Solid-state fermentation by Trichoderma viride for enhancing phenolic content, antioxidant and antimicrobial activities in ginger.

The phenolic content of methanol and water extracts of ginger fermented by Trichoderma spp. using solid-state fermentation (SSF) was evaluated and was compared with unfermented ginger. The total phenolic content in fermented ginger increased several times. The highest phenolic content in ginger was detected after SSF by T. viride. The optimal physiological conditions for the maximum production of phenolic compounds and ß-glucosidase activity of fermented ginger by T. viride were detected at day 7 incubation, pH 6·0, 30°C and 30% moisture. The SSF of ginger by T. viride greatly enhanced the antioxidant potency of phenolic compounds and was evaluated using DPPH and ABTS assays. A potent antibacterial activity of the phenolic compounds of fermented ginger was observed against all the tested human-pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to investigate the optimal physiological conditions of solid-state fermentation (SSF) of ginger by Trichoderma viride for enhancing its phenolic content and antioxidant capacity. In addition, the phenolic compounds of fermented ginger could be potentially used as a dietary adjunct and an antibacterial agent.

Neoadjuvant endocrine therapy with or without palbociclib in low-risk patients: a phase III randomized double-blind SAFIA trial.

BACKGROUND: The most prevalent subtype of breast cancer (BC) is luminal hormonal-positive breast cancer. The neoadjuvant chemotherapy regimens have side effects, emphasizing the need to identify new startegies. OBJECTIVE: Analyze the complete pathologic response (pCR) rate and overall response in a low-risk hormone-positive subset of patients receiving neoadjuvant hormone treatment (NAHT) with or without Palbociclib (a CDK4/CDK6 inhibitor) to boost NAHT effectiveness. MATERIALS AND METHODS: Based on the upfront 21-gene Oncotype DX or low-risk Breast Recurrence Score assay (RS™), the SAFIA trial is designed as a prospective multicenter international, double-blind neoadjuvant phase-III trial that selects operable with luminal BC patients that are HER2-negative for the induction hormonal therapy with Fulvestrant 500 mg ± Goserelin (F/G) followed by randomization of responding patients to palbociclib versus placebo. The pCR rate served as the study's main outcome, while the secondary endpoint was a clinical benefit. RESULTS: Of the 354 patients enrolled, 253 initially responded and were randomized to either F/G fulvestrant with palbociclib or placebo. Two hundred twenty-nine were eligible for the evaluation of the pathologic response. No statistically significant changes were observed in the pCR rates for the patients treated with the F/G therapy with placebo or palbociclib (7% versus 2%, respectively) per the Chevallier classification (Class1 + Class2) (p = 0.1464) and 3% versus 10% assessed per Sataloff Classification (TA, NA/NB) (p = 0.3108). Palbociclib did not increase the rate of complete pathological response. CONCLUSION: Neoadjuvant hormonal therapy is feasible in a selected population with a low RS score of < 31 CLINICAL TRIAL: NCT03447132.

Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi.

L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G - 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.

Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465.

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% ß-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

Quasi-Static Pull-in: an Instability in Electrostatic Actuators.

We identify a new instability in electrostatic actuators dubbed quasi-static pull-in. We report experimental evidence of the instability and study its characteristics in two types of micro actuators operating in ambient air. We found that the underlying mechanism is a fast-slow dynamic interaction between slowly-varying electrostatic excitation and fast resonator response that instigate large non-resonant oscillatory orbits and eventually disappears in a global Shilnikov bifurcation. Based on these findings, we formulate and present a new taxonomy of pull-in instabilities in electrostatic actuators.

