Response surface optimization of effective medium constituents for the production of alkaline protease from a newly isolated strain of Pseudomonas aeruginosa

Optimization of the fermentation medium for maximum alkaline protease production was carried out with a new strain of Pseudomonas aeruginosa (B-2). Replacing the protein source/inducer (albumin in place of casein) brought about significant increase in yield after 48 hr of inoculation. Three most effective medium constituents identified by initial screening method of Plackett-Burman were albumin, (NH4)2SO4 and glucose. Central Composite Design (CCD) and Response Surface Methodology (RSM) were used in the design of the experiment and in the analysis of the results. Optimum levels of the effective medium constituents were albumin (6.586%); (NH4)2SO4, 0.164%; and glucose, 6.72%. The alkaline protease production increased from 533460 to 793492 Ul(-1).     

Link between MnSOD Ala16Val (rs4880) polymorphism and asthma risk is insignificant from sequential meta-analysis

The mitochondrial manganese superoxide dismutase (MnSOD) enzyme protects lungs against oxidative stress by neutralizing the free radical superoxide produced in the respiratory function. This has relevance to asthma. Therefore, it is of interest to describe the potential effect of MnSOD Ala16Val genetic polymorphism to asthma risk. Known data in this context is inconclusive in nature. The possible link between MnSOD Ala16Val polymorphism and asthma is explored using sequence meta-analysis. Data from the pooled analysis of MnSOD Ala16Val polymorphism using five genetic models i.e., allelic (Val vs. Ala: p=0.846; OR=1.033, 95% CI=0.742 to 1.440) is discussed. Homozygous (Val Val vs. Ala Ala: p=0.517; OR=1.307, 95% CI=0.582 to 2.932) and heterozygous (Val Ala vs. Ala Ala: p=0.307; OR=1.138, 95% CI=0.888 to 1.459) data using the described models are documented. Data from the dominant model (Val Val + Val Ala vs. Ala Ala: p=0.301; OR=1.289, 95% CI=0.797 to 2.085) and the recessive model (Val Val vs. Val Ala + Ala Ala: p=0.761; OR=0.924, 95% CI=0.555 to 1.538) analyses for several ethnic subgroups in this context is reported.     

Feline Calicivirus,Murine Norovirus,Porcine Sapovirus,and Tulane Virus Survival on Postharvest Lettuce

Human norovirus (HuNoV) is the leading cause of foodborne illnesses, with an increasing number of outbreaks associated with leafy greens. Because HuNoV cannot be routinely cultured, culturable feline calicivirus (FCV), murine norovirus (MNV), porcine sapovirus (SaV), and Tulane virus (TV) have been used as surrogates. These viruses are generated in different cell lines as infected cell lysates, which may differentially affect their stability. Our objective was to uniformly compare the survival of these viruses on postharvest lettuce while evaluating the effects of cell lysates on their survival. Viruses were semipurified from cell lysates by ultrafiltration or ultracentrifugation followed by resuspension in sterile water. Virus survival was examined before and after semipurification: in suspension at room temperature (RT) until day 28 and on lettuce leaves stored at RT for 3 days or at 4°C for 7 and 14 days. In suspension, both methods significantly enhanced the survival of all viruses. On lettuce, the survival of MNV in cell lysates was similar to that in water, under all storage conditions. In contrast, the survival of FCV, SaV, and TV was differentially enhanced, under different storage conditions, by removing cell lysates. Following semipurification, viruses showed similar persistence to each other on lettuce stored under all conditions, with the exception of ultracentrifugation-purified FCV, which showed a higher inactivation rate than MNV at 4°C for 14 days. In conclusion, the presence of cell lysates in viral suspensions underestimated the survivability of these surrogate viruses, while viral semipurification revealed similar survivabilities on postharvest lettuce leaves.     

Integrating Bacterial and Viral Water Quality Assessment to Predict Swimming-Associated Illness at a Freshwater Beach: A Cohort Study

Background & Objective

Recreational waters impacted by fecal contamination have been linked to gastrointestinal illness in swimmer populations. To date, few epidemiologic studies examine the risk for swimming-related illnesses based upon simultaneous exposure to more than one microbial surrogate (e.g. culturable E. coli densities, genetic markers). We addressed this research gap by investigating the association between swimming-related illness frequency and water quality determined from multiple bacterial and viral genetic markers.

Methods

Viral and bacterial genetic marker densities were determined from beach water samples collected over 23 weekend days and were quantified using quantitative polymerase chain reaction (qPCR). These genetic marker data were paired with previously determined human exposure data gathered as part of a cohort study carried out among beach users at East Fork Lake in Ohio, USA in 2009. Using previously unavailable genetic marker data in logistic regression models, single- and multi-marker/multi-water quality indicator approaches for predicting swimming-related illness were evaluated for associations with swimming-associated gastrointestinal illness.

