Information theoretic perspective on genome clustering.

Shannon's information theoretic perspective of communication helps one to understand the storage and processing of information in one-dimensional sequences. An information theoretic analysis of 937 available completely sequenced prokaryotic genomes and 238 eukaryotic chromosomes is presented. Information content (Id) values were used to cluster these chromosomes. Chargaff's second parity rule i.e compositional self-complementarity, an empirical fact is observed in all the genomes, except for the proteobacteria Candidatus Hodgkinia cicadicola. High information content, arising out of biased base composition in all the 14 chromosomes of Plasmodium falciparum is found among two other genomes of prokaryotes viz. Buchnera aphidicola str. Cc (Cinara cedri) and Candidatus Carsonella ruddii PV. Despite size and compositional variations, both prokaryotic and eukaryotic genomes do not deviate significantly from an equiprobable and random situation. Eukaryotic chromosomes of an organism tend to have similar informational restraints as seen when a simple distance based method is used to cluster them. In eukaryotes, in certain cases, Id values are also similar for the two arms (p and q arm) of the chromosomes. The results of this current study confirm that the information content can provide insights into the clustering of genomes and the evolution of messaging strategies of the genomes. An efficient and robust Perl CGI standalone tool is created based on this information theory algorithm for the analysis of the whole genomes and is made available at https://github.com/AlagurajVeluchamy/InformationTheory.

Current Status and Future of End-Stage Kidney Disease in Gulf Cooperation Council Countries: Challenges and Opportunities.

There is a paucity of data on epidemiology along with an incomplete registry of end-stage kidney disease (ESKD), nephrologist workforce, and variability among the countries of Gulf Cooperation Council (GCC). The study is an observation, descriptive study which aimed to describe current ESKD burden, nephrologist density, and kidney care infrastructure in GCC. Responses to a questionnaire-based survey obtained from representatives of the Nephrology Societies of GCC countries were analyzed. The categorical variables were compared using Chi-square test. A P = 5% was considered as significant. The mean prevalence of ESKD per million populations (pmp) was 551, highest in Oman (1000/pmp), least in Qatar (347/pmp). Predominant etiology in GCC was diabetes mellitus (DM) and hypertension (HTN) (100%, each), followed by chronic glomerulonephritis (66.7%). A transplant registry was maintained by all GCC countries. Hemodialysis (HD) (67.2%) was the most opted modality of kidney replacement therapy (KRT), followed by kidney transplantation (22%) and peritoneal dialysis (9.6%); 1.0% of patients opted for conservative management. Unplanned initiation of HD was three times more common. The access distribution among incident and prevalent HD patients respectively was (i) nontunneled central catheter (nTCC) (58.7 ± 36.6 vs. 1.5 ± 1.5), (ii) tunneled central catheter (23.5 ± 29.9 vs. 33.6 ± 10.0), and (iii) arteriovenous fistula (17.3± 14.4 vs. 57.8 ± 11.86). Death and transplantation were the reasons for dropout from HD. GCC has adequate kidney care infrastructure. There are 1686 nephrologists [range: Bahrain 9, Kingdom of Saudi Arabia (KSA) 1279]. Qatar, KSA, and Kuwait provide training in kidney biopsy; all countries except Bahrain have formal training programs for nTCC placement. ESKD prevalence is high, DM, HTN; glome-rulonephritis (GN) is the most common causes. The need for KRT is expected to rise in GCC. HD is the predominant KRT modality with a high prevalence of dialysis catheters as vascular access.

The barriers to linkage and retention in care for women living with HIV in an high income setting where they comprise a minority group.

Women comprise a minority population of individuals living with HIV in Australia, and are often poorly represented in research and clinical trials so their needs remain largely unknown. Data suggests that they are diagnosed later than men and start antiretroviral therapy at a lower CD4 cell count. This raises the question whether there are sex specific barriers to linkage and retention in care. This study analyzed 484 surveys received from clinicians collecting demographic, virological, and reproductive health data along with perceived barriers to linkage and retention in care. Most women (67%) were estimated to have been linked into care within 28 days of diagnosis. For women who were not linked into care for more than 28 days, the most commonly reason cited was fear of disclosure to others, followed by fear of disclosure to their partner. The main reasons given for non-retention in care were related to transport, carer responsibilities, financial pressure, health beliefs and concern about stigma or disclosure.

