two cohorts of Irish and American Caucasians with pulmonary sarcoidosis. Furthermore, activation of TLR3 in primary lung fibroblasts from 412 F-homozygous pulmonary sarcoidosis patients resulted in reduced IFN-ß and TLR3 expression, reduced apoptosis- and dysregulated fibroproliferative-responses compared with TLR3 wild-type patients. DISCUSSION/CONCLUSION: This study identifies defective TLR3 function as a previously unidentified factor in persistent clinical disease in pulmonary sarcoidosis and reveals TLR3 L412F as a candidate biomarker.

DNA sequence of the cut A, B and C genes, encoding the molybdenum containing hydroxylase carbon monoxide dehydrogenase, from Pseudomonas thermocarboxydovorans strain C2.

Pseudomonas thermocarboxydovorans strain C2 is capable of using carbon monoxide as the sole source of carbon and energy. The key enzyme for CO utilisation is the molybdenum containing iron-flavoprotein carbon monoxide dehydrogenase (CODH). This paper reports the DNA sequencing of a 4.7 kb region of the C2 genome which appears to encode the CODH enzyme. The genes for the three subunits of CODH, which we have named cut A, B and C, have been identified and they appear to form an operon. The predicted protein sequences of the three subunits have homology to the structurally related protein, xanthine dehydrogenase, from Drosophila melanogaster. By comparison with xanthine dehydrogenase it can be predicted that the molybdenum cofactor binds to the large subunit of CODH, the small subunit of CODH contains the iron-sulphur centers and the medium subunit binds FAD/NAD+.

Microbial sulphate reduction during anaerobic digestion: EGSB process performance and potential for nitrite suppression of SRB activity.

The present study investigated mesophilic anaerobic treatment of sulphate-containing wastewater in EGSB reactors and assessed the inclusion of nitrite in the reactor influent as a method for control of biological sulphate reduction. Two EGSB reactors, R1 and R2, were operated for a period of 581 days at varying volumetric loading rates, COD/SO4(2-) ratios and influent nitrite concentrations (R2 only). COD removal efficiencies of > 93% were achieved in both reactors at influent sulphate concentrations of up to 3,000 mg l(-1). A two-fold increase in the influent sulphate concentration, giving an influent COD/SO4(2-) ratio of 2, resulted in a reduction in reactor COD removal efficiency to 84% and 89%, in R1 and R2, respectively. Despite inclusion of nitrite in the R2 influent at concentrations up to 500 mg NO2-N l(-1), sulphate reduction proceeded similarly in R2 and R1, suggesting the ineffectiveness of nitrite as a potential inhibitor of SRB

Purinoceptor activation of chloride transport in cystic fibrosis and CFTR-transfected pancreatic cell lines.

The regulation of chloride efflux from cystic fibrosis pancreatic adenocarcinoma cells (CFPAC-1) and wild-type CFTR-transfected CFPAC-1 cells (TPAC) was compared. Forskolin (10 microM) stimulated chloride efflux from the corrected TPAC cells but not from CFPAC-1 cells. Chloride efflux from both cell types was activated by thapsigargin (0.5 microM). The nucleotides ATP and UTP and the non-hydrolyzable ATP analogue, adenosine 5'-O-(3-thio) triphosphate (ATPgammaS), stimulated chloride efflux from both cell types. None of the other P2 purinoceptor agonists investigated elicited a response. The order of potency was ATP > or = UTP > or = ATPgammaS. Adenosine (10-100 microM) activated choride efflux from the TPAC but not the CFPAC cell line with no increase in intracellular cyclic AMP. Small but statistically significant inhibitions of the adenosine-(50 microM)-stimulated increase in chloride efflux were elicited by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX, 100 nM) and the A2 receptor antagonist 3,7-dimethyl-1-propylargylxanthine (DMPX, 10 microM). The A2A receptor antagonist 8-(3-chlorostyryl)caffeine (CSC, 100 nM) had no significant effect. These results provide evidence for the regulation of chloride efflux by P2Y2 purinoceptors in genetically-corrected and CF pancreatic cell lines. Studies with adenosine receptor antagonists indicate some possible involvement of A1 and A2 (but not A2A) receptors in the adenosine stimulation of chloride efflux, but the relatively small effects of the inhibitors coupled with lack of increase in cyclic AMP and a response only in the CFTR-transfected cells also suggests a possible direct effect of adenosine on CFTR.

