Contribución al conocimiento de la criptosporidiosis en diferntes regiones de Colombia a partir de aislados procedentes de humanos y diversas especies animales

En el presente trabajo se han investigado algunos aspectos de la epidemiología y el control de la criptosporidiosis en diversas regiones de Colombia. En un primer objetivo se estudió la presencia y diversidad genética de Cryptosporidium en muestras de origen humano y animal y se investigó su relación con diversas variables relacionadas con el hospedador como la edad, presencia de diarrea o procedencia geográfica. Para ello se analizaron muestras fecales de origen humano (663 individuos menores de 19 años), ganado vacuno (432 terneros menores de 35 días procedentes de 74 explotaciones) y ganado porcino (233 cerdos de diferentes fases de crianza procedentes de 19 explotaciones). En todas las muestras se investigó la presencia de ooquistes de Cryptosporidium mediante técnicas microscópicas como la tinción negativa de Heine (muestras de terneros) o la tinción de Ziehl-Neelsen modificada previa concentración por sedimentación (muestras de origen humano y porcino), seleccionando las que resultaron positivas para llevar a cabo su estudio molecular. En las muestras positivas se extrajo en primer lugar el ADN del parásito y se procedió a la amplificación por PCR y posterior análisis (restricción enzimática, secuenciación) de diversos marcadores para identificar la especie (gen SSu-rRNA) y subtipo (gen GP60) de Cryptosporidium. Asimismo, se llevó a cabo un análisis de fragmentos multilocus con cinco microsatélites (ML1, ML2, TP14, 5B12, CP47) y cuatro minisatélites (MSB, MSC6-7, cgd2_3850, cgd6_5400). El estudio molecular se hizo extensivo a los aislados de Giardia identificados en las muestras de origen humano, en los que se identificó la especie y ensamblaje mediante amplificación por PCR y posterior secuenciación de los genes ssu-rRNA, β-giardina y triosa fosfato isomerasa.El análisis microscópico permitió identificar ooquistes de Cryptosporidium en muestras de 115 terneros (26,6%) pertenecientes a 44 explotaciones (59,5%). La infección ha sido diagnosticada en terneros de 3 a 35 días de edad, aunque la prevalencia fue significativamente mayor en los de 8 – 14 días (40,7%). Los terneros menores de 21 días tenían hasta 4,3 veces más posibilidades de estar infectados que los mayores de 21 días, lo cual confirma que el riesgo de estar infectado está estrechamente asociado con la edad. Análogamente, la probabilidad de padecer diarrea se incrementó hasta 7 veces en los terneros parasitados en comparación con los no parasitados. La mayoría de aislados de origen bovino caracterizados a nivel molecular (71/73) fueron identificados como C. parvum y asignados a ocho subtipos de la familia IIa, con claro predominio de una variante no descrita previamente en la bibliografía científica (IIaA18G5R1). El estudio multilocus reveló una diversidad genética moderada (22 subtipos multilocus = SML) y una estructura de poblaciones predominantemente panmíctica. La mayoría de SMLs fueron únicos y distintivos de determinadas explotaciones bovinas. La infección por Cryptosporidium fue mucho menos prevalente en ganado