Optimised dosing of vancomycin in critically ill Indigenous Australian patients with severe sepsis.

Vancomycin is a commonly used antibiotic due to the high burden of methicillin-resistant Staphylococcus aureus infections. This study aimed to describe the pharmacokinetics (PK) of vancomycin in Australian Indigenous patients with severe sepsis, and advise an optimal dosing strategy. A population PK study was conducted in a remote Australian intensive care unit (ICU). Serial plasma samples were collected over one to two dosing intervals and assayed by validated chromatography. Concentration-time data collected were analysed using Pmetrics® software. The final population PK model was then used for Monte Carlo dosing simulations to determine optimal loading and intermittent maintenance doses. Fifteen Indigenous subjects were included for analysis with a median (interquartile range, IQR) age, weight and creatinine clearance (CrCL) of 43 (34-46) years, 73 (66-104) kg and 99 (56-139) ml/minute respectively. A two-compartment model described the data adequately. Vancomycin clearance (CL) and volume of distribution of the central compartment (Vc) were described by CrCL and patient weight respectively. Median (IQR) CL, Vc, distribution rate constants from central to peripheral, and from peripheral to central compartments were 4.6 (3.8-5.6) litres per hour, 25.4 (16.1-31.3) litres, 0.46 (0.28-0.52)/hour and 0.25 (0.12-0.37)/hour respectively. No significant interethnic PK differences were observed in comparison to published data. Therapeutic loading doses were significantly dependent on both weight and CrCL, whereas maintenance doses were dependent on CrCL. In the absence of severe renal impairment, initiation of maintenance dose eight hours post-loading dose achieved higher probability of target attainment at 24 hours. This is the first report of vancomycin PK in this patient group.

The rise of methicillin resistant Staphylococcus aureus: now the dominant cause of skin and soft tissue infection in Central Australia.

This study aimed to examine the epidemiology and treatment outcomes of community-onset purulent staphylococcal skin and soft tissue infections (SSTI) in Central Australia. We performed a prospective observational study of patients hospitalised with community-onset purulent staphylococcal SSTI (n = 160). Indigenous patients accounted for 78% of cases. Patients were predominantly young adults; however, there were high rates of co-morbid disease. Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) was the dominant phenotype, accounting for 60% of cases. Hospitalisation during the preceding 6 months, and haemodialysis dependence were significant predictors of CA-MRSA infection on univariate analysis. Clinical presentation and treatment outcomes were found to be comparable for methicillin-susceptible S. aureus (MSSA) and methicillin-resistant cases. All MRSA isolates were characterised as non-multi-resistant, with this term used interchangeably with CA-MRSA in this analysis. We did not find an association between receipt of an active antimicrobial agent within the first 48 h, and progression of infection; need for further surgical debridement; unplanned General Practitioner or hospital re-presentation; or need for further antibiotics. At least one adverse outcome was experienced by 39% of patients. Clindamycin resistance was common, while rates of trimethoprim-sulfamethoxazole resistance were low. This study suggested the possibility of healthcare-associated transmission of CA-MRSA. This is the first Australian report of CA-MRSA superseding MSSA as the cause of community onset staphylococcal SSTI.

Understanding the virulence of the entero-aggregative E. coli O104:H4.

O104:H4 is a new strain of E. coli that has caused an outbreak in Germany. It was isolated from patients with bloody diarrhoea and Haemolytic Uremic Syndrome (HUS). BGI (www.genomics.cn) sequenced and assembled this new strain. It was reported to show resistance to a number of drugs that are toxic to other E. coli and causes serious complications during infections, which ultimately lead to death. Multi-drug resistance and high virulence of this strain is thought to be acquired from different sources, by horizontal gene transfer. A total of 38 prophage elements were detected from the new strain by using three computational tools viz., DRAD, Prophage Finder and PHAST. Analysis on these prophage elements shows a number of virulence proteins like Shiga toxin and multi-drug resistance protein encoding genes. The high virulence of the strain could be attributed by the prophage elements acquired from its micro environment.

Analysis of dihedral angle preferences for alanine and glycine residues in alpha and beta transmembrane regions.

