Molecular cloning,over expression and characterization of thermoalkalophilic esterases isolated from <Emphasis Type="Italic">Geobacillus</Emphasis> sp.

Due to potential use for variety of biotechnological applications, genes encoding thermoalkalophilic esterase from three different Geobacillus strains isolated from thermal environmental samples in Balçova (Agamemnon) geothermal site were cloned and respective proteins were expressed in Escherichia coli (E.coli) and characterized in detail. Three esterases (Est1, Est2, Est3) were cloned directly by PCR amplification using consensus degenerate primers from genomic DNA of the strains Est1, Est2 and Est3 which were from mud, reinjection water and uncontrolled thermal leak, respectively. The genes contained an open reading frame (ORF) consisting of 741 bp for Est1 and Est2, which encoded 246 amino acids and ORF of Est3 was 729 bp encoded 242 amino acids. The esterase genes were expressed in E. coli and purified using His-Select HF nickel affinity gel. The molecular mass of the recombinant enzyme for each esterase was approximately 27.5 kDa. The three esterases showed high specific activity toward short chain p-NP esters. Recombinant Est1, Est2, Est3 have exhibited similar activity and the highest esterase activity of 1,100 U/mg with p-nitrophenyl acetate (pNPC₂) as substrate was observed with Est1. All three esterase were most active around 65°C and pH 9.5–10.0. The effect of organic solvents, several metal ions, inhibitors and detergents on enzyme activity for purified Est1, Est2, Est3 were determined separately and compared.     

Reverse phase high performance liquid chromatography of Escherichia coli ribosomal proteins: standardization of 70 S, 50 S, and 30 S protein chromatograms

We recently described the use of reverse phase high performance liquid chromatography for the separation of the proteins of the 30 S subunit of Escherichia coli ribosomes (Kerlavage, A. R., Kahan, L., and Cooperman, B. S. (1982) Anal. Biochem. 123, 342-348). In the present studies we report improvements in the technique and its extension to the separation of the proteins of the 50 S subunit and of 70 S ribosomes. Using an octadecasilyl silica column and a trifluoroacetic acid/acetonitrile solvent system, the 21 proteins of the 30 S subunit have been resolved into 17 peaks, the 33 proteins of the 50 S subunit into 22 peaks, and the 53 proteins of the 70 S ribosome into 31 peaks. The proteins present in each peak have been identified by polyacrylamide gel electrophoresis, by comparison with previously standardized chromatograms, and by calibration with authentic samples of purified proteins. All of the known ribosomal proteins have been identified on the chromatograms with the exception of L31 and its variant, L31'. Three protein peaks, not corresponding to known ribosomal proteins, have been observed in preparations from the total protein from 50 S subunits and 70 S ribosomes, but the significance of these peaks is unclear. The reverse phase high performance liquid chromatography technique has the potential for purifying all ribosomal proteins, as demonstrated by the increase in resolution we obtain when a peak isolated under standard gradient conditions and containing several proteins is reapplied to the column and eluted with a shallower gradient. Its utility in preparing proteins for functional studies is demonstrated by a reconstitution of active 30 S particles using 30 S proteins prepared by reverse phase high performance liquid chromatography.     

Differential nucleocytoplasmic shuttling of the nucleoprotein of influenza a viruses and association with host tropism

The nucleoprotein (NP) of influenza A virus plays a crucial role in virus replication, infectivity, and host adaptation. As a major component of the viral ribonucleoprotein complexes (vRNP), NP initiates vRNP shuttling between the nucleus and cytoplasm in the host cell. However, the characteristics of the nucleocytoplasmic shuttling of NP from H1N1 influenza A virus still remain unclear. In the present study, the subcellular localization and the related key residues of the H1N1 influenza virus NP were identified and evaluated. The NP of influenza virus A/WSN/33 (H1N1; WSN) displayed a more obvious nuclear accumulation than A/Anhui/1/2013 (H7N9; AH) and A/chicken/Shandong/lx1023/2007 (H9N2; SD). NP residue K4, located in NLS1, and residue F253, located in NES3, from WSN NP are not conserved in H7N9 and H9N2, which instead encode Q4 and I253, respectively. Crucially, these residues are involved in the regulation of NP nucleocytoplasmic shuttling through interactions with CRM1 and importin‐α. Moreover, residues at position 253 also play important roles in the replication of the virus, resulting in an increase in vRNP polymerase activity and an alteration of the cell tropism and pathogenicity in mice. The present data revealed a pivotal role of the Q4 and I253 residues of NP from H7N9 in enhancing the cytoplasmic accumulation of NP and vRNP activity compared to the K4 and F253 residues in WSN‐NP. In addition, an F253I substitution in the NP of WSN altered the survival ratio of infected mice and the growth curve in infected avian‐origin cells (DF‐1). The current data indicate that the F253I mutation results in attenuated pathogenicity of the virus in mice and altered cell tropism. The present study demonstrated the dissimilarity in subcellular NP transport processes between H1N1 virus WSN and other influenza A virus strains, as well as uncovered the mechanism responsible for this difference.     

