Genetic variability of human respiratory syncytial virus A strains circulating in Ontario: a novel genotype with a 72 nucleotide G gene duplication

Human respiratory syncytial virus (HRSV) is the main cause of acute lower respiratory infections in children under 2 years of age and causes repeated infections throughout life. We investigated the genetic variability of RSV-A circulating in Ontario during 2010-2011 winter season by sequencing and phylogenetic analysis of the G glycoprotein gene.Among the 201 consecutive RSV isolates studied, RSV-A (55.7%) was more commonly observed than RSV-B (42.3%). 59.8% and 90.1% of RSV-A infections were among children ≤12 months and ≤5 years old, respectively. On phylogenetic analysis of the second hypervariable region of the 112 RSV-A strains, 110 (98.2%) clustered within or adjacent to the NA1 genotype; two isolates were GA5 genotype. Eleven (10%) NA1-related isolates clustered together phylogenetically as a novel RSV-A genotype, named ON1, containing a 72 nucleotide duplication in the C-terminal region of the attachment (G) glycoprotein. The predicted polypeptide is lengthened by 24 amino acids and includes a23 amino acid duplication. Using RNA secondary structural software, a possible mechanism of duplication occurrence was derived. The 23 amino acid ON1 G gene duplication results in a repeat of 7 potential O-glycosylation sites including three O-linked sugar acceptors at residues 270, 275, and 283. Using Phylogenetic Analysis by Maximum Likelihood analysis, a total of 19 positively selected sites were observed among Ontario NA1 isolates; six were found to be codons which reverted to the previous state observed in the prototype RSV-A2 strain. The tendency of codon regression in the G-ectodomain may infer a decreased avidity of antibody to the current circulating strains. Further work is needed to document and further understand the emergence, virulence, pathogenicity and transmissibility of this novel RSV-A genotype with a72 nucleotide G gene duplication.     

Decoding the molecular design principles underlying Ca(2+) binding to βγ-crystallin motifs

Numerous proteins belonging to the recently expanded βγ-crystallin superfamily bind Ca(2+) at the double-clamp N/D-N/D-X(1)-X(2)-S/T-S motif. However, there have been no attempts to understand the intricacies involving Ca(2+) binding, such as the determinants of Ca(2+)-binding affinity and their contributions to gain in stability. This work is an in-depth analysis of understanding the modes and determinants of Ca(2+) binding to βγ-crystallin motifs. We have performed extensive naturally occurring substitutions from related proteins on the βγ-crystallin domains of flavollin, a low-affinity Ca(2+)-binding protein, and clostrillin, a moderate-affinity protein. We monitored the consequences of these modifications on Ca(2)(+) binding by isothermal titration calorimetry, thermal stability and conformational and crystal structure analyses. We demonstrate that Ca(2)(+) binding to the two sites of a βγ-domain is interdependent and that the presence of Arg at the fifth position disables a site. A change from Thr to Ser, or vice versa, influences Ca(2+)-binding affinity, highlighting the basis of diversity found in these domains. A subtle change in the first site has a greater influence on Ca(2)(+) binding than a similar alteration in the second site. Thus, the second site is more variable in nature. Replacing an acidic or hydrophobic residue in a binding site alters the Ca(2+)-binding properties drastically. While it appears from their binding site sequence that these domains have evolved randomly, our examination illustrates the subtlety in the design of these modules. Decoding such design schemes would aid in our understanding of the functional themes underlying differential Ca(2)(+) binding and in predicting these in emerging sequence information.     

Use of Ranks in Comparisons of Variability in the Non-Normal Populations

A method of analysis for comparing the variability of two samples drawn from two populations has been developed. The method is also suitable for the nonnumeric form of data. A test based on ordered observations for testing the null hypothesis of equality of two variances has been given. The test statistic is a function of the sum of ranks assigned to smaller size sample. Ranking procedure has been modified to depict the variability in the data by the sum of ranks. The null distribution of the test-statistic has been worked out for small samples and it turns out to be chi-square distribution for large samples. The analytical procedure has been explained by a numerical example on the productivity and production of rice and wheat in India from 1950–51 to 1983–84.     

Statistical Measures of Instability in Crop Output

Some aspects of methodological issues relating to statistical measures of instability in crop output has been discussed. Several existing techniques for detection and measurement of instability have been reviewed and some additions and modifications suggested. The application of distribution-free tests has been indicated to test the significance of variability in the crop output over space and time. Various procedures have been explained by numerical data on crop output.     

