Multilocus sequence typing of Salmonella strains by high-throughput sequencing of selectively amplified target genes

Rapid development of next generation sequencing (NGS) technologies in recent years has made whole genome sequencing of bacterial genomes widely accessible. However, it is often unnecessary or not feasible to sequence the whole genome for most applications of genetic analyses in bacteria. Selectively capturing defined genomic regions followed by NGS analysis could be a promising approach for high-resolution molecular typing of a large set of strains. In this study, we describe a novel and straightforward PCR-based target-capturing method, hairpin-primed multiplex amplification (HPMA), which allows for simultaneous amplification of numerous target genes. To test the feasibility of NGS-based strain typing using HPMA, 20 target gene sequences were simultaneously amplified with barcode tagging in each of 41 Salmonella strains. The amplicons were then pooled and analyzed by 454 pyrosequencing. Analysis of the sequence data, as an extension of multilocus sequence typing (MLST), demonstrated the utility and potential of this novel typing method, MLST-seq, as a high-resolution strain typing method. With the rapidly increasing sequencing capacity of NGS, MLST-seq or its variations using different target enrichment methods can be expected to become a high-resolution typing method in the near future for high-throughput analysis of a large collection of bacterial strains.     

Inactivation of phosphomannose isomerase gene abolishes sporulation and antibiotic production in Streptomyces coelicolor

Phosphomannose isomerases (PMIs) in bacteria and fungi catalyze the reversible conversion of D-fructose-6-phosphate to D-mannose-6-phosphate during biosynthesis of GDP-mannose, which is the main intermediate in the mannosylation of important cell wall components, glycoproteins, and certain glycolipids. In the present study, the kinetic parameters of PMI from Streptomyces coelicolor were obtained, and its function on antibiotic production and sporulation was studied. manA (SCO3025) encoding PMI in S. coelicolor was deleted by insertional inactivation. Its mutant (S. coelicolor?manA) was found to exhibit a bld-like phenotype. Additionally, S. coelicolor?manA failed to produce the antibiotics actinorhodin and red tripyrolle undecylprodigiosin in liquid media. To identify the function of manA, the gene was cloned and expressed in Escherichia coli BL21 (DE3). The purified recombinant ManA exhibited PMI activity (K(cat)/K(m) (mM(-1) s(-1) = 0.41 for D-mannose-6-phosphate), but failed to show GDP-D-mannose pyrophosphorylase [GMP (ManC)] activity. Complementation analysis with manA from S. coelicolor or E. coli resulted in the recovery of bld-like phenotype of S. coelicolor?manA. SCO3026, another ORF that encodes a protein with sequence similarity towards bifunctional PMI and GMP, was also tested for its ability to function as an alternate ManA. However, the purified protein of SCO3026 failed to exhibit both PMI and GMP activity. The present study shows that enzymes involved in carbohydrate metabolism could control cellular differentiation as well as the production of secondary metabolites.     

Applications of isothermal titration calorimetry in pure and applied research--survey of the literature from 2010

Isothermal titration calorimetry (ITC) is a biophysical technique for measuring the formation and dissociation of molecular complexes and has become an invaluable tool in many branches of science from cell biology to food chemistry. By measuring the heat absorbed or released during bond formation, ITC provides accurate, rapid, and label-free measurement of the thermodynamics of molecular interactions. In this review, we survey the recent literature reporting the use of ITC and have highlighted a number of interesting studies that provide a flavour of the diverse systems to which ITC can be applied. These include measurements of protein-protein and protein-membrane interactions required for macromolecular assembly, analysis of enzyme kinetics, experimental validation of molecular dynamics simulations, and even in manufacturing applications such as food science. Some highlights include studies of the biological complex formed by Staphylococcus aureus enterotoxin C3 and the murine T-cell receptor, the mechanism of membrane association of the Parkinson's disease-associated protein α-synuclein, and the role of non-specific tannin-protein interactions in the quality of different beverages. Recent developments in automation are overcoming limitations on throughput imposed by previous manual procedures and promise to greatly extend usefulness of ITC in the future. We also attempt to impart some practical advice for getting the most out of ITC data for those researchers less familiar with the method.     

Diversity and antagonistic potential of Bacillus spp. associated to the rhizosphere of tomato for the management of Rhizoctonia solani

Bacillus spp. has emerged as the most effective alternative to synthetic chemical fungicides. To get a better insight in the antagonistic potential of Bacillus strains, rhizospheric soil samples of healthy tomato plants from Indo-gangetic plain regions of India were analysed. A total of 108 Bacillus strains were obtained from preliminary screening. Potent strains identified on the basis of in vitro antagonistic and biochemical assays were subjected to diversity analysis using 16S-rDNA, BOX and ERIC-PCR. Furthermore, the four best performing antagonistic Bacillus strains under in vitro plant growth promotion and antagonistic assay were selected for pot experiment. In field study, Bacillus amyloliquefaciens MB101 and Bacillus subtilis MB14 showed drastic reduction in disease index by 55.7 and 41.74% with significant elevation in fruit yield up to 220 and 184 qha^–1, respectively. The present study was successful in selecting effective Bacillus strains by performing phenotypic and genotypic characterisation of Bacillus strains that can be used as an integral component of integrated disease management of tomato root rot and damping-off.     

