Expression of the extracellular domain of the human heat-stable enterotoxin receptor in Escherichia coli and generation of neutralizing antibodies

The entire extracellular domain of the human heat-stable enterotoxin (ST) receptor as well as a truncated N-terminal domain were cloned as glutathione S-transferase fusion proteins and expressed in Escherichia coli. The recombinant fusion proteins were purified from both the cytosol and the inclusion body fractions by selective detergent extraction followed by glutathione-agarose affinity chromatography. The purified protein, corresponding to the entire extracellular domain, bound the stable toxin peptide with an affinity comparable to that of the native receptor characterized from the human colonic T84 cell line. No binding was observed with the N-terminal truncated fragment of the receptor under similar conditions, Polyclonal antibodies were raised to the entire extracellular domain fusion protein as well as the truncated extracellular domain fusion protein, and the antibodies were purified by affinity chromatography. Addition of the purified antibodies to T84 cells inhibited ST binding and abolished ST-mediated cGMP production, indicating that critical epitopes involved in ligand interaction are present in the N-terminal fragment of the receptor, Purified antibodies recognized a single protein of M(r) 160,000 Da on Western blotting with T84 membranes, corresponding to a size of the native glycosylated receptor in T84 cells. These studies are the first report of the expression, purification, and characterization of any member of the guanylyl cyclase family of receptors in E. coli and show that binding of the toxin to the extracellular domain of the receptor is possible in the absence of any posttranslational modifications such as glycosylation. The recombinant fusion proteins as well as the antibodies that we have generated could serve as useful tools in the identification of critical residues of the extracellular domain involved in ligand interaction.

Time-frequency analysis of human motion during rhythmic exercises

Biomechanical signals due to human movements during exercise are represented in time-frequency domain using Wigner Distribution Function (WDF). Analysis based on WDF reveals instantaneous spectral and power changes during a rhythmic exercise. Investigations were carried out on 11 healthy subjects who performed 5 cycles of sun salutation, with a body-mounted Inertial Measurement Unit (IMU) as a motion sensor. Variance of Instantaneous Frequency (I.F) and Instantaneous Power (I.P) for performance analysis of the subject is estimated using one-way ANOVA model. Results reveal that joint Time-Frequency analysis of biomechanical signals during motion facilitates a better understanding of grace and consistency during rhythmic exercise.

Exploration of deleterious single nucleotide polymorphisms in the components of human P bodies: An in silico approach

P bodies are 100-300 nm sized organelles involved in mRNA silencing and degradation. A total of 60 human proteins have been reported to localize to P bodies. Several human SNPs contribute to complex diseases by altering the structure and function of the proteins. Also, SNPs alter various transcription factors binding, splicing and miRNA regulatory sites. Owing to the essential functions of P bodies in mRNA regulation, we explored computationally the functional significance of SNPs in 7 P body components such as XRN1, DCP2, EDC3, CPEB1, GEMIN5, STAU1 and TRIM71. Computational analyses of non-synonymous SNPs of these components was initiated using well utilized publicly available software programs such as the SIFT, followed by PolyPhen, PANTHER, MutPred, I-Mutant-2.0 and PhosSNP 1.0. Functional significance of noncoding SNPs in the regulatory regions were analysed using FastSNP. Utilizing miRSNP database, we explored the role of SNPs in the context that alters the miRNA binding sites in the above mentioned genes. Our in silico studies have identified various deleterious SNPs and this cataloguing is essential and gives first hand information for further analysis by in vitro and in vivo methods for a better understanding of maintenance, assembly and functional aspects of P bodies in both health and disease. (C) 2013 Elsevier B.V. All rights reserved.

Emergency Handling in MICS based Body Area Network

Body Area Network, a new wireless networking paradigm, promises to revolutionize the healthcare applications. A number of tiny sensor nodes are strategically placed in and around the human body to obtain physiological information. The sensor nodes are connected to a coordinator or a data collector to form a Body Area Network. The tiny devices may sense physiological parameters of emergency in nature (e.g. abnormality in heart bit rate, increase of glucose level above the threshold etc.) that needs immediate attention of a physician. Due to ultra low power requirement of wireless body area network, most of the time, the coordinator and devices are expected to be in the dormant mode, categorically when network is not operational. This leads to an open question, how to handle and meet the QoS requirement of emergency data when network is not operational? Emergency handling becomes more challenging at the MAC layer, if the channel access related information is unknown to the device with emergency message. The aforementioned scenarios are very likely scenarios in a MICS (Medical Implant Communication Service, 402-405 MHz) based healthcare systems. This paper proposes a mechanism for timely and reliable transfer of emergency data in a MICS based Body Area Network. We validate our protocol design with simulation in a C++ framework. Our simulation results show that more than 99 p ercentage of the time emergency messages are reached at the coordinator with a delay of 400ms.

Characterization of type I interferon pathway during hepatic differentiation of human pluripotent stem cells and hepatitis C virus infection

Pluripotent stem cells are being actively studied as a cell source for regenerating damaged liver. For long-term survival of engrafting cells in the body, not only do the cells have to execute liver-specific function but also withstand the physical strains and invading pathogens. The cellular innate immune system orchestrated by the interferon (IFN) pathway provides the first line of defense against pathogens. The objective of this study is to assess the innate immune function as well as to systematically profile the IFN-induced genes during hepatic differentiation of pluripotent stem cells. To address this objective, we derived endodermal cells (day 5 post-differentiation), hepatoblast (day 15) and hepatocyte-like cells (day 21) from human embryonic stem cells (hESCs). Day 5, 15 and 21 cells were stimulated with IFN-alpha and subjected to IFN pathway analysis. Transcriptome analysis was carried out by RNA sequencing. The results showed that the IFN-alpha treatment activated STAT-JAK pathway in differentiating cells. Transcriptome analysis indicated stage specific expression of classical and non-classical IFN-stimulated genes (ISGs). Subsequent validation confirmed the expression of novel ISGs including RASGRP3, CLMP and TRANK1 by differentiated hepatic cells upon IFN treatment. Hepatitis C virus replication in hESC-derived hepatic cells induced the expression of ISGs - LAMP3, ETV7, RASGRP3, and TRANK1. The hESC-derived hepatic cells contain intact innate system and can recognize invading pathogens. Besides assessing the tissue-specific functions for cell therapy applications, it may also be important to test the innate immune function of engrafting cells to ensure adequate defense against infections and improve graft survival. (C) 2015 The Authors. Published by Elsevier B.V.

Effect of potassium present in stratum corneum during non-invasive measurement of potassium in human subjects using reverse iontophoresis

Purpose: Reverse iontophoresis (RI) is one of the potential techniques used to monitor the concentration of various analytes in body fluids non -invasively. Transdermal extraction of potassium is investigated using RI. In the present work, the effect of potassium on stratum corneum (SC) during RI, feasibility of RI for continuous monitoring of potassium, and use of potassium as internal standard in RI, are investigated. Methods: Tape stripping experiment is carried out to find potassium concentration in SC. RI is carried out continuously for 180 min without passive diffusion and after passive diffusion for 60 min. Skin impedance measurements are done at 20 Hz and 20 kHz. Results: Potassium is found to be in the range 300-650 nmol/cm(2) on SC by tape stripping experiment. Correlation coefficient between blood potassium and extracted potassium through RI after passive diffusion (R-2 = 0.5870) is more than without passive diffusion (R-2 = 0.5117). The skin impedance measurement shows that RI has more effect on SC than superficial layer of SC during RI. Conclusion: The present investigations conclude that it is possible to monitor potassium continuously through RI and using potassium as internal standard in RI.