Structure-Function Studies of DNA Binding Domain of Response Regulator KdpE Reveals Equal Affinity Interactions at DNA Half-Sites

Expression of KdpFABC, a K+ pump that restores osmotic balance, is controlled by binding of the response regulator KdpE to a specific DNA sequence (kdpFABCBS) via the winged helix-turn-helix type DNA binding domain (KdpEDBD). Exploration of E. coli KdpEDBD and kdpFABCBS interaction resulted in the identification of two conserved, AT-rich 6 bp direct repeats that form half-sites. Despite binding to these half-sites, KdpEDBD was incapable of promoting gene expression in vivo. Structure-function studies guided by our 2.5 Å X-ray structure of KdpEDBD revealed the importance of residues R193 and R200 in the α-8 DNA recognition helix and T215 in the wing region for DNA binding. Mutation of these residues renders KdpE incapable of inducing expression of the kdpFABC operon. Detailed biophysical analysis of interactions using analytical ultracentrifugation revealed a 2∶1 stoichiometry of protein to DNA with dissociation constants of 200±100 and 350±100 nM at half-sites. Inactivation of one half-site does not influence binding at the other, indicating that KdpEDBD binds independently to the half-sites with approximately equal affinity and no discernable cooperativity. To our knowledge, these data are the first to describe in quantitative terms the binding at half-sites under equilibrium conditions for a member of the ubiquitous OmpR/PhoB family of proteins

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Detection of Rifampicin Resistance in HIV Seropositive Individuals with Suspected Pulmonary Tuberculosis by Using CBNAAT

Tuberculosis(TB) remains the most common opportunistic infection among people living with HIV (PLHIV) and diagnosing it becomes a challenging task as sputum microscopy is negative in more than half of the patients. Delayed treatment for TB in PLHIV is associated with increased mortality. Cartridge Based Nucleic Acid Amplification Test (CBNAAT) is a recently introduced Polymerase chain reaction based method for detection of Mycobacterium tuberculosis, which also detects Rifampicin resistance as it targets the rpoB gene of mycobacteria. To determine the prevalence of Rifampicin resistance in HIV seropositive patients with suspected pulmonary tuberculosis in a tertiary care hospital by using CBNAAT. HIV seropositive patients with clinically suspected tuberculosis were included in the study. Two sputum samples from each patient were collected and subjected to sputum microscopy by LED-fluorescent Microscope. Detection of mycobacteria and Rifampicin resistance was carried out by CBNAAT on Gene Xpert MTB/RIF. Out of the total 576 sputum, 74 (12.84%) patients were positive by sputum microscopy for acid fast bacilli and 137 (23.78%) were positive by CBNAAT. Rifampicin resistance was detected in 12 (8.75%) cases. CBNAAT helped in increased case detection in lesser time as compared to sputum microscopy. It also detects Rifampicin resistance with high specificity and can be used for screening for MDR-TB for the purpose of starting category IV anti-tubercular therapy (ATT) early

Performance of coriander under organic and chemical nutrient management practice

Coriander is one of the most important spice and fresh leaf vegetable which are used widely across the globe. Organic cultivation of this crop has wide scope as it is short duration and reduces the wastage of fertilizers as well as qualitative production. A field experiment was conducted at School of Agricultural Sciences and Forestry, RTU Bangalore in order to know the effective application rate of compost for economic production in comparison to chemical fertilizer. The research data reveals that, application of compost at 10t/ha produced significantly superior growth, yield parameters and herbage yield compared to control. But it was on par with RDF treatment. Thus application of compost at10t/ha might be an effective nutritional input in organic cultivation of coriander. &nbsp

Medication errors, what healthcare providers think? A Knowledge, Attitude and Practice survey.

