Prediction of MHC binding peptide using Gibbs motif sampler,weight matrix and artificial neural network

The identification of MHC restricted epitopes is an important goal in peptide based vaccine and diagnostic development. As wet lab experiments for identification of MHC binding peptide are expensive and time consuming, in silico tools have been developed as fast alternatives, however with low performance. In the present study, we used IEDB training and blind validation datasets for the prediction of peptide binding to fourteen human MHC class I and II molecules using Gibbs motif sampler, weight matrix and artificial neural network methods. As compare to MHC class I predictor based on sequence weighting (Aroc=0.95 and CC=0.56) and artificial neural network (Aroc=0.73 and CC=0.25), MHC class II predictor based on Gibbs sampler did not perform well (Aroc=0.62 and CC=0.19). The predictive accuracy of Gibbs motif sampler in identifying the 9-mer cores of a binding peptide to DRB1 alleles are also limited (40¢), however above the random prediction (14¢). Therefore, the size of dataset (training and validation) and the correct identification of the binding core are the two main factors limiting the performance of MHC class-II binding peptide prediction. Overall, these data suggest that there is substantial room to improve the quality of the core predictions using novel approaches that capture distinct features of MHC-peptide interactions than the current approaches.     

Nile Red is a fluorescent allosteric substrate of cytochrome P450 3A4

Cytochrome P450 3A4 (CYP3A4) plays a critical role in the metabolism of many drugs. CYP3A4 exhibits extraordinary substrate promiscuity and unusual allosteric kinetics. In addition, many CYPs catalyze sequential oxidations on a single substrate, but in most cases, mechanistic details of these processes are not well-established. As a result, in vivo clearance of many drugs and their metabolites is difficult to predict on the basis of the complex in vitro kinetics, and new in vitro probes are required to understand these behaviors. The near-IR fluorescent probe Nile Red, which has strong solvatochromic behavior, was investigated as a probe of allostery and sequential metabolism with CYP3A4. Nile Red binds with apparent Kd values of 0.05 and 2.3 muM, based on a sigmoidal dependence of heme spin state on Nile Red concentration, where the first equivalent of Nile Red increased the high-spin fraction by only 13% of the total change at saturation. Mass spectrometry analysis indicates that Nile Red is metabolized sequentially by CYP3A4 to the N-monoethyl and N-desethyl products, confirming that the immediate vicinity of the heme iron is one binding site. In the presence of CYP3A4, steady-state fluorescence emission and excitation spectra, as well as excited-state lifetimes at varying Nile Red concentrations, indicate a high-affinity site that modulates the fluorescent properties of Nile Red. The Nile Red binding site is competitively eliminated by itraconazole, which is a high-affinity ligand known to coordinate to the heme iron. Together, the data suggest that Nile Red binds to the active site with high affinity ( approximately 50 nM), where it is desolvated in a low-dielectric environment. In addition, Nile Red is sequentially oxidized at rates comparable to or faster than those of other in vitro probes, which emphasizes its utility in the further examination of this important kinetic phenomenon in vitro.     

The role of AMPK in psychosine mediated effects on oligodendrocytes and astrocytes: implication for Krabbe disease

Krabbe disease (KD) is an inherited neurological disorder caused by the deficiency of galactocerebrosidase activity resulting in accumulation of psychosine, which leads to energy depletion, loss of oligodendrocytes, induction of gliosis, and inflammation by astrocytes in CNS. In this study, for the first time, we report the regulation of 'cellular energy switch,' AMP-activated protein kinase (AMPK), by psychosine in oligodendrocytes and astrocytes. Psychosine treatment significantly down-regulated AMPK activity, resulting in increased biosynthesis of lipids including cholesterol and free fatty acid in oligodendrocytes cell line (MO3.13) and primary astrocytes. Pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) attenuated the psychosine-mediated down-regulation of AMPK and restored altered biosynthesis of lipids. AICAR treatment also down-regulated psychosine induced expression of proinflammatory cytokines and inducible nitric oxide synthase in primary astrocytes. However, AICAR treatment had no effect on psychosine induced-reactive oxygen species generation, arachidonic acid release, and death of oligodendrocytes; suggesting the specific role of AMPK in regulation of psychosine-mediated inflammatory response of astrocytes but not in cell death of oligodendrocytes. This study delineates an explicit role for AMPK in psychosine induced inflammation in astrocytes without directly affecting the cell death of oligodendrocytes. It also suggests that AMPK activating agents act as anti-inflammatory agents and can hold a therapeutic potential in Krabbe disease/twitcher disease, particularly when used in combination with drugs, which protect oligodendrocyte cell loss, such as sPLA2 inhibitor [ Giri et al. , J. Lipid Res. 47 (2006), 1478 ].     

