Isolation, Characterization and Molecular Cloning of DNase IIIb from Drosophila melanogaster

Nucleases are enzymes that breakdown nucleic acids; they are classified by their biochemical properties into different groups (Evans et al., 2003). The ββα-Me finger family of nucleases are enzymes that combine structurally different groups but they are defined by a highly evolutionary conserved active site, a stretch of 22 amino acids composed by a β-hairpin (ββ) and α-helix (α) that anchor a catalytic metal ion (Sokolowska et al, 2009). Within this family is found the DNA/RNA non-specific nucleases. Nucleases that belong to the DNA/RNA non-specific group hydrolyze both ds and ss DNA, as well as RNA at equal or similar rates (Rangarajan and Shankar, 2001). They all share a common structure, specifically, a DNA/RNA non-specific endonuclease (NUC) domain containing a conserved Asp-Arg-Gly-His (DRGH) motif with an active site histidine (Friedhoff et al., 1994, 1996, Meiss et al., 2000). In a recent phylogenetic study on Kamchatka crab duplex-specific nuclease (Par_DSN), a new classification of Par_DSN-like nucleases has been proposed. Conserved regions of the NUC domains from the alignment data were used for ML tree construction with TREE-PUZZLE software (Anisimova et al., 2008). The NUC domains examined were divided into two main subgroups, the first containing SFN members and two insect enzymes, and the second (that they called duplex-specific nucleases, DS_NUCs) consisting of other insect and Crustacean nucleases (Anisimova et al., 2008). Among this last group there are two nucleases that were uncover by our group in Drosophila melanogaster via microarray analysis annotated as: CG33346 and CG9989. Deoxyribonuclease II (DNase II) is an endonuclease with optimal activity at low pH. It is localized within the lysosomes of higher eukaryotes for degradation of DNA after phagocytosis. We have previously demonstrated that down regulation of the Drosophila melanogaster dnase II gene, using RNA interference (RNAi) resulted in increased susceptibility to bacterial infection. In addition, genome wide expression microarray analyses of infected DNase II-deficient flies revealed over expression of a DNase-like gene that has been tentatively named dnase IIIa. Interestingly, a second highly homologous gene, that we have named dnase IIIb, was found adjacent to dnase IIIa. This evidence suggests that dnase IIIa could be up-regulated to compensate for the lost of DNase II and function as antimicrobial response proteins. In this study we will focus on the dnase IIIb gene since this ORF seems to encode an authentic nuclease that can be up-regulated during bacterial infection. The putative dnase IIIb gene was cloned and modified to contain a GST and nine-Histidine-tag and inserted into the bacterial-expression vector pGEX-KG. DNase IIIβ was induced by addition of IPTG and the recombinant protein was then purified by GST affinity column and found to be enzymatically active. By characterizing this novel nuclease, we will be able to further determine the biological function of this enzyme during Gram-positive bacterial infection

Isolation, characterization, and molecular cloning of DNase IIIβ from Drosophila melanogaster

Nucleases are enzymes that breakdown nucleic acids; they are classified by their biochemical properties into different groups (Evans et al., 2003). The ββ-Me finger family of nucleases are enzymes that combine structurally different groups but they are defined by a highly evolutionary conserved active site, a stretch of 22 amino acids composed by a β-hairpin (ββ) and α-helix (α) that anchor a catalytic metal ion (Sokolowska et al, 2009). Within this family is found the DNA/RNA non-specific nucleases. Nucleases that belong to the DNA/RNA nonspecific group hydrolyze both ds and ss DNA, as well as RNA at equal or similar rates (Rangarajan and Shankar, 2001). They all share a common structure, specifically, a DNA/RNA non-specific endonuclease (NUC) domain containing a conserved Asp-Arg-Gly-His (DRGH) motif with an active site histidine (Friedhoff et al., 1994, 1996, Meiss et al., 2000). In a recent phylogenetic study on Kamchatka crab duplex-specific nuclease (Par_DSN), a new classification of Par_DSN-like nucleases has been proposed. Conserved regions of the NUC domains from the alignment data were used for ML tree construction with TREE-PUZZLE software (Anisimova et al., 2008). The NUC domains examined were divided into two main subgroups, the first containing SFN members and two insect enzymes, and the second (that they called duplex-specific nucleases, DS_NUCs) consisting of other insect and Crustacean nucleases (Anisimova et al., 2008). Among this last group there are two nucleases that were uncover by our group in Drosophila melanogaster via microarray analysis annotated as: CG33346 and CG9989. Deoxyribonuclease II (DNase II) is an endonuclease with optimal activity at low pH. It is localized within the lysosomes of higher eukaryotes for degradation of DNA after phagocytosis. We have previously demonstrated that down regulation of the Drosophila melanogaster dnase II gene, using RNA interference (RNAi) resulted in increased susceptibility to bacterial infection. In addition, genome wide expression microarray analyses of infected DNase II-deficient flies revealed over expression of a DNase-like gene that has been tentatively named dnase IIIa. Interestingly, a second highly homologous gene, that we have named dnase IIIb, was found adjacent to dnase IIIa. This evidence suggests that dnase IIIa could be up-regulated to compensate for the lost of DNase II and function as antimicrobial response proteins. In this study we will focus on the dnase IIIb gene since this ORF seems to encode an authentic nuclease that can be up-regulated during bacterial infection. The putative dnase IIIb gene was cloned and modified to contain a GST and nine-Histidine-tag and inserted into the bacterial-expression vector pGEX-KG. DNase IIIβ was induced by addition of IPTG and the recombinant protein was then purified by GST affinity column and found to be enzymatically active. By characterizing this novel nuclease, we will be able to further determine the biological function of this enzyme during Gram-positive bacterial infection

