Plasmid DNA/RNA separation by ultrafiltration: modeling and application study

RNA is one of the main soluble contaminants that needs to be separated from plasmid DNA (pDNA) during its recovery process from fermentation broths. Although significantly different in molecular size, pDNA and RNA are difficult to separate by membrane filtration due to the fact that both types of molecules are highly deformable and therefore suffer flow-induced elongation when permeating through porous membranes. The possibility of performing this separation by ultrafiltration is investigated here with the aid of a theoretical model describing the interactions between two electrically charged, flexible macromolecules that simultaneously permeate through a porous membrane. The results of the simulations, applied to pDNA and the different types of RNA present in cell lysates under typical process conditions, show that only by a careful choice of the membrane pore size and the imposed permeate flux one can achieve the required selectivity in this operation. Ultrafiltration tests using microfiltered lysates from the production of two different plasmids, pVAX1-LacZ (6050 bp) and pCAMBIA-1303 (12,361 bp), were carried out to check the validity of the theoretical predictions; the experimental results confirm these predictions and the idea that this technique can be used in practice for pDNA purification.Fundação para a Ciência e a Tecnologia (FCT

Is the Banana Ripe? Andean Bear–Human Conflict in a Protected Area of Colombia

The Andean bear (Tremarctos ornatus; bear) is endemic to the tropical Andes Mountains of South America. Previous assessments predict that bear populations will decline by \u3e30% in the next 30 years. The species may face the greatest threats within its historical distribution in Colombia where rapid agricultural expansion into natural habitats is increasing human–bear conflicts. Between April 2017 and March 2018, we studied bear feeding behavior on plantain (Musa sapientum) and banana (M. paradisiaca) crops within the Barbas-Bremen protected area in the central mountain range of Colombia to describe the magnitude of crop damage, economic losses, and spatial distribution of feeding sites where human–bear conflicts would most likely occur. We also identified all affected farmers and used structured interviews to determine their attitudes toward the bears and their conservation. We recorded 237 damaged plants and identified 57 bear feeding area locations on 9 farms. Bear damage consisted of bites to the trunk of each plant and consumption of the centers. The damage polygon covered 198 ha, and it was located in the northwestern portion of the protected area. Although we estimated that the magnitude of crop consumption by bears and social and economic dimensions of damage caused by the species in Colombia. Our research also provides insights on how human–bear conflicts may be mitigated in the study area

Plasmid DNA recovery from fermentation broths by a combined process of micro- and ultrafiltration : modeling and application

Microfiltration and ultrafiltration operations were used in tandem to isolate and purify a 6050 bp plasmid DNA (pDNA). To achieve primary isolation of the plasmid from fermentation broths, immediately after cell lysis, a 0.2 μm microfiltration membrane was selected for solid/liquid separation, which was performed in a diafiltration mode, as an alternative to centrifugation. Then to attain plasmid concentration and purification, an ultrafiltration membrane with a pore radius of 4.1 nm was selected. Permeation of pDNA and RNA in the two membrane steps was modeled using recently published mass transfer models applicable to the permeation of closed segmented chains and freely-jointed chains, respectively. The permeation of proteins and genomic DNA (gDNA) was also studied in these operations. The microfiltration operation allowed high plasmid and RNA permeation, as expected. It was observed that significant amounts of gDNA, previously precipitated during the cell lysis step, reappear in solution during the diafiltration step. The effect of the ionic strength on this apparent re-solubilization was investigated, by testing the addition of two different salts to the diafiltration buffer: CH3COOK and CaCl2. The results show that these salts can be used to control gDNA apparent re-solubilization. During the ultrafiltration operation high plasmid retention with low adsorption was obtained under low ionic strength conditions. The results also show that a significant removal proteins and the re-solubilized gDNA is achieved, as well as some purification in respect to low molecular weight RNA, since all these components permeate through the ultrafiltration membrane

Electrochemical Oxidation Pathways of Hydroxycoumarins on Carbon Electrodes Examined by LSCV and LC–MS/MS

