Prokaryotic communities adapted to microhabitats on the Indian lotus (Nelumbo nucifera) growing in the high-altitude urban Dal Lake

International Microbiology - Indian lotus (Nelumbo nucifera) is one of the dominant aquatic plants cultivated in Dal Lake, situated at 1586 m above mean sea level (MSL) in the northeast of...     

Localization of oxalacetate keto-enol-tautomerase.

The intracellular location of oxalacetate keto-enol-tautomerase (oxaloacetate keto-enolisomerase) (EC 5.3.2.2) has been determined in two types of animal cells, rat liver and pig kidney. Two fractionation procedures were adopted and modified to suit each type of tissue. One fractionation procedure gave the soluble phase, microsomal and mitochondrial fractions, while the other isolated the nuclear fraction. The tautomerase is distributed among the soluble phase, microsomes and mitochondria in both tissues. Fractionation efficiency was checked by determining percentage recoveries of enzymic activity and total protein after each step, by microscopy studies and by determining the distribution of several marker enzymes.     

Plasma membrane proteome of adhesion‐competent endometrial epithelial cells and its modulation by Rab11a

Human endometrial epithelium (EE) is composed of a multitude of proteins, amongst which those localized on the plasma membrane [plasma membrane proteins (PMPs)] are of critical relevance in the early stages of implantation. Evidence supports the key role of few PMPs in implantation. However, many remain unidentified, as efforts have not been made till date to generate the plasma membrane proteome of human EE cells, using a gel‐free approach. This study presents a protein catalog of the PMP enriched fraction of Ishikawa cell line; often used as an in vitro model for embryo‐adhesive EE. Liquid chromatography with tandem mass spectrometry identified 3,598 proteins. Of these, 1,963 proteins were annotated for their membrane localization. Of 1,963 proteins, 1,321 were found to have a transmembrane domain and 43 proteins had glycophosphatidylinositol (GPI) anchor. Extensive data mining revealed endometrial expression of 943 proteins reported in humans and/or rodents. Further, quantitative alterations were observed in the plasma membrane proteome on the perturbation of intracellular trafficking. Silencing of Rab11a (known for its role in plasma membrane organization) expression caused alteration in the abundance of 74 proteins. Caveolin‐1 and EpCAM levels were reduced whereas Rab4a abundance increased in the PMP extracts of Rab11a deficient cells, compared with control cells. Briefly, the study reports the identity of several novel plasma membrane‐localized proteins. A major spin‐off of the study is the identification of novel proteins trafficked by Rab11a to the plasma membrane. Targeted analysis of novel PMPs may reveal their specific roles in endometrial receptivity and implantation.     

High-throughput and automation advances for accelerating single-cell cloning,monoclonality and early phase clone screening steps in mammalian cell line development for biologics production

Mammalian cell line development is a multistep process wherein timelines for developing clonal cells to be used as manufacturing cell lines for biologics production can commonly extend to 9 months when no automation or modern molecular technologies are involved in the workflow. Steps in the cell line development workflow involving single-cell cloning, monoclonality assurance, productivity and stability screening are labor, time and resource intensive when performed manually. Introduction of automation and miniaturization in these steps has reduced the required manual labor, shortened timelines from months to weeks, and decreased the resources needed to develop manufacturing cell lines. This review summarizes the advances, benefits, comparisons and shortcomings of different automation platforms available in the market for rapid isolation of desired clonal cell lines for biologics production.     

A Central Role for Carbon-Overflow Pathways in the Modulation of Bacterial Cell Death

Similar to developmental programs in eukaryotes, the death of a subpopulation of cells is thought to benefit bacterial biofilm development. However mechanisms that mediate a tight control over cell death are not clearly understood at the population level. Here we reveal that CidR dependent pyruvate oxidase (CidC) and α-acetolactate synthase/decarboxylase (AlsSD) overflow metabolic pathways, which are active during staphylococcal biofilm development, modulate cell death to achieve optimal biofilm biomass. Whereas acetate derived from CidC activity potentiates cell death in cells by a mechanism dependent on intracellular acidification and respiratory inhibition, AlsSD activity effectively counters CidC action by diverting carbon flux towards neutral rather than acidic byproducts and consuming intracellular protons in the process. Furthermore, the physiological features that accompany metabolic activation of cell death bears remarkable similarities to hallmarks of eukaryotic programmed cell death, including the generation of reactive oxygen species and DNA damage. Finally, we demonstrate that the metabolic modulation of cell death not only affects biofilm development but also biofilm-dependent disease outcomes. Given the ubiquity of such carbon overflow pathways in diverse bacterial species, we propose that the metabolic control of cell death may be a fundamental feature of prokaryotic development.     