For the past 50 years, the Ramachandran map has been used effectively to study the protein structure and folding. However, though extensive analysis has been done on dihedral angle preferences of residues in globular proteins, related studies and reports of membrane proteins are limited. It is of interest to explore the conformational preferences of residues in transmembrane regions of membrane proteins which are involved in several important and diverse biological processes. Hence, in the present work, a systematic comparative computational analysis has been made on dihedral angle preferences of alanine and glycine in alpha and beta transmembrane regions (the two major classes of transmembrane proteins) with the aid of the Ramachandran map. Further, the conformational preferences of residues in transmembrane regions were compared with the non-transmembrane regions. We have extracted cation-pi interacting residues present in transmembrane regions and explored the dihedral angle preferences. From our observations, we reveal the higher percentage of occurrences of glycine in alpha and beta transmembrane regions than other hydrophobic residues. Further, we noted a clear shift in ψ-angle preferences of glycine residues from negative bins in alpha transmembrane regions to positive bins in beta transmembrane regions. Also, cation-pi interacting residues in beta transmembrane regions avoid preferring ψ-angles in the range of -59° to -30°. In this article, we insist that the studies on preferences of dihedral angles in transmembrane regions, thorough understanding of structure and folding of transmembrane proteins, can lead to modeling of novel transmembrane regions towards designing membrane proteins.

Scientific Insights in the Preparation and Characterisation of a Lead-based Naga Bhasma.

Naga bhasma is one of the herbo-metallic preparations used in Ayurveda, a traditional Indian System of Medicine. The preparation of Naga bhasma involves thermal treatment of 'Naga' (metallic lead) in a series of quenching liquids, followed by reaction with realgar and herbal constituents, before calcination to prepare a fine product. We have analysed the intermediates obtained during different stages of preparation to understand the relevance and importance of different steps involved in the preparation. Our results show that 'Sodhana' (purification process) removes heavy metals other than lead, apart from making it soft and amenable for trituration. The use of powders of tamarind bark and peepal bark maintains the oxidation state of lead in Jarita Naga (lead oxide) as Pb(2+). The repeated calcination steps result in the formation of nano-crystalline lead sulphide, the main chemical species present in Naga bhasma.

Human T-cell lymphotropic virus type 1 exposures following blood-borne virus incidents in central Australia, 2002-2012.

We retrospectively audited hospital occupational exposure events over a 10-year period, in a human T-cell lymphotropic virus type 1 (HTLV-1)-endemic area of Central Australia, and report on 53 individuals exposed to HTLV-1 with no transmissions documented (95% confidence interval, 0%-1.5%). This has important implications for the management of exposures including the role of postexposure prophylaxis.

Variations in physicochemical properties of a traditional mercury-based nanopowder formulation: need for standard manufacturing practices.

Rasasindura is a mercury-based nanopowder synthesized using natural products through mechanothermal processing. It has been used in the Ayurvedic system of medicine since time immemorial for various therapeutic purposes such as rejuvenation, treatment of syphilis and in genital disorders. Rasasindura is said to be composed of mercury, sulphur and organic moieties derived from the decoction of plant extracts used during its synthesis. There is little scientific understanding of the preparation process so far. Though metallic mercury is incorporated deliberately for therapeutic purposes, it certainly raises toxicity concerns. The lack of gold standards in manufacturing of such drugs leads to a variation in the chemical composition of the final product. The objective of the present study was to assess the physicochemical properties of Rasasindura samples of different batches purchased from different manufacturers and assess the extent of deviation and gauge its impact on human health. Modern characterization techniques were employed to analyze particle size and morphology, surface area, zeta potential, elemental composition, crystallinity, thermal stability and degradation. Average particle size of the samples observed through scanning electron microscope ranged from 5-100 nm. Mercury content was found to be between 84 and 89% from elemental analysis. Despite batch-to-batch and manufacturer-to-manufacturer variations in the physicochemical properties, all the samples contained mercury in the form of HgS. These differences in the physicochemical properties may ultimately impact its biological outcome.

The transition of prothrombin to thrombin.

