Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia

The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20% O2) and hypoxia (less than 5% O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50% lower under hypoxia, while the HRE plasmid was about 50% higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.

New multilocus sequence typing of MRSA in São Paulo, Brazil

An increased incidence of nosocomial and community-acquired infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has been observed worldwide. The molecular characterization of MRSA has played an important role in demonstrating the existence of internationally disseminated clones. The use of molecular biology methods in the surveillance programs has enabled the tracking of MRSA spread within and among hospitals. These data are useful to alert nosocomial infection control programs about the potential introduction of these epidemic clones in their areas. Four MRSA blood culture isolates from patients hospitalized at two hospitals in the city of São Paulo, Brazil, were analyzed; one of them was community acquired. The isolates were characterized as SCCmec, mecA and PVL by PCR, pulsed-field gel electrophoresis (PFGE) profile and molecular sequence typing (MLST) genotyping. The isolates presented type IV SCCmec, and none proved to be positive for PVL. The isolates showed a PFGE profile similar to the pediatric clone. MLST genotyping demonstrated that the isolates belonged to clonal complex 5 (CC5), showing a new yqiL allele gene, resulting in a new sequence typing (ST) (1176). Our results showed that strains of MRSA carrying a new ST are emerging in community and nosocomial infections, including bacteremia, in São Paulo, Brazil.

Cloning, bioinformatics analysis, and expression of the dust mite allergen Der f 5 of Dermatophagoides farinae

Crude extracts of house dust mites are used clinically for diagnosis and immunotherapy of allergic diseases, including bronchial asthma, perennial rhinitis, and atopic dermatitis. However, crude extracts are complexes with non-allergenic antigens and lack effective concentrations of important allergens, resulting in several side effects. Dermatophagoides farinae (Hughes; Acari: Pyroglyphidae) is one of the predominant sources of dust mite allergens, which has more than 30 groups of allergen. The cDNA coding for the group 5 allergen of D. farinae from China was cloned, sequenced and expressed. According to alignment using the VECTOR NTI 9.0 software, there were eight mismatched nucleotides in five cDNA clones resulting in seven incompatible amino acid residues, suggesting that the Der f 5 allergen might have sequence polymorphism. Bioinformatics analysis revealed that the matured Der f 5 allergen has a molecular mass of 13604.03 Da, a theoretical pI of 5.43 and is probably hydrophobic and cytoplasmic. Similarities in amino acid sequences between Der f 5 and allergens of other domestic mite species, viz. Der p 5, Blo t 5, Sui m 5, and Lep d 5, were 79, 48, 53, and 37%, respectively. Phylogenetic analysis indicated that Der f 5 and Der p 5 clustered together. Blo t 5 and Ale o 5 also clustered together, although Blomia tropicalis and Aleuroglyphus ovatus belong to different mite families, viz. Echimyopodidae and Acaridae, respectively.

Sequence analysis of the 5′ third of glycoprotein C gene of South American bovine herpesviruses 1 and 5

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) share high genetic and antigenic similarities, but exhibit marked differences in tissue tropism and neurovirulence. The amino-terminal region of glycoprotein C (gC), which is markedly different in each of the viruses, is involved in virus binding to cellular receptors and in interactions with the immune system. This study investigated the genetic and antigenic differences of the 5′ region of the gC (5′ gC) gene (amino-terminal) of South American BoHV-1 (n=19) and BoHV-5 (n=25) isolates. Sequence alignments of 374 nucleotides (104 amino acids) revealed mean similarity levels of 97.3 and 94.2% among BoHV-1 gC (gC1), respectively, 96.8 and 95.6% among BoHV-5 gC (gC5), and 62 and 53.3% between gC1 and gC5. Differences included the absence of 40 amino acid residues (27 encompassing predicted linear epitopes) scattered throughout 5′ gC1 compared to 5′ gC5. Virus neutralizing assays testing BoHV-1 and BoHV-5 antisera against each isolate revealed a high degree of cross-neutralization between the viruses, yet some isolates were neutralized at very low titers by heterologous sera, and a few BoHV-5 isolates reacted weakly with either sera. The virus neutralization differences observed within the same viral species, and more pronounced between BoHV-1 and BoHV-5, likely reflect sequence differences in neutralizing epitopes. These results demonstrate that the 5′ gC region is well conserved within each viral species but is divergent between BoHV-1 and BoHV-5, likely contributing to their biological and antigenic differences.