Results

Data pertaining to genetic marker exposure and 8- or 9-day health outcomes were available for a total of 600 healthy susceptible swimmers, and with this population we observed a significant positive association between human adenovirus (HAdV) exposure and diarrhea (odds ratio  = 1.6; 95% confidence interval: 1.1–2.3) as well as gastrointestinal illness (OR  = 1.5; 95% CI: 1.0–2.2) upon adjusting for culturable E. coli densities in multivariable models. No significant associations between bacterial genetic markers and swimming-associated illness were observed.

Conclusions

This study provides evidence that a combined measure of recreational water quality that simultaneously considers both bacterial and viral densities, particularly HAdV, may improve prediction of disease risk than a measure of a single agent in a beach environment likely influenced by nonpoint source human fecal contamination.     

Cytokine responses in porcine respiratory coronavirus-infected pigs treated with corticosteroids as a model for severe acute respiratory syndrome

The effectiveness and potential immunosuppressive effects of anti-inflammatory glucocorticoids in the lungs of severe acute respiratory syndrome (SARS) patients are undefined. We treated porcine respiratory coronavirus (PRCV)-infected conventional pigs with the corticosteroid dexamethasone (DEX) as a model for SARS. Innate and Th1 cytokines in bronchoalveolar lavage (BAL) and serum were elevated in PRCV-infected pigs compared to controls, but were decreased after DEX treatment in the PRCV-infected, DEX-treated (PRCV/DEX) pigs. Although decreased in BAL, Th2 cytokine levels were higher in serum after DEX treatment. Levels of the proinflammatory cytokine interleukin-6 in BAL and serum were decreased in PRCV/DEX pigs early but increased later compared to those in phosphate-buffered saline-treated, PRCV-infected pigs, corresponding to a similar trend for lung lesions. PRCV infection increased T-cell frequencies in BAL, but DEX treatment of PRCV-infected pigs reduced frequencies of T cells; interestingly B and SWC3a(+) (monocytes/macrophages/granulocytes) cell frequencies were increased. DEX reduced numbers of PRCV-stimulated Th1 gamma interferon-secreting cells in spleen, tracheobroncheolar lymph nodes, and blood. Our findings suggest that future glucocorticoid treatment of SARS patients should be reconsidered in the context of potential local immunosuppression of immune responses in lung and systemic Th1 cytokine-biased suppression.     

Markers of atopic dermatitis,allergic rhinitis and bronchial asthma in pediatric patients: correlation with filaggrin,eosinophil major basic protein and immunoglobulin E

Background

Allergic reactions have been implicated as contributions in a number of atopic disorders, including atopic dermatitis (AD), allergic rhinitis (AR) and bronchial asthma (BA). However, the potential for filaggrin protein, eosinophil major basic protein (MBP) and immunoglobulin E (IgE) to elicit allergic response or to contribute to atopic disorders remains largely unexplored in pediatric patients. This study was undertaken to investigate the status and contribution of filaggrin protein, eosinophil MBP and total IgE in pediatric patients with AD, AR and BA.

Methods

Sera from 395 pediatric patients of AD, AR or BA with varying levels of disease activity according to the disease activity index and 410 age-matched non-atopic healthy controls were evaluated for serum levels of atopic markers, including filaggrin, eosinophil MBP and IgE.

Results

Serum analysis showed that filaggrin levels were remarkably high in pediatric patients with AD, followed by BA and AR, whereas its levels were low in non-atopic pediatric controls. Eosinophil MBP levels in sera of atopic patients were significantly high as compared with their respective controls, but its levels were highest in AR patients, followed by AD and BA. Total IgE in sera of AD patients was markedly high, followed by AR and BA patients, whereas its levels were low in non-atopic pediatric controls. Interestingly, not only was an increased number of subjects positive for filaggrin protein, eosinophil MBP or total IgE, but also their levels were statistically significantly higher among those atopic patients whose disease activity scores were higher as compared with atopic patients with lower disease activity scores.

Conclusions

These findings strongly support a role of filaggrin protein, eosinophil MBP and IgE in the onset of allergic reactions in pediatric patients with AD, AR and BA. The data suggest that filaggrin, eosinophil MBP or IgE might be useful in evaluating the progression of AD, AR or BA and in elucidating the mechanisms involved in the pathogenesis of these pediatric disorders.

     

Detection of whole genome selection signatures of Pakistani Teddy goat

Background

Natural and artificial selection tend to cause variability that contributes to shape the genome of livestock in a way that differentiates them among the animal kingdom. The particular aim here is to identify positive selection signatures with whole genome pooled-sequence data of Pakistani Teddy goat.

