Evaluation of Hybridization Capture Versus Amplicon‐Based Methods for Whole‐Exome Sequencing

Next‐generation sequencing has aided characterization of genomic variation. While whole‐genome sequencing may capture all possible mutations, whole‐exome sequencing remains cost‐effective and captures most phenotype‐altering mutations. Initial strategies for exome enrichment utilized a hybridization‐based capture approach. Recently, amplicon‐based methods were designed to simplify preparation and utilize smaller DNA inputs. We evaluated two hybridization capture‐based and two amplicon‐based whole‐exome sequencing approaches, utilizing both Illumina and Ion Torrent sequencers, comparing on‐target alignment, uniformity, and variant calling. While the amplicon methods had higher on‐target rates, the hybridization capture‐based approaches demonstrated better uniformity. All methods identified many of the same single‐nucleotide variants, but each amplicon‐based method missed variants detected by the other three methods and reported additional variants discordant with all three other technologies. Many of these potential false positives or negatives appear to result from limited coverage, low variant frequency, vicinity to read starts/ends, or the need for platform‐specific variant calling algorithms. All methods demonstrated effective copy‐number variant calling when evaluated against a single‐nucleotide polymorphism array. This study illustrates some differences between whole‐exome sequencing approaches, highlights the need for selecting appropriate variant calling based on capture method, and will aid laboratories in selecting their preferred approach.     

Evaluation of Caesalpinia bonducella flower extract for anti-inflammatory action in rats and its high performance thin layer chromatography chemical fingerprinting

Objective:

The study is aimed to evaluate anti-inflammatory activity of Caesalpinia bonducella Fleming (Caesalpiniaceae) flower extract (CBFE) and to study its effect on radiographic outcome in adjuvant induced arthritis and authentication by high performance thin layer chromatography (HPTLC) chemical fingerprinting.

Materials and Methods:

CBFE was administered orally (30, 100, and 300 mg/kg b.wt.) and tested for its anti-inflammatory activity in carrageenan-induced inflammation, cotton pellet induced chronic granulomatous inflammation and autacoids-induced inflammation. Effect on radiographic outcome was tested in adjuvant-induced arthritis. CBFE was HPTLC fingerprinted in suitable solvent system.

Result:

In carrageenan-induced inflammation, CBFE produced significant inhibition in edema volume at all the doses (30, 100 and 300 mg/kg b.wt.) and percentage of inhibition was 28.68, 31.00, and 22.48, respectively as compared to control at 5 h of its administration. In cotton pellet granuloma assay, CBFE significantly decreased the granuloma weight at 300 mg/kg dose level by 22.53%. CBFE (300 mg/kg) caused significant inhibition by 37.5, 44.44, and 35.29% edema volume, at ½, 1 and 3 h after 5-hydroxytryptamine injection, respectively. Radiographic score of animals treated with 300 mg/kg CBFE was significantly decreased when compared to arthritic control animals.

Conclusion:

The extract was found to possess significant anti-inflammatory activity. CBFE treatment improved the bony architecture in adjuvant-induced arthritis in rats. The developed HPTLC fingerprint would be helpful in the authentication of C. bonducella flower extract.KEY WORDS: 5-hydroxy tryptamine, Caesalpinia bonducella, carrageenan-induced inflammation, cotton pellet granuloma, high performance thin layer chromatography, radiography     

Does hand involvement in systemic sclerosis limit completion of patient-reported outcome measures?

Clinical Rheumatology - The objective of this analysis is to examine whether the severity of systemic sclerosis (SSc)-hand involvement influences patient-reported outcome measure (PROM) completion...     

Utility of urinary progranulin in patients with type 2 diabetic nephropathy and its correlation with renal function

International Journal of Diabetes in Developing Countries - Urinary progranulin is an inflammatory marker that may indicate renal damage at an early stage of diabetic nephropathy. To determine...     

Statistical Optimization of Bioprocess Parameters for Improved Production of L-Asparaginase from Lactobacillus plantarum

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences - L-asparaginase (ASNase), a tetrameric enzyme, holds comprehensive applications in food industries as a...     