Amiloride-sensitive sodium uptake into human placental brush border membrane vesicles.

Sodium transport into human placental brush border membrane vesicles was examined in the presence of an outwardly directed sodium gradient leading to the formation of an intravesicular negative charge. 22Na entered the vesicles in a time dependent fashion. The activation energy of the uptake process was calculated and was found to be 11.2 kcal/mol, similar to the value of ionic diffusion in free solution. Amiloride inhibited Na uptake in a concentration dependent fashion with an IC50 value of 3.08 microM. Neither ouabain nor bumetanide had an effect on Na uptake at concentrations up to 100 or 1000 microM, respectively. The system presented here indicates Na transport via channels without involvement of the Na-K-ATPase or the Na-K-Cl cotransporter. The system may be useful in investigating Na transport defects in cystic fibrosis.

Role of ions and membrane potential in uptake of serotonin into plasma membrane vesicles from mouse brain.

Plasma membrane vesicle preparations from mouse cerebral cortex actively accumulated [3H]serotonin upon the imposition of a K+ gradient (in greater than out), a Na+ gradient (out greater than in), and the presence of external Cl-. Maximal stimulation of uptake by internal K+ occurred at 15 mM and half-maximal stimulation at 2 mM. Internal K+ did not enhance uptake merely via generation of a membrane potential because simultaneous parallel increases in internal and external K+ concentration also stimulated uptake. External Cl- increased serotonin uptake with a Km of 18 mM and a Hill number of 1.0, suggesting a requirement for one chloride ion for transport. Uptake could not be driven by internal H+ instead of K+. Estimation of the membrane potential by the distribution of triphenylmethylphosphonium ion showed a modest effect of valinomycin (1-20 microM) in increasing the potential from -19 to -31 mV accompanied by an increase in serotonin uptake. Proton ionophores prevented this effect of valinomycin and, by themselves, reduced the potential to -6 mV, but did not affect serotonin transport. A model is proposed for serotonin transport in brain plasma membrane vesicles that is similar to the model for porcine blood platelet vesicles as far as electroneutrality and stimulation by K+, Na+, and Cl- are concerned, but that is different in substitution of internal H+ for K+.

Myasthenia gravis: the role of immunological deficiency.

To evaluate further the association between myasthenia gravis and humoral immune deficiency, 92 sera from myasthenic patients were tested so as to determine titers against commensal E. coli, isohemagglutinin titers, and IgG and IgM concentrations. Results were compared with those obtained from normals, disease controls, and W27 positive arthropathy. On the basis of these three investigations it is concluded that subtle immunodeficiency is common in myasthenia gravis, and it is suggested that an immune defect might explain such diverse associations as thymic disease, HL-A antigens, and various autoimmune phenomena.

Expression of the cyanide hydratase enzyme from Fusarium lateritium in Escherichia coli and identification of an essential cysteine residue.

The filamentous fungus Fusarium lateritium is cyanide tolerant, due partly to the induction of the enzyme cyanide hydratase in the presence of cyanide. This enzyme catalyses the hydration of cyanide to formamide. The expression in Escherichia coli of a cDNA clone encoding cyanide hydratase is described. The cDNA cloned was expressed as a transcriptional fusion in the expression vector pKK233-2 and a high level of activity of cyanide hydratase was detected in E. coli. Site-directed mutagenesis of the cys-163 residue inactivated the enzyme.

Cloning and expression of the carbon monoxide dehydrogenase genes from Pseudomonas thermocarboxydovorans strain C2.

Carbon monoxide dehydrogenase (CODH) from Pseudomonas thermocarboxydovorans strain C2 is composed of three non-identical subunits. A gene library of C2 DNA in lambda vector L47.1 was generated and screened using anti-CODH serum. Western blotting experiments revealed a protein which co-migrated with and had the same immunological reaction as the large subunit of CODH in some of the clones isolated from the library. The coding region was pinpointed to a 4 kb fragment which was subcloned into plasmid. Western blotting experiments showed that all three subunits of CODH were coded for by the subclone. However, no CODH activity was detected.

A practical vibration isolation workstation for electrophysiology.