Enhancement of Annexin V in response to combination of epigallocatechin gallate and quercetin as a potent arrest the cell cycle of colorectal cancer / Aumento da anexina V em resposta à combinação de galato de epigalocatequina e quercetina como uma potente parada do ciclo celular do câncer colorretal

Colorectal cancer (CRC) is one of the most common cancers leading to comorbidities and mortalities globally. The rational of current study was to evaluate the combined epigallocatechin gallate and quercetin as a potent antitumor agent as commentary agent for therapeutic protocol. The present study investigated the effect of epigallocatechin Gallate (EGCG) (150mg) and quercetin (200mg) at different proportions on proliferation and induction of apoptosis in human colon cancer cells (HCT-116). Cell growth, colonogenic, Annexin V in addition cell cycle were detected in response to phytomolecules. Data obtained showed that, the colony formation was inhibited significantly in CRC starting from the lowest concentration tested of 10 µg/mL resulting in no colonies as visualized by a phase-contrast microscope. Data showed a significant elevation in the annexin V at 100 µg/mL EGCG(25.85%) and 150 µg/mL quercetin (48.35%). Moreover, cell cycle analysis showed that this combination caused cell cycle arrest at the G1 phase at concentration of 100 µg/mL (72.7%) and 150 µg/mL (75.25%). The combined effect of epigallocatechin Gallate and quercetin exert antiproliferative activity against CRC, it is promising in alternative conventional chemotherapeutic agent.

O câncer colorretal (CCR) é um dos cânceres mais comuns, levando a comorbidades e mortalidade em todo o mundo. O racional do presente estudo foi avaliar a combinação de galato de epigalocatequina e quercetina como um agente antitumoral potente como agente de comentário para protocolo terapêutico. O presente estudo investigou o efeito de galato de epigalocatequina (EGCG) (150 mg) e quercetina (200 mg) em diferentes proporções na proliferação e indução de apoptose em células de câncer de cólon humano (HCT-116). O crescimento celular, colonogênico, anexina V, além do ciclo celular foram detectados em resposta a fitomoléculas. Os dados obtidos mostraram que a formação de colônias foi inibida significativamente no CRC a partir da concentração mais baixa testada de 10 µg/mL, resultando em nenhuma colônia conforme visualizado por um microscópio de contraste de fase. Os dados mostraram uma elevação significativa na anexina V a 100 µg/mL de EGCG (25,85%) e 150 µg/mL de quercetina (48,35%). Além disso, a análise do ciclo celular mostrou que essa combinação causou parada do ciclo celular na fase G1 na concentração de 100 µg/mL (72,7%) e 150 µg/mL (75,25%). O efeito combinado da epigalocatequina galato e quercetina exerce atividade antiproliferativa contra o CCR, é promissor como agente quimioterápico alternativo convencional.

Optimal abdominal CT protocol for obese patients.

INTRODUCTION: This study investigated the impact of different protocols on radiation dose and image quality for obese patients undergoing abdominal CT examinations. METHODS: Five abdominal/pelvis CT protocols employed across three scanners from a single manufacturer in a single centre used a variety of parameters (kV: 100/120, reference mAs: 150/190/218/250/300, image reconstruction: filtered back projection (FBP)/iterative (IR)). The routine protocol employed 300 reference mAs and 120 kV. Data sets resulting from obese patient examinations (n = 42) were assessed for image quality using visual grading analysis by three experienced radiologists. Objective assessment (noise, signal/contrast-noise ratios) and radiation dose was compared to determine optimal protocols for prospective testing on a further sample of patients (n = 47) for scanners using FBP and IR techniques. RESULTS: Compared to the routine protocol, mean radiation dose was reduced by 60% when using 100 kV and SAFIRE technique strength 3 (p = 0.001). Reduction of up to 30% in radiation dose was noted for the FBP protocol: 120 kV and 190 reference mAs (p = 0.008). Subjective and objective image quality for both protocols were comparable to that of the routine protocol (p > 0.05). An overall improvement in image quality with increasing strength of SAFIRE was noted. Upon clinical implementation of the optimal dose protocols, local radiology consensus deemed image quality to be acceptable for the participating obese patient cohort. CONCLUSION: Radiation dose for obese patients can be optimised whilst maintaining image quality. Where iterative reconstruction is available relatively low kV and quality reference mAs are also viable for imaging obese patients at 30-60% lower radiation doses.

Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi / Estudos sobre a produção recombinante e atividade anticancerígena da L asparaginase I termoestável de Pyrococcus abyssi

L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G – 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.

