Circulating platelet-neutrophil aggregates characterize the development of type 1 diabetes in humans and NOD mice.

Platelet-neutrophil aggregates (PNAs) facilitate neutrophil activation and migration and could underpin the recruitment of neutrophils to the pancreas during type 1 diabetes (T1D) pathogenesis. PNAs, measured by flow cytometry, were significantly elevated in the circulation of autoantibody-positive (Aab+) children and new-onset T1D children, as well as in pre-T1D (at 4 weeks and 10-12 weeks) and T1D-onset NOD mice, compared with relevant controls, and PNAs were characterized by activated P-selectin+ platelets. PNAs were similarly increased in pre-T1D and T1D-onset NOD isolated islets/insulitis, and immunofluorescence staining revealed increased islet-associated neutrophil extracellular trap (NET) products (myeloperoxidase [MPO] and citrullinated histones [CitH3]) in NOD pancreata. In vitro, cell-free histones and NETs induced islet cell damage, which was prevented by the small polyanionic drug methyl cellobiose sulfate (mCBS) that binds to histones and neutralizes their pathological effects. Elevated circulating PNAs could, therefore, act as an innate immune and pathogenic biomarker of T1D autoimmunity. Platelet hyperreactivity within PNAs appears to represent a previously unrecognized hematological abnormality that precedes T1D onset. In summary, PNAs could contribute to the pathogenesis of T1D and potentially function as a pre-T1D diagnostic.

Comparative evaluation of the effect of electrolyzed oxidizing water on surface detail reproduction, dimensional stability and Surface texture of poly vinyl siloxane impressions.

AIM: The aim of this in vitro study was to comparatively evaluate the effect of chemical disinfectants on the surface detail reproduction, dimensional stability and surface texture of polyvinyl siloxane (PVS) impressions. MATERIALS AND METHODS: The impressions were then divided into five groups (fifteen samples per group) and subjected to a ten minutes immersion with 2% glutaraldehyde (Group I), 1% sodium hypochlorite (Group II), freshly prepared electrolyzed oxidizing water (EOW) with different pH values - acidic (Group III), alkali (Group IV) and neutral (Group V). The samples were examined pre and post-immersion under visual observation for surface detail reproduction, travelling microscope for measurement of dimensional stability and surface profilometer (3D) for evaluation of surface texture. A standardized master die was fabricated and seventy-five PVS test samples were made. The samples were subjected to immersion disinfection and studied for surface detail reproduction, dimensional stability and surface texture. Post-hoc test, paired t test and ANOVA were used to analyze dimensional stability statistically both within and between the test groups. RESULTS: The surface detail reproduction was satisfactory with both pre and post-immersion test samples. A statistically significant dimensional change was observed post-immersion in Groups II, III and V test samples and a statistically insignificant dimensional change was observed in Groups I and IV test samples. There was a negligible change in surface texture post-immersion in Groups I, III, IV and V test samples with a slight increase in surface roughness post-immersion in Group II samples. CONCLUSION: In this study, all the test disinfectants produced satisfactory surface detail reproduction on Polyvinyl siloxane impressions. 2% glutaraldehyde and electrolyzed oxidizing water (alkali) have resulted in statistically insignificant dimensional change, while 1% sodium hypochlorite, electrolyzed oxidizing water (acidic) and electrolyzed oxidizing water (neutral) have resulted in statistically significant dimensional changes. All the test disinfectants except 1% sodium hypochlorite showed a reduction in surface roughness (Ra) values.

Evaluating the impact of diabetes and diabetic cardiomyopathy rat heart on the outcome of ischemia-reperfusion associated oxidative stress.