Magnetic core–shell Fe3O4@Cu2O and Fe3O4@Cu2O–Cu materials as catalysts for aerobic oxidation of benzylic alcohols assisted by TEMPO and N-methylimidazole

In this work, core–shell Fe₃O₄@Cu₂O and Fe₃O₄@Cu₂O–Cu nanomaterials for aerobic oxidation of benzylic alcohols are reported with 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) and N-methylimidazole (NMI) as the co-catalysts. To anchor Cu₂O nanoparticles around the magnetic particles under solvothermal conditions, the magnetic material Fe₃O₄ was modified by grafting a layer of l-lysine (l-Lys) to introduce –NH₂ groups at the surface of the magnetic particles. With amine groups as the anchor, Cu(NO₃)₂ was used to co-precipitate the desired Cu₂O by using ethylene glycol as the reducing agent. Prolonging the reaction time would lead to over-reduced forms of the magnetic materials in the presence of copper, Fe₃O₄@Cu₂O–Cu. The nanomaterials and its precursors were fully characterized by a variety of spectroscopic techniques. In combination with both TEMPO and NMI, these materials showed excellent catalytic activities in aerobic oxidation of benzylic alcohols under ambient conditions. For most of the benzylic alcohols, the conversion into aldehydes was nearly quantitative with aldehydes as the sole product. The materials were recyclable and robust. Up to 7 repeat runs, its activity dropped less than 10%. The over-reduced materials, Fe₃O₄@Cu₂O–Cu, exhibited slightly better performance in durability. The magnetic properties allowed easy separation after reaction by simply applying an external magnet.

Robust core–shell magnetic materials catalyse quantitatively the aerobic oxidation of a wide range of benzylic alcohols into corresponding aldehydes at room temperature showing excellent tolerance towards the substituents on the phenyl ring.     

Engineering Escherichia coli for efficient conversion of glucose to pyruvate

Escherichia coli TC44, a derivative of W3110, was engineered for the production of pyruvate from glucose by combining mutations to minimize ATP yield, cell growth, and CO2 production (DeltaatpFH DeltaadhE DeltasucA) with mutations that eliminate acetate production [poxB::FRT (FLP recognition target) DeltaackA] and fermentation products (DeltafocA-pflB DeltafrdBC DeltaldhA DeltaadhE). In mineral salts medium containing glucose as the sole carbon source, strain TC44(DeltafocA-pflB DeltafrdBC DeltaldhA DeltaatpFH DeltaadhE DeltasucA poxB::FRT DeltaackA) converted glucose to pyruvate with a yield of 0.75 g of pyruvate per g of glucose (77.9% of theoretical yield; 1.2 g of pyruvate liters(-1).h(-1)). A maximum of 749 mM pyruvate was produced with excess glucose. Glycolytic flux was >50% faster for TC44 producing pyruvate than for the wild-type W3110 during fully aerobic metabolism. The tolerance of E. coli to such drastic changes in metabolic flow and energy production implies considerable elasticity in permitted pool sizes for key metabolic intermediates such as pyruvate and acetyl-CoA. In strain TC44, pyruvate yield, pyruvate titer, and the rate of pyruvate production in mineral salts medium were equivalent or better than previously reported for other biocatalysts (yeast and bacteria) requiring complex vitamin feeding strategies and complex nutrients. TC44 offers the potential to improve the economics of pyruvate production by reducing the costs of materials, product purification, and waste disposal.     

Induction of deletion mutation on ompR gene of Salmonella enterica serovar Typhi isolates from asymptomatic typhoid carriers to evolve attenuated strains for vaccine development

Objective:To develop allenualed slrains of Salmonella enterica serorar Typhi(S.typhi) for the candidate vaccine by osmolar stress.Mothods:S.typhi SS3 and SS5 strains were isolated from asymptomatic typhoid carriers in Mamakkal,Tamil Nadu.India.Both strains were grown in LB(Luria Bertani) medium supplemented with various concentration of NaCl(0.1- 0.7M) respectively.The effecl of osmolar stress was determined at molecular level by PCR using MCR 06 and MCR07 primers corresponding to ompR with chromosomal DNA of S.typhi SS3 and SS5 strains.Attenuation by osmolar stress results in deletion mutation of the.S.typhi slrains was determined by agglutination assays,precipitation method.SDS PAGE analysis and by animal models.Results:The 799 bp amplified ompR gene product from wild type S.typhi SS3 and SS5 illustrate the presence of virulent gene.Interestingly,there was only a 282 bp amplified product from S.typhi SS3 and SS5 grown in the presence of 0.5.0.6 and 0.7 M NaCl.This illustrates the occurrence of deletion mutation in ompR gene al high concentration of NaCl.Furthermore,both the wildtype and mutant S.typhi outer membrane SDS-PAGF.profile reveals the differences in the expression of ompF.ompC and ompA proteins.In mice,wild type and mutant strains lethal dose(LD_(50)) were determined.The mice died within 72 h when both the wild type strains were injected intraperitoneally with 3 log CFU-mL~(-1).When the mice were injected with the mutants in same dosage,no clinical symptoms were observed;whereas the serum antibodv litre was elicited within two weeks indicated that the mutants have the ability to induce protective humoral immune response.These results suggest that S.typhi SS3 and SS5 may bo used as good candidate strains for the development of live attenuated vaccine against salmonellosis.Conclusions:This study demonstrates that the S.typhi strains were allenualed and could be good vaccine candidates in future.