Fludarabine-based reduced intensity regimen for matched related donor hematopoietic stem cell transplantation in acquired severe aplastic anemia.

Different conditioning regimens have been evaluated in matched-related donor allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acquired severe aplastic anemia (SAA) with varying results. In this manuscript, we report our experience with fludarabine (120mg/m2), very low dose cyclophosphamide (1200mg/m2) and antithymocyte globulin (7.5mg/kg). Low dose total body irradiation (2Gy) was added to the conditioning regimen for patients older than 15 years. Nineteen patients (median age 23years) underwent transplant between 2008 and 2015. The majority (89%) were younger than 40 years. Stem cell source was BM (n=11) or PBSC (n=8). GvHD prophylaxis consisted of cyclosporine and either a short course of methotrexate (n=9) or mycophenolate mofetil (n=10). Eighteen (94.7%) patients achieved sustained engraftment. The median times to neutrophil and platelet engraftments were 19 (range: 14-34) and 17.1 (range: 12-25) days, respectively. The day-30 cumulative incidence of neutrophil and platelet engraftment was 89.4% and 94.7%, respectively. No secondary graft rejection was observed. The 1-year cumulative incidence of aGvHD (grade II-IV) and cGvHD was 11.7% and 0%, respectively. The 2-year GvHD-free survival rate was 78.6% (95% CI: 52.5-91.4%). Fludarabine-based reduced intensity regimen for MRD allo-HSCT in SAA compares favorably to other available regimens. This regimen deserves further investigations with larger cohort of patients.

Amplification of the Escherichia coli lacZ gene in Bacillus subtilis and its expression on a by-product growth medium.

A Bacillus subtilis wild type strain and a kinA (spoIIJ) isogenic mutant were compared as hosts for the expression of the Escherichia coli beta-galactosidase gene, lacZ, driven by the B. subtilis aprE promoter in a chromosomal system. The 2 x SG sporulation formula, with some modifications, was used as a basal medium. The specific activity values recorded by the mutant strain at the stationary phase were markedly higher than those achieved by the wild type host. Exposure of the cells to increasing levels of chloramphenicol resulted in significant amplifications of the lacZ region. Gene copy numbers of 19 and 11 were estimated in the amplified wild type and kinA strains, respectively, with high segregational stability records. The magnitude of beta-galactosidase over-expression was dependent on, and roughly proportional to antibiotic resistance levels. Among five examined by-products, a 2.3-times diluted concentration of neutralized cheese whey was successfully used as a sole medium component for over-expression of the recombinant beta-galactosidase gene in B. subtilis.

Application of the response surface methodology for optimizing the activity of an aprE-driven gene expression system in Bacillus subtilis.

The major targets for improvement of recombinant expression systems in microbial cells are gene dosage, transcriptional control machinery and, to some extent, translation. Here we show that optimization of fermentation conditions by applying statistically designed, multifactorial experiments offers an additional method for potential enhancement of gene expression systems. A chromosomally encoded fusion between the Bacillus subtilis aprE regulatory region and the E. coli lacZ gene carried by the B. subtilis host cells was used. The 2 x SG sporulation medium was used as a basal medium. Among the 11 fermentation factors we examined, the most significant variables influencing beta-galactosidase expression were statistically elucidated for optimization and included peptone, MgSO4.7H2O, and KCl. The optimum concentrations of these variables were predicted by using a second-order polynomial model fitted to the results obtained by applying the Box-Behnken design, a response surface method. Calculated optimum concentrations were predicted to confer a maximum yield of 2,423.5 beta-galactosidase specific activity units. A verification experiment performed under optimal conditions yielded 96% of the predicted specific activity value with an increase by a factor of almost 5 compared with the results obtained under basal conditions.

Effect of nutrients on alginate synthesis in Azotobacter vinelandii and characterization of the produced alginate.

The role of nutrients on alginate production by Azotobacter vinelandii was studied in batch cultures. The largest amount of bacterial alginate was obtained in presence of: 0.3 g/l MgSO4.7H2O. 0.4 g/l NaCl, 42 mg/l CaCl2.2H2O,.4 mg/l KH2PO4, 16 mg/l K2HPO4, 2.5 mg/l FeSO4.7H2O, 2.9 mg/l H3BO3, 2 mg/l ZnSO4.7H2O, 2 mg/l Na2MoO4.2H2O, 0.3 mg/l CuSO4.5H2O, 0.2 mg/l MnCl2.4H2O. Alginate production was not enhanced by natural additives or inducing agents, except for acetate, which increased alginate yield. The pure alginate contained 0.36% ash and 0.4% protein. It is similar to algal alginate, but it has an extra acetyl group. It contains 69.5% M-M block, 27.5% M-G block and 3% G-G block.

