Molecular expression, purification and structural characterization of recombinant L-Glutaminase from Streptomyces roseolus.

The present research reports the anti-cancer potential of recombinant L-Glutaminase from Streptomyces roseolus. L-Glutaminase gene was synthesized by codon-optimization, cloned and successfully expressed in E. coli BL21 (DE3). Affinity purified recombinant L-Glutaminase revealed a molecular mass of 32 kDa. Purified recombinant L-Glutaminase revealed stability at pH 7.0-8.0 with optimum activity at 70 °C further indicating its thermostable nature based on thermodynamic characterization. Recombinant L-Glutaminase exhibited profound stability in the presence of several biochemical parameters and demonstrated its metalloenzyme nature and was also found to be highly specific towards favorable substrate (l-Glutamine) based on kinetics. It demonstrated antioxidant property and pronounced cytotoxic effect against breast cancer (MCF-7 cell lines) in a dose dependent behavior with IC50 of 40.68 µg/mL. Matrix-assisted laser desorption ionization-time of flight-mass spectroscopy (MALDI-TOF-MS) analysis of desired mass peaks ascertained the recombinant L-Glutaminase identity. N-terminal amino acid sequence characterization through Edman degradation revealed highest resemblance for L-glutaminase within the Streptomyces sp. family. The purified protein was characterized structurally and functionally by employing spectroscopic methods like Raman, circular dichroism and nuclear magnetic resonance. The thermostability was assessed by thermogravimetric analysis. The outcomes of the study, suggests the promising application of recombinant L-Glutaminase as targeted therapeutic candidate for breast cancer.

Optimization of subtilisin production from Bacillus subtilis strain ZK3 and biological and molecular characterization of synthesized subtilisin capped nanoparticles.

The increase and dissemination of multi-drug resistant bacteria have presented a major healthcare challenge, making bacterial infections a significant concern. The present research contributes towards the production of bioactive subtilisin from a marine soil isolate Bacillus subtilis strain ZK3. Custard apple seed powder (raw carbon) and mustard oil cake (raw nitrogen) sources showed a pronounced effect on subtilisin production. A 7.67-fold enhancement in the production was evidenced after optimization with central composite design-response surface methodology. Subtilisin capped silver (AgNP) and zinc oxide (ZnONP) nanoparticles were synthesized and characterized by UV-Visible spectroscopy. Subtilisin and its respective nanoparticles revealed significant biological properties such as, antibacterial activity against all tested pathogenic strains with potential against Escherichia coli and Pseudomonas aeruginosa. Prospective antioxidant behavior of subtilisin, AgNP and ZnONP was evidenced through radical scavenging assays with ABTS and DPPH. Subtilisin, AgNP and ZnONP revealed cytotoxic effect against cancerous breast cell lines MCF-7 with IC50of 83.48, 3.62 and 7.57 µg/mL respectively. Characterizations of nanoparticles were carried out by Fourier transform infrared spectroscopy, scanning electron microscopy with energy dispersive X-ray, X-ray diffraction, thermogravimetric analysis and atomic force microscopy analysis to elucidate the structure, surface and thermostability properties. The study proposes the potential therapeutic applications of subtilisin and its nanoparticles, a way forward for further exploration in the field of healthcare.

Propiedades biológicas de cepas de dengue virus-3 aisladas durante la epidemia ocurrida en La Habana, 2001-2002 / Biological properties of virus dengue-3 strains isolated during the epidemic ocurred in Havana, 2001-2002

Introducción: durante epidemias de dengue ocurridas en Cuba se ha observado de forma reiterada, un incremento de severidad clínica con la progresión de las epidemias en el tiempo, particularmente en infecciones secundarias. Se considera que este incremento pudiera estar relacionado con cambios genéticos del virus circulante. Objetivo: estudiar algunas propiedades biológicas de cepas aisladas en diferentes momentos de la epidemia de La Habana, 2001-2002. Métodos: se estudiaron 9 cepas de dengue virus-3, se evaluó el efecto citopatogénico y el crecimiento viral en las líneas celulares C6/36 HT y Vero, tamaño de las placas virales, sensibilidad a la temperatura, neurovirulencia en ratones lactantes y la influencia del pH en la unión del virus a la célula, así como en el medio de multiplicación de este. Resultados: las cepas de dengue virus-3 resultaron más citopatogénicas en células Vero, sin embargo, en C6/36 HT se obtuvieron títulos superiores. El conjunto de cepas mostró reducción del título viral y del tamaño de placa al aumentar la temperatura y fueron poco neurovirulentas. En cuanto a la unión del virus a la célula, a pH básico se observaron los mejores títulos, mientras que a pH ácido solo se observó el crecimiento de algunas cepas aisladas al final de la epidemia. El medio de multiplicación del virus a pH 6,5-7 favoreció el crecimiento de las cepas del inicio de la epidemia, mientras que las cepas del final tuvieron títulos superiores a pH 7-8. Conclusiones: se pudo comprobar la existencia de cambios fenotípicos entre cepas de diferentes momentos de la epidemia, que pudieran estar asociados con diferencias en cuanto a su adecuación viral o en su potencial virulento. No obstante, algunas de las propiedades biológicas estudiadas sugieren que las cepas de dengue virus-3 son menos virulentas que las cepas cubanas de dengue virus-2 de 1997.

