Comparative characterization of commercially important xylanase enzymes

Xylanase is an industrially important enzyme having wide range of applications especially in paper industry. It is crucial to gain an understanding about the structure and functional aspects of various xylanases produced from diverse sources. In this study, a bioinformatics and molecular modeling approach was adopted to explore properties and structure of xylanases. Physico-chemical properties were predicted and prediction of motifs, disulfide bridges and secondary structure was performed for functional characterization. Apart from these analyses, three dimensional structures were constructed and stereo-chemical quality was evaluated by different structure validation tools. Comparative catalytic site analysis and assessment was performed to extract information about the important residues. Asn72 was found to be the common residue in the active sites of the proteins {"type":"entrez-protein","attrs":{"text":"P35809","term_id":"549462","term_text":"P35809"}}P35809 and {"type":"entrez-protein","attrs":{"text":"Q12603","term_id":"2494332","term_text":"Q12603"}}Q12603.     

Rubidium chloride tolerant callus cultures of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) accumulate more potassium and cross tolerate to other salts

Callus cultures from salt tolerant (CSR-10) and susceptible (Swarnadhan) varieties of Oryza sativa L. were established in Murashige and Skoog’s (MS) medium containing lethal concentrations (50 mM) of rubidium chloride (RbCl) as a selective agent. While 95–100% cells were viable in callus cultures grown without RbCl, viability was 75% in 50 mM RbCl selected cultures. Growth of RbCl selected calli in presence of salt was comparable to that of callus grown without it. Cells tolerant to RbCl showed more vacuoles and accumulated more K⁺ in comparison with their corresponding controls. Suspension cultures were established and uptake of ⁸⁶Rb⁺ was measured at 10 and 20 min intervals, which revealed a linear relationship between the absorption of K⁺ and time. Callus cultures (560-day-old) tolerant to 50 mM RbCl regenerated shoots with 35–40% frequencies in both the varieties, but the same age-old callus grown in the medium devoid of RbCl did not show any organogenesis. Callus cultures that are tolerant to 50 mM RbCl when exposed to 25 mM LiCl, 50 mM NaCl, 50 mM KCl and 25 mM CsCl also exhibited cross tolerance in both the varieties. This is the first time that a callus line of rice resistant to RbCl was raised and shown to accumulate a major cation K⁺ and also an increased influx of it.     

High quality methylome-wide investigations through next-generation sequencing of DNA from a single archived dry blood spot

The potential importance of DNA methylation in the etiology of complex diseases has led to interest in the development of methylome-wide association studies (MWAS) aimed at interrogating all methylation sites in the human genome. When using blood as biomaterial for a MWAS the DNA is typically extracted directly from fresh or frozen whole blood that was collected via venous puncture. However, DNA extracted from dry blood spots may also be an alternative starting material. In the present study, we apply a methyl-CpG binding domain (MBD) protein enrichment-based technique in combination with next generation sequencing (MBD-seq) to assess the methylation status of the ~27 million CpGs in the human autosomal reference genome. We investigate eight methylomes using DNA from blood spots. This data are compared with 1,500 methylomes previously assayed with the same MBD-seq approach using DNA from whole blood. When investigating the sequence quality and the enrichment profile across biological features, we find that DNA extracted from blood spots gives comparable results with DNA extracted from whole blood. Only if the amount of starting material is ≤ 0.5µg DNA we observe a slight decrease in the assay performance. In conclusion, we show that high quality methylome-wide investigations using MBD-seq can be conducted in DNA extracted from archived dry blood spots without sacrificing quality and without bias in enrichment profile as long as the amount of starting material is sufficient. In general, the amount of DNA extracted from a single blood spot is sufficient for methylome-wide investigations with the MBD-seq approach.     

Improved experimental data processing for UHPLC–HRMS/MS lipidomics applied to nonalcoholic fatty liver disease

Introduction

Untargeted metabolomics workflows include numerous points where variance and systematic errors can be introduced. Due to the diversity of the lipidome, manual peak picking and quantitation using molecule specific internal standards is unrealistic, and therefore quality peak picking algorithms and further feature processing and normalization algorithms are important. Subsequent normalization, data filtering, statistical analysis, and biological interpretation are simplified when quality data acquisition and feature processing are employed.

Objectives

Metrics for QC are important throughout the workflow. The robust workflow presented here provides techniques to ensure that QC checks are implemented throughout sample preparation, data acquisition, pre-processing, and analysis.

Methods

The untargeted lipidomics workflow includes sample standardization prior to acquisition, blocks of QC standards and blanks run at systematic intervals between randomized blocks of experimental data, blank feature filtering (BFF) to remove features not originating from the sample, and QC analysis of data acquisition and processing.

Results

The workflow was successfully applied to mouse liver samples, which were investigated to discern lipidomic changes throughout the development of nonalcoholic fatty liver disease (NAFLD). The workflow, including a novel filtering method, BFF, allows improved confidence in results and conclusions for lipidomic applications.

Conclusion

Using a mouse model developed for the study of the transition of NAFLD from an early stage known as simple steatosis, to the later stage, nonalcoholic steatohepatitis, in combination with our novel workflow, we have identified phosphatidylcholines, phosphatidylethanolamines, and triacylglycerols that may contribute to disease onset and/or progression.

     

Single-Cell Mapping of Progressive Fetal-to-Adult Transition in Human Naive T Cells

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