Artificial chaperone mediated refolding of xylanase from an alkalophilic thermophilic Bacillus sp. Implications for in vitro protein renaturation via a folding intermediate.

To gain insight into the molecular aspects of unfolding/refolding of enzymes from extremophilic organisms, we have used xylanase from an alkalophilic thermophilic Bacillus as the model system. Kinetics of denaturation/renaturation were monitored using intrinsic fluorescence studies. The protein fluorescence measurements suggested a putative intermediate state present in 0.08 M guanidine hydrochloride with an emission maximum of 345 nm; the far-UV circular dichroism spectra revealed content of secondary structure similar to the native enzyme. Studies with the fluorescent apolar probe 1-anilinonapthalene-8-sulfonate (1,8-ANS) were consistent with the presence of increased hydrophobic surfaces as compared with the native or fully unfolded protein. The refolding of Xyl II, was attempted by a relatively new strategy using an artificial chaperone assisted two-step method. The unfolded xylanase was found to bind to the detergent transiently and the subsequent addition of methyl-beta-cyclodextrin helped to strip the detergent and assist in the folding. Our findings suggested that the detergent stabilized a putative intermediate in the folding pathway seemingly equivalent to the folding state described as molten globule. The reactivation of Xyl II was affected by ionic as well as nonionic detergents. However, the cationic detergent cetyltrimethylammonium bromide (CTAB) provided a maximum reactivation (threefold) of the enzyme. The 'delayed detergent addition' experiments revealed that the detergent acts by suppressing the initial aggregate formation and not by dissolving aggregates. The relevance of our findings to the role of artificial chaperones in vivo is discussed.     

Exploring Indus crop processing: combining phytolith and macrobotanical analyses to consider the organisation of agriculture in northwest India c. 3200–1500 <Emphasis Type="SmallCaps">bc</Emphasis>

This paper presents a preliminary study combining macrobotanical and phytolith analyses to explore crop processing at archaeological sites in Haryana and Rajasthan, northwest India. Current understanding of the agricultural strategies in use by populations associated with South Asia’s Indus Civilisation (3200–1900 bc) has been derived from a small number of systematic macrobotanical studies focusing on a small number of sites, with little use of multi-proxy analysis. In this study both phytolith and macrobotanical analyses are used to explore the organisation of crop processing at five small Indus settlements with a view to understanding the impact of urban development and decline on village agriculture. The differing preservation potential of the two proxies has allowed for greater insights into the different stages of processing represented at these sites: with macrobotanical remains allowing for more species-level specific analysis, though due to poor chaff presentation the early stages of processing were missed; however these early stages of processing were evident in the less highly resolved but better preserved phytolith remains. The combined analyses suggests that crop processing aims and organisation differed according to the season of cereal growth, contrary to current models of Indus Civilisation labour organisation that suggest change over time. The study shows that the agricultural strategies of these frequently overlooked smaller sites question the simplistic models that have traditionally been assumed for the time period, and that both multi-proxy analysis and rural settlements are deserving of further exploration.     

Protein,enzyme and carbohydrate quantification using smartphone through colorimetric digitization technique

In this paper the utilization of smartphone as a detection platform for colorimetric quantification of biological macromolecules has been demonstrated. Using V‐channel of HSV color space, the quantification of BSA protein, catalase enzyme and carbohydrate (using D‐glucose) have been successfully investigated. A custom designed android application has been developed for estimating the total concentration of biological macromolecules. The results have been compared with that of a standard spectrophotometer which is generally used for colorimetric quantification in laboratory settings by measuring its absorbance at a specific wavelength. The results obtained with the designed sensor is found to be similar when compared with the spectrophotometer data. The designed sensor is low cost, robust and we envision that it could promote diverse fields of bio‐analytical investigations.

Schematic illustration of the smartphone sensing mechanism for colorimetric analysis of biomolecular samples.     