An extremely rapid and simple DNA-release method for detection of M. tuberculosis from clinical specimens

A single-step, 5-min lysis method was investigated as a rapid technique to extract genomic DNA from mycobacteria for PCR detection of M. tuberculosis directly from clinical specimens. Of 67 smear-positive clinical specimens, 64 (95.5%) were positive by PCR after this rapid extraction method.     

Exogenous H<Subscript>2</Subscript>S restores ischemic post-conditioning-induced cardioprotection through inhibiting endoplasmic reticulum stress in the aged cardiomyocytes

Background

A gasotransmitter hydrogen sulfide (H₂S) plays an important physiological and pathological role in cardiovascular system. Ischemic post-conditioning (PC) provides cardioprotection in the young hearts but not in the aged hearts. Exogenous H₂S restores PC-induced cardioprotection by inhibition of mitochondrial permeability transition pore opening and oxidative stress and increase of autophagy in the aged hearts. However, whether H₂S contributes to the recovery of PC-induced cardioprotection via down-regulation of endoplasmic reticulum stress (ERS) in the aged hearts is unclear.

Methods

The aged H9C2 cells (the cardiomyocytes line) were induced using H₂O₂ and were exposed to H/R and PC protocols. Cell viability was observed by CCK-8 kit. Apoptosis was detected by Hoechst 33342 staining and flow cytometry. Related protein expressions were detected through Western blot.

Results

In the present study, we found that 30 μM H₂O₂ induced H9C2 cells senescence but not apoptosis. Supplementation of NaHS protected against H/R-induced apoptosis, the expression of cleaved caspase-3 and cleaved caspase-9 and the release of cytochrome c. The addition of NaHS also counteracted the reduction of cell viability caused by H/R and decreased the expression of GRP 78, CHOP, cleaved caspase-12, ATF 4, ATF 6 and XBP-1 and the phosphorylation of PERK, eIF 2α and IRE 1α. Additionally, NaHS increased Bcl-2 expression. PC alone did not provide cardioprotection in H/R-treated aged cardiomyocytes, which was significantly restored by the supplementation of NaHS. The beneficial role of NaHS was similar to the supply of 4-PBA (an inhibitor of ERS), GSK2656157 (an inhibitor of PERK), STF083010 (an inhibitor of IRE 1α), respectively, during PC.

Conclusion

Our results suggest that the recovery of myocardial protection from PC by exogenous H₂S is associated with the inhibition of ERS via down-regulating PERK-eIF 2α-ATF 4, IRE 1α-XBP-1 and ATF 6 pathways in the aged cardiomyocytes.

     

The influence of nuptial feeding and sperm transfer on the immunological cost of reproduction in the ground cricket Allonemobius socius

Mating is often accompanied by decreased female immune function across numerous animals systems, suggesting that immune suppression is a widespread reproductive cost. However, the trade‐off between immunity and reproduction can be minimized when females have access to abundant nutrient resources. This observation suggests that the nuptial gifts provided by males in many insect systems may help to offset the common immunological cost to reproduction. In the present study, this hypothesis is tested in the ground cricket Allonemobius socius (Scudder), whose females receive a sizeable haemolymph‐based gift. Accordingly, male gift donation is controlled by covering the tibial spur (the source of the gift) of randomly chosen males with clear nail polish. The influence of sperm transfer on female immunity is disentangled from that of the nuptial gift by also examining females who fail to receive sperm during mating (spermatophore transfer has a 40% failure rate in virgin males). It is predicted that females who receive a nuptial gift will exhibit superior immune function compared with those who receive no gift. The results show that sperm transfer reduces female immune function, which is an expected immunological cost of reproduction. By contrast to the prediction, nuptial gifts do not minimize the immunological cost of reproduction in this system. Unexpectedly, the receipt of a gift appears to decrease female immune function independent of sperm transfer. The findings suggest that the nuptial gift, similar to sperm, signals the female to begin her reproductive investment, causing limited resources to be reallocated from immune function.     