Earlier literature underlines that oxidative stress plays a major role in the pathology of myocardial ischemia-reperfusion (I/R) injury, diabetic cardiomyopathy (DCM), diabetes mellitus (DM), fibrosis and hypertrophy which could adversely affect the normal cardiac function. However, the contributory role of oxidative stress in I/R pathology of heart with pre-existing abnormalities or diseases like DM and DCM remains to be explored. I/R injury was induced in normal (normal diet), DM (normal diet + streptozotocin: multiple low dose of 30 mg/kg) and DCM (high fat diet (40% fat) + streptozotocin: multiple low dose of 30 mg/kg) rat hearts using Langendorff isolated heart perfusion apparatus. Cardiac physiological recovery after I/R was assessed by hemodynamic parameters like LVDP, and LVSP, whereas cardiac injury was measured by tissue infarct size, and apoptosis, LDH, and CK release in coronary effluent. The oxidative stress was evaluated in myocardial homogenate, mitochondrial subpopulation, and microsomes. Reperfusing the ischemic DCM heart significantly deteriorated cardiac physiological recovery and elevated the cardiac injury (infarct size: 60%), compared to the control. But in DM heart, physiological recovery was prominent in the initial phase of reperfusion but deteriorated towards the end of reperfusion, supported by less infarct size. In addition, elevated lipid peroxidation (70% in DCM-I/R vs Sham) and impaired antioxidant enzymes (% decline vs Sham: GSH - 56% (DM), 63% (DCM); Catalase - 58% (DM), 35% (DCM); GPx - 19% (DM), 27% (DCM) and GR - 28% (DCM)) was observed in myocardial tissue from both DM and DCM. Interestingly, upon reperfusion, only normal heart showed significant deterioration in the antioxidant defense system. Collectively these results demonstrated that I/R induced oxidative stress is minimal in DM and DCM rat heart, despite high infarct size and low cardiac performance. This may be due to the prior adaptive modification in the antioxidant system associated with disease pathology.

Dimensional change in soft tissues with complete dental prosthesis and its effect on airway space and natural head position.

BACKGROUND: Insertion of the intraoral prosthesis causes deflection of the tongue, soft palate affecting the patency of the airway. AIMS: To evaluate the cephalometric soft tissue variation in tongue, soft palate, and its effect on posterior airway space in edentulous patients with the insertion of the denture. SETTINGS AND DESIGN: Randomized controlled trial. MATERIALS AND METHODS: Complete denture (CD) prosthesis was fabricated for first time denture wearers comprising 22 males and 18 females within the age range of 50-65 years. Lateral skull radiographs were taken for each of the subjects in their resting position without CDs (T0), with CDs (T1) at least 1-week postinsertion and after 6 months of usage (T2), respectively. The reference points, lines, and angles on the cephalometric films were used to evaluate the position or inclination of the head, variations in tongue position and length, the changes in the anteroposterior dimension of the soft palate, and posterior airway space variations. Intra-investigator error variance was not found to be statistically significant (P < 0.05). STATISTICAL ANALYSIS USED: Using Statistical Package for the Social Sciences (SPSS) v16.0 the significant difference in the paired samples (without CD and with CD) was found using the paired t-test. The probability value P = 0.05 is considered as a significant level. RESULTS: On comparison of T1 and T0, the heads were more extended with the downward position of the tongue with an increase in its length and the posterior airway space was decreased in T1. Significant differences in values were not observed between T1 and T2. CONCLUSIONS: With CDs, the change in airway space was related to alteration in soft palate and tongue dimension. There were no changes in dimension at 6 months follow-up.

Expression, purification and molecular modeling of the NIa protease of Cardamom mosaic virus.

The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8 M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease.

Homology modeling of Cry10Aa toxin from B. thuringiensis israelensis and B. thuringiensis subsp. LDC-9.

