Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems

Pathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% for Escherichia coli and human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such as Legionella pneumophila, Shigella flexneri, and Campylobacter fetus were detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment.     

A predictive method for the evaluation of peptide binding in pocket 1 of HLA-DRB1 via global minimization of energy interactions

Human leukocyte antigens (HLA) or histocompatibility molecules are glycoproteins that play a pivotal role in the development of an effective immune response. An important function of the HLA molecules is the ability to bind and present antigen peptides to T lymphocytes. Presently there is no comprehensive way of predicting and energetically evaluating peptide binding on HLA molecules. To quantitatively determine the binding specificity of a class II HLA molecule interacting with peptides, a novel decomposition approach based on deterministic global optimization is proposed that takes advantage of the topography of HLA binding grove, and examined the interactions of the bound peptide with the five different pockets. In particular, the main focus of this paper is the study of pocket 1 of HLA DR1 (DRB1*0101 allele). The determination of the minimum energy conformation is based on the ECEPP/3 potential energy model that describes the energetics of the atomic interactions. The minimization of the total potential energy is formulated on the set of peptide dihedral angles, Euler angles, and translation variables to describe the relative position. The deterministic global optimization algorithm, αBB, which has been shown to be ϵ-convergent to the global minimum potential energy through the solution of a series of nonlinear convex optimization problems, is utilized. The PACK conformational energy model that utilizes the ECEPP/3 model but also allows the consideration of protein chain interactions is interfaced with αBB. MSEED, a program used to calculate the solvation contribution via the area accessible to the solvent, is also interfaced with αBB. Results are presented for the entire array of naturally occurring amino acids binding to pocket 1 of the HLA DR1 molecule and very good agreement with experimental binding assays is obtained. Proteins 29:87–102, 1997. © 1997 Wiley-Liss, Inc.     

Analysis of the signals for polarized transport of influenza virus (A/WSN/33) neuraminidase and human transferrin receptor, type II transmembrane proteins.

In polarized MDCK cells influenza virus (A/WSN/33) neuraminidase (NA) and human transferrin receptor (TR), type II glycoproteins, when expressed from cloned cDNAs, were transported and accumulated preferentially on the apical and basolateral surfaces, respectively. We have investigated the signals for polarized sorting by constructing chimeras between NA and TR and by making deletion mutants. NATR delta 90, which contains the cytoplasmic tail and transmembrane domain of NA and the ectodomain of TR, was found to be localized predominantly on the apical membrane, whereas TRNA delta 35, containing the cytoplasmic and transmembrane domains of TR and the ectodomain of NA, was expressed preferentially on the basolateral membrane. TR delta 57, a TR deletion mutant lacking 57 amino acids in the TR cytoplasmic tail, did not exhibit any polarized expression and was present on both apical and basolateral surfaces, whereas a deletion mutant (NA delta 28-35) lacking amino acid residues from 28 to 35 in the transmembrane domain of NA resulted in secretion of the NA ectodomain predominantly from the apical side. These results taken together indicate that the cytoplasmic tail of TR was sufficient for basolateral transport, but influenza virus NA possesses two sorting signals, one in the cytoplasmic or transmembrane domain and the other within the ectodomain, both of which are independently able to transport the protein to the apical plasma membrane.     

Signal processing, glycosylation, and secretion of mutant hemagglutinins of a human influenza virus by Saccharomyces cerevisiae.