The proteolytic conversion of prothrombin to thrombin catalyzed by prothrombinase is one of the more extensively studied reactions of blood coagulation. Sophisticated biophysical and biochemical insights into the players of this reaction were developed in the early days of the field. Yet, many basic enzymological questions remained unanswered. I summarize new developments that uncover mechanisms by which high substrate specificity is achieved, and the impact of these strategies on enzymic function. Two principles emerge that deviate from conventional wisdom that has otherwise dominated thinking in the field. (i) Enzymic specificity is dominated by the contribution of exosite binding interactions between substrate and enzyme rather than by specific recognition of sequences flanking the scissile bond. Coupled with the regulation of substrate conformation as a result of the zymogen to proteinase transition, novel mechanistic insights result for numerous aspects of enzyme function. (ii) The transition of zymogen to proteinase following cleavage is not absolute and instead, thrombin can reversibly interconvert between zymogen-like and proteinase-like forms depending on the complement of ligands bound to it. This establishes new paradigms for considering proteinase allostery and how enzyme function may be modulated by ligand binding. These insights into the action of prothrombinase on prothrombin have wide-ranging implications for the understanding of function in blood coagulation.

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.

Asymmetric pore occupancy in crystal structure of OmpF porin from Salmonella typhi.

OmpF is a major general diffusion porin of Salmonella typhi, a Gram-negative bacterium, which is an obligatory human pathogen causing typhoid. The structure of S. typhi Ty21a OmpF (PDB Id: 3NSG) determined at 2.8 Å resolution by X-ray crystallography shows a 16-stranded ß-barrel with three ß-barrel monomers associated to form a trimer. The packing observed in S. typhi Ty21a rfOmpF crystals has not been observed earlier in other porin structures. The variations seen in the loop regions provide a starting point for using the S. typhi OmpF for structure-based multi-valent vaccine design. Along one side of the S. typhi Ty21a OmpF pore there exists a staircase arrangement of basic residues (20R, 60R, 62K, 65R, 77R, 130R and 16K), which also contribute, to the electrostatic potential in the pore. This structure suggests the presence of asymmetric electrostatics in the porin oligomer. Moreover, antibiotic translocation, permeability and reduced uptake in the case of mutants can be understood based on the structure paving the way for designing new antibiotics.

Characterization of DLP12 Prophage Membrane Associated Protein: HolinGFP.

Lysis cassette genes from phages determine the final lytic event of the host cells. The lysis cassette genes are conserved in phages and prophages. The membrane associated holin from DLP12 prophage, available as a GFP fusion construct, was shown to be overexpressed, using confocal microscopy analysis, in bacterial cells. The protein expression caused cell death in E. coli AG1 strain suggesting the protein was functional. The His-tag HolinGFP protein was purified using cobalt affinity column and was eluted in the presence of different non-ionic detergents DDM (n-dodecyl-ß-d-maltoside), LDAO (Lauryldimethylamine-oxide), OG (n-octyl ß-d-glucopyranoside) and C12E9 (dodecyl nonaoxyethylene ether). HolinGFP existed predominantly as a dimer in LDAO in Superdex S200 gel filtration chromatography. Circular dichroism and fluorescence spectroscopy of the fluorescent HolinGFP in all four detergents (C12E9, DDM, LDAO, and OG) confirmed the folded state. Both dithiobis succinimidyl propionate and gluteraldehyde crosslinking revealed the existence of higher order oligomers and dimers. HolinGFP has been functionally and biophysically characterised and is being explored for crystallographic structure determination.

Molecular modeling and conformational analysis of native and refolded viral genome-linked protein of cardamom mosaic virus.

The viral genome-linked protein (VPg) of Potyviruses is covalently attached to the 5' end of the genomic RNA. Towards biophysical characterization, the VPg coding region of Cardamom mosaic virus (CdMV) was amplified from the cDNA and expressed in E. coli. Most of the expressed VPg aggregated as inclusion bodies that were solubilized with urea and refolded with L-arginine hydrochloride. The various forms of CdMV VPg (native, denatured and refolded) were purified and the conformational variations between these forms were observed with fluorescence spectroscopy. Native and refolded CdMV VPg showed unordered secondary structure in the circular dichroism (CD) spectrum. The model of CdMV VPg was built based on the crystal structure of phosphotriesterase (from Pseudomonas diminuta), which had the maximum sequence homology with VPg to identify the arrangement of conserved amino acids in the protein to study the functional diversity of VPg. This is the first report on the VPg of CdMV, which is classified as a new member of the Macluravirus genus of the Potyviridae family.

