The PE-PPE domain in mycobacterium reveals a serine α/β hydrolase fold and function: an in-silico analysis

The PE and PPE proteins first reported in the genome sequence of Mycobacterium tuberculosis strain H37Rv are now identified in all mycobacterial species. The PE-PPE domain (Pfam ID: PF08237) is a 225 amino acid residue conserved region located towards the C-terminus of some PE and PPE proteins and hypothetical proteins. Our in-silico sequence analysis revealed that this domain is present in all Mycobacteria, some Rhodococcus and Nocardia farcinica genomes. This domain comprises a pentapeptide sequence motif GxSxG/S at the N-terminus and conserved amino acid residues Ser, Asp and His that constitute a catalytic triad characteristic of lipase, esterase and cutinase activity. The fold prediction and comparative modeling of the 3-D structure of the PE-PPE domain revealed a "serine α/β hydrolase" structure with a central β-sheet flanked by α-helices on either side. The structure comprises a lid insertion with a closed structure conformation and has a solvent inaccessible active site. The oxyanion hole that stabilizes the negative charge on the tetrahedral intermediate has been identified. Our findings add to the growing list of serine hydrolases in mycobacterium, which are essential for the maintenance of their impermeable cell wall and virulence. These results provide the directions for the design of experiments to establish the function of PE and PPE proteins.     

Pathogen Induced Changes in the Protein Profile of Human Tears from Fusarium Keratitis Patients

Fusarium is the major causative agent of fungal infections leading to corneal ulcer (keratitis) in Southern India and other tropical countries. Keratitis caused by Fusarium is a difficult disease to treat unless antifungal therapy is initiated during the early stages of infection. In this study tear proteins were prepared from keratitis patients classified based on the duration of infection. Among the patients recruited, early infection (n = 35), intermediate (n = 20), late (n = 11), samples from five patients in each group were pooled for analysis. Control samples were a pool of samples from 20 patients. Proteins were separated on difference gel electrophoresis (DIGE) and the differentially expressed proteins were quantified using DeCyder software analysis. The following differentially expressed proteins namely alpha-1-antitrypsin, haptoglobin α2 chain, zinc-alpha-2-glycoprotein, apolipoprotein, albumin, haptoglobin precursor - β chain, lactoferrin, lacrimal lipocalin precursor, cystatin SA III precursor, lacritin precursor were identified using mass spectrometry. Variation in the expression level of some of the proteins was confirmed using western blot analysis. This is the first report to show stage specific tear protein profile in fungal keratitis patients. Validation of this data using a much larger sample set could lead to clinical application of these findings.     

in silico identification of cross affinity towards Cry1Ac pesticidal protein with receptor enzyme in Bos taurus and sequence,structure analysis of crystal proteins for stability

Any novel protein introduced into the GM crops need to be evaluated for cross affinity on living organisms. Many researchers are currently focusing on the impact of Bacillus thuringiensis cotton on soil and microbial diversity by field experiments. In spite of this, in silico approach might be helpful to elucidate the impact of cry genes. The crystal a protein which was produced by Bt at the time of sporulation has been used as a biological pesticide to target the insectivorous pests like Cry1Ac for Helicoverpa armigera and Cry2Ab for Spodoptera sp. and Heliothis sp. Here, we present the comprehensive in silico analysis of Cry1Ac and Cry2Ab proteins with available in silico tools, databases and docking servers. Molecular docking of Cry1Ac with procarboxypeptidase from Helicoverpa armigera and Cry1Ac with Leucine aminopeptidase from Bos taurus has showed the 125^th amino acid position to be the preference site of Cry1Ac protein. The structures were compared with each other and it showed 5% of similarity. The cross affinity of this toxin that have confirmed the earlier reports of ill effects of Bt cotton consumed by cattle.     