A three dimensional model was developed for Cry10Aa protein sequence of B. thuringiensis LDC-9 and B. thuringiensis israelensis that has not been solved empirically by X-ray crystallography or NMR. Homology modeling was employed for the structure prediction using Cry2Aa as template protein, a high-resolution X-ray crystallography structure. The model predicted for the B. thuringiensis LDC-9 Cry10Aa protein reveals a partial N-terminal domain only due to its partial sequence of 104 amino acids. B. thuringiensis israelensis Cry10Aa model contains three domains such as domain I, a bundle of eight alpha helices with the central relatively hydrophobic helix surrounded by amphipathic helices while domain II and III contain mostly beta-sheets. Significant structural differences within domain II in this model among all Cry protein structures indicates that it is involved in recognition and binding to cell surfaces. Comparison of B. thuringiensis israelensis predicted structure with available experimentally determined Cry structures reveals identical folds. The distribution of electrostatic potential on the surface of the molecules in the model is non-uniform and identifies one side of the alpha-helical domain as negatively charged indicating orientation of toxic molecules toward the cell membrane during the initial binding with a cell surface receptor. The collective knowledge of Cry toxin structures will lead to a more critical understanding of the structural basis for receptor binding and pore formation, as well as allowing the scope of diversity to be better appreciated. This model will serve as a starting point for the design of mutagenesis experiments aimed to improve the toxicity and to provide a new tool for the elucidation of the mechanism of action of these mosquitocidal proteins.

Molecular modeling and characterization of the B. thuringiensis and B. thuringiensis LDC-9 cytolytic proteins.

The Cyt toxins are able to lyse a wide range of cell types in vitro, unlike the Cry delta-endotoxins. It exerts its activity by the formation of pores within target cell membranes. The structural information available for Cyt2Aa (PDB id: 1CBY) consists of a single domain in which two outer layers of alpha-helix wrap around a mixed beta-sheet. Beta-barrel was suggested as a possible structure of the pores. Hence, this study seeks to investigate the structural properties of other Cytolytic proteins by predicting the three-dimensional (3D) model using Cyt2Aa as template. The predicted models are expected to be significantly more accurate as all the Cyt proteins showed significant similarity with the template (PDB id: 1CBY). The refined homology models revealed similar secondary structures (alpha-helices and beta-sheets) and tertiary features as Cyt2Aa. The variation in the loop regions of the tertiary structure accounts for the differential toxicity.

Identification of novel indigenous Bacillus thuringiensis isolates.

Internally Transcribed Spacers (ITS) characterization and distribution of Repetitive Extragenic Palindromic (REP) sequences were studied in the genome of 223 field isolates of Bacillus thuringiensis from Madurai, India. They were characterized by morphological, biochemical and molecular methods. One hundred and twenty four of a total 223 isolates fitted ITS characterization of B. thuringiensis varieties known. Significant genomic variation was observed among seven isolates using REP primers. The ITS PCR product (EMBL accession number AJ639659) exhibited 98% nucleotide sequence homology with B. thuringiensis and placed the origin of indigenous isolate LDC-7 closer to B. thuringiensis on the basis of phylogenetic analysis.

Effect of water stress and heavy metals on induction of somatic embryogenesis in wheat leaf base cultures.

In vitro cultures of plant tissues are known to mimic the response of field-grown plants when subjected to stress treatments. This investigation on Triticum aestivum explores the effect of drought stress on somatic embryogenesis and endogenous proline content. Leaf bases were cultured on MS medium supplemented with 2,4-D (10 microM) and different concentrations of PEG (2.5, 5, 7.5%) or mannitol (0.25 and 0.5 M) and also subjected to different periods of aerial drying in the laminar flow for one-day and subsequently transferred to MS basal medium. PEG treatment induced a high percentage (up to 50%) of embryoid formation. However, with mannitol and aerial drying, percentage of embryoid formation decreased with increasing concentrations and duration. After ten days, the endogenous proline content of explants treated with different concentrations of PEG, mannitol and different durations of aerial drying increased with increasing concentration and increasing duration of the treatment, thus, corroborating the role of proline as an osmolyte during stress conditions. Similarly, addition of metals such as cadmium and cobalt caused a reduction in percentage explants depicting embryogenesis. However, when cadmium was employed alone, 22% explants displayed somatic embryogenesis as compared to 54% in 2,4-D treated cultures.