Genome annotation and comparative genomic analysis of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> MJ01, a new bio-degradation strain isolated from oil-contaminated soil

One of the main challenges in elimination of oil contamination from polluted environments is improvement of biodegradation by highly efficient microorganisms. Bacillus subtilis MJ01 has been evaluated as a new resource for producing biosurfactant compounds. This bacterium, which produces surfactin, is able to enhance bio-accessibility to oil hydrocarbons in contaminated soils. The genome of B. subtilis MJ01 was sequenced and assembled by PacBio RS sequencing technology. One big contig with a length of 4,108,293 bp without any gap was assembled. Genome annotation and prediction of gene showed that MJ01 genome is very similar to B. subtilis spizizenii TU-B-10 (95% similarity). The comparison and analysis of orthologous genes carried out between B. subtilis MJ01, reference strain B. subtilis subsp. subtilis str. 168, and close relative spizizenii TU-B-10 by microscope platform and various bioinformatics tools. More than 88% of 4269 predicted coding sequences in MJ01 had at least one similar sequence in genome of reference strain and spizizenii TU-B-10. Despite this high similarity, some differences were detected among encoding sequences of non-ribosome protein and bacteriocins in MJ01 and spizizenii TU-B-10. MJ01 has unique nucleotide sequences and a novel predicted lasso-peptide bacteriocin; it also has not any similar nucleotide sequence in non-redundant nucleotide data base.     

Do nitroxides protect cardiomyocytes from hydrogen peroxide or superoxide?

The aim of the research was to study the role played by extracellular O ₂ ^.- radicals, which are implicated in cardiac cell damage and the protective effect by cell-permeable, nitroxide, superoxide dismutase-mimics. Cardiomyocytes cultures from 1-day-old rats served as the test-system. Experiments were performed since 5th day in culture when >80% of the cells were beating myocardial cells. Oxidative damage was induced by 0.5 mM hypoxanthine and 0.06 U/ml xanthine oxidase or by 10 mM glucose and 0.15 U/ml glucose oxidase. The parameters used to evaluate damages were spontaneous beating, lactate dehydrogenase release and ATP level. The rhythmic pulsation was followed microscopically. To determine the kinetics of cytosolic enzyme release from the cells, media samples were collected at various points of time and assayed for enzyme activity. To determine the cellular ATP, cells were washed with sodium phosphate buffer, scraped off and boiled for 3 min with sodium phosphate buffer. Following centrifugation the supernatant was collected and ATP was determined by the chemiluminogenic assay using firefly tails. The present results indicate that nitroxide stable free radicals, in the millimolar concentration range, provide full protection without toxic side-effect. Unlike exogenously added SOD that failed to protect, exogenous catalase provided almost full protection. In addition, the metal-chelating agent dipyridyl, but not diethylene-triamine-pentaacetate or desferrioxamine, protected the cultured cells. The present results suggest that H₂O₂ is the predominant toxic species mediating the oxidative damage whereas extracellular superoxide radical does not contribute to cultured cardiomyocyte damage. Since nitroxides do not remove H₂O₂ they can protect the cells possibly by oxidizing the metal ions and inhibiting the Fenton reaction. The superoxide dismutase-mimic activity of nitroxides does not seem to underlie their protective effect, however, the involvement of intracellular O ₂ ^.- cannot be excluded.Abbreviations CHDO 2-spirocyclohexane doxyl (2-cyclohexane-5,5-demethyl-3-oxazolidinoxyl) - DF desferrioxamine - DTPA diethylene-triamine-pentaacetate - EPR electron paramagnetic resonance - HX hypoxanthine - LDH lactate dehydrogenase - SOD superoxide dismutase - SEM standard error of mean: TEMPOL, 4-hydroxy-2,2,6,6-tetramethyl-piperidinoxyl - TEMPAMINE 4-amino-2,2,6,6-tetramethyl-piperidinoxyl - XO xanthine oxidase - CAT catalase     