Dry-reagent gold nanoparticle-based lateral flow biosensor for the simultaneous detection of Vibrio cholerae serogroups O1 and O139

Cholera is a communicable disease caused by consumption of contaminated food and water. This potentially fatal intestinal infection is characterised by profuse secretion of rice watery stool that can rapidly lead to severe dehydration and shock, thus requiring treatment to be given immediately. Epidemic and pandemic cholera are exclusively associated with Vibrio cholerae serogroups O1 and O139. In light of the need for rapid diagnosis of cholera and to prevent spread of outbreaks, we have developed and evaluated a direct one-step lateral flow biosensor for the simultaneous detection of both V. cholerae O1 and O139 serogroups using alkaline peptone water culture. Serogroup specific monoclonal antibodies raised against lipopolysaccharides (LPS) were used to functionalize the colloidal gold nanoparticles for dual detection in the biosensor. The assay is based on immunochromatographic principle where antigen-antibody reaction would result in the accumulation of gold nanoparticles and thus, the appearance of a red line on the strip. The dry-reagent dipstick format of the biosensor ensure user-friendly application, rapid result that can be read with the naked eyes and cold-chain free storage that is well-suited to be performed at resource-limited settings.     

Modal analysis of the deep-water solitary scleractinian, <Emphasis Type="Italic">Desmophyllum dianthus</Emphasis>, on SW Pacific seamounts: inferred recruitment periodicity,growth, and mortality rates

Little is known about the demography of corals inhabiting deep-sea features due to the logistical difficulties of working at the extreme depths they inhabit. To obtain basic information about growth, mortality, and recruitment dynamics for such a coral, we applied modal analysis to the size frequency distributions of live-caught and sub-fossil specimens of the widely distributed solitary cup coral, Desmophyllum dianthus, collected on SW Pacific seamounts. Comparison of live-caught material collected in 1997 and 2007–2009 indicated modal progression over time and an implied maximum age of approximately 190 years, which is similar to ages determined previously for D. dianthus using radiometric techniques. A log-linear decline in the number of individuals with increasing size further implies a constant adult mortality rate, of 15.1% per annum in 1997 and 9.2% per annum in 2007–2009. The spacing of size modes in the 2007–2009 samples suggests regularly episodic recruitment events, at 22- to 32-year intervals, which may relate to periodic variability in large-scale Southern Ocean circulation. Preliminary analyses of size frequency distributions of the sub-fossil material suggest that the trophodynamics, growth, and adult mortality schedules of D. dianthus in the SW Pacific have remained basically similar throughout the Holocene.     

Immune Response in Goats to Different Payloads of FMDV Monovalent Vaccine: Protection Against Virulent Challenge and Development of Carrier Status

The relationship of Foot-and-Mouth Disease virus antigen payload and number of dose of vaccine conferring protection against virus challenge in goats was studied. Goats vaccinated with oil adjuvant Foot-and-Mouth Disease vaccines containing different antigen payloads with or without booster resisted virulent challenge at 21 days post-vaccination or 7 days after booster respectively. However, localized sub-clinical infection was observed in two vaccinated goats on 35 days post-challenge. RNA could be detected from 31.8% of vaccinated goats (10^2.69–10^4.99 viral RNA copies per cotton swab of nasal secretions) on day 35 post-challenge. Since no live virus could be isolated after 5 days post-challenge, the risk of these animals transmitting the disease was probably very low. The finding showed that oil adjuvant Foot-and-Mouth Disease vaccines containing antigen payload of 1.88 μg may prevent or reduce the local virus replication at the oropharynx and shedding of virus from nasal secretions and thereby reduce the amount of virus released into the environment subsequent to exposure to live virus. This study also showed that goats with poor sero conversion to vaccination can be infected without overt clinical signs and became carriers like sheep.     