Hydrogen production by Rhodobacter sphaeroides strain O.U.001 using spent media of Enterobacter cloacae strain DM11

Combined dark and photo-fermentation was carried out to study the feasibility of biological hydrogen production. In dark fermentation, hydrogen was produced by Enterobacter cloacae strain DM11 using glucose as substrate. This was followed by a photo-fermentation process. Here, the spent medium from the dark process (containing unconverted metabolites, mainly acetic acid etc.) underwent photo-fermentation by Rhodobacter sphaeroides strain O.U.001 in a column photo-bioreactor. This combination could achieve higher yields of hydrogen by complete utilization of the chemical energy stored in the substrate. Dark fermentation was studied in terms of several process parameters, such as initial substrate concentration, initial pH of the medium and temperature, to establish favorable conditions for maximum hydrogen production. Also, the effects of the threshold concentration of acetic acid, light intensity and the presence of additional nitrogen sources in the spent effluent on the amount of hydrogen produced during photo-fermentation were investigated. The light conversion efficiency of hydrogen was found to be inversely proportional to the incident light intensity. In a batch system, the yield of hydrogen in the dark fermentation was about 1.86 mol H₂ mol⁻¹ glucose; and the yield in the photo-fermentation was about 1.5–1.72 mol H₂ mol⁻¹ acetic acid. The overall yield of hydrogen in the combined process, considering glucose as the preliminary substrate, was found to be higher than that in a single process.     

Differences in morphology of phagosomes and kinetics of acidification and degradation in phagosomes between the pathogenic Entamoeba histolytica and the non-pathogenic Entamoeba dispar

Phagocytosis plays an important role in the pathogenicity of the intestinal protozoan parasite Entamoeba histolytica. We compared the morphology of phagosomes and the kinetics of phagosome maturation using conventional light and electron microscopy and live imaging with video microscopy between the virulent E. histolytica and the closely-related, but non-virulent E. dispar species. Electron micrographs showed that axenically cultivated trophozoites of the two Entamoeba species revealed morphological differences in the number of bacteria contained in a single phagosome and the size of phagosomes. Video microscopy using pH-sensitive fluorescein isothiocynate-conjugated yeasts showed that phagosome acidification occurs within 2 min and persists for >12 h in both species. The acidity of phagosomes significantly differed between two species (4.58 +/- 0.36 or 5.83 +/- 0.38 in E. histolytica or E. dispar, respectively), which correlated well with the differences in the kinetics of degradation of promastigotes of GFP-expressing Leishmania amazonensis. The acidification of phagosomes was significantly inhibited by a myosin inhibitor, whereas it was only marginally inhibited by microtubules or actin inhibitors. A specific inhibitor of vacuolar ATPase, concanamycin A, interrupted both the acidification and degradation in phagosomes in both species, suggesting the ubiquitous role of vacuolar ATPase in the acidification and degradation in Entamoeba. In contrast, inhibitors against microtubules or cysteine proteases (CP) showed distinct effects on degradation in phagosomes between these two species. Although depolymerization of microtubules severely inhibited degradation in phagosomes of E. histolytica, it did not affect degradation in E. dispar. Similarly, the inhibition of CP significantly reduced degradation in phagosomes of E. histolytica, but not in E. dispar. These data suggest the presence of biochemical or functional differences in the involvement of microtubules and proteases in phagosome maturation and degradation between the two species.     

Nature of stress: differential effects on brain acetylcholinesterase activity and memory in rats

Effect of acute, chronic-predictable and chronic-unpredictable stress on memory and acetylcholinesterase (AChE) was investigated in rats. The animals were subjected to 3 type of stressors--(1) acute immobilization stress, (2) chronic-predictable stress i.e., immobilization daily for 5 consecutive days and (3) chronic-unpredictable stress that included reversal of light/dark cycle, over-night fasting, forced-swimming, immobilization and forced exercise in random unpredictable manner daily for 5 consecutive days. Learning and memory function was studied by single trial Passive avoidance test. AChE activity was assayed spectrophotometrically in the detergent (DS) and salt (SS) soluble fractions in different brain regions. Learning was obtained in acute and chronic-predictable stress groups but not in chronic-unpredictable group. Acute, chronic-predictable and chronic-unpredictable stress caused significant decrease in AChE activity in the DS fraction of cortex, hippocampus and hypothalamus as compared to control. Results indicate that AChE in DS fraction is predominantly affected in stressed and stressed-trained group but cognition is affected only by chronic-unpredictable stress. In acute and chronic-predictable groups the decreased AChE activity in the hippocampal DS fraction during learning may be responsible to maintain cognitive function by enhancing the cholinergic activity.