A L-asparaginase, que catalisa a degradação da L-asparagina em L-aspartato e amônia, é uma enzima de importância terapêutica no tratamento do câncer, especialmente dos linfomas e da leucemia. O presente estudo descreve a produção recombinante, propriedades e potencial anticancerígeno da enzima de Pyrococcus abyssi, um archaeon hipertermofílico. Existem dois genes que codificam para a asparaginase no genoma desse organismo. Um gene de 918 bp, que codifica 305 aminoácidos, foi amplificado por PCR e clonado na cepa BL21 (DE3) de E. coli usando o plasmídeo pET28a (+). A produção da enzima recombinante foi induzida sob 0,5mM de IPTG, purificada por desnaturação seletiva por calor e cromatografia de troca iônica. A enzima purificada foi analisada quanto à cinética, estrutura in silico e propriedades anticancerígenas. A enzima recombinante apresentou peso molecular de 33 kDa, atividade específica de 1.175 U / mg, valor de KM 2,05 mM, temperatura ótima de 80º C e pH 8. Nenhuma atividade enzimática detectável foi encontrada quando a L-glutamina foi usada como substrato. Estudos in silico mostraram que a enzima existe como um homodímero, com Arg11, Ala87, Thr110, His112, Gln142, Leu172 e Lys232 sendo os resíduos do local ativo putativo. A mudança de energia livre calculada por estudos de docking molecular da enzima e do substrato foi encontrada como ∆G – 4,5 kJ / mol, indicando a afinidade da enzima com o substrato. Valores de IC50 de 5U / mL a 7,5U / mL foram determinados para células FB, células caco2 e células HepG2. Uma quantidade de enzima (5U / mL) apresentou inibição de crescimento de 78% a 55% das células caco2 e HepG2, respectivamente. Em conclusão, a enzima recombinante produzida e caracterizada no presente estudo é uma boa possibilidade para o tratamento do câncer. Os procedimentos adotados na presente pesquisa podem ser aplicados para estudos in vivo.

Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi

Abstract L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as G 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.

Resumo A L-asparaginase, que catalisa a degradação da L-asparagina em L-aspartato e amônia, é uma enzima de importância terapêutica no tratamento do câncer, especialmente dos linfomas e da leucemia. O presente estudo descreve a produção recombinante, propriedades e potencial anticancerígeno da enzima de Pyrococcus abyssi, um archaeon hipertermofílico. Existem dois genes que codificam para a asparaginase no genoma desse organismo. Um gene de 918 bp, que codifica 305 aminoácidos, foi amplificado por PCR e clonado na cepa BL21 (DE3) de E. coli usando o plasmídeo pET28a (+). A produção da enzima recombinante foi induzida sob 0,5mM de IPTG, purificada por desnaturação seletiva por calor e cromatografia de troca iônica. A enzima purificada foi analisada quanto à cinética, estrutura in silico e propriedades anticancerígenas. A enzima recombinante apresentou peso molecular de 33 kDa, atividade específica de 1.175 U / mg, valor de KM 2,05 mM, temperatura ótima de 80º C e pH 8. Nenhuma atividade enzimática detectável foi encontrada quando a L-glutamina foi usada como substrato. Estudos in silico mostraram que a enzima existe como um homodímero, com Arg11, Ala87, Thr110, His112, Gln142, Leu172 e Lys232 sendo os resíduos do local ativo putativo. A mudança de energia livre calculada por estudos de docking molecular da enzima e do substrato foi encontrada como G 4,5 kJ / mol, indicando a afinidade da enzima com o substrato. Valores de IC50 de 5U / mL a 7,5U / mL foram determinados para células FB, células caco2 e células HepG2. Uma quantidade de enzima (5U / mL) apresentou inibição de crescimento de 78% a 55% das células caco2 e HepG2, respectivamente. Em conclusão, a enzima recombinante produzida e caracterizada no presente estudo é uma boa possibilidade para o tratamento do câncer. Os procedimentos adotados na presente pesquisa podem ser aplicados para estudos in vivo.

Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi / Estudos sobre a produção recombinante e atividade anticancerígena da L-asparaginase I termoestável de Pyrococcus abyssi

L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G ­ 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.