We investigated the nature of signal recognition, transport, and secretion of mutant hemagglutinins (HAs) of a human influenza virus by the yeast Saccharomyces cerevisiae. The cDNA sequences encoding variant forms of influenza HA were expressed in S. cerevisiae. The HA polypeptides (HA500 and HA325) that were synthesized with their N-terminal signal peptides were correctly targeted to the membrane compartment where they were glycosylated. In contrast, the HA polypeptides (HA484 and HA308) lacking the signal peptide were expressed in the cytoplasm and did not undergo any glycosidic modification, demonstrating the importance of the heterologous signal sequence in the early steps of translocation in S. cerevisiae. The analysis of the N-terminal amino acid sequence of HA500 and HA325 polypeptides demonstrated the correct cleavage of the signal peptide, indicating the structural compatibility of a heterologous signal peptide for efficient recognition and processing by the yeast translocation machinery. The membrane-sequestered and glycosylated HA polypeptides were relatively stable in S. cerevisiae compared with the signal-minus, nonglycosylated HA molecules. Although both the anchor-minus HA (HA500) and HA1 (HA325) polypeptides were targeted efficiently to the membrane, their glycosylation and transport patterns were shown to be different. During pulse-chase, the HA500 remained cell-associated with no detectable secretion into the extracellular medium, whereas the HA325 secreted into the medium. Furthermore, only the cell-associated and secreted forms of HA325 and not HA500 appeared to have undergone hyperglycosylation with the extensive addition of high-molecular-weight outer-chain mannans. Possible reasons for the observed phenotypic behavior of these two mutant HAs are discussed.     

Rapid and robust generation of functional oligodendrocyte progenitor cells from epiblast stem cells

Myelin-related disorders such as multiple sclerosis and leukodystrophies, for which restoration of oligodendrocyte function would be an effective treatment, are poised to benefit greatly from stem cell biology. Progress in myelin repair has been constrained by difficulties in generating pure populations of oligodendrocyte progenitor cells (OPCs) in sufficient quantities. Pluripotent stem cells theoretically provide an unlimited source of OPCs, but current differentiation strategies are poorly reproducible and generate heterogenous populations of cells. Here we provide a platform for the directed differentiation of pluripotent mouse epiblast stem cells (EpiSCs) through defined developmental transitions into a pure population of highly expandable OPCs in 10 d. These OPCs robustly differentiate into myelinating oligodendrocytes in vitro and in vivo. Our results demonstrate that mouse pluripotent stem cells provide a pure population of myelinogenic oligodendrocytes and offer a tractable platform for defining the molecular regulation of oligodendrocyte development and drug screening.     

Phytoremediation of 137Cs: factors and consequences in the environment

Radionuclide contamination is a concerning threat due to unexpected nuclear disasters and authorized discharge of radioactive elements, both in the past and in present times. Use of atomic power for energy generation is associated with unresolved issues concerning storage of residues and contaminants. For example, the nuclear accidents in Chernobyl 1986 and Fukushima 2011 resulted in considerable deposition of cesium (Cs) in soil, along with other radionuclides. Among Cs radioactive variants, the anthropogenic radioisotope ¹³⁷Cs (t_½?=?30.16 years) is of serious environmental concern, owing to its rapid incorporation into biological systems and emission of β and γ radiation during the decaying process. To remediate contaminated areas, mostly conventional techniques are applied that are not eco-friendly. Hence, an alternative green technology, i.e., phytoremediation, should in future be considered and implemented. This sustainable technology generates limited secondary waste and its objectives are to utilize hyper-accumulating plants to extract, stabilize, degrade, and filter the radionuclides. The review highlights plant mechanisms for up-taking radionuclides and influences of different environmental factors involved in the process, while considering its long-term effects.

     

Rational design and synthesis of 2-anilinopyridinyl-benzothiazole Schiff bases as antimitotic agents

Based on our previous results and literature precedence, a series of 2-anilinopyridinyl-benzothiazole Schiff bases were rationally designed by performing molecular modeling experiments on some selected molecules. The binding energies of the docked molecules were better than the E7010, and the Schiff base with trimethoxy group on benzothiazole moiety, 4y was the best. This was followed by the synthesis of a series of the designed molecules by a convenient synthetic route and evaluation of their anticancer potential. Most of the compounds have shown significant growth inhibition against the tested cell lines and the compound 4y exhibited good antiproliferative activity with a GI₅₀ value of 3.8 µM specifically against the cell line DU145. In agreement with the docking results, 4y exerted cytotoxicity by the disruption of the microtubule dynamics by inhibiting tubulin polymerization via effective binding into colchicine domain, comparable to E7010. Detailed binding modes of 4y with colchicine binding site of tubulin were studied by molecular docking. Furthermore, 4y induced apoptosis as evidenced by biological studies like mitochondrial membrane potential, caspase-3, and Annexin V-FITC assays.