Vibration control is a major concern in electrophysiological research, particularly during intracellular recording where movements of only a few micrometers may disrupt the cell membrane. The workstation described here is based on the concept of a table nested within a second table. The inner table supports a vibration-isolated surface while the outer table provides protection and a bench-top for equipment. The work surface is supported on squash balls in order to avoid the substantial cost of passive commercial isolators. The properties of the squash ball isolators were found to be similar to a set commercial isolators when compared with respect to impulse response, compliance and transmissibility. The table has been successfully used for both intra- and extracellular recording over a 3-year period. Work efficiency was enhanced by the close proximity of the bench-top and isolated work surface, while perimeter protection helped maintain stable recordings.

Inhibition of serotonin uptake into mouse brain synaptosomes by ionophores and ion-channel agents.

[3H]Serotonin uptake into mouse cerebrocortical synaptosomes was decreased by the K+ ionophore valinomycin, the K+ and Na+ ionophore gramicidin, and the proton ionophore carbonylcyanide m-chlorophenylhydrazone. The Na+/H+ exchanger monensin reduced uptake at non-depolarizing concentrations. Uptake was also decreased by inhibition of the Na+, K(+)-ATPase with ouabain and by tetrodotoxin-sensitive activation of voltage-dependent sodium channels with veratridine, batrachotoxin and scorpion venom. In contrast, the Ca2+ channel agents BAY K8644 and nimodipine were ineffective. The effect of reducing the Na+ gradient depended upon whether the internal Na+ concentration was raised (i.e. by scorpion venom, monensin) or the external Na+ concentration was lowered (37 mM NaCl in the medium).

Assessing depression in fibromyalgia patients.

PURPOSE: This study investigated the relationships among four methods of detecting depression in patients with fibromyalgia. METHODS: Data were obtained from 100 women (mean age 43 years) who had been diagnosed with fibromyalgia. Instruments included a computerized Diagnostic Interview Schedule (C-DIS), Beck Depression Inventory (BDI), an adjusted "disease-free" BDI (BDI-A), and Minnesota Multiphasic Personality Inventory depression subscale (MMPI-D). Chance-corrected concordance, sensitivity, specificity, and accuracy among the four methods were calculated. RESULTS: The C-DIS detected 22% and BDI-A 29% with current major depression. The BDI and MMPI-D yielded higher estimates of 55% of the 44%, respectively. Agreement on the diagnosis among the four methods was significantly greater than chance. When compared with the C-DIS, the BDI was the most sensitive instrument and the BDI-A most specific.

A nonseparation, time-resolved fluoroimmunoassay to monitor ovarian function and predict potential fertility in women.

The authors describe a nonseparation, time-resolved fluoroimmunoassay involving the use of monoclonal antibodies for the measurement of estrone-3-glucuronide in urine. The method has appropriate sensitivity (12 nmol/l), better specificity than a conventional radioimmunoassay with polyclonal antibodies, and the advantages of speed, simplicity, less imprecision, and improved clinical effectiveness. The labeled antigen is a novel fluorescent europium chelate covalently linked to estrone glucuronide at carbon 6 of the glucuronide moiety. The antibodies were raised against estrone-3-glucuronyl-6-bovine serum albumin. The antibody binding reaction is performed in microtiter wells (or tubes) and involves the addition of labeled antigen in buffer (2 ng/100 microliters), a limited concentration of antibodies in buffer (100 microliters), and standard or urine sample (10 microliters). The mixture is incubated for 15 minutes at room temperature. Time-resolved fluorescence from the unbound label is proportional to the concentration of estrone glucuronide. The method may be used to monitor ovarian function and potential fertility in women.

PIP: In the study and measurement of analytes which indicate the status of reproduction, immunoassays are still being updated and utilized. Nonseparation immunoassays have been specifically developed in the study of drugs in serum or urine and most likely can be developed for the measure of urinary estrone glucuronide. The advantages of this process include accuracy, simplicity, speed and the possibility of full automation. However, nonseparation immunoassays are affected by nonspecific interference from the attributes within biologic materials, assay buffers, reagents and plastics. The use of time-resolved fluorescence has been implemented and has been proven to be successful. The variety of assays used in the measurement of haptens and large molecular weight analytes has been greatly improved. The technique of using time-resolved fluorescence gives us the greatest potential in the production of the most sensitive separation and nonseparation immunoassays. This method has more appropriate sensitivity and a better specificity than typical radioimmunoassays with polyclonal antibodies. This process may be used in the study of ovarian functions and future fertility in women.