Damage to cultured lens epithelial cells of squirrels and rabbits by UV-A (99.9%) plus UV-B (0.1%) radiation and alpha tocopherol protection

The purpose of this research is to observe the near-UV radiation induced damage to cultured rabbit and squirrel lens epithelial cells as related to destruction and alterations of specific biochemical targets in the cells and to determine protective effects on the cells and targets that are provided by -tocopherol.Confluent monolayers of cultured rabbit and squirrel lens epithelial cells were exposed to black light (BL) lamps, which emit predominantly UV-A radiation. These cells received a mixture 3 J/cm² of UV-A and 4 mJ/cm² of UV-B per h. This mixture is termed near UV_A (ie:predominantly UV-A). Cells were exposed in Tyrode's or in MEM without or with -tocopherol added at 2.5–10 g/ml. Analyses of cell viability and survival, the physical state of cytoskeletal actin, and the activities of Na-K-ATPase and catalase were made.Exposure to near UV_A damaged these cells as measured by vital staining and colony forming ability. Pretreatment with -tocopherol decreased the magnitude of near UV_A cytotoxicity. Near UV_A exposure in MEM always produced more damage to the cells and biochemical targets than in Tyrode's. Cytoskeletal actin was degraded and the activities of Na-K-ATPase and catalase were markedly inhibited by UV-exposure. All of these targets were at least partially protected by -tocopherol in the medium. Without -tocopherol added to the media, the viability and survival of the cells did not recover even after 25 h of incubation. Cell viability was better protected from near UV_A by -tocopherol than was the ability to grow into colonies. This indicates that -tocopherol protects actin, catalase, and Na-K-ATPase from near UV_A damage.     

A comparative analysis of methylation status of tumor suppressor genes in paired biopsy and serum samples from cervical cancer patients among north indian population

Tumor-specific genetic or epigenetic alterations have been detected in serum DNA in case of various types of cancers. In breast cancer, the detection of tumor suppressor gene hypermethylation has been reported in several body fluids. Promoter hypermethylation of some genes like MYOD1, CALCA, hTERT, etc. has also been detected in serum samples from cervical cancer. The present study is the first report on the comparison of promoter hypermethylation of tumor suppressor genes like p14, p15, p16, p21, p27, p57, p53, p73, RARβ2, FHIT, DAPK, STAT1, and RB1 genes in paired biopsy and serum samples from cervical cancer patients among north Indian population. This is also the first report on the hypermethylation of these genes in serum samples from cervical cancer patients among north Indian population. According to the results of the present study, promoter hypermethylation of these genes can also be detected in serum samples of cervical cancer patients. The sensitivity of detection of promoter hypermethylalion in serum samples of cervical cancer patients as compared to paired biopsy samples was found to be around 83.3%. It was observed that promoter hypermethylation was mainly observed in the serum samples in the higher stages and very rarely in the lower stages. The present study clearly showed that serum of patients with cervical cancer can also be used to study methylated genes as biomarkers.     

TB trifusion antigen adsorbed on calcium phosphate nanoparticles stimulates strong cellular immunity in mice

Among various vaccine candidates, subunit vaccines play an important role in immune protection against tuberculosis (TB). Calcium phosphate (CP) is considered as a strong inorganic adjuvant due to its great potential in increasing immune responses. The purpose of this study was to evaluate specific immune responses following the administration of trifusion-CP nanoparticles. The physiochemical properties of these nanoparticles, including morphology, particle size, zeta potential and adsorption rate, were measured in vitro. Subcutaneous immunization was performed three times on days 0, 14, and 28. Two weeks after the last administration, IFN-gamma, IL-4, and TGF-beta levels were measured by indirect enzyme linked immunosorbent assay (ELISA). The trifusion protein was successfully adsorbed onto calcium phosphate nanoparticles. The mean sizes of the resultant trifusion- CPN and CPN were 97.84 ± 12.08 and 67 ± 11.85 nm, respectively. CPN containing trifusion had stronger ability to induce IFN-gamma than the control groups. IL-4 and TGF-beta secretions in trifusion and trifusion-CPN groups were higher than those in the PBS group. However, there was no significant (p > 0.05) difference in IL-4 and TGFbeta concentrations between trifusion group and trifusion- CPN group. Therefore, calcium phosphate nanoparticles are good candidates for immunization against TB because antigen can be easily adsorbed onto CPN and strong cellular immune responses against CPN-antigen can be stimulated.     