It is reported the voltammetric behavior and the identification of the oxidation products of four different hydroxycoumarins, namely, 3-, 4-, 6- and 7-hydroxycoumarin (3HC, 4HC, 6HC and 7HC), on carbon electrodes by performing bulk electrolyses in buffered solutions. LC–MS/MS in high resolution mode is used for the product identification. It was found that the voltammetric behaviours of the four compounds are quite different, as well as the corresponding oxidation products. For 3HC the end product was 2 hydroxymandelic acid [2-hydroxy-2-(2-hydroxyphenyl)acetic acid]: for 4HC, salicylic acid [2 hydroxybenzoic acid]; for 6HC, 2H-1-benzopyran-2,6(8aH)-dione and for 7HC no low-molecular weight products were identified. Based on these products and on the voltammetric results, the overall oxidation pathways are formulated as four-electron processes for 3HC and 4HC, and two-electron processes for 6HC. 7HC oxidation is proposed to be an electropolymerization reaction.Ministerio de Economía y Competitividad (research project CTQ-2015-68813R)Departamento de Ingeniería Química, Química Física y Ciencias de los Materiale

Análisis de la investigación científica de los Congresos Internacionales de Ingeniería de Proyectos de AEIPRO.

El artículo presenta la actividad científica desarrollada en los congresos internacionales de ingeniería de proyectos organizados por AEIPRO. Analizando y visualizando la información a través del análisis de dominios científicos y del análisis de redes de la literatura científica desarrollada desde el II Congreso Internacional del 1998 hasta el XVI Congreso Internacional del 2012. Los resultados permiten identificar los frentes de investigación y la base de conocimientos científica en Ingeniería de Proyectos desarrollada en los congresos internacionales de AEIPRO, proporcionando resultados estadísticos sobre la distribución del aporte internacional, el grado de integración de la investigación y la colaboración científica entre universidades, instituciones científicas y profesionales. Finalmente, se realiza una comparación entre la distribución de la investigación según la temática actual de los congresos y las áreas de conocimientos que gestionan el ciclo de vida del proyecto, alcance, tiempo, costes, calidad, recursos humanos, comunicación, riesgos y adquisiciones

Thalamic innervation of the direct and indirect basal ganglia pathways in the rat: Ipsi- and contralateral projections

The present study describes the thalamic innervation coming from the rat parafascicular nucleus (PF) onto striatal and subthalamic efferent neurons projecting either to the globus pallidus (GP) or to the substantia nigra pars reticulata (SNr) by using a protocol for multiple neuroanatomical tracing. Both striatofugal neurons targeting the ipsilateral SNr (direct pathway) as well as striatal efferent neurons projecting to the ipsilateral GP (indirect pathway) were located within the terminal fields of the thalamostriatal afferents. In the subthalamic nucleus (STN), both neurons projecting to ipsilateral GP as well as neurons projecting to ipsilateral SNr also appear to receive thalamic afferents. Although the projections linking the caudal intralaminar nuclei with the ipsilateral striatum and STN are far more prominent, we also noticed that thalamic axons could gain access to the contralateral STN. Furthermore, a small number of STN neurons were seen to project to both the contralateral GP and PF nuclei. These ipsi- and contralateral projections enable the caudal intralaminar nuclei to modulate the activity of both the direct and the indirect pathway

Estudio de insecticidas para el control de mosca blanca Bemisia tabaci Genn enn el cultivo del frijol Phaseolus vulgaris L.

Se evaluaron nuevos plaguicidas para reducir las poblaciones de la mosca blanca (Bemisia tabaci Genn), vector del Mosaico Dorado del frijol (BGMV), en el Valle de Zapotitán y la Estación Experimental de San Andrés a 460 mnsm, durante la época seca a partir del 22 de diciembre de 1989 a mayo de 1990. Las variedades de frijol utilizadas fueron Rojo de Seda, Sangre de toro y CENTACuscatleco. Se utilizó un diseño experimental de bloques al azar, con 4 y 5 repeticiones, y un total de 4 ensayos. Se encontró en el primer y segundo ensayo que bifentrin 100 EC (60 g.i.a./ha) y fenpropatrin 375 FC (203 g.i.a./ha) fueron eficaces para el control de la mosca blanca, y redujeron la incidencia del mosaico dorado. Cuando B. tabaci no se controló eficazmente la incidencia de BGMV alcanzó un 100% entre los 36 y 32 días después de la siembra, en las variedades susceptibles Rojo de Seda y Sangre de Toro. En los ensayos tres y cuatro se utilizaron además tratadores de semilla y suelo combinados con aspersiones al follaje. Los mejores rendimientos se produjeron con los tratamientos de carbosulfan + bifentrin (1519,9 kg/ha), acephato + bifentrin (1428 kg/ha) en la variedad resistente CENIA Cuscatleco, acephato + fenpropatrin (756,6 kg/ha), acephato + bifentrin (725,1 kg/ha) y carbofuran + fenpropatrin (636,3 g/ha) en la variedad susceptible Rojo de Seda. De los productos evaluables, fenpropatrin 375 EC, fue el que mostró las poblaciones más bajas de huevos y ninfas de B. tabaci. Existió una correlación significativa entre la época de aparición de los síntomas de la enfermedad BGMV y la producción. El tratamiento acephato 95% + bifentrin 100 EC el mayor beneficio neto seguido por carbofuran 5% g + bifentrin 100 EC