A L-asparaginase, que catalisa a degradação da L-asparagina em L-aspartato e amônia, é uma enzima de importância terapêutica no tratamento do câncer, especialmente dos linfomas e da leucemia. O presente estudo descreve a produção recombinante, propriedades e potencial anticancerígeno da enzima de Pyrococcus abyssi, um archaeon hipertermofílico. Existem dois genes que codificam para a asparaginase no genoma desse organismo. Um gene de 918 bp, que codifica 305 aminoácidos, foi amplificado por PCR e clonado na cepa BL21 (DE3) de E. coli usando o plasmídeo pET28a (+). A produção da enzima recombinante foi induzida sob 0,5mM de IPTG, purificada por desnaturação seletiva por calor e cromatografia de troca iônica. A enzima purificada foi analisada quanto à cinética, estrutura in silico e propriedades anticancerígenas. A enzima recombinante apresentou peso molecular de 33 kDa, atividade específica de 1.175 U / mg, valor de KM 2,05 mM, temperatura ótima de 80º C e pH 8. Nenhuma atividade enzimática detectável foi encontrada quando a L-glutamina foi usada como substrato. Estudos in silico mostraram que a enzima existe como um homodímero, com Arg11, Ala87, Thr110, His112, Gln142, Leu172 e Lys232 sendo os resíduos do local ativo putativo. A mudança de energia livre calculada por estudos de docking molecular da enzima e do substrato foi encontrada como ∆G ­ 4,5 kJ / mol, indicando a afinidade da enzima com o substrato. Valores de IC50 de 5U / mL a 7,5U / mL foram determinados para células FB, células caco2 e células HepG2. Uma quantidade de enzima (5U / mL) apresentou inibição de crescimento de 78% a 55% das células caco2 e HepG2, respectivamente. Em conclusão, a enzima recombinante produzida e caracterizada no presente estudo é uma boa possibilidade para o tratamento do câncer. Os procedimentos adotados na presente pesquisa podem ser aplicados para estudos in vivo.

Escherichia coli expression and characterization of alfa-amylase from Geobacillus thermodenitrificans DSM-465 / Expressão e caracterização de Escherichia coli de alfa-amilase de Geobacillus thermodenitrificans DSM-465

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% β-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

A alfa-amilase, que catalisa a hidrólise do amido, é uma enzima ubíqua com imensas aplicações industriais. Um gene de 1698 pb que codifica a amilase de 565 aminoácidos foi amplificado por PCR, a partir de Geobacillus thermodenitrificans DSM-465, clonado no plasmídeo pET21a (+), expresso na cepa BL21 (DE3) de E. coli e caracterizado. A enzima recombinante exibiu peso molecular de 63 kDa, pH ótimo igual a 8, temperatura ótima de 70° C e valor KM de 157,7 µM. Em escala piloto, a enzima purificada removeu com eficiência até 95% de amido do tecido de algodão, indicando sua capacidade de desengomagem em alta temperatura. O modelo 3D da enzima construída por Raptor-X e validada por Ramachandran plot apareceu como um monômero com 31% de hélices alfa, 15% de folhas beta e 52% de loops. Os estudos de docking mostraram melhor afinidade de ligação da enzima com amilopectina (∆G: - 10,59). De acordo com nossos resultados, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276 e Arg175 constituem o sítio ativo potencial da enzima.

Escherichia coli expression and characterization of -amylase from Geobacillus thermodenitrificans DSM-465

Abstract Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% -helices, 15% -sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

Resumo A alfa-amilase, que catalisa a hidrólise do amido, é uma enzima ubíqua com imensas aplicações industriais. Um gene de 1698 pb que codifica a amilase de 565 aminoácidos foi amplificado por PCR, a partir de Geobacillus thermodenitrificans DSM-465, clonado no plasmídeo pET21a (+), expresso na cepa BL21 (DE3) de E. coli e caracterizado. A enzima recombinante exibiu peso molecular de 63 kDa, pH ótimo igual a 8, temperatura ótima de 70° C e valor KM de 157,7 µM. Em escala piloto, a enzima purificada removeu com eficiência até 95% de amido do tecido de algodão, indicando sua capacidade de desengomagem em alta temperatura. O modelo 3D da enzima construída por Raptor-X e validada por Ramachandran plot apareceu como um monômero com 31% de hélices alfa, 15% de folhas beta e 52% de loops. Os estudos de docking mostraram melhor afinidade de ligação da enzima com amilopectina (G: - 10,59). De acordo com nossos resultados, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276 e Arg175 constituem o sítio ativo potencial da enzima.

Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465 / Expressão e caracterização de Escherichia coli de alfa-amilase de Geobacillus thermodenitrificans DSM-465

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% ß-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

A alfa-amilase, que catalisa a hidrólise do amido, é uma enzima ubíqua com imensas aplicações industriais. Um gene de 1698 pb que codifica a amilase de 565 aminoácidos foi amplificado por PCR, a partir de Geobacillus thermodenitrificans DSM-465, clonado no plasmídeo pET21a (+), expresso na cepa BL21 (DE3) de E. coli e caracterizado. A enzima recombinante exibiu peso molecular de 63 kDa, pH ótimo igual a 8, temperatura ótima de 70° C e valor KM de 157,7 µM. Em escala piloto, a enzima purificada removeu com eficiência até 95% de amido do tecido de algodão, indicando sua capacidade de desengomagem em alta temperatura. O modelo 3D da enzima construída por Raptor-X e validada por Ramachandran plot apareceu como um monômero com 31% de hélices alfa, 15% de folhas beta e 52% de loops. Os estudos de docking mostraram melhor afinidade de ligação da enzima com amilopectina (∆G: - 10,59). De acordo com nossos resultados, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276 e Arg175 constituem o sítio ativo potencial da enzima.

Arthrogryposis multiplex congenita, craniofacial, and ophthalmological abnormalities and normal intelligence: a new syndrome?

We report on an 8-year-old boy with clinical manifestations suggestive of a new arthrogryposis syndrome. These included characteristic craniofacial abnormalities, cleft palate, arthrogryposis multiplex congenita, pulmonary hypoplasia, cryptorchidism, and unusual ophthalmological findings. There was no intrauterine growth retardation or decreased fetal movements. Despite the poor prognosis expected in early life, the patient presented with normal mental capability on follow-up. Family data showed that a maternal first cousin of the mother (mother's brother's son) had similar findings and died in infancy. Differential diagnosis included Pena-Shokeir syndrome or phenotype, Gordon syndrome, Marden-Walker syndrome, and the syndrome of arthrogryposis with ophthalmoplegia and retinopathy. The possibility of autosomal dominant inheritance with reduced penetrance is suggested for this apparently new syndrome.

The anti-inflammatory, analgesic and antipyretic activity of Nigella sativa.

The aqueous extract of Nigella sativa (N. sativa) was investigated for anti-inflammatory, analgesic and antipyretic activities in animal models. The extract has an anti-inflammatory effect demonstrated by its inhibitory effects on Carrageenan induced paw edema. It also produced significant increase in the hot plate reaction time in mice indicating analgesic effect. However, N. sativa crude suspension had no effect on yeast induced pyrexia. This study therefore, supports its use in folk medicine both as analgesic and anti-inflammatory agent and calls for further investigations to elucidate its mechanism of action.

Tenoxicam-induced gastropathy in the rat: a comparison with piroxicam and diclofenac sodium, and the inhibitory effects of ranitidine and sucralfate.

The ulcer-inducing potential, indicated by the oral dose that induced gastric ulcers in 50% of rats, was higher for tenoxicam (10.2 mg/kg) than for diclofenac sodium (34 mg/kg, equivalent to 6.8 mg/kg tenoxicam) or piroxicam (6.2 mg/kg). The mean lesion scores, a measure of the intensity of ulceration, using 16 and 32 mg/kg tenoxicam given orally were 3.6 +/- 3.4 and 8.7 +/- 7.3, respectively, compared with 9.6 +/- 6.4 and 24.7 +/- 10.5, respectively, for similar oral doses of piroxicam; the differences were statistically significant (P less than 0.05 and P less than 0.001, respectively). The mean lesion score for 32 mg/kg tenoxicam was also significantly (P less than 0.05) less than that for 160 mg/kg diclofenac sodium: 8.7 +/- 7.3 compared with 14.8 +/- 8.1. Ranitidine (20 40 mg/kg) and 260-520 mg/kg sucralfate but not 4 mg/kg ranitidine strongly inhibited ulceration induced by 32 mg/kg tenoxicam.