The consensus sequence of FAMLF alternative splice variants is overexpressed in undifferentiated hematopoietic cells

The familial acute myeloid leukemia related factor gene (FAMLF) was previously identified from a familial AML subtractive cDNA library and shown to undergo alternative splicing. This study used real-time quantitative PCR to investigate the expression of the FAMLF alternative-splicing transcript consensus sequence (FAMLF-CS) in peripheral blood mononuclear cells (PBMCs) from 119 patients with de novo acute leukemia (AL) and 104 healthy controls, as well as in CD34+cells from 12 AL patients and 10 healthy donors. A 429-bp fragment from a novel splicing variant of FAMLF was obtained, and a 363-bp consensus sequence was targeted to quantify total FAMLF expression. Kruskal-Wallis, Nemenyi, Spearman's correlation, and Mann-Whitney U-tests were used to analyze the data. FAMLF-CS expression in PBMCs from AL patients and CD34+ cells from AL patients and controls was significantly higher than in control PBMCs (P<0.0001). Moreover,FAMLF-CS expression in PBMCs from the AML group was positively correlated with red blood cell count (rs=0.317, P=0.006), hemoglobin levels (rs=0.210, P=0.049), and percentage of peripheral blood blasts (rs=0.256, P=0.027), but inversely correlated with hemoglobin levels in the control group (rs=–0.391, P<0.0001). AML patients with high CD34+ expression showed significantly higherFAMLF-CS expression than those with low CD34+ expression (P=0.041). Our results showed thatFAMLF is highly expressed in both normal and malignant immature hematopoietic cells, but that expression is lower in normal mature PBMCs.

Sequence-characterized amplified region (SCAR) technique used for variety discrimination in vinca (Catharanthus roseus (L.) G.Don)

Sequence-Characterized Amplified Region (SCAR) appears as a useful technique for genetic purity testing and variety discrimination, applicable to species in which some other techniques have failed. In particular, this technique is very attractive with species in which RAPD results were not consistent. The RAPD polymorphic bands were cloned, sequenced and from the sequence information, primers pairs for normal PCR were developed. Since the probability of obtaining successful SCAR primers from RAPD polymorphic bands was about 50%, a larger number of RAPD polymorphic bands are needed to develop sufficient SCAR primers for varietal discrimination in vinca. In addition, the efficiency of the SCAR technique is strongly affected by the quality of DNA extracted from seeds. The SCAR banding patterns obtained from vinca seed were consistent and repeatable making the results reliable for genetic purity testing and variety discrimination. The SCAR technique is simple, fast, relatively inexpensive and allows the use of DNA extracted from dry seeds, which is very important in a seed-quality evaluating program

Investigating the Existence of Business-IT Alignment Trap in Finnish IT Organizations