Riboflavin production by Aspergillus terreus from beet-molasses.

Aspergillus terreus was used for riboflavin (vitamin B2) production in a medium containing beet molasses as the sole carbon source. Growth and the vitamin production of the fungus were markedly affected by the composition of the culture medium. A maximum riboflavin yield was achieved at the late exponential growth phase (16 day-old cultures) in the presence of (g/l): centrifuged beet-molasses, 90; L-asparagine, 1; MgSO4.7H2O, 0.5; K2HPO4/kH2PO4 (1:1), 5; and the medium initially adjusted to pH 8.

Some nutritional requirements of a mixed culture transforming Reichstein's compound S into prednisolone.

Reichstein's compound S was successfully converted to prednisolone in a single-step fermentation using a mixed culture of Curvularia lunata and Mycobacterium smegmatis. Introducing additional medium at the time of bacterial inoculation and increasing the M. smegmatis inoculum to 8% were necessary for maximal dehydrogenation of cortisol to prednisolone (86%). However, beef extract, corn-steep solids, and malt extract were inhibitory to the dehydrogenase activity and stimulatory to hydroxylase. Of the vitamins tested, nicotinic acid and riboflavin at 0.2 and 1.13 mg/L, respectively, resulted in maximum transformation of Reichstein's compound S (100%) and optimized prednisolone yields (92%) in the mixed culture. The trace elements present in the medium were sufficient for maximal transformation, and there was no need for an exogenous supply. Addition of ATP, sodium acetate, and NAD inhibited the dehydrogenation reaction.

Single cell protein production from beet pulp by mixed culture.

Different mixed cultures of Trichoderma reesei and a yeast were cultivated on beet pulp (BP)-containing medium. T. reesei and Kluyveromyces marxianus offered a combination that gave high SCP yields (51%) and efficiently converted BP into proteins (39.4%). The yeast extract in the basal medium could be substituted and the BP level multiplicated from 2 to 4% upon using mixed culture of T. reesei and K. marxianus. Under these conditions the protein yields reached to a maximum value of 54% and highest efficiency of BP conversion into proteins (41.8%). The obtained protein proved to contain all essential amino acids, which compared favourably with those of the FAO guideline and soy bean oil meal.

Physiological study on riboflavin production by hydrocarbon-utilizing Candida guilliermondii Wickerham.

The production of riboflavin (vitamin B2) by Candida guilliermondii Wickerham cultivated on solar-containing medium was stimulated in the presence of corn oil (0.1 g%), arginine, phenylalanine (1 m mole/l) or CoSO4.7H2O (1000 micrograms/l). On the other hand, emulsifying agents strongly inhibited yeast growth and vitamin production. Similarly, FeSO4.7H2O and MnSO4.4H2O at 50 and 200 micrograms/l levels, respectively, also showed inhibitory effect. Among the tested purines, xanthine enhanced vitamin production.

Protein-enriched feedstuff from beet pulp.

Of five moulds that were cultivated in a medium containing beet pulp, Trichoderma reesel was superior in protein production (34%) and in conversion of pulp to proteins (18% w/w). Protein ylelds were improved by up to 49% by pre-treatment of the beet pulp, adjustment of the solld/llquid and liquld/solid ratios, the incubation period, nitrogen level and addition of some trace elements as well as with inoculum size. The protein obtained contained all essential amino acids and compared favourably with those of the FAO guldeline and of soy bean oll meal.

Utilization of beet molasses for sterol production by some moulds.

Different moulds were cultivated in beet molasses (BM)-containing medium. Penicillium crustosum Thom was superior to the other moulds in total sterols production (4% on dry weight basis), efficiency of convertibility of the BM sugars to sterols (2%), total lipids (19.4%) and unsaponified lipids (13.4%). The treatment of BM with H2SO4- followed by centrifugation allowed maximum fermentation yields. The highest unsaponified lipids (16.5%) and total sterols level (7.4%) were obtained with a medium composed of (g/1): NaNO3, 3; K2HPO4, 3; MgSO4.7H2O, 1.5; K2SO4, 0.11; ZnSO4.7H2O, 0.05; FeCl3.6H2O, 0.16; H2SO4-treated BM, 60. Maximal sterol yields (8.4%) and high growth rate were achieved at the accelerated growth phase (8 days old cultures), when the initial pH value of the medium was adjusted to 7.0.