Bioprocess optimization of laccase production through solid substrate fermentation using Perenniporia tephropora-L168 and its application in bioremediation of triaryl-methane dye

Laccases are multi copper oxidases that can oxidize both phenolic and nonphenolic lignin related compounds. Consequently, there has been continuous demand for laccases for the oxidative degradation of phenolic dyes in effluents. In view of this, the present work was focused on laccase production by solid substrate fermentation using a newly isolated fungus Perenniporia tephropora-L168. To intensify the laccase production, the process parameters pH, nitrogen, inducer, and substrate: water ratio were optimized by using statistical model. A set of optimal conditions noted were pH 3, nitrogen 0.001 g/L; inducer 0.5% and substrate: water ratio (1:10), which yielded laccase 1,160 U/g. The crude laccase exhibited noteworthy potential to degrade a triaryl-methane dye especially Malachite green. Also, during bioremediation studies, the statistical process optimization could achieve 81% decolourization within 180 min. The laccase treatment brought chemical transformation in malachite green as evident from UV–Visible spectra, FTIR, HPLC while toxicity against bacteria and fungi was also reduced. During phytotoxicity study, effect of treated and untreated dye on germination of seed was analyzed. Interestingly, the germination index for Vigna aconitifolia and Vigna radiata was increased by two and fourfold, respectively. Overall, this work demonstrates optimized production of laccase using Perenniporia tephropora-L168 and its efficient bioremediation potential for triaryl-methane dye.     

Cellular Uptake and In Vitro Drug Release Studies on Paclitaxel-Loaded Poly(caprolactone)-Grafted Dextran Copolymeric Nanoparticles

Biodegradable and biocompatible polymers play a key role to provide a solution for sustained chemotherapy, when engineered to nanostructure. One such effort has been put forward to engineer self-assembled poly(caprolactone)-grafted dextran (PGD) core–shell micellar vehicle for anticancer drug (paclitaxel) and presented in this study. Paclitaxel-loaded PGD nanoparticles (NPs) were prepared by a modified oil/water emulsion method and characterized by laser light scattering, atomic force microscopy, and zeta potential measurements. The effects of the copolymeric compositions of PGD NPs on drug encapsulation efficiency, in vitro drug release, cellular uptake, and cell viability of NP formulation with paclitaxel were investigated. The drug encapsulation efficiency was determined spectrophotometrically, and in vitro drug release was estimated using dialysis bag. Human gastric cancer cell line (SNU-638) were used to image and measure the cellular uptake of fluorescent PGD NPs. Cancer cell viability of the drug-loaded PGD NPs was measured by crystal violet staining method. From the results obtained on various aspects, we inferred that the above formulated drug-loaded PGD NPs have significant drug encapsulation efficiency, cellular uptake, and the cancer cell mortality.     

Covalent binding of an intermediate(s) in prostaglandin biosynthesis to guinea pig lung microsomal protein

We have investigated the possible covalent binding of intermediates in prostaglandin (PG) biosynthesis to tissue macromolecules. Following incubation of arachidonic acid -1-[14]C (AA) with guinea pig lung microsomes, radioactivity was associated with the microsomal protein which was not dissociated from the protein by exhaustive solvent extraction. Furthermore, filtration of the protein complex through a Sephadex G-25 column failed to dissociate the radioactivity from the protein. This probably indicates covalent binding of AA metabolite(s) to protein. [3]H-PGE₂, [3]H-PGF_2α, and [3]H-thromboxane B₂ (TXB₂) did not show this high affinity binding to microsomal protein. The covalent binding of AA metabolites was greatly reduced in denatured microsomes and was inhibited by the addition of glutathione (GSH) or indomethacin to the incubation mixtures. Chromatographic analysis of the water layers obtained from microsomal incubations with either [3]H-AA or [3]H-GSH suggested the presence of one or more glutathione conjugates derived from AA. These studies indicate that most likely an intermediate formed during PG synthesis from AA covalently binds to tissue macromolecules. This covalent binding may be of physiological and pathological significance.