The overall purpose of this master's thesis is to investigate the existence of business-IT alignment trap in Finnish IT organizations. The alignment trap refers to the inability of IT investments to deliver the expected business benefits. The basis for this investigation is due to the previous knowledge that high level of IT alignment practice with very low efficacy in an organization can lead to alignment trap. The theory which was established on the dimensions of IT-alignment and efficacy of IT as a whole with considerations for cost reduction and revenue growth benefits. This study explored the same dimensions with the previous study but identified additional benefit (profitability). The study was conducted using the Finnish IT barometer data from different IT organizations. A quantitative research method was used in conducting this study which was built on positivist philosophical stance. The empirical data is based on survey data, an excerpt from the Finnish IT barometer data that captured the annual survey results of IT significance to Finnish organizations as evaluated by business and IT professionals. The survey data comprised of 249 respondents and their responses were categorized into high and low IT intensive which form the basis of the statistical analysis conducted. Overall, five analyses were conducted using the variables of cost reductions, revenue growth and profitability in the 2x2 matrix dimensions of IT alignment and efficacy of IT, grouped into alignment trap, maintenance zone, well-oiled IT and IT-enabled growth. The empirical results, revealed a partial existence of alignment trap in Finnish IT organizations. This is due to a very minute number of organizations that were ensnared in the alignment trap zone on the analyses conducted. Although they recorded considerable high performances in terms of revenue growth rate with IT spending below the average companies, their profitability was considered very low. Generally it was observed that Finnish IT organizations with high efficacy of IT practices had good performances, while those with low efficacy of IT experienced low performances, especially in the aspect of profitability, regardless of the degree of IT alignment. The study proposes that organizations should improve on practices that enhance effectiveness of IT more in order for them to realize the full benefits of IT and to avoid alignment trap.

Probing MST1 Kinase Sequence and Structure for Rational Drug Discovery (Targeting diabetes by blocking protein-protein interactions)

Apoptotic beta cell death is an underlying cause majorly for type I and to a lesser extent for type II diabetes. Recently, MST1 kinase was identified as a key apoptotic agent in diabetic condition. In this study, I have examined MST1 and closely related kinases namely, MST2, MST3 and MST4, aiming to tackle diabetes by exploring ways to selectively block MST1 kinase activity. The first investigation was directed towards evaluating possibilities of selectively blocking the ATP binding site of MST1 kinase that is essential for the activity of the enzymes. Structure and sequence analyses of this site however revealed a near absolute conservation between the MSTs and very few changes with other kinases. The observed residue variations also displayed similar physicochemical properties making it hard for selective inhibition of the enzyme. Second, possibilities for allosteric inhibition of the enzyme were evaluated. Analysis of the recognized allosteric site also posed the same problem as the MSTs shared almost all of the same residues. The third analysis was made on the SARAH domain, which is required for the dimerization and activation of MST1 and MST2 kinases. MST3 and MST4 lack this domain, hence selectivity against these two kinases can be achieved. Other proteins with SARAH domains such as the RASSF proteins were also examined. Their interaction with the MST1 SARAH domain were evaluated to mimic their binding pattern and design a peptide inhibitor that interferes with MST1 SARAH dimerization. In molecular simulations the RASSF5 SARAH domain was shown to strongly interact with the MST1 SARAH domain and possibly preventing MST1 SARAH dimerization. Based on this, the peptidic inhibitor was suggested to be based on the sequence of RASSF5 SARAH domain. Since the MST2 kinase also interacts with RASSF5 SARAH domain, absolute selectivity might not be achieved.

GIS-based approach for optimization of onshore wind park infrastructure alignment in Finland

Wind power is a rapidly developing, low-emission form of energy production. In Fin-land, the official objective is to increase wind power capacity from the current 1 005 MW up to 3 500–4 000 MW by 2025. By the end of April 2015, the total capacity of all wind power project being planned in Finland had surpassed 11 000 MW. As the amount of projects in Finland is record high, an increasing amount of infrastructure is also being planned and constructed. Traditionally, these planning operations are conducted using manual and labor-intensive work methods that are prone to subjectivity. This study introduces a GIS-based methodology for determining optimal paths to sup-port the planning of onshore wind park infrastructure alignment in Nordanå-Lövböle wind park located on the island of Kemiönsaari in Southwest Finland. The presented methodology utilizes a least-cost path (LCP) algorithm for searching of optimal paths within a high resolution real-world terrain dataset derived from airborne lidar scannings. In addition, planning data is used to provide a realistic planning framework for the anal-ysis. In order to produce realistic results, the physiographic and planning datasets are standardized and weighted according to qualitative suitability assessments by utilizing methods and practices offered by multi-criteria evaluation (MCE). The results are pre-sented as scenarios to correspond various different planning objectives. Finally, the methodology is documented by using tools of Business Process Management (BPM). The results show that the presented methodology can be effectively used to search and identify extensive, 20 to 35 kilometers long networks of paths that correspond to certain optimization objectives in the study area. The utilization of high-resolution terrain data produces a more objective and more detailed path alignment plan. This study demon-strates that the presented methodology can be practically applied to support a wind power infrastructure alignment planning process. The six-phase structure of the method-ology allows straightforward incorporation of different optimization objectives. The methodology responds well to combining quantitative and qualitative data. Additional-ly, the careful documentation presents an example of how the methodology can be eval-uated and developed as a business process. This thesis also shows that more emphasis on the research of algorithm-based, more objective methods for the planning of infrastruc-ture alignment is desirable, as technological development has only recently started to realize the potential of these computational methods.