porcino, puesto que únicamente se identificaron ooquistes en 15 animales (6,4%) de 7 explotaciones, aunque este hallazgo constituye la primera denuncia de este protozoo en cerdos en Colombia. La infección fue más frecuente en animales en la etapa de precebo (21-63 días) (14,9%) y no estuvo asociada estadísticamente con la presencia de diarrea. El análisis molecular únicamente proporcionó un resultado positivo en dos aislados que fueron identificados como C. parvum. El estudio coprológico en población humana confirmó la infección por Cryptosporidium en 42 personas (6,3%), siendo más frecuente en niños procedentes de colegios (10,7%) que en muestras de origen hospitalario, así como en individuos de procedencia rural (12%) en comparación con los de origen urbano. C. parvum fue la especie identificada en la mayoría aislados correctamente caracterizados (32/34), siendo C. hominis muy poco prevalente. Cabe destacar el alto porcentaje de portadores asintomáticos y la escasa diversidad genética de ambas especies en el locus GP60, ya que todos los aislados fueron asignados al mismo alelo (C. parvum: 351pb; C. hominis: 384 pb). El estudio multilocus fue realizado con base a la combinación del locus GP60 y los dos únicos microsatélites que amplificaron de manera regular en la mayoría de aislados (TP14, MSB) y reveló cinco MLTs, tres en C. parvum y dos en C. hominis. El estudio molecular también se pudo realizar con 20 aislados de Giardia duodenalis de origen humano, que fueron asignados a los ensamblajes/subensamblajes B (n: 11), A (n: 2) y AII (n: 7).En un segundo objetivo del trabajo de investigación se utilizó un modelo experimental de ratones con el fin estudiar la capacidad de diversos péptidos sintéticos de C. parvum para estimular la producción de anticuerpos neutralizantes y valorar su utilidad como candidatos para una vacuna frente a la criptosporidiosis. Para ello se diseñaron in silico y sintetizaron un total de siete péptidos derivados de tres proteínas de C. parvum (P23, CP15 y CSL), que fueron inoculados en diversos grupos de ratones siguiendo un protocolo establecido. La producción de anticuerpos séricos en los ratones inoculados fue analizada mediante una técnica ELISA y su capacidad neutralizante fue investigada mediante amplificación por PCR del extracto de cultivos celulares inoculados con ooquistes incubados con el suero de los diferentes grupos experimentales.Los siete péptidos sintetizados estimularon la producción de anticuerpos en los ratones, especialmente cuando eran inoculados con un inmunoestimulante (FIS) y adyuvante. Las mejores respuestas se observaron en los animales inoculados con cuatro péptidos, dos derivados de la proteína CSL y otros dos derivados de la proteína CP15-1, aunque sólo estos últimos mostraron la capacidad de bloquear la infección de las células por parte de los esporozoítos de C. parvum, por lo que podrían considerarse candidatos para una vacuna frente a la criptosporidiosis. Las lesiones tisulares que se observaron en algunos ratones inoculados fueron consecuencia de la formulación utilizada y no del péptido, por lo que se aconseja utilizar adyuvantes menos nocivos en posteriores investigaciones.<br /