Empirical treatment of uncomplicated urinary tract infection by community pharmacist in the Eastern province of Saudi Arabia.

OBJECTIVE: It is a well known phenomenon in the Kingdom of Saudi Arabia that prescription drugs are dispensed over the counter in the community pharmacies. The aim of this study is to document the attitude of community pharmacists to fulfill the concept of pharmaceutical care and to evaluate how they manage a case of acute uncomplicated lower urinary tract infection. METHODS: Eighty-eight community pharmacists in the Eastern province of Saudi Arabia were presented with a patient claiming urinary tract infection and seeking medical treatment. RESULTS: Only one attendant pharmacist refused to dispense medications without prescription. Fifteen others (17%) dispensed urinary antiseptic only and 72 (82%) gave antibacterial agents. Fluoroquinolones were the most commonly dispensed (69%) as first choice and 87% as an alternative) followed by co-trimoxazole, penicillins, cephalosporins and tetracyclines. The number of drugs dispensed ranged from a single agent at 52 (59%), 2 drugs at 31 (35%) and 3 drugs at 4 (4.5%) pharmacies. The average cost was Saudi Riyal (SR) 45.8 ($12.2) for first choice drugs and SR 31.5 ($ 8.4) for the alternatives. CONCLUSION: The heavy dispensing of fluoroquinolones over the counter could eventually lead to increased resistance of the pathogenic bacteria to these drugs. However, despite the lack of pharmacist's adherence to the pharmaceutical law, this study does not indicate that they had abused their patients. It is rather demonstrating the urgent needs for successful implementation of the pharmaceutical law taking into consideration better integration between governmental health providers and the private retail pharmacies. In addition, it supports calls to educate pharmacists to perform basic clinical assessment in the community pharmacy, as a vital tool to effectively manage their patients' health status. The Ministry of health should credit such educational activity for the renewal of pharmacist's license in the Kingdom.

Utilization of digoxin and theophylline assays in the eastern province.

The utilization of therapeutic drug monitoring (TDM) for serum digoxin and theophylline was assessed with respect to the indication, time of sample collection and contribution to patient care. A total of 585 serum drug level determinations of digoxin (275) and theophylline (328) were done in a four month period. Reasons for requests were subtherapeutic response (27.5%), suspected toxicity (3.8%), baseline data (26.7%), patients at risk for toxicity (7.7%), and for the remaining 34.4% of orders, no reason was stated. The highest number of requests 246 (42.1%) were from the inpatient ward (INP), followed by emergency room (ER) 32.6%, outpatient department (OPD) 13% and intensive care unit (ICU) 12.3%. Repeated assays accounted for 363 determinations of which 55.9% had levels similar to the previous ones. In this study, we found a high incidence of inappropriateness in patient selection (34.4%), time of serum sample collection (28.4%), and dosage adjustment (46.8%). However, when these audit criteria were analyzed together, the overall appropriateness was as low as 33.5%. This indicated that serum drug monitoring was poorly utilized and did not contribute much to the patient's care. This results in an estimated financial loss per year (for inappropriate use of digoxin and theophylline levels) of about 37,344 Saudi Riyals (US $9,956.00). Corrective educational programs for the staff, based on standard guidelines for TDM, have been initiated and this study is a baseline for future prospective audits.

A study on diarrheal diseases in children under five years of age.

A cross sectional house-to-house survey on diarrheal disease in children less than five-years-of-age was conducted in the Eastern Province of Saudi Arabia. The main objective was to determine diarrheal disease morbidity and its relationship with different variable factors and management practices during the episode of diarrhea. The survey population included 1,105 households with 2,005 children under five-years-of-age selected from the entire Eastern Province. The prevalence observed was 7.6% during a two-week period giving an annual incidence of two episodes per child per year. Diarrheal morbidity was less prevalent among with age, water storage, and feeding patterns. Diarrhea morbidity was less prevalent among children receiving breast milk. The majority of cases with diarrhea were treated in health facilities where 27% were given drugs only. A solution containing sugar and salt in addition to home fluids were given to 46.4% while 23% received oral rehydration solution (ORS).