Author: Ana Paulina Barba de la Rosa
ANA PAULINA BARBA DE LA ROSA (2015)
"Diabetes is an increasing worldwide problem leading to major associated health issues and increased health care costs. In 2012, 9.3% of the American population was affected by diabetes, according to the American Diabetes Association, with 1.7 million of new cases since during the year (www.diabetes.org). Proteome initiatives can provide a deeper understanding of the biology of this disease and help develop more effective treatments. The collaborative effort of the Human Diabetes Proteome Project (HDPP) brings together a wide variety of complementary resources to increase the existing knowledge about both type 1 and type 2 diabetes and their related complications. The goals are to identify proteins and protein isoforms associated with the pathology and to characterize underlying disease-related pathways and mechanisms. Moreover, a considerable effort is being made on data integration and network biology. Sharing these data with the scientific community will be an important part of the consortium. Here we report on: the content of the HDPP session held at the 12th HUPO meeting in Yokohama; recent achievements of the consortium; discussions of several HDPP workshops; as well as future HDPP directions as discussed at the 13th HUPO congress in Madrid, with a special attention given to the lists of prioritized, diabetes-related proteins and the proteomic means to study them."
"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."
"11S globulin is one of the major seed storage proteins in amaranth. Recombinant protein was produced as up to similar to 80% of the total bacterial protein using Escherichia coli Rosetta-gami (DE3) containing pET21d with amaranth 11S globulin cDNA. The best expression condition was at 302 K for 20 h using LB medium containing 0.5 M NaCl. The recombinant protein was easily separated from most of the Escherichia coli proteins by precipitation with 0-40% ammonium sulfate solution. It formed aggregates at low temperature and at low salt concentrations. This behaviour may imply that it has a more hydrophobic nature than other 11S seed globulins. The crystals diffracted to 6 A resolution and belonged to space group P6(3), with unit-cell parameters a = b = 97.6, c = 74.8 A, gamma = 120.0 degrees. One subunit of a trimer was estimated to be present in the asymmetric unit, assuming a V (sol) of 41%. To obtain the complete structure solution, experiments to improve crystallization and flash-cooling conditions are in progress."
"The expression of recombinant Trichoderma atroviride endochitinase ech42 (rech42) in Pichia pastoris driven by the constitutive PGAP promoter was tested at low (3%) and high (40%) dissolved oxygen tension (DOT) conditions. Maximum volumetric enzyme activities of 18,600±1053 and 12,330±1000 U1−1 were attained at 3% and 40% DOT, respectively. Whereas maximum specific enzyme activities were 89.4±4.6 y 88.8±4.0 U mg−1protein. We found that conjunction of low DOT and the overexpression of rech42 affected the cell size of the P. pastoris but no the rech42 specific enzyme activity."
"La expresión de la Endoquitinasa de 42 kDa de Trichoderma atroviride recombinante (rech42) en Pichia pastoris bajo la dirección del promotor constitutivo, PGAP, fue analizada a baja (3%) y alta (40%) tensión de oxígeno disuelto (TOD). Las actividades enzimáticas volumétricas máximas logradas en cada condición fueron 18,600±1053 y 12,330±1000 U 1−1, a TOD de 3% y 40%, respectivamente. Mientras que las actividades específicas máximas fueron 89.4±4.6 y 88.8±4.0 U mg−1proteína. Se encontró que la conjunción de una baja TOD y la sobreexpresión de rech42 afectó el tamaño de las células de Pichia pastoris pero no afectó la actividad enzimática específica de rech42."
"Aims: To identify the yeast and bacteria present in the mezcal fermentation from Agave salmiana.
Methods and Results: The restriction and sequence analysis of the amplified region, between 18S and 28S rDNA and 16S rDNA genes, were used for the identification of yeast and bacteria, respectively. Eleven different micro‐organisms were identified in the mezcal fermentation. Three of them were the following yeast: Clavispora lusitaniae, Pichia fermentans and Kluyveromyces marxianus. The bacteria found were Zymomonas mobilis subsp. mobilis and Zymomonas mobilis subsp. pomaceae, Weissella cibaria, Weissella paramesenteroides, Lactobacillus pontis, Lactobacillus kefiri, Lactobacillus plantarum and Lactobacillus farraginis.
Conclusions: The phylogenetic analysis of 16S rDNA and ITS sequences showed that microbial diversity present in mezcal is dominated by bacteria, mainly lactic acid bacteria species and Zymomonas mobilis. Pichia fermentans and K. marxianus could be micro‐organisms with high potential for the production of some volatile compounds in mezcal.