A Novel Electrochemical Sensor for Probing Doxepin Created on a Glassy Carbon Electrode Modified with Poly(4-Amino- benzoic Acid)/Multi-Walled Carbon Nanotubes Composite Film

A novel electrochemical sensor for sensitive detection of doxepin was prepared, which was based on a glassy carbon electrode modified with poly(4-aminobenzoic acid)/multi-walled carbon nanotubes composite film [poly(4-ABA)/MWNTs/GCE]. The sensor was characterized by scanning electron microscopy and electrochemical methods. It was observed that poly(4-ABA)/MWNTs/GCE showed excellent preconcentration function and electrocatalytic activities towards doxepin. Under the selected conditions, the anodic peak current was linear to the logarithm of doxepin concentration in the range from 1.0 × 10−9 to 1.0 × 10−6 M, and the detection limit obtained was 1.0 × 10−10 M. The poly(4-ABA)/MWNTs/GCE was successfully applied in the measurement of doxepin in commercial pharmaceutical formulations, and the analytical accuracy was confirmed by comparison with a conventional ultraviolet spectrophotometry assay

Identification of eight spliceogenic variants in BRCA2 exon 16 by minigene assays

Genetic testing of BRCA1 and BRCA2 identifies a large number of variants of uncertain clinical significance whose functional and clinical interpretations pose a challenge for genetic counseling. Interestingly, a relevant fraction of DNA variants can disrupt the splicing process in cancer susceptibility genes. We have tested more than 200 variants throughout 19 BRCA2 exons mostly by minigene assays, 54% of which displayed aberrant splicing, thus confirming the utility of this assay to check genetic variants in the absence of patient RNA. Our goal was to investigate BRCA2 exon 16 with a view to characterizing spliceogenic variants recorded at the mutational databases. Seventy-two different BIC and UMD variants were analyzed with NNSplice and Human Splicing Finder, 12 of which were selected because they were predicted to disrupt essential splice motifs: canonical splice sites (ss; eight variants) and exonic/intronic splicing enhancers (four variants). These 12 candidate variants were introduced into the BRCA2 minigene with seven exons (14-20) by site-directed mutagenesis and then transfected into MCF-7 cells. Seven variants (six intronic and one missense) induced complete abnormal splicing patterns: c.7618-2A>T, c.7618-2A>G, c.7618-1G>C, c.7618-1G>A, c.7805G>C, c.7805+1G>A, and c.7805+3A>C, as well as a partial anomalous outcome by c.7802A>G. They generated at least 10 different transcripts: Δ16p44 (alternative 3'ss 44-nt downstream; acceptor variants), Δ16 (exon 16-skipping; donor variants), Δ16p55 (alternative 3'ss 55-nt downstream), Δ16q4 (alternative 5'ss 4-nt upstream), Δ16q100 (alternative 5'ss 4-nt upstream), ▾16q20 (alternative 5'ss 20-nt downstream), as well as minor (Δ16p93 and Δ16,17p69) and uncharacterized transcripts of 893 and 954 nucleotides. Isoforms Δ16p44, Δ16, Δ16p55, Δ16q4, Δ16q100, and ▾16q20 introduced premature termination codons which presumably inactivate BRCA2. According to the guidelines the American College of Medical Genetics and Genomics these eight variants could be classified as pathogenic or likely pathogenic whereas the Evidence-based Network for the Interpretation of Germline Mutant Alleles rules suggested seven class 4 and one class 3 variants. In conclusion, our study highlights the relevance of splicing functional assays by hybrid minigenes for the clinical classification of genetic variations. Hence, we provide new data about spliceogenic variants of BRCA2 exon 16 that are directly correlated with breast cancer susceptibility.EV’s lab was supported by grants from the Spanish Ministry of Economy and Competitivity, Plan Nacional de I+D+I 2013–2016, ISCIII (Grants: PI13/01749 and PI17/00227) co-funded by FEDER from European Regional Development Funds (European Union), and grant CSI090U14 from the Consejería de Educación (ORDEN EDU/122/2014) and Junta de Castilla y León. EF-B was supported by a predoctoral fellowship from the University of Valladolid and Banco Santander (2015–2019).We acknowledge the support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Electroanalytical behaviour of a nanoarray self-assembled thiocholesterol gold electrode

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Treatment of Activated Carbon with HO. Effect on the Porous Texture

The effect of the oxidation of activated carbon (AC) with aqueous solutions of H 2 O 2 on the porous texture of the material has been investigated. In such treatments of AC, H 2 O 2 solutions of varying concentration and pH (i.e., unchanged pH and pH values of 2.5 and 11.5) were used. Oxidizing solutions containing Fe 2+ , Fe 3+ , ethanol or ether were also employed. Initial outgassing of AC and contact of the intermediate product with the oxidizing solution were effected under controlled conditions of temperature and time. The resulting samples were characterized texturally by gas adsorption (N 2 , 77 K) and mercury porosimetry. The mass of sample significantly increased when H 2 O 2 solutions at pH 2.5 were used. Such treatment of AC produced a loss of microporosity or none depending on the method of preparation of the samples. Thus, a loss of microporosity occurred, for example, when contact between the H 2 O 2 solution and the AC was effected at 0°C, whereas no loss of microporosity occurred when the temperature was 70°C. For a few samples the unfavourable effect of oxidation of the AC on its porous texture not only concerned the micropores but also the mesopores and macropores