Development of novel α-chitin/nanobioactive glass ceramic composite scaffolds for tissue engineering applications

Bioactive glass ceramic nanoparticles (nBGC) were prepared by sol–gel technique. The novel chitin/nBGC composite scaffolds were prepared using chitin gel with nBGC by lyophilization technique. The prepared nBGC and composite scaffolds were characterized using Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Fourier Transformed Infrared Spectroscopy (FT-IR) and X-ray diffraction (XRD). The composite scaffolds showed adequate porosity where the nBGC nanoparticles were homogenously distributed on the pore walls. The swelling, density, degradation and in vitro biomineralization capability of the composite scaffolds were also evaluated. The developed composite scaffolds showed adequate swelling and degradation properties along with its ability to become bioactive. Cytocompatability of the scaffolds was assessed using MTT assay, direct contact test and cell attachment studies. Results indicated no sign of toxicity and cells found to be attached to the pore walls offered by the scaffolds. These results suggested that the developed composite scaffold possess the prerequisites for tissue engineering scaffolds and it can be used for tissue engineering applications.     

Responses of wild type and abscisic acid mutants ofArabidopsis thaliana to cadmium

Heavy metal-induced inhibitory effects are reported to be concomitant with an increase in endogenous abscisic acid (ABA) levels in plant tissues indicating the possibility of this phytohormone mediating a part of the metal-imposed phytotoxicity. We examined this possibility by comparing the seed germination and seedling growth responses of ABA-deficient (aba-1, aba-3 and aba-4) and ABA-insensitive (abi2-1 and abi3-1) mutants of Arabidopsis thaliana to Cd with those of the wild type (Landsberg erecta, Ler). Assuming that Cd imposed a part of its toxic influence via affecting a rise in endogenous ABA level, all ABA mutants studied could be predicted to exhibit reduced responsiveness to Cd exposure in comparison to the wild type. However, the data obtained both in germination and growth assays were not consistent with this prediction. In germination assays, all ABA mutants proved consistently more sensitive than the wild type to Cd. In case of growth (root length and seedling fresh weight), the magnitude of Cd-induced inhibition in ABA mutants (aba-1, abi2-1 and abi3-1) was generally comparable to that in the wild type. Based on these observations a direct mediatory role of ABA in Cd-imposed phytotoxic effects on early growth could be excluded. The possible significance of heavy metal-dependent increase in endogenous ABA levels in plant tissues is discussed.     

Tumor environment dictates medulloblastoma cancer stem cell expression and invasive phenotype

The neural precursor surface marker CD133 is thought to be enriched in brain cancer stem cells and in radioresistant DAOY medulloblastoma-derived tumor cells. Given that membrane type-1 matrix metalloproteinase (MT1-MMP) expression is a hallmark of highly invasive, radioresistant, and hypoxic brain tumor cells, we sought to determine whether MT1-MMP and other MMPs could regulate the invasive phenotype of CD133(+) DAOY cells. We found that when DAOY medulloblastoma or U87 glioblastoma cells were implanted in nude mice, only those cells specifically implanted in the brain environment generated CD133(+) brain tumors. Vascular endothelial growth factor and basic fibroblast growth factor gene expression increases in correlation with CD133 expression in those tumors. When DAOY cultures were induced to generate in vitro neurosphere-like cells, gene expression of CD133, MT1-MMP, MMP-9, and MDR-1 was induced and correlated with an increase in neurosphere invasiveness. Specific small interfering RNA gene silencing of either MT1-MMP or MMP-9 reduced the capacity of the DAOY monolayers to generate neurospheres and concomitantly abrogated their invasive capacity. On the other hand, overexpression of MT1-MMP in DAOY triggered neurosphere-like formation which was further amplified when cells were cultured in neurosphere medium. Collectively, we show that both MT1-MMP and MMP-9 contribute to the invasive phenotype during CD133(+) neurosphere-like formation in medulloblastoma cells. Increases in MMP-9 may contribute to the opening of the blood-brain barrier, whereas increased MT1-MMP would promote brain tumor infiltration. Our study suggests that MMP-9 or MT1-MMP targeting may reduce the formation of brain tumor stem cells.     

Interaction of nuclear proteins with repeat sequences in the 5' flanking region of mouse muscle creatine kinase gene during aging

The 5' flanking region of the mouse muscle creatine kinase (MCK) gene contains two repeat sequences-a mononucleotide repeat, (A)(22) (-2694 to -2673), and a tetranucleotide repeat, (GTTT)(8) (-2962 to -2931). We show here that these repeats in the mouse MCK gene bind to specific nuclear protein factors. Some of the factors interacting with these sequences are tissue-specific and show age-related decrease in the binding activity. Nonspecific competitor and heterologous DNA probes failed to compete out the complexes showing that the interaction is specific to the repeat sequences. These proteins may have a role in the expression of the gene during aging.     

Kinetics study of endogenous heat shock protein 70 expression

The purpose of this study was to determine the kinetics of HSP70 expression in response to mild thermal stress. The rationale is to produce a basis for design of optimal heating methods to induce HSP70 expression for preconditioning in cardiac surgery. Bovine aortic endothelial cells were heated at 42 degrees C for 0.5 to 5 hours followed by 37 degrees C recovery for 1 to 48 hours. Quantitative analysis of western blot results showed HSP70 expression kinetics is a coupled function of heating temperature and time and of post-heating duration. Bimodal HSP70 expression kinetics were identified which may be an important cause of the "second window of protection" observed by other researchers.