Sequential optimization of bioprocess nutritional conditions for production of glutaminase-near-free L-asparaginase by Aspergillus candidus UCCM 00117 was conducted under shake flask laboratory conditions. Catalytic and anti-cancer activities of the poly-peptide were evaluated using standard in vitro biochemical methods. Medium nutrients were selected by one-factor-at-a-time (OFAT) approach while Plackett–Burman design (PBD) screened potential factors for optimization. Path of steepest ascent (PSA) and response surface methodology (RSM) of a Min-Run-Res V fractional factorial of a central composite rotatable design (CCRD) were employed to optimize factor levels towards improved enzyme activity. A multi-objective approach using desirability function generated through predictor importance and weighted coefficient methodology was adopted for optimization. The approach set optimum bioprocess conditions as 49.55 g/L molasses, 64.98% corn steep liquor, 44.23 g/L asparagine, 1.73 g/L potassium, 0.055 g/L manganese and 0.043 g/L chromium (III) ions, at a composite desirability of 0.943 and an L-asparaginase activity of 5216.95U. The Sephadex-200 partially-purified polypeptide had a specific activity of 476.84 U/mg; 0.087U glutaminase activity, 36.46% yield and 20-fold protein purification. Anti-cancer activity potentials of the catalytic poly-peptide were dose-dependent with IC 50 (µg/mL): 4.063 (HL-60), 13.75 (HCT-116), 15.83 (HeLa), 11.68 (MCF-7), 7.61 (HepG-2). The therapeutic enzyme exhibited 15-fold more cytotoxicity to myeloid leukemia cell line than to normal (HEK 238 T) cell. Optimum temperature and pH for activity were within physiological range. However, significant interactions between exposure time and levels of each of temperature and pH made interpretations of residual enzyme activities difficult. The manganese-dependent L-asparaginase from Aspergillu s candidus UCCM 00117 is recommended for further anticancer drug investigations.
Background. Sickle cell anaemia (SCA) is an inherited condition whose clinical manifestations arise from the tendency of haemoglobin to polymerize and deform red blood cells into characteristic sickle shape. Allogeneic bone marrow transplantation offers a cure. The aim of this study was to determine the level of awareness, knowledge, and acceptance of this beneficial procedure in Nigeria. Materials and Methods. This multicentre cross-sectional study was conducted in 7 tertiary hospitals in Nigeria in 2015. Approval was obtained from each institution's research and ethics committee. A pretested structured questionnaire was administered to respondents aged 18 years and above and to the parents or guardians of those below 18 years of age. Results. There were 265 respondents comprising 120 males and 145 females. One hundred and seventy-one (64.5%) respondents were aware of BMT for the treatment of SCA. About 67.8% (116 of 171) of those who were aware believed SCA can be cured with BMT (p = 0.001) and 49.7% (85 of 171) of the respondents accepted BMT (p = 0.001). Conclusion. Awareness of BMT in Nigeria is low when compared with reports from developed countries. The knowledge is poor and acceptance is low. With adequate information, improved education, and psychological support, more Nigerians will embrace BMT.
Sickle cell disorder (SCD) is a multisystem disease with heterogeneous phenotypes. Although all patients have the mutated hemoglobin (Hb) in the SS phenotype, the severity and frequency of complications are variable. When exposed to low oxygen tension, the Hb molecule becomes dense and forms tactoids, which lead to the peculiar sickled shapes of the affected red blood cells, giving the disorder its name. This sickle cell morphology is responsible for the profound and widespread pathologies associated with this disorder, such as vaso-occlusive crisis (VOC). How much of the clinical manifestation is due to sickled erythrocytes and what is due to the relative contributions of other elements in the blood, especially in the microcapillary circulation, is usually not visualized and quantified for each patient during clinical management. Here, we used a microfluidic microcirculation mimetic (MMM), which has 187 capillary-like constrictions, to impose deformations on erythrocytes of 25 SCD patients, visualizing and characterizing the morpho-rheological properties of the cells in normoxic, hypoxic (using sodium meta-bisulfite) and treatment conditions (using hydroxyurea). The MMM enabled a patient-specific quantification of shape descriptors (circularity and roundness) and transit time through the capillary constrictions, which are readouts for morpho-rheological properties implicated in VOC. Transit times varied significantly (p < 0.001) between patients. Our results demonstrate the feasibility of microfluidics-based monitoring of individual patients for personalized care in the context of SCD complications such as VOC, even in resource-constrained settings.
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