Classification of 15 new BRCA2 exons2-9 splicing variants by hybrid minigenes

Trabajo presentado a la European Human Genetics (ESHG) Conference, celebrada en Milan (Italia) del 16 al 19 de junio de 2018.The clinical classification of BRCA2 variants of uncertain significance (VUS) poses a challenge in human genetics. Historically, variants have been catalogued from the protein outlook, however, upstream gene-expression mechanisms, such as splicing, could be impaired by changes in the DNA sequence. Disrupted splicing variants could alter the ORF provoking the BRCA2 truncation and be involved in cancer development. Here, we have evaluated the splicing effect of 83 BRCA2 exons 2-9 variants by functional assays using the minigene MGBR2_2-9. The MGBR2_2-9 was built based on the splicing vector pSAD (Patent-P201231427, CSIC). In silico selected BRCA2 variants, from BIC and UMD databases, were introduced into MGBR2_2-9 and assayed in MCF-7 cells. Transcripts were analyzed by capillary electrophoresis and sequenced. After the bioinformatic analysis of 302 BRCA2 variants, 83 were functionally tested. Results showed that 53 variants impaired splicing (26 intronic and 27 exonic). Among them, 36 provoked ¿2/3 of aberrant transcripts. According to the ACMG and ENIGMA criteria, our results support the re-classification of 12 variants from VUS to pathogenic (c.67+1G>T, c.67+3A>G, c.426-12del5, c.441A>G, c.451G>A, c.475+3A>T, c.516+4delAA, c.517-1G>A, c.517G>T, c.631+1G>A, c.632+3A>G and c.632-3G>C) and 3 from VUS to likely pathogenic (c.97G>A, c.100G>A, c.476-3C>A). The reproducibility of this technique was supported by previous studies based on minigenes or/and patient RNA. MGBR2_2-9 is a robust tool which provides RNA data for the clinical interpretation of splicing variants.Projects FIS PI13/01749 and PI17/00227 (ISCIII, Spanish Ministry of Economy and Competitivity), BIO/VA34/15 (Junta Castilla-León); EF-B is supported by a predoctoral fellowship (University of Valladolid/Banco Santander).Peer reviewe

Novel combination of non-aqueous capillary electrophoresis and multivariate curve resolution-alternating least squares to determine phenolic acids in virgin olive oil

This paper presents the development of a non-aqueous capillary electrophoresis method coupled to UV detection combined with multivariate curve resolution-alternating least-squares (MCR-ALS) to carry out the resolution and quantitation of a mixture of six phenolic acids in virgin olive oil samples. p-Coumaric, caffeic, ferulic, 3,4-dihydroxyphenylacetic, vanillic and 4-hydroxyphenilacetic acids have been the analytes under study. All of them present different absorption spectra and overlapped time profiles with the olive oil matrix interferences and between them. The modeling strategy involves the building of a single MCR-ALS model composed of matrices augmented in the temporal mode, namely spectra remain invariant while time profiles may change from sample to sample. So MCR-ALS was used to cope with the coeluting interferences, on accounting the second order advantage inherent to this algorithm which, in addition, is able to handle data sets deviating from trilinearity, like the data herein analyzed. The method was firstly applied to resolve standard mixtures of the analytes randomly prepared in 1-propanol and, secondly, in real virgin olive oil samples, getting recovery values near to 100% in all cases. The importance and novelty of this methodology relies on the combination of non-aqueous capillary electrophoresis second-order data and MCR-ALS algorithm which allows performing the resolution of these compounds simplifying the previous sample pretreatment stages.Fil: Godoy Caballero, María del Pilar. Universidad de Extremadura; EspañaFil: Culzoni, Maria Julia. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe; ArgentinaFil: Galeano Diaz, Teresa. Universidad de Extremadura; EspañaFil: Acedo Valenzuela, María Isabel. Universidad de Extremadura; Españ

A global assessment of primate responses to landscape structure

Land-use change modifies the spatial structure of terrestrial landscapes, potentially shaping the distribution, abundance and diversity of remaining species assemblages. Non-human primates can be particularly vulnerable to landscape disturbances, but our understanding of this topic is far from complete. Here we reviewed all available studies on primates' responses to landscape structure. We found 34 studies of 71 primate species (24 genera and 10 families) that used a landscape approach. Most studies (82%) were from Neotropical forests, with howler monkeys being the most frequently studied taxon (56% of studies). All studies but one used a site-landscape or a patch-landscape study design, and frequently (34% of studies) measured landscape variables within a given radius from the edge of focal patches. Altogether, the 34 studies reported 188 responses to 17 landscape-scale metrics. However, the majority of the studies (62%) quantified landscape predictors within a single spatial scale, potentially missing significant primate–landscape responses. To assess such responses accurately, landscape metrics need to be measured at the optimal scale, i.e. the spatial extent at which the primate–landscape relationship is strongest (so-called ‘scale of effect’). Only 21% of studies calculated the scale of effect through multiscale approaches. Interestingly, the vast majority of studies that do not assess the scale of effect mainly reported null effects of landscape structure on primates, while most of the studies based on optimal scales found significant responses. These significant responses were primarily to landscape composition variables rather than landscape configuration variables. In particular, primates generally show positive responses to increasing fore