Discovery of potent, selective and orally bioavailable triaryl-sulfonamide based PTP1B inhibitors

A novel series of pTyr mimetics containing triaryl-sulfonamide derivatives (5a-r) are reported as potent and selective PTP1B inhibitors. Some of the test compounds (5o and 5p) showed excellent selectivity towards PTP1B over various PTPs, including TCPTP (in vitro). The lead compound 5o showed potent antidiabetic activity (in vivo), along with improved pharmacokinetic profile. These preliminary results confirm discovery of highly potent and selective PTP1B inhibitors for the treatment of T2DM.     

Synthesis, study on anti-arthritic, anti-inflammatory activity and toxicity of some novel bis-oxy cyclophane diamides

A series of bis-oxy cyclophane diamides with bis(aminomethyl)m-terphenyl as spacer have been synthesized and characterized from spectral and analytical data. All the cyclophane diamides exhibit better anti-arthritic activity than the reference drug viz. diclofenac sodium. Some of the cyclophane diamides exhibit good anti-inflammatory activity. The cyclophane amide 4 and 5 do not show any evidence of mutagenicity and cytotoxicity.     

Simple, fast and efficient synthesis of β-keto esters from the esters of heteroaryl compounds, its antimicrobial study and cytotoxicity towards various cancer cell lines

A series of β-keto esters were synthesized from heteroaryl esters and ethyl acetate using LiHMDS as base at -50 to -30 °C. The increase in yields of cross condensed product were observed and the percentage of self condensed product was reduced drastically by applying the suitable base (LiHMDS), solvent and the minimum amount of ethyl acetate. All these β-keto esters were characterized using (1)H NMR, (13)C NMR and mass spectral data. A plausible mechanism is also depicted to prove the formation of trans-esterified products. All the synthesized compounds were subjected to test for their cytotoxicity towards various cancer cell lines and also tested for their antimicrobial activity towards various bacterial and fungal strains and some of them were found to have promising activity.     

Discovery of liver selective non-steroidal glucocorticoid receptor antagonist as novel antidiabetic agents

Series of benzyl-phenoxybenzyl amino-phenyl acid derivatives (8a-q) are reported as non-steroidal GR antagonist. Compound 8g showed excellent h-GR binding and potent antagonistic activity (in vitro). The lead compound 8g exhibited significant oral antidiabetic and antihyperlipidemic effects (in vivo), along with liver selectivity. These preliminary results confirm discovery of potent and liver selective passive GR antagonist for the treatment of T2DM.     

InCl3-catalysed synthesis of 2-aryl quinazolin-4(3H)-ones and 5-aryl pyrazolo[4,3-d]pyrimidin-7(6H)-ones and their evaluation as potential anticancer agents

A convenient and practical methodology for the synthesis of 2-aryl quinazolin-4(3H)-ones by the condensation of o-aminobenzamides with aromatic aldehydes under mild conditions using catalytic InCl(3) with good yields and high selectivities. This method has been extended for the synthesis of 5-aryl pyrazolo[4,3-d]pyrimidin-7(6H)-ones which have potential applications in medicinal chemistry. Many of these compounds were evaluated for their anti-proliferative properties in vitro against four cancer cell lines and several compounds were found to be active. Further in vitro studies indicated that inhibition of sirtuins could be the possible mechanism of action of these molecules.     

Factors associated with physician agreement and coding choices of cause of death using verbal autopsies for 1130 maternal deaths in India

Background

The Indian Sample Registration System (SRS) with verbal autopsy methods provides estimations of cause specific mortality for maternal deaths, where the majority of deaths occur at home, unregistered. We aim to examine factors that influence physician agreement and coding choices in assigning causes of death from verbal autopsies.

Methodology/Principal Findings

Among adult deaths identified in the SRS, pregnancy-related deaths recorded in 2001–2003 were assigned ICD-10 codes by two independent physicians. Inter-rater reliability was estimated using Landis Koch Kappa classification – poor to fair agreement; > – moderate agreement; > – substantial agreement; >– high agreement. We identified factors associated with physician agreement using multivariate logistic regression. A central consensus panel reviewed cases for errors and reclassified as needed based on 2011 ICD-10 coding guidelines. Of 1130 pregnancy-related deaths, 1040 were assigned ICD-10 codes by two physicians. We found substantial agreement regardless of the woman''s residence, whether the death was registered, religion, respondent''s or deceased''s education, age, hospital admission or gestational age. Physician agreement was not influenced by the above variables, with the exception of greater agreement in cases where the respondent did not live with the deceased, or early gestational age at the time of death. A central consensus panel reviewed all cases and recoded 10% of cases due to insufficient use of information in the verbal autopsy by the coding physicians and rationale for this reclassification are discussed.

Conclusion

In the absence of complete vital registration and universal healthcare services, physician coded verbal autopsies continues to be heavily relied upon to ascertain pregnancy-related death. From this study, two independent physicians had good inter-rater reliability for assigning pregnancy-related causes of death in a nationally-represented sample, and physician coding does not appear to be heavily influenced by case characteristics or demographics.