Introduction: during dengue epidemics in Cuba, an increase in clinical severity with the epidemics progression in time, particularly in secondary infections, have been frequently observed. It is considered that this increase could be related with genetic changes in the circulating virus. Objective: to study some biological attributes related to strains isolated at different points of time during the dengue epidemic occurred in Havana city, 2001-2002. Methods: nine DENV-3 strains were studied. Cytopathogenic effect, viral growth in C6/36 HT and Vero cell lines, viral plaque sizes, temperature sensitivity, neurovirulence in newborn mice and pH influence in the binding of the virus and the cell as well as in the multiplication medium were evaluated. Results: DENV-3 strains were more cytopathogenic in Vero Cells. However, higher titres were obtained in C6/36 HT cells. All the strains showed reduction of viral titres and plaque size with temperature increasing and low neurovirulence. Basic pH favoured virus-cell binding whereas acid pH was only permissive for some strains isolated at the end of the epidemic. On the other hand, at pH 6.5-7, the viral multiplication medium favoured the growth of strains isolated at the beginning of the epidemic whereas the growth of those isolated at the endof the epidemic was noticeable at pH 7-8. Conclusions: this study proved the phenotypical changes among strains isolated at different points of time in the epidemic. They might be related to differences in viral fitness or in virulent potential. Nevertheless, some of the studied biological properties suggest that dengue virus-3 strains are less virulent than the Cuban dengue virus 2 strains isolated in 1997.

Propiedades biológicas de cepas de virus dengue serotipo 2 aisladas durante la epidemia en Santiago de Cuba, 1997 / Biological properties of dengue virus serotype 2 strains isolated from the epidemic occurred in Santiago de Cuba in 1997

Introducción: durante la epidemia cubana de dengue ocurrida en Santiago de Cuba, 1997, se evidenció un incremento de la severidad en el tiempo en términos de proporción de casos graves y muertes por dengue hemorrágico, que pudiera estar dado por la aparición de mutantes de escape a la neutralización, con mayor potencial virulento. Objetivos: estudiar algunas propiedades biológicas de cepas aisladas en diferentes momentos de la epidemia de Santiago de Cuba en 1997. Métodos: se estudiaron 9 cepas de DENV-2, se evaluó el efecto citopatogénico y el crecimiento viral en las líneas celulares C6/36 HT y Vero, tamaño de las placas virales, sensibilidad a la temperatura, neurovirulencia en ratones lactantes y la influencia del pH en la unión del virus a la célula, así como en el medio de multiplicación. Resultados: las cepas del final de la epidemia mostraron propiedades que las diferencian de las aisladas al inicio, entre las que se encuentran un mayor efecto citopatogénico en células C6/36 HT, títulos virales superiores y una mayor neurovirulencia en ratones lactantes. Por otra parte, en la unión del virus a la célula las cepas del inicio de la epidemia resultaron favorecidas por un pH ácido mientras que a las cepas del final de la epidemia las favoreció un pH básico. Conclusiones: pudo demostrarse que además de los cambios genotípicos observados previamente, existen diferencias fenotípicas entre cepas de distintos momentos de la epidemia, que pudieran estar asociados con diferencias en cuanto a la adecuación viral de estas y(o) en su potencial virulento.

Introduction: during the Cuban epidemic that occurred in Santiago de Cuba in 1997, there was observed increasing severity in the course of time, in terms of proportion of serious dengue haemorrhagic cases and deaths that could be due to the emergence of escape mutants to neutralization with greater virulent potential. Objective: to study some biological attributes of a group of strains isolated at different points of time during the Santiago de Cuba epidemic in 1997. Methods: nine DENV-2 strains were studied. The cytopathogenic effect, the viral growth in C6/36 HT and VERO cell lines, the virus plaque sizes, the sensitivity to temperatures, the neurovirulence in newborn mice and the influence of the pH in the union of the virus to the cell as well as in the multiplication medium were all evaluated. Results: the strains isolated at the end of the epidemic differed from those of the beginning showing increased neurovirulence in newborn mice and higher viral titers and greater cytopathogenic effect in HT C6/36 cells. On the other hand, the virus and the cell union was favored by acid pH when testing strains from the beginning of the epidemic, whereas this union was favored by the basic PH in the strains isolated at the end of the epidemic Conclusions: the present study managed to show that in addition to the previously observed genotypical changes, there were phenotypical differences among the strains isolated at different points of time in the epidemic; all these aspects may be associated with differences in the viral fitness and/or in the virulent potential of these strains.