RESUMEN

The emergence of antibiotic resistance (AR) in bacteria is becoming an alarming health concern because it allows them to adapt themselves to changing environments. It is possible to prevent the spread of AR in many ways, such as reducing antibiotic misuse in human and veterinary medicine. Streptococcus pseudopneumoniae is one of these AR bacterial species that can cause pneumonia in humans and is responsible for high mortality and morbidity rates. It is oval shaped gram-positive bacterium that shows resistance to several antibiotics like penicillin, tetracycline, ciprofloxacin, erythromycin, and co-trimoxazale and no approved vaccine is available to overcome diseases of the pathogen. Thus, substantial efforts are necessary to select protective antigens from a whole genome of pathogens that are easily tested experimentally. The in silico designed vaccine was safe and potent in immunizing individuals against the aforementioned pathogens. Herein, we utilized a subtractive genomic approach to identify potential epitope-based vaccine candidates against S. pseudopneumoniae. In total, 50850 proteins were retrieved from the NCBI, representing the complete genome of S. pseudopneumoniae. Out of the total, CD-HIT analysis identified 1022 proteins as non-redundant and 49828 proteins as redundant and further subjected for subcellular localization in which bulk of proteins was located in the cytoplasm, with seven extracellular proteins (penicillin-binding protein, alpha-amylase, solute-binding protein, hypothetical protein, CHAP domain-containing protein, polysaccharide deacetylase family protein, hypothetical protein). Six immune cells epitopes (SNLQSENDRL, RNDSLQKQAR, NPTTTSEGF, KVKKKNNKK, AYSQGSQKEH, and SVVDQVSGDF) were predicted with the help of the IEDB server. To design a multi-epitopes vaccine these immune cell epitopes were together by GPGPG and adjuvant linker to enhance immune response efficacy. The 3D structure of the designed vaccine was modeled and conducted molecular docking and dynamic simulation studies were to check the binding efficacy with immune cells receptor and dynamic behavior of the docked complex. Finally, we concluded that the designed vaccine construct can provoke a proper and protective immune response against S. pseudopneumoniae.

O surgimento de resistência a antibióticos (AR) em bactérias tem se tornando uma preocupação sanitária alarmante, uma vez que permite que elas se adaptem a ambientes em constante alteração. É possível prevenir a disseminação da RA de várias maneiras, como reduzir o uso indevido de antibióticos na medicina humana e veterinária. O Streptococcus pseudopneumoniae é uma dessas espécies bacterianas de AR que podem causar pneumonia em humanos e são responsáveis por altas taxas de mortalidade e morbidade. É uma bactéria gram-positiva de forma oval que mostra resistência a diversos antibióticos como penicilina, tetraciclina, ciprofloxacina, eritromicina e cotrimoxazale, além disso, nenhuma vacina aprovada está disponível para superar as doenças do patógeno. Assim, esforços substanciais são necessários para selecionar antígenos protetores de todo um genoma de patógenos que são facilmente testados experimentalmente. A vacina projetada in silico foi considerada segura e potente na imunização de indivíduos contra os patógenos mencionados. Neste trabalho, utilizamos uma abordagem genômica subtrativa para identificar potenciais candidatos a vacinas baseadas em epítopos contra S. pseudopneumoniae. No total, 50.850 proteínas foram recuperadas do NCBI, representando o genoma completo de S. pseudopneumoniae. Do total, a análise de CD-HIT identificou 1.022 proteínas como não redundantes e 49.828 proteínas como redundantes e posteriormente submetidas à localização subcelular na qual a maior parte das proteínas estava localizada no citoplasma, com sete proteínas extracelulares (proteína de ligação à penicilina, alfa- amilase, proteína de ligação a soluto, proteína hipotética, proteína contendo domínio CHAP, proteína da família polissacarídeo desacetilase, proteína hipotética). Seis epítopos de células imunes (SNLQSENDRL, RNDSLQKQAR, NPTTTSEGF, KVKKKNNKK, AYSQGSQKEH e SVVDQVSGDF) foram previstos com a ajuda do servidor IEDB. Para projetar uma vacina de múltiplos epítopos, esses epítopos de células imunes foram reunidos por GPGPG e um ligante adjuvante para aumentar a eficácia da resposta imune. A estrutura 3D da vacina projetada foi modelada e conduzido estudos de docking molecular e simulação dinâmica para verificar a eficácia da ligação com o receptor de células imunes e o comportamento dinâmico do complexo docked. Finalmente, concluímos que a construção da vacina projetada pode provocar uma resposta imune adequada e protetora contra S. pseudopneumoniae.

