Expansion of CD34+ human hematopoietic cells from umbilical cord blood using roller bottles

"El transplante de células madre hematopoyéticas está limitado por la cantidad de células CD34+ presentes en las unidades de sangre de cordón umbilical; el objetivo de este trabajo fue obtener la expansión in vitro de células madre hematopoyéticas. Se establecieron cultivos de células CD34+ de sangre de cordón umbilical en medio IMDM suplementado con citocinas para promover la expansión de progenitores hematopoyéticos. En 5 días de cultivo en frascos giratorios se obtuvo la máxima expansión de unidades formadoras de colonias (UFC) totales de 16.94±2.82 veces, mientras que en cultivo estático fue de 17.28±4.47. La máxima expansión de células totales fue de 20.31±6.18 veces en frasco giratorio y de 26.45±9.89 veces en cultivo estático a los 10 días de cultivo. Se demostró la eficacia del sistema de frascos giratorios con medio IMDM para el cultivo a corto plazo de células enriquecidas CD34+ provenientes de sangre de cordón umbilical y para la expansión de progenitores hematopoyéticos, potencializando el uso de este sistema para otros experimentos y aplicaciones clínicas a futuro."Hematopoietic stem cell (HSC) transplantation is limited by the initial CD34+ cell content in cord blood units; the aim of this work was the in vitro expansion of HSC to overcome this issue. Supplemented IMDM roller bottle cultures of CD34+ cells from human umbilical cord blood were established for hematopoietic progenitor expansion. The maximum total colony forming cells (CFC) expansion was achieved after 5 days of culture, being 16.94±2.82 folds in roller bottles and 17.28±4.47 in static cultures. However, the maximum total cell fold expansion was attained after 10 days of culture. It was of 20.31±6.18 for the roller bottles and 26.45±9.89 for the static cultures. The efficacy of the roller bottles system for short-term cultures of CD34+ cells and expanding hematopoietic progenitors in IMDM was demonstrated; encouraging the use of this culture system for other experiments and may be used for future clinical applications.

Optimization of fermentation conditions for the production of the mezcal from Agave salmiana using response surface methodology

"Response surface methodology was applied to optimize the fermentative phase for the mezcal production from Agave salmiana. A 3k factorial design was used to obtain models describing the relationship between the ethanol production, process productivity, and product yield with respect to the fermentation temperature and the initial sugar concentration. The results showed that the fermentative conditions affected the composition of higher alcohols (referred as a quality indicator) in the mezcal as well as the amount of ethanol. The highest ethanol production was attained by employing the following predicted optimum operational conditions: temperature of 28 °C and an initial sugar concentration of 105 g/l. However, the maximum productivity process was attained with 34.6 °C and 90 g/l, whereas the maximum product yield and the best quality mezcal at 28 °C and 77 g/l. Results show that the simultaneous optimization for high ethanol production and fast production rate are not compatible, since high ethanol production requires a high substrate concentration, which in turn inhibit the growth rate.

Expansion of human hematopoietic cells from umbilical cord blood using roller bottles in CO2 and CO2-free atmosphere

"In this work, we evaluated the expansion of human hematopoietic stem cells from umbilical cord blood in roller bottles. The Iscove's modified Dulbecco's medium, the Stem Pro 34-SFM medium, and the L-15 Leibovitz's medium for cultures in CO2-free atmosphere were assessed. At day 5 of culture, total colony forming unit expansions of 14.44 ± 3.74, 11.20 ± 6.37, and 17.25 ± 3.65-folds were attained, respectively. The expansion reached using L-15 medium in roller bottles was around 10 times higher than that achieved in the static control cultures. To our knowledge, this is the first report of cultures in CO2-free atmosphere to expand cord blood human hematopoietic stem cells and it opens a new branch of possibilities for culturing and clinical applications.

Proteomic analysis of amaranth (Amaranthus hypochondriacus L.) leaves under drought stress

"Amaranth (Amaranthus hypochondriacus L.) is a plant that produces seed with high protein content, is rich on nutraceutical compounds, and can grow under environmental conditions where most of the basic crops are not able to develop. But little is know about the amaranth stress-responsive genes/proteins. The aim of this work was to apply the comparative proteomics approach to study the differential expression of amaranth leaf proteins under drought stress. However, the protein extraction from amaranth tissues is difficult as a result of high endogenous concentrations of interfering compounds; we have made some modifications of the classical trichloroacetic acid-acetone precipitation method to improve the quantity and quality of extracted proteins. Satisfactory and reproducible two-dimensional electrophoresis protein profiles were obtained; the method was also tested forAgave tequilana and Opuntia spp., two more examples of plants that are tolerant to drought stress. Drought-responsive proteins in amaranth leaves were identified by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). The upregulated proteins identified included chloroplast chaperonins involved in refolding and protein complexes protection. Downregulated proteins include Rubisco large subunit, cytochrome b6f, oxygen evolving complexes, and the ascorbate peroxidase mitochondrial. The results have shown that chloroplasts and mitochondria may play a central role in amaranth adaptation to abiotic stress, and further studies should be done at the subcellular level.

