In silico assessment of EpCAM transcriptional expression and determination of the prognostic biomarker for human lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC)

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein which is involved in cell signaling, proliferation, maturation, and movement, all of which are crucial for the proper development of cells and tissues. Cleavage of the EpCAM protein leads to the up-regulation of c-myc, e-fabp, and cyclins A and E which promote tumorigenesis. EpCAM can act as potential diagnostic and prognostic biomarker for different types of cancers as it is also found to be expressed in epithelia and epithelial-derived neoplasms. Hence, we aimed to analyze the EpCAM gene expression and any associated feedback in the patients of two major types of lung cancer (LC) i.e., lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), based on the publicly available online databases. In this study, server-based gene expression analysis represents the up-regulation of EpCAM in both LUAD and LUSC subtypes as compared to the corresponding normal tissues. Besides, the histological sections revealed the over-expression of EpCAM protein in cancerous tissues by depicting strong staining signals. Furthermore, mutation analysis suggested missense as the predominant type of mutation both in LUAD and LUSC in the EpCAM gene. A significant correlation (P-value < 0.05) between the higher EpCAM expression and lower patient survival was also found in this study. Finally, the co-expressed genes were identified with their ontological features and signaling pathways associated in LC development. The overall study suggests EpCAM to be a significant biomarker for human LC prognosis.     

A gemcitabine sensitivity screen identifies a role for NEK9 in the replication stress response

The Replication Stress Response (RSR) is a signaling network that recognizes challenges to DNA replication and coordinates diverse DNA repair and cell-cycle checkpoint pathways. Gemcitabine is a nucleoside analogue that causes cytotoxicity by inducing DNA replication blocks. Using a synthetic lethal screen of a RNAi library of nuclear enzymes to identify genes that when silenced cause gemcitabine sensitization or resistance in human triple-negative breast cancer cells, we identified NIMA (never in mitosis gene A)-related kinase 9 (NEK9) as a key component of the RSR. NEK9 depletion in cells leads to replication stress hypersensitivity, spontaneous accumulation of DNA damage and RPA70 foci, and an impairment in recovery from replication arrest. NEK9 protein levels also increase in response to replication stress. NEK9 complexes with CHK1, and moreover, NEK9 depletion impairs CHK1 autophosphorylation and kinase activity in response to replication stress. Thus, NEK9 is a critical component of the RSR that promotes CHK1 activity, maintaining genome integrity following challenges to DNA replication.     

Production of an anti‐Candida peptide via fed batch and ion exchange chromatography

Interest in peptides as diagnostic and therapeutic materials require their manufacture via either a recombinant or synthetic route. This study examined the former, where a recombinant fusion consisting of an antifungal peptide was expressed and isolated from Escherichia coli. Fed batch fermentation with E. coli harboring an arabinose‐inducible plasmid produced the 12 residue anti‐Candida peptide fused to the N‐terminal of Green Fluorescent Protein (GFP_UV). The purification of the fusion protein, using ion‐exchange chromatography, was monitored by using the intrinsic fluorescence of GFP_UV. The recombinant antifungal peptide was successfully released by cyanogen bromide‐induced cleavage of the fusion protein. The recombinant peptide showed the expected antifungal activity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:865–871, 2016     

Bio‐based extraction and stabilization of anthocyanins

This work reports a novel method of recovering anthocyanin compounds from highly‐pigmented grapes via a fermentation based approach. It was hypothesized that batch growth of Zymomonas mobilis on simple medium would produce both ethanol and enzymes/biomass‐acting materials, the combination of which will provide a superior extraction when compared to simple alcohol extraction. To examine this hypothesis, Z. mobilis was fermented in a batch consisting of mashed Vitis vinifera and glucose, and the recovered anthocyanin pool was compared to that recovered via extraction with ethanol. Data indicated higher amounts of anthocyanins were recovered when compared to simple solvent addition. Additionally, the percent polymeric form of the anthocyanins could be manipulated by the level of aeration maintained in the fermentation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:601–605, 2016     

Sequence- or Position-Specific Mutations in the Carboxyl-Terminal FL Motif of the Kidney Sodium Bicarbonate Cotransporter (NBC1) Disrupt Its Basolateral Targeting and α-Helical Structure

The sodium-bicarbonate cotransporter NBC1 is targeted exclusively at the basolateral membrane. Mutagenesis of a dihydrophobic FL motif (residues 1013–1014) in the C-terminal domain disrupts the targeting of NBC1. In the present study, we determined the precise constraints of the FL motif required for basolateral targeting of NBC1 by expressing epitope-tagged wild-type and mutant NBC1 in MDCK cells and RNA-injected Xenopus oocytes and examining their subcellular localization. We assayed the functional activity of the mutants by measuring bicarbonate-induced currents in oocytes. Wild-type NBC1 (containing PFLS) was expressed exclusively on the basolateral membrane in MDCK cells. Reversal of the FL motif (PLFS) had no effect on basolateral targeting or activity. Shifting the FL motif one residue upstream (FLPS) resulted in mistargeting of the apical membrane but the FLPS mutant retained its functional activity in oocytes. Shifting the FL motif one residue downstream resulted in a mutant (PSFL) that did not efficiently translocate to the plasma membrane and was instead colocalized with the ER marker, protein disulfide isomerase (PDI). Analysis of circular dichroism (CD) revealed that a short peptide, 20 amino acid residues, of wild-type NBC1 contained a significant α-helical structure, whereas peptides in which the FL motif was reversed or C-terminally shifted were disordered. We therefore propose that the specific orientation and the precise location of the FL motif in the primary sequence of NBC1 are strict requirements for the α-helical structure of the C-terminal cytoplasmic domain and for targeting of NBC1 to the basolateral membrane.     

Characterization of domain-specific interaction of synthesized dye with serum proteins by spectroscopic and docking approaches along with determination of in vitro cytotoxicity and antiviral activity

The interaction between a synthesized dye with proteins, bovine, and human serum albumin (BSA, HSA, respectively) under physiological conditions has been characterized in detail, by means of steady-state and time-resolved fluorescence, UV–vis absorption, and circular dichroism (CD) techniques. An extensive time-resolved fluorescence spectroscopic characterization of the quenching process has been undertaken in conjugation with temperature-dependent fluorescence quenching studies to divulge the actual quenching mechanism. From the thermodynamic observations, it is clear that the binding process is a spontaneous molecular interaction, in which van der Waals and hydrogen bonding interactions play the major roles. The UV–vis absorption and CD results confirm that the dye can induce conformational and micro-environmental changes of both the proteins. In addition, the dye binding provokes the functionality of the native proteins in terms of esterase-like activity. The average binding distance (r) between proteins and dye has been calculated using FRET. Cytotoxicity and antiviral effects of the dye have been found using Vero cell and HSV-1F virus by performing MTT assay. The AutoDock-based docking simulation reveals the probable binding location of dye within the sub-domain IIA of HSA and IB of BSA.