Genetic studies in sunflower broomrape

Much research has been conducted to identify sources of genetic resistance to sunflower broomrape (Orobanche cumana Wallr.) and to study their mode of inheritance. However, studies on the parasite have been scarce. This manuscript reviews three genetic studies in sunflower broomrape. First, the inheritance of the absence of pigmentation in a natural mutant of this species with yellow plant color phenotype was studied. In a first stage, lines from the unpigmented mutant and a normally pigmented population were developed by several generations of self-pollination. Plants of both lines were crossed and the F1, F2, and F3 generations were evaluated. The results indicated that plant pigmentation is controlled by a partially dominant allele at a single locus. Second, the unpigmented mutant was used to evaluate outcrossing potential of the species. Two experiments in which single unpigmented plants were surrounded by normally pigmented plants were conducted under pot and field conditions. The cross-fertilization rate was estimated as the percentage of F1 hybrids in the progenies of unpigmented plants, which averaged 21.5% in the pot and 28.8% in the field experiment. The results indicated that, under the conditions of this study, the species was not strictly self-pollinated. Finally, the inheritance of avirulence was studied in crosses of plants from lines of O. cumana races E and F, developed by several generations of self-pollination. The F1 and F3 generations were evaluated on the differential line P-1380 carrying the race-E resistance gene Or5. The results suggested that race E avirulence and race F virulence on P-1380 are allelic and controlled by a single locus, which confirmed the gene-for-gene theory for the O. cumana-sunflower interaction.The manuscript reviews research partially funded by Fundación Ramón Areces, Madrid. The contribution of Dr. Enrique Quesada Moraga, entomologist from the University of Córdoba, Spain, to taxonomic classification of pollinators is gratefully acknowledged. R. Pineda-Martos was the recipient of a PhD fellowship from the Spanish National Research Council (CSIC) (JAEPre_08_00370)Peer Reviewe

Using integrated wildlife monitoring to prevent future pandemics through one health approach

In the One Health context, Integrated Wildlife Monitoring (IWM) merges wildlife health monitoring (WHM) and host community monitoring to early detect emerging infections, record changes in disease dynamics, and assess the impact of interventions in complex multi-host and multi-pathogen networks. This study reports the deployment and results obtained from a nationwide IWM pilot test in eleven sites representing the habitat diversity of mainland Spain. In each study site, camera-trap networks and sampling of indicator species for antibody and biomarker analysis were used to generate information. The results allowed identifying differences in biodiversity and host community characteristics among the study sites, with a range of 8 to 19 relevant host species per point. The Eurasian wild boar (Sus scrofa) was the most connected and central species of the host communities, becoming a key target indicator species for IWM. A negative relationship between biodiversity and disease risk was detected, with a lower number and prevalence of circulating pathogens in the sites with more species in the community and larger network size. However, this overall trend was modified by specific host-community and environmental factors, such as the relative index of wild boar - red deer interactions or the proximity to urban habitats, suggesting that human-driven imbalances may favour pathogen circulation. The effort of incorporating wildlife population monitoring into the currently applied WHM programs to achieve effective IWM was also evaluated, allowing to identify population monitoring as the most time-consuming component, which should be improved in the future. This first nationwide application of IWM allowed to detect drivers and hotspots for disease transmission risk among wildlife, domestic animals, and humans, as well as identifying key target indicator species for monitoring. Moreover, anthropogenic effects such as artificially high wildlife densities and urbanisation were identified as risk factors for disease prevalence and interspecific transmission