Establishment of callus from Opuntia robusta Wendl., a wild and medicinal cactus, for phenolic compounds production

In this work, a protocol for the establishment of callus cultures from Opuntia robusta, a wild and medicinal cactus, was developed. The effects of plant growth regulators and culture media composition on callus development were evaluated.The best response was observed on Murashige and Skoog medium added with 2,4-dichlorophenoxyacetic acid, benzyladenine, biotin, casein hydrolysate and proline. The exposure of O. robusta callus to jasmonic acid increased 1.3-fold and 3-fold total phenolic acids and flavonoids concentration, respectively. The in vitro culture from O. robusta could be a new approach for the obtainment of metabolites with pharmaceutical and/or nutraceutical value.Keywords: Callus, flavonoids, jasmonic acid, wild Opuntia, phenolsAfrican Journal of Biotechnology Vol. 12(21), pp. 3204-320

Replicative and integrative plasmids for production of human interferon gamma in Bacillus subtilis

"Integrative and replicative plasmids for the expression driven by the P43 promoter and secretion of recombinant proteins in Bacillus subtilis were constructed. The plasmids named pInt and pRep respectively were tested for the production of recombinant human interferon gamma (rhIFN-γ). A synthetic hIFN-γ gene employing the optimized B. subtilis codon usage was fused with the Bacillus licheniformis α-amylase signal peptide (sp-amyL) encoding sequence. The integrative construct produced 2.5 ± 0.2 mg l−1 and the replicative system produced 20.3 ± 0.8 mg l−1 of total recombinant rhIFN-γ. The results showed that secretion of hIFN-γ was the bottleneck for the overexpression of mature rhIFN-γ by B. subtilis.

HLA-C genotype and TCR vβ expression analysis in Mexican patients with Psoriasis

"Genetic background and T-cell expansion have been associated as the most important factors for psoriasis susceptibility in the Caucasian population. This study was performed to identify the T cell receptor Vβ repertoire and HLA-Cw genotype in two Mexican groups with severe chronic plaque-type psoriasis. HLA-C typing was performed to detect the allele pattern associated with the disease by sequence-specific primer-polymerase chain reaction. In parallel, RT-PCR and Western blot were used for the identification of the TCR Vβ repertoire. We found a wide variety of HLA-C alleles displayed with a preference to HLA-Cw *07 as the most representative allele in the group of patients. TCR Vβ-2 and Vβ-7 clone-type frequencies were statistically significant (p of 0.0280) when compared to other TCR Vβ expressed in the two groups. We found notable differences both in the HLA-C genotype and TCR Vβ repertoire in the groups of patients studied. Since Mexican individuals are genetically different from the Caucasian population, we suggest that due to these differences the susceptibility to disease and activation of T-cells for a proper immune response may be affected.

Molecular and biochemical characterization of extracellular tannin acyl hydrolase activity from a Mexican isolate of Aspergillus niger

"Microbial tannase, a hydrolysable tannin-degrading enzyme, is extensively used in manufacture of instant tea, beer, wine, and gallic acid. Aspergillus niger strain, obtained from a Mexican tannery wastewaters rich in gallic acid [Quebracho Phenolics-rich Tannery Wastewaters, (QPTW)], displayed a good growth and tannase activity in a minimal medium added with 1% (w/v) QPTW (Kr= 0.451 mm.h-1). Using PCR and RACE 3´ and 5´methodologies, a complete cDNA of a tannase was cloned from this isolate.Nucleotide sequence of complete cDNA was of 4690 bp with a complete ORF of 1833 bp encoding 611 amino acids. Transcriptionalinduction was observed in mineral medium added with carbon sources as tannic acid alone (1 and 10 g/l), as well as mix of glucose(1 and 10 g/l) and tannic acid (1 g/l) in the media. However, neither glucose (1 and 10 g/l) and sucrose (1 and 10 g/l) nor (+)-catechin(1 and 10 g/l) as sole carbon sources displayed gene induction in in vitro assays. A. niger-GTO is a new strain with interesting characteristics for industrial tannase production purposes.

Modified penicillin acylase signal peptide allows the periplasmic production of soluble human interferon-γ but not of soluble human interleukin-2 by the Tat pathway in Escherichia coli

"Production of periplasmic human interferon-γ (hINF-γ) and human interleukin-2 (hIL-2) by the Tat translocation pathway in Escherichia coli BL21-SI was evaluated. The expression was obtained using the pEMR vector which contains the Tat-dependent modified penicillin acylase signal peptide (mSPpac) driven by the T7 promoter. The mSPpac-hINF-γ was processed and the protein was transported to periplasm. Up to 30.1% of hINF-γ was found in the periplasmic soluble fraction, whereas only 15% of the mSPpac-hIL-2 was processed, but hIL-2 was not found in the periplasmic soluble fraction.