Optimization of growth regulators in organogenesis of Bletia purpurea (Lam.) using response surface design and genetic evaluation

This paper reports the optimal concentrations of indol acetic acid (IAA) and benzylaminopurine to stimulate morphogenetic induction Bletia purpurea (Lam.) and the application of random amplification of polymorphic deoxyribonucleic acid (DNA) (RAPD) for genetic evaluation of micropropagated plantlets. Organogenesis was induced from B. purpurea (Lam.) explant pseudobulbs in Murashige and Skoog (MS) medium at full ionic strength and supplemented with 20 g/l sucrose, 2 g/l activated carbon and 2.2 g/l Gel Rite. Optimum growth regulator concentrations were determined by response surface design, according to Box-Benhken. At 15 days after morphogenic induction was observed, direct shoots formation on the explant was attained, and optimum concentrations for the greatest number of shoots was 1.8 mg/l Indol acetic acid and no benzylaminopurine. Comparison of RAPD genetic profiles for mother plantlets and regenerated daughter plantlets showed no changes in the genome based on the oligonucleotides used. The conditions applied in this protocol produces plantlets which maintain the genetic traits of the first generation.Keywords: Genetic evaluation, polymerase chain reaction (PCR), random amplification of polymorphic DNA (RAPD) analysis, Bletia purpurea, pseudobulbs, direct organogenesis, response surface design, in vitr

Progressive Shifts in the Gut Microbiome Reflect Prediabetes and Diabetes Development in a Treatment-Naive Mexican Cohort.

Type 2 diabetes (T2D) is a global epidemic that affects more than 8% of the world\u27s population and is a leading cause of death in Mexico. Diet and lifestyle are known to contribute to the onset of T2D. However, the role of the gut microbiome in T2D progression remains uncertain. Associations between microbiome composition and diabetes are confounded by medication use, diet, and obesity. Here we present data on a treatment-naive cohort of 405 Mexican individuals across varying stages of T2D severity. Associations between gut bacteria and more than 200 clinical variables revealed a defined set of bacterial genera that were consistent biomarkers of T2D prevalence and risk. Specifically, gradual increases in blood glucose levels, beta cell dysfunction, and the accumulation of measured T2D risk factors were correlated with the relative abundances of four bacterial genera. In a cohort of 25 individuals, T2D treatment-predominantly metformin-reliably returned the microbiome to the normoglycemic community state. Deep clinical characterization allowed us to broadly control for confounding variables, indicating that these microbiome patterns were independent of common T2D comorbidities, like obesity or cardiovascular disease. Our work provides the first solid evidence for a direct link between the gut microbiome and T2D in a critically high-risk population. In particular, we show that increased T2D risk is reflected in gradual changes in the gut microbiome. Whether or not these T2D-associated changes in the gut contribute to the etiology of T2D or its comorbidities remains to be seen

Development, characterization, and stability of O/W pepper nanoemulsions produced by high-pressure homogenization

Interest in the utilization of bioactive plant compounds in foods has increased due to their biochemical activities (antioxidant, antimicrobial, etc.), and as alternatives in the reduction of the use of high concentrations of chemical substances. However, some of these additives are hydrophobic, thus being harder to disperse into the food matrix, which is generally water-based. A good alternative is the use of low concentrations of these compounds as nanoemulsions. The objective of the present study was to develop oil-in-water nanoemulsions containing dedo-de-moça pepper extract for food applications. Research in the development of these nanoemulsions was carried out using a high-speed homogenizer, followed by a high-pressure homogenizer. The influence of the following parameters was assessed: type and concentration of surfactants, hidrophilic-lipophilic balance, lipid/aqueous phase ratio, surfactant/oil ratio, pepper extract composition in nanoemulsion, and processing conditions. Nanoemulsions were evaluated by environmental (centrifugal and thermal) and storage stabilities, characterized by average droplet size and -potential measurements, color, interfacial tension, atomic force, and cryo-scanning electron microscopy. Those with average droplet size between 132 ± 2.0 and 145 ± 1.0 nm were developed depending on working pressure and number of cycles; -potential was around 36.71 ± 0.62 mV and the best nanoemulsion was stable to centrifugation and most of the thermal stresses. Droplets were characterized with cryo-scanning electron microscopy as being spherical, homogeneous, and stable, and remained stable when stored at 4 °C and room temperature for over 120 days. The pepper nanoemulsion, developed in the present study, has potential applications in the food industry.The first author gratefully acknowledges the CNPq and CAPES (National Council for Scientific and Technological Development, Program Science without Boarder) for the BSWE^ PhD (Process 236877/2012-1) fellowship, and CAPES for the national PhD fellowship. The last author acknowledges the São Paulo Research Foundation (FAPESP) Brazil, for the grant (CEPID-FoRC, 2013/07914-8).info:eu-repo/semantics/publishedVersio

