With the growing interest in developing biologics for pulmonary delivery, systematic fast screening methods are needed for rapid development of formulations. Due to the labile nature of macromolecules, the development of stable, biologically active formulations with desired aerosol performance imposes several challenges both from a formulation and processing perspective. In this study, spray-freeze-drying was used to develop respirable protein powders. In order to systematically map the selected design space, lysozyme aqueous pre-formulations were prepared based on a constrained mixture design of experiment. The physicochemical properties of the resulting powders were characterized and the effects of formulation factors on aerosol performance and protein stability were systematically screened using a logic flow chart. Our results elucidated several relevant formulation attributes (density, total solid content, protein:sugars ratio) required to achieve a stable lysozyme powder with desirable characteristics for pulmonary delivery. A similar logical fast screening strategy could be used to delineate the appropriate design space for different types of proteins and guide the development of powders with pre-determined aerodynamic properties.
Cuba está ubicada en una zona de países con alta incidencia de dengue. En los últimos 10 años ha sido afectada por varias epidemias, es por ello que se diseñó, implementó y evaluó una estrategia participativa, basada en el enfoque de ecosalud, la cual estuvo dirigida a propiciar acciones intersectoriales en la gestión del ecosistema para disminuir las poblaciones del mosquito Aedes aegypti y prevenir la transmisión de dengue en el municipio Cotorro de Ciudad de La Habana, Cuba. Para el desarrollo de este trabajo se utilizó la metodología de investigación acción participativa. Como resultado del proceso se pudo describir una estrategia que garantiza la participación activa de la comunidad, los sectores y el gobierno en la producción de ecosistemas saludables, se desarrollaron acciones de prevención y control oportunas e integrados que disminuyeron los riesgos para la proliferación del vector y la transmisión local de la enfermedad. Este enfoque permitió el análisis holístico de los problemas, su priorización y la gestión de sus soluciones; la estrategia se sostiene dos años después de concluido el proceso.
SUMMARYRandom amplified polymorphic DNA (RAPD) technique is a simple and reliable method to detect DNA polymorphism. Several factors can affect the amplification profiles, thereby causing false bands and non-reproducibility of assay. In this study, we analyzed the effect of changing the concentration of primer, magnesium chloride, template DNA and Taq DNA polymerase with the objective of determining their optimum concentration for the standardization of RAPD technique for genetic studies of Cuban Triatominae. Reproducible amplification patterns were obtained using 5 pmoL of primer, 2.5 mM of MgCl 2 , 25 ng of template DNA and 2 U of Taq DNA polymerase in 25 µL of the reaction. A panel of five random primers was used to evaluate the genetic variability of T. flavida. Three of these (OPA-1, OPA-2 and OPA-4) generated reproducible and distinguishable fingerprinting patterns of Triatominae. Numerical analysis of 52 RAPD amplified bands generated for all five primers was carried out with unweighted pair group method analysis (UPGMA). Jaccard's Similarity Coefficient data were used to construct a dendrogram. Two groups could be distinguished by RAPD data and these groups coincided with geographic origin, i.e. the populations captured in areas from east and west of Guanahacabibes, Pinar del Río. T. flavida present low interpopulation variability that could result in greater susceptibility to pesticides in control programs. The RAPD protocol and the selected primers are useful for molecular characterization of Cuban Triatominae.
SUMMARYThe Triatominae (Hemiptera:Reduviidae) contains the principal and potential Chagas disease vectors present in Mexico, Central America and South America. Triatoma flavida and T. bruneri are Cuban species. These species are closely related according to morphology and were considered synonyms until 1981, when they were separated on the grounds of external characters of the body and the morphology of male genitalia. The present study seeks to analyze genetic polymorphism of T. flavida and T. bruneri populations using RAPD techniques, and to assess the genetic relationship between these species. Ten random primers were used to evaluate the genetic variability among species using RAPD-PCR. The genetic flow among them was calculated. The dendrogram based on calculated Jaccard distances showed two clearly distinguishable clusters which coincided with the studied species. Within each species, moderate genetic differentiation (Fst 0.05-0.15) and migration rates (N > 1) were found among populations, that reveal gene flow and genetic homogeneity. Between species, the Fst value showed a high genetic differentiation and the migration rate was insufficient to maintain genetic homogeneity, and confirmed the absence of gene flow between them. Our results confirm the genetic variability among T. flavida and T. bruneri species.
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