Asunto(s)

RESUMEN

The emergence of antibiotic resistance (AR) in bacteria is becoming an alarming health concern because it allows them to adapt themselves to changing environments. It is possible to prevent the spread of AR in many ways, such as reducing antibiotic misuse in human and veterinary medicine. Streptococcus pseudopneumoniae is one of these AR bacterial species that can cause pneumonia in humans and is responsible for high mortality and morbidity rates. It is oval shaped gram-positive bacterium that shows resistance to several antibiotics like penicillin, tetracycline, ciprofloxacin, erythromycin, and co-trimoxazale and no approved vaccine is available to overcome diseases of the pathogen. Thus, substantial efforts are necessary to select protective antigens from a whole genome of pathogens that are easily tested experimentally. The in silico designed vaccine was safe and potent in immunizing individuals against the aforementioned pathogens. Herein, we utilized a subtractive genomic approach to identify potential epitope-based vaccine candidates against S. pseudopneumoniae. In total, 50850 proteins were retrieved from the NCBI, representing the complete genome of S. pseudopneumoniae. Out of the total, CD-HIT analysis identified 1022 proteins as non-redundant and 49828 proteins as redundant and further subjected for subcellular localization in which bulk of proteins was located in the cytoplasm, with seven extracellular proteins (penicillin-binding protein, alpha-amylase, solute-binding protein, hypothetical protein, CHAP domain-containing protein, polysaccharide deacetylase family protein, hypothetical protein). Six immune cells epitopes (SNLQSENDRL, RNDSLQKQAR, NPTTTSEGF, KVKKKNNKK, AYSQGSQKEH, and SVVDQVSGDF) were predicted with the help of the IEDB server. To design a multi-epitopes vaccine these immune cell epitopes were together by GPGPG and adjuvant linker to enhance immune response efficacy. The 3D structure of the designed vaccine was modeled and conducted molecular docking and dynamic simulation studies were to check the binding efficacy with immune cells receptor and dynamic behavior of the docked complex. Finally, we concluded that the designed vaccine construct can provoke a proper and protective immune response against S. pseudopneumoniae.

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Several polymorphisms in the DNA repair gene are thought to have significant effects on cancer risk. We investigated the association of polymorphisms in the DNA repair genes XRCC1 Arg399Gln, XRCC3 Thr241Met, XPD Lys751Gln, XPG Asp1104His, APE1 Asp148Glu, and HOGG1 Ser326Cys with endometriosis risk. Genotypes were determined by PCR-RFLP assays in 52 patients with endometriosis and 101 age-matched healthy controls. Although there were no significant (P > 0.05) differences in the frequencies of genotypes or alleles of APE1, XRCC1, XPD, XPG, and HOGG1 genes between patients and controls, the frequency of the XRCC3 Thr/Thr genotype was significantly greater in endometriosis patients compared with controls (P = 0.005). XRCC3 Thr/Met genotypes (P = 0.022), and the Met allele (P = 0.005) seem to have a protective role against endometriosis. The distributions of genotypes and alleles of the genes APE1, XRCC1, XRCC3, XPD, XPG, and HOGG1 were not significantly associated with the different stages of endometriosis (P > 0.05). We conclude that the XRCC3 Thr/Thr genotype is associated with endometriosis in Turkish women.

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The presence of insulin binding sites in a clonal line of cells from rat pituitary tumor (GH1 cells) is described. The binding of insulin was a reversible and specific process. Binding of 125I-insulin to GH1 cells was inhibited by unlabeled insulin and to a minor extent, by proinsulin and desalanine insulin in direct proportion to their biological activities. Trypsin abolished the insulin binding to the cells. Twelve hours incubation of cells with insulin (5 microM) reduced in a 45% of the number of binding sites for the hormone. 125I-insulin bound to GH1 cells dissociated with a t 1/2 of 8 min. At 24 C labeled insulin was degraded by GH1 cells. No degradation was detected at 6 C. Unlabeled insulin inhibited most of the degradation of 125I-insulin, suggesting that degradation resulted from a saturable process. At steady-state, radioactive degradation products as well as 125I-insulin were recovered from GH1 cells.

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RESUMEN

The presence of insulin binding sites in a clonal line of cells from rat pituitary tumor (GH1 cells) is described. The binding of insulin was a reversible and specific process. Binding of 125I-insulin to GH1 cells was inhibited by unlabeled insulin and to a minor extent, by proinsulin and desalanine insulin in direct proportion to their biological activities. Trypsin abolished the insulin binding to the cells. Twelve hours incubation of cells with insulin (5 microM) reduced in a 45

of the number of binding sites for the hormone. 125I-insulin bound to GH1 cells dissociated with a t 1/2 of 8 min. At 24 C labeled insulin was degraded by GH1 cells. No degradation was detected at 6 C. Unlabeled insulin inhibited most of the degradation of 125I-insulin, suggesting that degradation resulted from a saturable process. At steady-state, radioactive degradation products as well as 125I-insulin were recovered from GH1 cells.

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