RESUMEN
CONTEXT: Plant peptides garner attention for their potential antimicrobial properties amid the rising concern over antibiotic-resistant bacteria. OBJECTIVE: This study investigates the antibacterial potential of crude peptide extracts from 27 Thai plants collected locally. MATERIALS AND METHODS: Peptide extracts from 34 plant parts, derived from 27 Thai plants, were tested for their antimicrobial efficacy against four highly resistant bacterial strains: Streptococcus aureus MRSA, Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli. The stability of these peptide extracts was examined at different temperatures, and the synergistic effects of two selected plant peptide extracts were investigated. Additionally, the time-kill kinetics of the individual extracts and their combination were determined against the tested pathogens. RESULTS: Peptides from Allium sativum L. and Allium oschaninii O. Fedtsch (Amaryllidaceae) were particularly potent, inhibiting bacterial growth with MICs ranging from 1.43 to 86.50 µg/mL. The consistent MICs and MBCs of these extracts across various extraction time points highlight their reliability. Stability tests reveal that these peptides maintain their antimicrobial activity at -20 °C for over a month, emphasizing their durability for future exploration and potential applications in addressing antibiotic resistance. Time-kill assays elucidate the time and concentration-dependent nature of these antimicrobial effects, underscoring their potent initial activity and sustained efficacy over time. DISCUSSION AND CONCLUSIONS: This study highlights the antimicrobial potential of Allium-derived peptides, endorsing them for combating antibiotic resistance and prompting further investigation into their mechanisms.
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Allium , Antibacterianos , Ajo , Pruebas de Sensibilidad Microbiana , Extractos Vegetales , Ajo/química , Extractos Vegetales/farmacología , Extractos Vegetales/aislamiento & purificación , Antibacterianos/farmacología , Antibacterianos/aislamiento & purificación , Allium/química , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/crecimiento & desarrollo , Pseudomonas aeruginosa/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Farmacorresistencia Bacteriana , Tailandia , Péptidos/farmacología , Péptidos/aislamiento & purificación , Sinergismo FarmacológicoRESUMEN
Accurate measurement of glucose-6-phosphate dehydrogenase (G6PD) activity is critical for malaria treatment as misclassification of G6PD deficiency could cause serious harm to patients. G6PD activity should be assessed in blood samples on the day of collection. Otherwise, specimens should be stored under suitable conditions to prevent loss of G6PD activity. Here, we assessed stability and integrity of G6PD testing in samples from normal controls, heterozygous females, and G6PD deficient individuals using water-soluble tetrazolium salts (WST-8) assay. Specimens were stored as ethylenediaminetetraacetic acid (EDTA) whole blood and dried blood spots (DBS) at various temperatures (37⯰C, room temperature, 4⯰C and -20⯰C) and under different humidity conditions (with and without desiccant). G6PD normal samples were stable for up to 1 year when stored at -20⯰C under controlled conditions, with 85% and 91% G6PD activity in EDTA whole blood and DBS in the presence of desiccant, respectively. Specimens from heterozygous females showed greater G6PD activity when stored as DBS, with 85% enzyme activity after 1 year of storage at -20⯰C under controlled conditions in the presence of desiccant. G6PD deficient samples rapidly lost enzyme activity in all storage conditions tested. However, the reduction in G6PD enzyme activity in G6PD deficient samples did not interfere with G6PD classification. Samples stored under suitable conditions for G6PD testing will allow accurate measurement of enzyme activity, prevent misclassification of G6PD deficiency and enable safe and effective use of antimalarial drugs such as primaquine and tafenoquine.
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Recolección de Muestras de Sangre/métodos , Deficiencia de Glucosafosfato Deshidrogenasa/sangre , Deficiencia de Glucosafosfato Deshidrogenasa/diagnóstico , Tamizaje Masivo/métodos , Sales de Tetrazolio , Femenino , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Heterocigoto , Humanos , Masculino , TemperaturaRESUMEN
Recently, several studies have examined possible applications of nanoparticles for the development of electronic and optical sensors. The plasmon absorbance of gold nanoparticles has been used extensively to study biomolecular processes, including nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate-dependent enzymatic reactions. In this report, we describe the development of gold nanoparticles as a new colorimetric and sensitive detection method of glucose-6-phosphate dehydrogenase deficiency by means of controlled reversible assembly of gold nanoparticles. 3-nm polyvinylpyrrolidone/N,N'-dimethylaminopyridine-stabilized gold nanoparticles were synthesized, characterized and applied for an in vitro activity assay of 11 recombinant human glucose-6-phosphate dehydrogenase variants. Differences in the activity of the glucose-6-phosphate dehydrogenase variants from different deficiency classes were readily detected using the synthesized gold nanoparticles. The developed method can be easily distinguished with color change by naked eye for the detection of glucose-6-phosphate dehydrogenase deficiency. Moreover, we are the first to propose the segregation mechanism of polyvinylpyrrolidone/N,N'-dimethylaminopyridine-stabilized gold nanoparticles by reduced nicotinamide adenine dinucleotide phosphate. The method enables visual detection of glucose-6-phosphate dehydrogenase deficiency, which could be further developed for diagnostic testing of glucose-6-phosphate dehydrogenase deficiency.