Methods and results

Paired-end alignment of 635,357,043 reads of Teddy goat with (ARS1) reference genome assembly was carried out. Pooled-Heterozygosity (Hp) and Tajima’s D (TD) are applied for validation and getting better hits of selection signals, while pairwise F_ST statistics is conducted on Teddy vs. Bezoar (wild goat ancestor) for genomic differentiation, moreover annotation of regions under positive selection was also performed. Hp score with???ZHp?>?5 detected six windows having highest hits on Chr. 29, 9, 25, 15 and 14 that harbor HRASLS5, LACE1 and AXIN1 genes which are candidate for embryonic development, lactation and body height. Secondly, ? ZTD value of?>?3.3 showed 4 windows with very strong hits on Chr.5 & 9 which harbor STIM1 and ADM genes related to body mass and weight. Lastly, ? ZF_ST?<?? 5 generated four strong signals on Chr.5 & 12 harbor LOC102183233 gene. Other significant selection signatures encompass genes associated with wool production, prolificacy and coat colors traits in this breed.

Conclusions

In brief, this study identified the genes under selection in Pakistani Teddy goat that will be helpful to refining the marker-assisted breeding policies and converging required production traits within and across other goat breeds and to explore full genetic potential of this valued species of livestock.

     

A Modified Lumry–Eyring Analysis for the Determination of the Predominant Mechanism Underlying the Diminution of Protein Aggregation by Glycerol

Aggregation of aspartate-β-semialdehyde dehydrogenase (ASD) was analyzed by applying modified Lumry–Eyring with nucleated polymerization (LENP) model. Intrinsic nucleation time scales were determined. In absence of glycerol, ASD undergoes concentration and time-dependent polymerization into low-molecular weight soluble aggregates and thereafter condensation into insoluble aggregates. In the presence of increasing solvent glycerol concentration, the aggregation becomes more and more nucleation dominated, with slower polymerization to low-molecular weights soluble aggregates, without any condensation into insoluble aggregates. Effective nucleus size as well as the number of monomers in each irreversible growth event were sensitive to the changes in solvent glycerol concentration. Glycerol-directed diminution of aggregation appears to be largely due to the inhibition of rearrangement (decreased nucleation rearrangement rate coefficient, K _r,x ) because of compaction induced due to preferential hydration, thus, preventing the soluble aggregates from locking into irreversible soluble nuclei. Appreciably decreased K _r,x (as compared to nucleation dissociation constant, K _d,x ), appears to be responsible for increased nucleus size at higher solvent glycerol concentration. This study explains how modified LENP model can be applied to determine the predominant mechanism responsible for the diminution of aggregation by polyhydric alcohols (glycerol).     

Purification and characterization of adult diarrhea rotavirus: identification of viral structural proteins.

Adult diarrhea rotavirus (ADRV) is a newly identified strain of noncultivable human group B rotavirus that has been epidemic in the People's Republic of China since 1982. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western (immuno-) blot analysis to examine the viral proteins present in the outer and inner capsids of ADRV and compared these with the proteins of a group A rotavirus, SA11. EDTA treatment of double-shelled virions removed the outer capsid and resulted in the loss of three polypeptides of 64, 61, and 41, kilodaltons (kDa). Endo-beta-N-acetylglucosaminidase H digestion of double-shelled virions identified the 41-kDa polypeptide as a glycoprotein. CaCl2 treatment of single-shelled particles removed the inner capsid and resulted in the loss of one polypeptide with a molecular mass of 47 kDa. The remaining core particle had two major structural proteins of 136 and 113 kDa. All of the proteins visualized on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were antigenic by Western blot analysis when probed with convalescent-phase human and animal antisera. A 47-kDa polypeptide was most abundant and was strongly immunoreactive with human sera, animal sera raised against ADRV and against other group B animal rotaviruses (infectious diarrhea of infant rat virus, bovine and porcine group B rotavirus, and bovine enteric syncytial virus) and a monoclonal antibody prepared against infectious diarrhea of infant rat virus. This 47-kDa inner capsid polypeptide contains a common group B antigen and is similar to the VP6 of the group A rotaviruses. Human convalescent-phase sera also responded to a 41-kDa polypeptide of the outer capsid that seems similar to the VP7 of group A rotavirus. Other polypeptides have been given tentative designations on the basis of similarities to the control preparation of SA11, including a 136-kDa polypeptide designated VP1, a 113-kDa polypeptide designated VP2, 64- and 61-kDa polypeptides designated VP5 and VP5a, and several proteins in the 110- to 72-kDa range that may be VP3, VP4, or related proteins. The lack of cross-reactivity on Western blots between antisera to group A versus group B rotaviruses confirmed that these viruses are antigenically quite distinct.