Identification of biochemical and putative biological role of a xenolog from Escherichia coli using structural analysis.

YagE is a 33 kDa prophage protein encoded by CP4-6 prophage element in Escherichia coli K12 genome. Here, we report the structures of YagE complexes with pyruvate (PDB Id 3N2X) and KDGal (2-keto-3-deoxy galactonate) (PDB Id 3NEV) at 2.2A resolution. Pyruvate depletion assay in presence of glyceraldehyde shows that YagE catalyses the aldol condensation of pyruvate and glyceraldehyde. Our results indicate that the biochemical function of YagE is that of a 2-keto-3-deoxy gluconate (KDG) aldolase. Interestingly, E. coli K12 genome lacks an intrinsic KDG aldolase. Moreover, the over-expression of YagE increases cell viability in the presence of certain bactericidal antibiotics, indicating a putative biological role of YagE as a prophage encoded virulence factor enabling the survival of bacteria in the presence of certain antibiotics. The analysis implies a possible mechanism of antibiotic resistance conferred by the over-expression of prophage encoded YagE to E. coli.

Polymorphisms in an intronic region of the myocilin gene associated with primary open-angle glaucoma--a possible role for alternate splicing.

PURPOSE: To examine the possible role of alternate splicing leading to aggregation of myocilin in primary open-angle glaucoma. METHODS: Several single nucleotide variations found in the myocilin (MYOC) genomic region were collected and examined for their possible role in causing splice-site alterations. A model for myocilin built using a knowledge-based consensus method was used to map the altered protein products. A total of 150 open-angle glaucoma patients and 50 normal age-matched control subjects were screened for the predicted polymorphisms, and clustering was performed. RESULTS: A total of 124 genomic variations were screened, and six polymorphisms that lead to altered protein products were detected as possible candidates for the alternative splicing mechanism. Five of these lay in the intronic regions, and the one that lay in the exon region corresponded to the previously identified polymorphism (Tyr347Tyr) implicated in primary open-angle glaucoma. Experimentally screening the intronic region of the MYOC gene showed the presence of the predicted g.14072G>A polymorphism, g.1293C/T heterozygous polymorphism, instead of our predicted g.1293C/- polymorphism. Other than the prediction, two novel SNPs (g.1295G>T and g.1299T>G) and two reported SNPs (g.1284G>T and g.1286G>T) were also identified. Cluster analysis showed the g.14072G>A homozygous condition was more common in this cohort than the heterozygous condition. CONCLUSIONS: We previously proposed that the disruption of dimer or oligomer formation by the C-term region allows greater chances of nucleation for aggregation. Here we suggest that polymorphisms in the myocilin genomic region that cause synonymous codon changes or those that occur in the intron regions can possibly lead to altered myocilin protein products through altered intron-exon splicing.

Trichloroacetic acid-induced protein precipitation involves the reversible association of a stable partially structured intermediate.

Sample preparation for proteomic analysis involves precipitation of protein using 2,2,2-trichloroacetic acid (TCA). In this study, we examine the mechanism of the TCA-induced protein precipitation reaction. TCA-induced protein precipitation curves are U-shaped and the shape of the curve is observed to be independent of the physicochemical properties of proteins. TCA is significantly less effective in precipitating unfolded states of proteins. Results of the 1-anilino-8-napthalene sulfonate (ANS) and size-exclusion chromatography, obtained using acidic fibroblast growth factor (aFGF), show that a stable "molten globule-like" partially structured intermediate accumulates maximally in 5% (w/v) of trichloroacetate. Urea-induced unfolding and limited proteolytic digestion data reveal that the partially structured intermediate is significantly less stable than the native conformation. (1)H-(15)N chemical shift perturbation data obtained using NMR spectroscopy indicate that interactions stabilizing the beta-strands at the N- and C- terminal ends (of aFGF) are disrupted in the trichloroacetate-induced "MG-like" state. The results of the study clearly demonstrate that TCA-induced protein precipitation occurs due to the reversible association of the "MG-like" partially structured intermediate state(s). In our opinion, the findings of this study provide useful clues toward development of efficient protocols for the isolation and analysis of the entire proteome.