Expresión de interleucina -10 humana recombinante en Bifidobacterium longum y Escherichia coli.

Tesis (Doctorado en Ciencias en Biología Molecular)."Bifidobacterium longum es una bacteria Gram positiva no patógena que forma parte de un grupo de microorganismos conocidos como probióticos. Recientemente, el interés en esta bacteria ha aumentado considerablemente no solo por sus cualidades probióticas, ya que también se ha considerado como un sistema de producción de proteínas recombinantes y como vehículo para dirigir pro-fármacos hacia superficies tumorales. La bacteria Gram-negativa Escherichia coli, es el microorganismo más comúnmente empleado en la expresión de proteínas recombinantes a nivel industrial, sin embargo su sistema de secreción de proteínas hacia medio extracelular es más complejo y son pocas las proteínas que se exportan hasta el medio de cultivo. El objetivo de este trabajo fue el diseño y construcción de un plásmido estable y funcional en Bifidobacterium longum para la expresión de Interleucina-10 humana, proteína que se ha propuesto para el tratamiento de enfermedades de carácter inflamatorio. El gen IL-10 se optimizó en paralelo para su expresión en B. longum y en E. coli, y se fusionó al péptido señal BIF3 de la β galactosidasa de B. bifidum. El vector de expresión obtenido se denominó pLR y se utilizó para transformar células de B. longum y de E. coli. La presencia de hIL-10 en medio de cultivo y células se confirmó por Western blot y se cuantificó por ELISA. El vector pLR fue funcional en ambas cepas y el péptido BIF3 resultó eficiente para la secreción de hIL-10 en E. coli.""Bifidobacterium longum is a Gram-positive, non-pathogenic bacteria considered as probiotic. In last years the importance of Bifidobacterium spp has grown for their probiotic properties and recently has been proposed as expression system for recombinant proteins and for delivery pro-drugs target to tumor surfaces as well. Escherichia coli, is a Gram-negative bacteria frequently used for recombinant protein expression. Nevertheless, the secretory system for proteins in Gramnegative bacteria is complex and there are only few proteins exported at extracellular medium. The main objective of this work was the design and construction of a stable and functional vector for Bifidobacterium longum for the expression of human interleukine-10, which is considered as therapeutic treatment of inflammatory diseases. The IL-10 gene was optimized for both, Bifidobacterium longum and Escherichia coli, and was fused to the signal peptide BIF3 from a β- galactosidase exported by Bifidobacterium bifidum. The novel expression vector was named pLR and was used to transform Bifidobacterium longum ATCC 15707 and Escherichia coli strains. hIL-10 was confirmed by Western blot and ELISA assays. pLR vector was functional in both strains and BIF3 led the secretion of hIL- 10 in Escherichia coli.

Chemical and Pharmacological Aspects of Capsaicin

Capsaicin is a unique alkaloid found primarily in the fruit of the Capsicum genus and is what provides its spicy flavor. Generally extracted directly from fruit, high demand has driven the use of established methods to increase production through extraction and characterization. Over time these methods have improved, usually be applying existing techniques in conjunction. An increasingly wide range of potential applications has increased interest in capsaicin. Especially compelling are the promising results of medical studies showing possible beneficial effects in many diseases. Capsaicin’s pungency has limited its use in clinical trials to support its biological activity. Characterization and extraction/ synthesis of non-pungent analogues is in progress. A review is made of capsaicin research focusing mainly on its production, synthesis, characterization and pharmacology, including some of its main potential clinical uses in humans

PLR plasmid for expressing recombinant proteins in bacteria of the genus bifidobacterium

"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.