Low molecular weight chitosans—Preparation with the aid of pronase,characterization and their bactericidal activity towards Bacillus cereus and Escherichia coli

The homogeneous low molecular weight chitosans (LMWC) of molecular weight 9.5–8.5 kDa, obtained by pronase catalyzed non-specific depolymerization (at pH 3.5, 37 °C) of chitosan showed lyses of Bacillus cereus and Escherichia coli more efficiently (100%) than native chitosan (< 50%). IR and ¹H-NMR data showed decrease in the degree of acetylation (14–19%) in LMWC compared to native chitosan (∼ 26%). Minimum inhibitory concentration of LMWC towards 10⁶ CFU ml^− 1 of B. cereus was 0.01% (w/v) compared to 0.03% for 10⁴ CFU ml^− 1 of E. coli. SEM revealed pore formation as well as permeabilization of the bacterial cells, as also evidenced by increased carbohydrate and protein contents as well as the cytoplasmic enzymes in the cell-free supernatants. N-terminal sequence analyses of the released proteins revealed them to be cytoplasmic/membrane proteins. Upon GLC, the supernatant showed characteristic fatty acid profiles in E. coli, thus subscribing to detachment of lipopolysaccharides into the medium, whereas that of B. cereus indicated release of surface lipids. The mechanism for the observed bactericidal activity of LMWC towards both Gram-positive and Gram-negative bacteria has been discussed.     

Utilization of meat industry by products: protein hydrolysate from sheep visceral mass

Protein hydrolysate was prepared from pre-treated sheep visceral mass (including stomach, large and small intestines) by enzymatic treatment at 43+/-1 degrees C (at the in situ pH 7.1+/-0.2 of the visceral mass) using fungal protease. The enzyme readily solubilized the proteins of the visceral mass as indicated by the degree of hydrolysis (34%) and nitrogen recovery (>64%). Hydrolysis with an enzyme level of 1% (w/w of total solids) at 43+/-1 degrees C with a pH around 7.0 for 45 min was found to be the optimum condition. The yield of protein hydrolysate was about 6% (w/w). The amino acid composition of the protein hydrolysate that was very hygroscopic, was comparable to that of casein.     

Acyl dipeptides as reversible caspase inhibitors. Part 1: initial lead optimization

Parallel synthesis was used to explore the SAR of a peptidomimetic caspase inhibitor. The most potent compound had nanomolar activity against caspases 1, 3, 6, 7, and 8.     

The Association of the Triglyceride-to-HDL Cholesterol Ratio with Insulin Resistance in White European and South Asian Men and Women

Introduction

There is recent interest surrounding the use of the triglyceride-to-HDL cholesterol ratio as a surrogate marker of insulin resistance in clinical practice, as it may identify people at high risk of developing diabetes or its complications. However, it has been suggested using this lipid ratio may not be appropriate for measuring insulin resistance in African-Americans, particularly women. We investigated if this inconsistency extended to South Asian women in a UK multi-ethnic cohort of White Europeans and South Asians.

Methods

Cross-sectional analysis was done of 729 participants from the ADDITION-Leicester study from 2005 to 2009. The association between tertiles of triglyceride-to-HDL cholesterol ratio to fasting insulin, homeostatic model of assessment for insulin resistance (HOMA1-IR), quantitative insulin sensitivity check index (QUICKI) and glucose: insulin ratio was examined with adjustment for confounding variables.

Results

Incremental tertiles of the triglyceride-to-HDL cholesterol ratio demonstrated a significant positive association with levels of fasting insulin, HOMA1-IR, glucose: insulin ratio and a negative association with QUICKI in White European men (n = 255) and women (n = 250) and South Asian men (n = 124) (all p<0.05), but not South Asian women (n = 100). A significant interaction was demonstrated between sex and triglyceride-to-HDL cholesterol ratio tertiles in South Asians only (p<0.05). The area under the receiver operating characteristic curve for triglyceride-to-HDL cholesterol ratio to detect insulin resistance, defined as the cohort HOMA1-IR≥75^th percentile (3.08), was 0.74 (0.67 to 0.81), 0.72 (0.65 to 0.79), 0.75 (0.66 to 0.85) and 0.67 (0.56 to 0.78) in White European men and women, South Asian men and women respectively. The optimal cut-points for detecting insulin resistance were 0.9–1.7 in mmol/l (2.0–3.8 in mg/dl) for the triglyceride-to-HDL ratio.

Conclusion

In South Asian women the triglyceride-to-HDL cholesterol ratio was not associated with insulin resistance; therefore there may be limitations in its use as a surrogate marker in this group.