Objective: Evaluation of knowledge, attitude and practice of healthcare providers about Medication errors.   Method: It was a KAP survey conducted with the help of specially designed and pre-validated questionnaire. Doctors', nurses' and pharmacists' awareness and perception about Medication errors were assessed. KAP survey questionnaire was analyzed and their question-wise percentage value was calculated. Data was analysed with One-way ANOVA followed by Dunnets post-hoc test and descriptive statistics were applied whenever necessary.   Results: Response rate amongst healthcare providers was satisfactory. Doctors' knowledge and practice were significantly higher compared to the nurses and the pharmacists. Their perception when analysed, revealed that most of the participants consider medication error as an important problem which can be prevented. There is a difference of opinions as regards to the liability for legal punishment to the personnels responsible for such incidences, thus considering it as a crime.   Conclusion: There is awareness regarding Medication errors, yet certain circumstances which significantly contribute in occurrence of these events need to be overcome. Regardless of the availability of few solutions, as this area of practice is least approached, further research is warranted in various interventions to deal with the same. Key words: Awareness, Doctors, Medication errors, Nurses, Pharmacists

Household Water Storage Management, Hygiene Practices, and Associated Drinking Water Quality in Rural India

Household drinking water storage is commonly practiced in rural India. Fecal contamination may be introduced at the water source, during collection, storage, or access. Within a trial of a community-level water supply intervention, we conducted five quarterly household-level surveys to collect information about water, sanitation, and hygiene practices in rural India. In a random subsample of households, we tested stored drinking water samples for Escherichia coli, concurrently observing storage and access practices. We conducted 9961 surveys and collected 3296 stored water samples. Stored water samples were frequently contaminated with E. coli (69%), and E. coli levels were the highest during the wet season. Most households contributing two or more drinking water samples had detectable E. coli in some (47%) or all (44%) samples. Predictors of stored water contamination with E. coli included consumption of river water and open defecation; consumption of reverse osmosis-treated water and safe water access practices appeared to be protective. Until households can be reached with on-premises continuous safe water supplies, suboptimal household water storage practices are likely to continue. Improvements to source water quality alone are unlikely to prevent exposure to contaminated drinking water unless attention is also given to improving household water storage, access, and sanitation practices

A stepped wedge cluster-randomized trial assessing the impact of a riverbank filtration intervention to improve access to safe water on health in rural India

Sustainable and low-cost methods for delivery of safe drinking water in resource-limited settings remain suboptimal, which contributes to global diarrhea morbidity. We aimed to assess whether delivery of riverbank filtration-treated water to newly installed water storage tanks (improved quality and access, intervention condition) reduced reported diarrhea in comparison to delivery of unfiltered river water (improved access alone, control condition) in rural Indian villages. We used a stepped wedge cluster-randomized trial (SW-CRT) design involving four clusters (villages). Selection criteria included village size, proximity to a river, and lack of existing or planned community-level safe water sources. All adults and children were eligible for enrollment. All villages started in the control condition and were sequentially randomized to receive the intervention at 3-month intervals. Our primary outcome was 7-day-period prevalence of self- or caregiver-reported diarrhea, measured at 3-month intervals (five time points). Analysis was by intention to treat. Because blinding was not possible, we incorporated questions about symptoms unrelated to water consumption to check response validity (negative control symptoms). We measured outcomes in 2,222 households (9,836 participants). We did not find a measurable reduction in diarrhea post-intervention (RR: 0.98 [95% CI: 0.24-4.09]); possible explanations include low intervention uptake, availability of other safe water sources, low baseline diarrheal prevalence, and reporting fatigue. Our study highlights both the difficulties in evaluating the impact of real-world interventions and the potential for an optimized SW-CRT design to address budgetary, funding, and logistical constraints inherent in such evaluations

Highly Efficient Regioselective Synthesis of 2‑Imino-4-oxothiazolidin-5-ylidene Acetates via a Substitution-Dependent Cyclization Sequence under Catalyst-Free Conditions at Ambient Temperature

A green and efficient method for the synthesis of newer 2-imino-4-oxothiazolidin-5-ylidene acetate derivatives under catalyst-free conditions by simply stirring symmetrical and unsymmetrical 1,3-diarylthioureas with dialkyl acetylenedicarboxylates in ethanol at room temperature has been developed. Interestingly, the regioselective synthesis affords the 2-imino-4-oxothiazolidin-5-ylidene acetate derivatives: the amine nitrogen bonded to an electron-withdrawing substituent becomes part of the imino component, and the amine nitrogen bonded to an electron-donating substituent becomes the heterocyclic nitrogen. This is the first report wherein the impact of substituents in directing the regiocyclization has been explained and the structure conflict resolved by single-crystal X-ray analysis