Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars

Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 was mutagenized using UV-C irradiation to produce yeast strains for anaerobic conversion of lignocellulosic sugars to ethanol. UV-C irradiation potentially produces large numbers of random mutations broadly and uniformly over the whole genome to generate unique strains. Wild-type cultures of S. stipitis NRRL Y-7124 were subjected to UV-C (234 nm) irradiation targeted at approximately 40% cell survival. When surviving cells were selected in sufficient numbers via automated plating strategies and cultured anaerobically on xylose medium for 5 months at 28°C, five novel mutagenized S. stipitis strains were obtained. Variable number tandem repeat analysis revealed that mutations had occurred in the genome, which may have produced genes that allowed the anaerobic utilization of xylose. The mutagenized strains were capable of growing anaerobically on xylose/glucose substrate with higher ethanol production during 250- to 500-h growth than a Saccharomyces cerevisiae yeast strain that is the standard for industrial fuel ethanol production. The S. stipitis strains resulting from this intense multigene mutagenesis strategy have potential application in industrial fuel ethanol production from lignocellulosic hydrolysates.     

Will an Unsupervised Self-Testing Strategy for HIV Work in Health Care Workers of South Africa? A Cross Sectional Pilot Feasibility Study

Background

In South Africa, stigma, discrimination, social visibility and fear of loss of confidentiality impede health facility-based HIV testing. With 50% of adults having ever tested for HIV in their lifetime, private, alternative testing options are urgently needed. Non-invasive, oral self-tests offer a potential for a confidential, unsupervised HIV self-testing option, but global data are limited.

Methods

A pilot cross-sectional study was conducted from January to June 2012 in health care workers based at the University of Cape Town, South Africa. An innovative, unsupervised, self-testing strategy was evaluated for feasibility; defined as completion of self-testing process (i.e., self test conduct, interpretation and linkage). An oral point-of-care HIV test, an Internet and paper-based self-test HIV applications, and mobile phones were synergized to create an unsupervised strategy. Self-tests were additionally confirmed with rapid tests on site and laboratory tests. Of 270 health care workers (18 years and above, of unknown HIV status approached), 251 consented for participation.

Findings

Overall, about 91% participants rated a positive experience with the strategy. Of 251 participants, 126 evaluated the Internet and 125 the paper-based application successfully; completion rate of 99.2%. All sero-positives were linked to treatment (completion rate:100% (95% CI, 66.0–100). About half of sero-negatives were offered counselling on mobile phones; completion rate: 44.6% (95% CI, 38.0–51.0). A majority of participants (78.1%) were females, aged 18–24 years (61.4%). Nine participants were found sero-positive after confirmatory tests (prevalence 3.6% 95% CI, 1.8–6.9). Six of nine positive self-tests were accurately interpreted; sensitivity: 66.7% (95% CI, 30.9–91.0); specificity:100% (95% CI, 98.1–100).

Interpretation

Our unsupervised self-testing strategy was feasible to operationalize in health care workers in South Africa. Linkages were successfully operationalized with mobile phones in all sero-positives and about half of the sero-negatives sought post-test counselling. Controlled trials and implementation research studies are needed before a scale-up is considered.     

Stepwise development of structure–activity relationship of diverse PARP-1 inhibitors through comparative and validated in silico modeling techniques and molecular dynamics simulation

Inhibitors of poly (ADP-ribose) polymerase-1 (PARP-1) enzyme are useful for the treatment of various diseases including cancer. Comparative in silico studies were performed on different ligand-based (2D-QSAR, Kernel-based partial least square (KPLS) analysis, Pharmacophore Search Engine (PHASE) pharmacophore mapping), and structure-based (molecular docking, MM-GBSA analyses, Gaussian-based 3D-QSAR analyses on docked poses) modeling techniques to explore the structure–activity relationship of a diverse set of PARP-1 inhibitors. Two-dimensional (2D)-QSAR highlighted the importance of charge topological index (JGI7), fractional polar surface area (Jurs_FPSA3), and connectivity index (CIC2) along with different molecular fragments. Favorable and unfavorable fingerprints were demonstrated in KPLS analysis, whereas important pharmacophore features (one acceptor, one donor, and two ring aromatic) along with favorable and unfavorable field effects were demonstrated in PHASE-based pharmacophore model. MM-GBSA analyses revealed significance of different polar, non-polar, and solvation energies. Docking-based alignment of ligands was used to perform Gaussian-based 3D-QSAR study that further demonstrated importance of different field effects. Overall, it was found that polar interactions (hydrogen bonding, bridged hydrogen bonding, and pi–cation) play major roles for higher activity. Steric groups increase the total contact surface area but it should have higher fractional polar surface area to adjust solvation energy. Structure-based pharmacophore mapping spotted the positive ionizable feature of ligands as the most important feature for discriminating highly active compounds from inactives. Molecular dynamics simulation, conducted on highly active ligands, described the dynamic behaviors of the protein complexes and supported the interpretations obtained from other modeling analyses. The current study may be useful for designing PARP-1 inhibitors.     

Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis.

The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and pH optima) identical to those of the native enzyme purified from C. thermosulfurogenes. Simple heat treatment of crude extracts from E. coli and B. subtilis cells carrying the recombinant plasmid at 85 degrees C for 15 min generated 80% pure glucose isomerase. The maximum conversion yield of glucose (35%, wt/wt) to fructose with the thermostable glucose isomerase (10.8 U/g of dry substrate) was 52% at pH 7.0 and 70 degrees C.     

