METHODS: Effects of APC on expressions of genes encoding catalase (katA), superoxide dismutases (SODs), including sodA and sodM, and alkyl hydroperoxide reductase (ahpC) in S· aureus were quantitated by RT-qPCR in reference to gyrA and 16S rRNA. Corresponding activities of the enzymes were also investigated. The Livak analysis was performed for verification of gene-fold expression data. Effects of APC on intracellular and extracellular reactive oxygen species (ROS) levels were determined using the nitroblue tetrazolium (NBT) reduction assay.
RESULTS: APC-treated S· aureus cells had higher sodA and sodM transcripts at 1.5-fold and 0.7-fold expressions respectively with corresponding increase in total SOD activity of 12.24 U/mL compared to untreated cells, 10.85 U/mL (P<0.05). Expression of ahpC was highest in APC-treated cells with 5.5-fold increased expression compared to untreated cells (P<0.05). Correspondingly, ahpC activity was higher in APC-treated cells at 0.672 (A310nm) compared to untreated cells which was 0.394 (A310nm). In contrast, katA expression was 1.48-fold and 0.33-fold lower respectively relative to gyrA and 16S rRNA. Further, APC-treated cells showed decreased catalase activity of 1.8 ×10-4 (U/L or μmol/(min·L)) compared to untreated cells, which was 4.8 ×10-4 U/L (P<0.05). Absorbance readings (A575nm) for the NBT reduction assay were 0.709 and 0.695 respectively for untreated and treated cells, which indicated the presence of ROS. APC-treated S· aureus cells had lower ROS levels both extracellularly and intracellularly, but larger amounts remained intracellularly compared to extracellular levels with absorbances of 0.457 and 0.137 respectively (P<0.05).
CONCLUSION: APC induced expressions of both sodA and sodM, resulting in increased total SOD activity in S· aureus. Higher sodA expression indicated stress induced intracellularly involving O2- , presumably leading to higher intracellular pools of H2O2. A concommittant decrease in katA expression and catalase activity possibly induced ahpC expression, which was increased the highest in APC-treated cells. Our findings suggest that in the absence of catalase, cells are propelled to seek an alternate pathway involving ahpC to reduce stress invoked by O2- and H2O2. Although APC reduced levels of ROS, significant amounts eluded its antioxidative action and remained intracellularly, which adds to oxidative stress in treated cells.
METHODOLOGY: We conducted a longitudinal observational study in gut microbiota profile in a group of paediatric patients diagnosed with ALL using 16 s ribosomal RNA sequencing and compared these patients' microbiota pattern with age and ethnicity-matched healthy children. Temporal changes of gut microbiota in these patients with ALL were also examined at different time-points in relation to chemotherapy.
RESULTS: Prior to commencement of chemotherapy, gut microbiota in children with ALL had larger inter-individual variability compared to healthy controls and was enriched with bacteria belonging to Bacteroidetes phylum and Bacteroides genus. The relative abundance of Bacteroides decreased upon commencement of chemotherapy. Restitution of gut microbiota composition to resemble that of healthy controls occurred after cessation of chemotherapy. However, the microbiota composition (beta diversity) remained distinctive and a few bacteria were different in abundance among the patients with ALL compared to controls despite completion of chemotherapy and presumed restoration of normal health.
CONCLUSION: Our findings in this pilot study is the first to suggest that gut microbiota profile in children with ALL remains marginally different from healthy controls even after cessation of chemotherapy. These persistent microbiota changes may have a role in the long-term wellbeing in childhood cancer survivors but the impact of these changes in subsequent health perturbations in these survivors remain unexplored.
METHODS: A subcohort of 201 children with behavioural outcome measures was identified within a longitudinal, Australian birth-cohort study. The faecal microbiota were analysed at 1, 6, and 12 months of age. Behavioural outcomes were measured at 2 years of age.
FINDINGS: In an unselected birth cohort, we found a clear association between decreased normalised abundance of Prevotella in faecal samples collected at 12 months of age and increased behavioural problems at 2 years, in particular Internalizing Problem scores. This association appeared independent of multiple potentially confounding variables, including maternal mental health. Recent exposure to antibiotics was the best predictor of decreased Prevotella.
INTERPRETATION: Our findings demonstrate a strong association between the composition of the gut microbiota in infancy and subsequent behavioural outcomes; and support the importance of responsible use of antibiotics during early life.
FUNDING: This study was funded by the National Health and Medical Research Council of Australia (1082307, 1147980, 1129813), The Murdoch Children's Research Institute, Barwon Health, Deakin University, Perpetual Trustees, and The Shepherd Foundation. The funders had no involvement in the data collection, analysis or interpretation, trial design, recruitment or any other aspect pertinent to the study.
RESULTS: A thermostable organic solvent-tolerant protease producer had been identified as Bacillus subtilis strain Rand, based on the 16S rRNA analysis conducted, as well as the morphological characteristics and biochemical properties. The production of the thermostable organic solvent-tolerant protease was optimized by varying various physical culture conditions. Inoculation with 5.0% (v/v) of (AB600 = 0.5) inoculum size, in a culture medium (pH 7.0) and incubated for 24 h at 37 degrees C with 200 rpm shaking, was the best culture condition which resulted in the maximum growth and production of protease (444.7 U/ml; 4042.4 U/mg). The Rand protease was not only stable in the presence of organic solvents, but it also exhibited a higher activity than in the absence of organic solvent, except for pyridine which inhibited the protease activity. The enzyme retained 100, 99 and 80% of its initial activity, after the heat treatment for 30 min at 50, 55, and 60 degrees C, respectively.
CONCLUSION: Strain Rand has been found to be able to secrete extra-cellular thermostable organic solvent-tolerant protease into the culture medium. The protease exhibited a remarkable stability towards temperature and organic solvent. This unique property makes it attractive and useful to be used in industrial applications.