"Attitude is a little thing that makes a big difference" : further exploration of attitude alignment in the context of close relationship dyads /

One hundred and five primarily Caucasian undergraduate couples were tested to study the phenomenon of attitude alignment, or the way in which individuals change their opinions to achieve greater attitudinal congruence with their romantic partners, and the hypotheses that relationship closeness, affect, attachment, social desirability, and centrality are each related to attitude alignment for individuals in close couples. The couples filled out an attitude questionnaire consisting of 50 issues in which they were asked to give their attitude on a scale from 1 to 9 as well as to rate the centrality, or importance, of that issue on a scale from 1 to 9. Before discussing four of these issues with each other - 2 were more central to the man and less central, or peripheral, to the woman, whereas the other 2 were central to the woman and peripheral to the man - the individuals completed established measures of relationship closeness, affect, attachment, and social desirability. The couples then filled out the identical attitudes questionnaire again, and their answers on the four discussion issues were compared to their prediscussion answers to determine whether attitude alignment had occurred. There were two experimental groups: a social desirability group, where the couples were told it was natural and normal to disagree with their partners, and a control group. Results indicated that attitude alignment did significantly occur across all couples, but most other variables - including centrality, relationship closeness, and affect - did not predict attitude alignment behaviour. As well, the social desirability experimental groups did not significantly differ on attitude alignment behaviours, but higher scores on the social desirability scale, specifically self-deception, significantly predicted higher attitude alignment scores across all couples. Large differences between individuals' frequency.

THE ASSOCIATION OF SEQUENCE OF HIRING PRACTICE AND BIOPHYSICAL COMPONENTS IN SCREENING PROBATIONARY FIREFIGHTERS

Variation in hiring procedures occurs within fire service human resource departments. In this study, City 1 and City 2 applicants were required to pass their biophysical assessments prior to being hired as firefighters at the beginning and end of the screening process, respectively. City 1 applicants demonstrated significantly lower resting heart rate (RHR), resting diastolic blood pressure (RDBP), body fat% (BF) and higher z-scores for BF, trunk flexibility (TF) and overall clinical assessment (p<0.05). Regression analysis found that age and conducting the biophysical assessment at the end of the screening process explained poorer biophysical assessment results in BF% (R2=21%), BF z-score (R2=22%), TF z-score (R2=10%) and overall clinical assessment z-score (R2=7%). Each of RHR (OR=1.06, CI=1.01-1.10), RDBP (OR=1.05, CI=1.00-1.11) and BF% (OR=1.20, CI=1.07-1.37) increased the odds of being a City 2 firefighter (p<0.05). Biophysical screening at the end of the hiring process may result in the hiring of a less healthy firefighter.

Complete Genome Sequence of Erwinia amylovora Bacteriophage

The complete genome of an Erwinia amylovora bacteriophage, vB_EamM_Ea35-70 (Ea35-70), is 271,084 bp, encodes 318 putative proteins, and contains one tRNA. Comparative analysis with other Myoviridae genomes suggests that Ea35-70 is related to the Phikzlikevirus genus within the family Myoviridae, since 26% of Ea35-70 proteins share homology to proteins in Pseudomonas phage φKZ.

AnaBench: a Web/CORBA-based workbench for biomolecular sequence analysis

Affiliation: Département de biochimie, Faculté de médecine, Université de Montréal