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Colorimetría , Deficiencia de Glucosafosfato Deshidrogenasa/diagnóstico , Glucosafosfato Deshidrogenasa/análisis , Oro/química , Nanopartículas del Metal/química , Glucosafosfato Deshidrogenasa/metabolismo , Deficiencia de Glucosafosfato Deshidrogenasa/metabolismo , HumanosRESUMEN
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common polymorphism and enzymopathy in humans, affecting approximately 400 million people worldwide. It is responsible for various clinical manifestations, including favism, hemolytic anemia, chronic non-spherocytic hemolytic anemia, spontaneous abortion, and neonatal hyperbilirubinemia. Understanding the molecular mechanisms underlying the severity of G6PD deficiency is of great importance but that of many G6PD variants are still unknown. In this study, we report the construction, expression, purification, and biochemical characterization in terms of kinetic properties and stability of five clinical G6PD variants-G6PD Bangkok, G6PD Bangkok noi, G6PD Songklanagarind, G6PD Canton+Bangkok noi, and G6PD Union+Viangchan. G6PD Bangkok and G6PD Canton+Bangkok noi showed a complete loss of catalytic activity and moderate reduction in thermal stability when compared with the native G6PD. G6PD Bangkok noi and G6PD Union+Viangchan showed a significant reduction in catalytic efficiency, whereas G6PD Songklanagarind showed a catalytic activity comparable to the wild-type enzyme. The Union+Viangchan mutation showed a remarkable effect on the global stability of the enzyme. In addition, our results indicate that the location of mutations in G6PD variants affects their catalytic activity, stability, and structure. Hence, our results provide a molecular explanation for clinical manifestations observed in individuals with G6PD deficiency.
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Biocatálisis , Deficiencia de Glucosafosfato Deshidrogenasa/enzimología , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/metabolismo , Estabilidad de Enzimas , Glucosafosfato Deshidrogenasa/genética , Cinética , Modelos Moleculares , Mutación , Multimerización de Proteína , Estructura Cuaternaria de ProteínaRESUMEN
Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is an X-linked hereditary genetic defect that is the most common polymorphism and enzymopathy in humans. To investigate functional properties of two clinical variants, G6PDViangchan and G6PDViangchan+Mahidol, these two mutants were created by overlap-extension PCR, expressed in Escherichia coli and purified to homogeneity. We describe an overexpression and purification method to obtain substantial amounts of functionally active protein. The KM for G6P of the two variants was comparable to the KM of the native enzyme, whereas the KM for NADP(+) was increased 5-fold for G6PDViangchan and 8-fold for G6PDViangchan+Mahidol when compared with the native enzyme. Additionally, kcat of the mutant enzymes was markedly reduced, resulting in a 10- and 18-fold reduction in catalytic efficiency for NADP(+) catalysis for G6PDViangchan and G6PDViangchan+Mahidol, respectively. Furthermore, the two variants demonstrated significant reduction in thermostability, but similar susceptibility to trypsin digestion, when compared with the wild-type enzyme. The presence of NADP(+) is shown to improve the stability of G6PD enzymes. This is the first report indicating that protein instability and reduced catalytic efficiency are responsible for the reduced catalytic activity of G6PDViangchan and G6PDViangchan+Mahidol and, as a consequence, contribute to the clinical phenotypes of these two clinical variants.
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Deficiencia de Glucosafosfato Deshidrogenasa/enzimología , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Biocatálisis , Dicroismo Circular , Estabilidad de Enzimas , Eritrocitos/enzimología , Glucosafosfato Deshidrogenasa/aislamiento & purificación , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Humanos , Cinética , Proteínas Mutantes/aislamiento & purificación , Mutación , Estructura Secundaria de Proteína , Temperatura , Tripsina/químicaRESUMEN
and young children, but rotavirus gastroenteritis in adults is uncommon. In this study, 260 stool samples collected in Thailand from January 2006 to February 2007 from patients, of all ages with acute gastroenteritis, were tested for group A rotavirus and compared with rotavirus infections in children and adults. Rota- virus was detected in 42% of the patients' samples, but children (< 18 years old) have a significantly higher prevalence (57%) of rotavirus infection than adults (≥ 18 years old) (27%) (OR 3.55; 95% CI: 2.11-5.96; p < 0.001). The highest attack rate was found in the age group of < 2 years old (14%), followed by 2-4 years of age (9%), 18-59 years of age (8%), 5-17 years of age (6%) and ≥ 60 years of age (5%). The dominant genotype was G1P[8] (27%), followed by G2P[4] (7%), G3P[8] (1%), and G9P[8] (1%). The rare genotypes identified were G1P[4], G1P[6], G2P[6], G2P[8], and G3P[6]. Mixed infections mostly occurred in children, comprising G1P[4]/P[8], G1P[4]/P[6], G1P[6]/P[8], G1/G2P[4], G1/G3P[4], and G1/G3P[4]/P[8]. Rotaviruses G3, G9, and P[4] were found only in children and genotype P[6] was found in adults (75%) at a higher frequency than in children (25%) (p < 0.001). The number of rotavirus in children was 1.99x10(8)/ml and in adult patients was 7.32x10(6)/ ml. The present study highlights the higher prevalence of rotavirus infection in children compared to adults and rotavirus genetic heterogeneity. Rotaviruses are the most important cause of severe diarrhea in infants