Significance and Impact of the Study: We identified the community of bacteria and yeast present in mezcal fermentation from Agave salmiana."
"Opuntia spp. are plants native from Mexico where the largest varieties of wild and semi-domesticated species are found. Nowadays the most domesticated species, O. ficus-indica is widely distributed worldwide. Since pre-Hispanic times, young cladodes or nopalitos have been used as a source of food and in folk medicine. Reports indicate that dietary intake of Opuntia spp. exhibit antioxidant, anti-inflammatory, and antiatherogenic properties, which emphasizes their nutritional benefit for preventing cardiovascular and most chronic non-infectious diseases. However the Opuntia biological properties may fluctuate depending on the cladode phytochemical composition, variations that can be quantitative and qualitative as function of the plant growing conditions. For these reasons cell tissue cultures represent an excellent alternative for the control of Opuntia metabolite production."
"La presente invención se refiere a la construcción y uso de un vector de expresión para el género Bifidobacterium. Particularmente se refiere plásmido pLR-hIL-10 en el cual se incluyeron un promotor, un terminador y un péptido señal BIF3, además del gen que codifica para la interleucina-10 humana. Se usa Bifidobacterium longum, como vehículo para la IL-10, debido a las ventajas que ofrece sobre otros sistemas de expresión como E. coli, entre las que destacan la ausencia de lipopolisacáridos de superficie que son causantes de patogenicidad y el contar de eficientes sistemas de exporte de proteínas hacia el medio extracelular. La IL-10, es una proteína que ayuda al tratamiento de enfermedades inflamatorias, como son: colitis ulcerosa, mieloma múltiple, melanoma, linfoma no-Hodking´s, enfermedad de crhon, artritis reumatoide, esclerosis múltiple, lupus eritematoso, psoriasis, dermatitis atópica, asma, entre otras.El resultado es un producto que puede utilizarse para la producción de proteínas recombinantes para aplicación terapéutica y biotecnológica. Por otro lado, este vector puede ser utilizado para la producción de proteínas inestables debido a que la misma bacteria podría funcionar como vehículo para dirigir la proteína al sitio en que se requiere su acción, sin ser necesario pasar por un proceso de purificación de proteínas."
"The present invention refers to the construction and use of an expression vector for the genus Bifidobacterium. The invention mainly refers to a pLR-hIL-10 plasmid, wherein a promoter, a terminator and a BIF3 signal peptide are included, as well as a gene that codifies for the human interleukin-10. Bifidobacterium longum is used as a vehicle for IL-10 due to the advantages offered over further expression systems like E.coli, amongst which are the absence of surface lipopolysaccharides which are know to be the cause of pathogenicity, and the presence of efficient systems for exporting proteins to the extracellular medium. The IL-10, is a protein useful for treating inflammatory diseases such as: ulcerative colitis, multiple myeloma, melanoma, no-Hodgkin s lymphoma, Crohn s disease, rheumatoid arthritis, multiple sclerosis, lupus eritematous, psoriasis, atopic dermatitis, asthma, amongst others. The result is a product useful for the production of recombinant proteins to be therapeutica lly and biotechnologically applied. The inventive vector may be used for producing unstable proteins since the same bacterium may work as a vehicle for leading proteins to the place where the action thereof is required, without being subjected to a protein purification process."
"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."
"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."
"We present here the structural modeling and biochemical characterization of a recombinant superoxide dismutase (SOD) from Deschampsia antarctica E. Desv. [Poaceae] produced in Escherichia coli. The recombinant protein was purified by affinity chromatography nickel-nitrilotriacetic acid (Ni-NTA), and its identity was demonstrated by immunoblotting and inhibition by H2O2 and KCN. Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis confirmed the presence of Cu and Zn. Modeling of the D. antarctica Cu/Zn-SOD (DaSOD) amino acid sequence using the SWISS-MODEL and 2Q2L_B monomer of the psychrophilic Cu/Zu-SOD from Potentilla atrosanguinea (PaSOD) as template produced a structure similar to that of the typical eukaryotic Cu/Zn-SODs. Activity assays using the p-nitro blue tetrazolium chloride (NBT) solution method showed that the purified DaSOD had a specific activity of 5818 U/mg at 25 °C and pH 7.2 and that it was active in a pH interval of 5–8 and a temperature interval of 0–40 °C. Furthermore, DaSOD was still active at −20 °C as observed by a zymogram assay. We found 100 % activity when it was heated at 80 °C for 60 min, indicating a high thermostability. DaSOD properties suggest that this enzyme could be useful for preventing the oxidation of refrigerated or frozen foods, as well as in the preparation of cosmetic and pharmaceutical products."