Suited Ground Vacuum Chamber Testing Decompression Sickness Tiger Team Report

Suited vacuum chamber testing is critical to flight crew training, sustaining engineering, and development engineering. Most suited vacuum chamber testing at NASAs Johnson Space Center (JSC) involves crewmembers or human test subjects working at a hypobaric pressure of 4.3 psia, which requires that an oxygen prebreathe be performed prior to decompression to reduce the risk of decompression sickness (DCS). Since 1986, NASAs policy has been to require a 4-hour resting prebreathe for hypobaric chamber exposures of 4.2 psia lasting greater than 30 minutes. There have been no reports of Type II (i.e., serious, potentially life-threatening) DCS at NASA while using this prebreathe protocol. Several chamber runs, believed to be approximately 5% of all runs, are believed to have been terminated due to Type I DCS symptoms that were performance impairing; however, detailed records of DCS symptoms during suited vacuum chamber runs are not available. The adequacy of the 4-hour prebreathe protocol, as well as the processes by which prebreathe protocols and policies are established, became the subject of significant discussion in April 2018 when medical planning was initiated for chamber runs that were scheduled to occur later in 2018 that would last 8 hours or more with high metabolic rates

Laboratory-based surveillance of streptococcus pneumoniae invasive disease in children in 10 latin American countries: A SIREVA II project, 2000-2005

Durante los últimos 14 años la Organización Panamericana de la Salud ha estado promoviendo la vigilancia de la enfermedad neumocócica invasiva en niños latinoamericanos para comprender mejor las tendencias de la enfermedad en cuanto a la circulación de tipos capsulares en cada país y la susceptibilidad a los antimicrobianos. Métodos: Se analizaron los datos de vigilancia de laboratorio de 10 países latinoamericanos recopilados entre 2000 y 2005, incluida la distribución de serotipos y la susceptibilidad a los antibióticos betalactámicos. Resultados: Aunque se identificaron 61 tipos capsulares diferentes durante la vigilancia de 6 años, 13 serotipos representaron el 86% de todos los aislamientos. Estos fueron consistentemente los más prevalentes durante todo el período de estudio con predominio del serotipo 14. Se detectó una disminución de la susceptibilidad a la penicilina en el 38% de todos los aislados de Streptococcus pneumoniae, con mayor prevalencia en República Dominicana y México. La menor susceptibilidad a la penicilina aumentó en Brasil y Colombia, mientras que en Chile se registró una disminución de las tasas de alta resistencia. Conclusiones: Estos datos indican que 10 países de la Región continúan teniendo vigilancia de laboratorio de alta calidad para la enfermedad neumocócica generando información valiosa para que los tomadores de decisiones en salud puedan priorizar las intervenciones. La vacuna heptavalente cubrirá potencialmente del 52,4% al 76,5% de las cepas que causan la enfermedad neumocócica invasiva y la 13 valente del 76,7% al 88,3%. Copyright © 2009 de Lippincott Williams y Wilkins. Estos datos indican que 10 países de la Región continúan teniendo vigilancia de laboratorio de alta calidad para la enfermedad neumocócica generando así información valiosa para que los tomadores de decisiones en salud puedan priorizar las intervenciones. La vacuna heptavalente cubrirá potencialmente del 52,4% al 76,5% de las cepas que causan la enfermedad neumocócica invasiva y la 13 valente del 76,7% al 88,3%. Copyright © 2009 de Lippincott Williams y Wilkins. Estos datos indican que 10 países de la Región continúan teniendo vigilancia de laboratorio de alta calidad para la enfermedad neumocócica generando así información valiosa para que los tomadores de decisiones en salud puedan priorizar las intervenciones. La vacuna heptavalente cubrirá potencialmente del 52,4% al 76,5% de las cepas que causan la enfermedad neumocócica invasiva y la 13 valente del 76,7% al 88,3%.For the last 14 years the Pan American Health Organization has been promoting surveillance of invasive pneumococcal disease in Latin American children for better understanding of the disease tendencies regarding capsular types circulation in each country and susceptibility to antimicrobials. Methods: Laboratory-based surveillance data from 10 Latin American countries collected from 2000 to 2005 were analyzed, including serotype distribution and susceptibility to beta-lactam antibiotics. Results: Although 61 different capsular types were identified during the 6-year surveillance, 13 serotypes accounted for 86% of all isolates. These were consistently the most prevalent throughout the study period with serotype 14 predominating. Diminished susceptibility to penicillin was detected in 38% of all Streptococcus pneumoniae isolates, with the highest prevalence in Dominican Republic and Mexico. Decreased susceptibility to penicillin increased in Brazil and Colombia whereas decreased high resistance rates was recorded in Chile. Conclusions: These data indicate that 10 countries of the Region continue to have high quality laboratory-based surveillance for pneumococcal disease thus generating valuable information so that healthcare decision makers may prioritize interventions. The heptavalent vaccine will potentially cover from 52.4% to 76.5% of strains causing invasive pneumococcal disease and the 13 valent from 76.7% to 88.3%