Sandwich microgravimetric immunoassay: sensitive and specific detection of low molecular weight analytes using piezoelectric quartz crystal

A multi-channel sandwich microgravimetric immunoassay (sMIA), using the quartz crystal microbalance (QCM) principle, has been developed to quantify low molecular weight substances in standard solutions. An antigen is sandwiched between two antigen-specific antibodies: the first antibody is coated on the quartz crystal surface and the second antibody is used for the detection of analyte. The concentration of low molecular weight antigen (insulin was used in this study, M _r6000 Da) was correlated with the shift of resonant frequency of QCM system before and after second antibody binding to insulin. The developed assay is highly specific showing low cross-reactivity, and is sensitive to approx. 1 ng insulin ml^–1 with a linear response for insulin from 10 g ml^–1 to 10 ng ml^–1 in standard solutions. The technique may also be applied for the detection of other small biomolecules.     

Piriformospora indica?rescues growth diminution of rice seedlings during high salt stress

Piriformospora indica association has been reported to increase biotic as well as abiotic stress tolerance of its host plants. We analyzed the beneficial effect of P. indica association on rice seedlings during high salt stress conditions (200 and 300 mM NaCl). The growth parameters of rice seedlings such as root and shoot lengths or fresh and dry weights were found to be enhanced in P. indica-inoculated rice seedlings as compared with non-inoculated control seedlings, irrespective of whether they are exposed to salt stress or not. However, salt-stressed seedlings performed much better in the presence of the fungus compared with non-inoculated control seedlings. The photosynthetic pigment content [chlorophyll (Chl) a, Chl b, and carotenoids] was significantly higher in P. indica-inoculated rice seedlings under high salt stress conditions as compared with salt-treated non-inoculated rice seedlings, in which these pigments were found to be decreased. Proline accumulation was also observed during P. indica colonization, which may help the inoculated plants to become salt tolerant. Taken together, P. indica rescues growth diminution of rice seedlings under salt stress.     

Bimodal oxygen uptake in juveniles and adults amphibious fish,Channa (= Ophiocephalus) marulius

Oxygen uptake of Channa marulius was studied under water with and without access to air. There was a significant increase in the oxygen uptake through the gills when access to air was prevented. However, this value (0.863 ± 0.058 mlO₂/indiv./h) was quite low in comparison to the total bimodal oxygen uptake (2.04 ± 0.14 mlO₂/indiv./h) in juveniles. In adult fish the oxygen uptake per unit time increased appreciably (4.673 ± 0.404 mlO₂/indiv./h). In juveniles as well as in adults the air breathing dominated over aquatic breathing. This fish showed a definite circadian rhythm in the bimodal oxygen uptake during different hours of the day.This work was performed in the Ichthyology Laboratory, P. G. Dept. of Zoology, Bhagalpur University, and was supported by a research grant from Bhagalpur University     

Microscopic and spectroscopic study of the corona formation and unfolding of human haemoglobin in presence of ZnO nanoparticles

The interaction of zinc oxide nanoparticles (ZnO NPs) with human haemoglobin (Hb) is studied for the biologically safe application of ZnO NPs in the human body. The Hb corona is formed around the ZnO nanoparticles, directly observed from high‐resolution transmission electron microscopy (HRTEM) images. Hb formed ‘hard corona' on the surface of ZnO NPs from an exponential association mechanism over a very short duration, as well as unfolding of Hb that occurred over a long lifetime. Dynamic light scattering measurements demonstrated that the ZnO NPs were completely covered by Hb with shell thickness of c. 6 nm that formed a ‘hard corona'. Zeta potential measurements represented that the ZnO NPs were fully covered by Hb molecules using an exponential association mechanism. Tryptophans (TRY), as well as heme‐porphyrin moieties of Hb, are the major binding sites for ZnO NPs. The nature of the interaction between ZnO NPs and Hb was analysed from the fluorescence quenching of TRYs. Electrostatic interaction, along with the hydrophobic interaction between ZnO NPs and Hb, is responsible for the conformational change in Hb due to increase in the percentage of β‐sheets together with a decrease in α‐helices.     

Flavone-resistant Leishmania donovani Overexpresses LdMRP2 Transporter in the Parasite and Activates Host MRP2 on Macrophages to Circumvent the Flavone-mediated Cell Death

In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries, and in many instances, it is due to overexpressed ABC efflux pumps. Progressively adapted baicalein (BLN)-resistant parasites (pB²⁵R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or Leishmania donovani multidrug resistance protein 2 (LdMRP2). The protein is primarily localized in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be cured by treatment of macrophages with BLN. Amastigote resistance is concomitant with the overexpression of macrophage MRP2 transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element 1 is activated upon infection. The expression of this phase II detoxifying gene is regulated by NFE2-related factor 2 (Nrf2)-mediated antioxidant response element activation. In view of the fact that the signaling pathway of phosphoinositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with the intricate pathway of phosphoinositol 3-kinase-mediated nuclear translocation of Nrf2, which activates MRP2 expression in macrophages upon infection by the parasites. In contrast, phalloidin, an agent that prevents depolymerization of actin filaments, inhibits Nrf2 translocation and Mrp2 gene activation by pB²⁵R infection. Taken together, these results provide insight into the mechanisms by which resistant clinical isolates of L. donovani induce intracellular events relevant to drug resistance.