Kani: a QoS-aware hypervisor-level scheduler for cloud computing environments

Cloud computing environments (CCEs) are expected to deliver their services with qualities in service level agreements. On the other hand, they typically employ virtualization technology to consolidate multiple workloads on the same physical machine, thereby enhancing the overall utilization of physical resources. Most existing virtualization technologies are, however, unaware of their delivered quality of services (QoS). For example, the Xen hypervisor merely focuses on fair sharing of processor resources. We believe that CCEs have got married with traditional virtualization technologies without many traits in common. To bridge the gap between these two technologies, we have designed and implemented Kani, a QoS-aware hypervisor-level scheduler. Kani dynamically monitors the quality of delivered services to quantify the deviation between desired and delivered levels of QoS. Using this information, Kani determines how to allocate processor resources among running VMs so as to meet the expected QoS. Our evaluations of Kani scheduler prototype in Xen show that Kani outperforms the default Xen scheduler namely the Credit scheduler. For example, Kani reduces the average response time to requests to an Apache web server by up to \(93.6\,\%\); improves its throughput by up to \(97.9\,\%\); and mitigates the call setup time of an Asterisk media server by up to \(96.6\,\%\).     

Development of a novel engineered antibody targeting human CD123

Antibody engineering involves a range of custom modifications of immunoglobulins to improve their affinity, valency, and pharmacokinetics, ensuring a better target therapy achievement. A number of therapeutic antibodies have been used for cell surface receptor blockage, interfering with the ligand binding and inhibiting receptor-driven activation of cells. Here we describe the construction and characterization of a recombinant bivalent single-chain Fv (biscFv) that targets CD123. On conversion of anti-CD123 scFv to biscFv format, the recognition of the cognate ligand is not altered. Moreover, the increased overall efficacy of the anti-CD123 biscFv in binding and inhibition of CD123/IL-3 (interleukin-3) interactions in TF-1 cells is demonstrated.     

Multiway analysis applied to time‐resolved chemiluminescence for simultaneous determination of paracetamol and codeine in pharmaceuticals

This method is based on the enhancing effect of codeine (COD) and paracetamol (PAR) on the chemiluminescence (CL) reaction of Ru(phen)₃²⁺ with Ce(IV). In the batch mode, COD gives a relatively sharp peak with the highest CL intensity at 4.0 s, whereas the maximum CL intensity of the PAR appears at ~60 s after injection of Ce(IV) solution. Whole CL time profiles allowed use of the time‐resolved CL data in combination with multiway calibration techniques, as multiway partial least squares (N‐PLS), for the quantitative determination of both COD and PAR in binary mixtures. In this work, we found that the impact of Ce(IV) concentration on the CL intensity was different for COD and PAR. Therefore, a Ce(IV) concentration mode was added to the time and sample modes to obtain 3D data. The percent relative standard deviation (%RSD) values for 10 determinations of 1.0 × 10^?5 mol/L of COD and 1.0 × 10^?4 mol/L of PAR were 6.1% and 8.7%, respectively. The limit of detection (LOD) values (S/N = 3) were 0.9 × 10^?8 mol/L and 1.0 × 10^?6 mol/L for COD and PAR, respectively. The proposed method was successfully applied to the determination of PAR and COD in commercial pharmaceutical formulations. Acceptable recoveries (90–110%) were obtained for the quantification of these drugs in the real samples. Copyright © 2016 John Wiley & Sons, Ltd.