Assessment of Total and Organic Mercury Levels in Blue Sharks (Prionace glauca) from the South and Southeastern Brazilian Coast

Mercury occurrence was evaluated in samples of edible muscle tissue of 27 blue sharks (Prionace glauca) caught in the Atlantic Ocean, adjacent to the south and southeastern Brazilian coast, indicating a slight increase in comparison with previous data obtained for the same studied area and being higher than those fish caught at different sites of the Atlantic Ocean. Total Hg concentrations ranged from 0.46 to 2.40 mg kg^?1 with the organic Hg fraction ranging between 0.44 and 2.37 mg kg^?1. A negative correlation between total Hg concentration in muscle tissue and blue shark size was obtained, and 40 % of samples analyzed had Hg concentrations higher than 1.0 mg kg^?1 Hg, the maximum concentration permitted in Brazilian predator fish. Data obtained showed that total Hg can be used as a reliable predictor of organic Hg in blue shark muscle because 95 to 98 % of the total Hg measured was found to be organic mercury. The wide range of Hg concentrations obtained for our set of samples can be explained by the heterogeneity of sampled population and the large size of the studied area. Given the adverse toxicological effects of Hg on animals and humans, a regular monitoring program of Hg contamination in Brazilian marine ecosystem can be recommended.     

Computer aided prediction and identification of potential epitopes in the receptor binding domain (RBD) of spike (S) glycoprotein of MERS-CoV

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) belongs to the coronaviridae family. In spite of several outbreaks inthe very recent years, no vaccine against this deadly virus is developed yet. In this study, the receptor binding domain (RBD) ofSpike (S) glycoprotein of MERS-CoV was analyzed through Computational Immunology approach to identify the antigenicdeterminants (epitopes). In order to do so, the sequences of S glycoprotein that belong to different geographical regions werealigned to observe the conservancy of MERS-CoV RBD. The immune parameters of this region were determined using different insilico tools and Immune Epitope Database (IEDB). Molecular docking study was also employed to check the affinity of the potentialepitope towards the binding cleft of the specific HLA allele. The N-terminus RBD (S367-S606) of S glycoprotein was found to beconserved among all the available strains of MERS-CoV. Based on the lower IC₅₀ value, a total of eight potential T-cell epitopes and19 major histocompatibility complex (MHC) class-I alleles were identified for this conserved region. A 9-mer epitope CYSSLILDYdisplayed interactions with the maximum number of MHC class-I molecules and projected the highest peak in the B-cellantigenicity plot which concludes that it could be a better choice for designing an epitope based peptide vaccine against MERSCoVconsidering that it must undergo further in vitro and in vivo experiments. Moreover, in molecular docking study, this epitopewas found to have a significant binding affinity of -8.5 kcal/mol towards the binding cleft of the HLA-C*12:03 molecule.     

Diagnostic detection of intended bacteria associated with respiratory tract infections among Kelantanese Malaysian Hajj pilgrims by a ready-to-use,thermostable multiplex PCR assay

Bacterial respiratory tract infections (RTIs) are prone to be associated with serious health problems during the annual Hajj pilgrimage and are a public health concern due to the potential of pathogens transmission across continents. This study aimed to perform a diagnostic screening of intended bacteria associated with RTIs among Malaysian Hajj pilgrims by using a newly developed PCR assay. Expectorated sputum specimens (n = 202) and sociodemographic characteristics of the returning Hajj pilgrims were collected upon arrival in Kelantan, Malaysia. Diagnostic screening of bacterial respiratory pathogens was performed using a thermostabilized multiplex PCR assay in parallel with the sputum culture. Of the six intended bacteria: Haemophilus influenzae, Klebsiella pneumoniae, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pneumoniae, the sputum specimens were found positive for H. influenzae (n = 139), K. pneumoniae (n = 20), and S. pneumoniae (n = 19) by the multiplex PCR assay. The sensitivity, specificity, positive- and negative predictive values (PPV and NPV) of this assay were 100% (95% confidence interval (CI): 97.85% to 100.00%), 92.23% (95% CI: 85.27% to 96.59%), 95.51% (95% CI: 91.61% to 97.64%) and 100.00%, respectively. The accuracy of this assay was 97.07% (95% CI: 94.31% to 98.73%). Overall, H. influenzae was found to be the predominant organism in the pilgrims’ sputa by both molecular and microbial culture methods. The multiplex PCR assay would enable a simple, faster and reliable means for the massive screening of intended bacteria compared to the sputum culture, especially during the Hajj pilgrimage.