A Prospective Study on the Diagnostic Value of Hyperbilirubinemia as a Predictive Factor for Appendicular Perforation in Acute Appendicitis.

Background Appendicitis is one of the most common surgical emergency in general surgical practices. Early and prompt diagnosis is necessary to avoid life-threatening complications associated with it. The diagnosis is mainly clinically aided by imaging techniques. The physiological obstruction of the bile flow associated with appendicular pathology leads to hyperbilirubinemia, which can be used as a predictive factor of appendicular perforation. Method This prospective study was conducted in the department of general surgery in Madras Medical College and Rajiv Gandhi Government Hospital, Chennai, from January 2012 to November 2012. A total of 378 patients with the features of acute appendicitis or appendicular perforation admitted in the emergency surgical ward were included. Results Out of 378 of the study population, 18% had appendicular perforation and 82% had acute appendicitis. Out of 67 perforations, 60 patients have hyperbilirubinemia (90%) whereas out of 311 patients with appendicitis, only 89 (29%) of them had elevated bilirubin. Hyperbilirubinemia with a cutoff point of 0.9 mg% for appendicitis patients has a sensitivity of 89.6%, specificity of 71.4%, a positive predictive value of 27%, and a negative predictive value of 96.9%. Hyperbilirubinemia with a cutoff point of >1.3 mg% for appendicular perforation has a sensitivity of 80%, specificity of 89%, a positive predictive value of 93%, and a negative predictive value of 96%. Conclusions Hyperbilirubinemia with bilirubin levels more than 1.3 mg% are highly predictive of appendicular perforation and, hence, aid in prompt diagnosis. This can be combined with a clinical diagnosis and imaging for an accurate and precise diagnosis.

A novel C-terminal region within the multicargo type III secretion chaperone CesT contributes to effector secretion.

The enteropathogenic Escherichia coli (EPEC) multicargo chaperone CesT interacts with at least 10 effector proteins and is central to pathogenesis. CesT has been implicated in coordinating effector hierarchy, although the mechanisms behind this regulation are poorly understood. To address this question, we set out to functionally characterize CesT with respect to roles in (i) effector binding, (ii) effector recruitment to the type III secretion system (T3SS), and (iii) effector translocation into host cells. A CesT variant expression library was screened in EPEC using a newly developed semi-high-throughput secretion assay. Among many deficient CesT variants, a predominant number were localized to a novel CesT C-terminal region. These CesT C-terminal variants exhibited normal effector binding yet reduced effector secretion levels. Structural correlation and thermal spectroscopy analyses of purified CesT variants implicated multiple surface-exposed residues, a terminal helix region, and a flexible C-terminal triple-serine stretch in effector secretion. Site-directed mutagenesis of the flexible CesT C-terminal triple-serine sequence produced differential effector secretion, implicating this region in secretion events. Infection assays further indicated that the C-terminal region of CesT was important for NleA translocation into host cells but was dispensable for Tir translocation. The findings implicate the CesT C terminus in effector secretion and contribute to a model for multiple-cargo chaperone function and effector translocation into host cells during infection.

Ca2+ increases the specific coenzyme Q10 content in Agrobacterium tumefaciens.

Of various metal ions (Ca2+, Cr3+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+ and Zn2+) added to the culture medium of Agrobacterium tumefaciens at 1 mM, only Ca2+ increased Coenzyme Q10 (CoQ(10)) content in cells without the inhibition of cell growth. In a pH-stat fed-batch culture, supplementation with 40 mM of CaCO3 increased the specific CoQ(10) content and oxidative stress by 22.4 and 48%, respectively. Also, the effect of Ca2+ on the increase of CoQ(10) content was successfully verified in a pilot-scale (300 L) fermentor. In this study, the increased oxidative stress in A. tumefaciens culture by the supplementation of Ca2+ is hypothesized to stimulate the increase of specific CoQ(10) content in order to protect the membrane against lipid peroxidation. Our results improve the understanding of Ca2+ effect on CoQ(10) biosynthesis in A. tumefaciens and should contribute to better industrial production of CoQ(10) by biological processes.

Identification and characterization of a novel nitrilase from Pseudomonas fluorescens Pf-5.

Nitrile groups are catabolized to the corresponding acid and ammonia through one-step reaction involving a nitrilase. Here, we report the use of bioinformatic and biochemical tools to identify and characterize the nitrilase (NitPf5) from Pseudomonas fluorescens Pf-5. The nitPf5 gene was identified via sequence analysis of the whole genome of P. fluorescens Pf-5 and subsequently cloned and overexpressed in Escherichia coli. DNA sequence analysis revealed an open-reading frame of 921 bp, capable of encoding a polypeptide of 307 amino acids residues with a calculated isoelectric point of pH 5.4. The enzyme had an optimal pH and temperature of 7.0 degrees C and 45 degrees C, respectively, with a specific activity of 1.7 and 1.9 micromol min(-1) mg protein(-1) for succinonitrile and fumaronitrile, respectively. The molecular weight of the nitrilase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography was 33,000 and 138,000 Da, respectively, suggesting that the enzyme is homotetrameric. Among various nitriles, dinitriles were the preferred substrate of NitPf5 with a K (m) = 17.9 mM and k (cat)/K (m) = 0.5 mM(-1) s(-1) for succinonitrile. Homology modeling and docking studies of dinitrile and mononitrile substrate into the active site of NitPf5 shed light on the substrate specificity of NitPf5. Although nitrilases have been characterized from several other sources, P. fluorescens Pf-5 nitrilase NitPf5 is distinguished from other nitrilases by its high specific activity toward dinitriles, which make P. fluorescens NitPf5 useful for industrial applications, including enzymatic synthesis of various cyanocarboxylic acids.

Identification of a molecular marker for genotyping human lymphatic filarial nematode parasite Wuchereria bancrofti.

In India, Mass Drug Administration is on going towards elimination of lymphatic filariasis in many areas, which might lead to intense selection pressure on the parasite populations and their genetic restructuring. This calls for molecular finger printing of Wuchereria bancrofti parasite populations at national level and monitoring genetic changes in the future. For this purpose a reliable, less expensive, rapid, and reproducible molecular tool is necessary, which is not available for W. bancrofti at this time. We identified robust molecular markers based on the comparison of random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) profiles and the genetic data generated from parasite populations collected from areas in Northern (Varanasi, Uttar Pradesh state), Southern (Kozhikode, Kerala State) and Central regions (Jagdalpur, Chattisgarh state) of India, where lymphatic filariasis is endemic for many decades. RAPD profiles for these parasite populations were generated using three different primers and the dendrograms constructed using the profiles were all different. In order to identify appropriate RAPD primer(s), we compared the results of RAPD with the fingerprint profile and genetic data obtained by the more reliable AFLP technique, using the parasite populations from the same areas. RAPD marker (OP8) primer produced phylogenetic data almost similar to that of AFLP analysis. The marker was able to reveal variations between the parasite populations collected from Varanasi, Kozhikode, and Jagdalpur. Most importantly, RAPD primer OP8 produced reproducible results, when tested in three different trials. In view of the limited availability of W. bancrofti parasite DNA, along with a lower cost and ease of performance, RAPD appears to be more suitable compared to AFLP at the present juncture, since complete genome information of this parasite is still not available. Thus, RAPD primer OP8 can be a very useful molecular maker for DNA finger printing of W. bancrofti populations at present.