Estabilidad e interacciones del dominio de unión del receptor de LDL

El receptor de lipoproteínas de baja densidad (rLDL) es una proteína de membrana que juega un papel crucial en el mantenimiento de concentraciones adecuadas de colesterol en el organismo. Su disfunción se asocia con la aparición de Hipercolesterolemia Familiar (HF), una enfermedad común caracterizada por altos niveles de colesterol en los vasos sanguíneos. El mecanismo por el cual el receptor lleva a cabo la internalización de las lipoproteínas a las células y su posterior liberación en los endosomas sigue siendo hoy en día de gran interés para entender en segundo lugar disfunciones del receptor asociadas a HF. Entre los distintos dominios del receptor, se encuentra el dominio de unión a ligando, compuesto a su vez por 7 repeticiones homólogas de unos 40 aminoácidos, conocidas como módulos LR (1-7) de los cuales los números 4 y 5 son los directamente implicados en la unión con las lipoproteínas. Todos ellos requieren la presencia de Ca2+ para adoptar una conformación estable y mantener a las LDL unidas. En este trabajo se expone un estudio de las propiedades del módulo 4 y el tándem 4-5 con objeto de estudiar sus diferencias conformacionales en presencia y ausencia de Ca2+ y Mg2+ tanto en condiciones extracelulares (pH 7) como endosomales (pH 5.5), donde se produce la liberación. Entre los estudios llevados a cabo en este trabajo, se encuentran en su mayoría ensayos cinéticos de asociación y disociación, realizado mediante el sistema de flujo detenido, de los módulos LR4 y LR5 con Ca2+y Mg2+en ambas condiciones que en algunos casos se complementan con otras técnicas termodinámicas. Por otro lado, en el trabajo también se lleva a cabo la purificación y una caracterización espectroscópica preliminar del dominio de unión a ligando completo LR (1-7)

Complete sequence and genomic annotation of carrot torradovirus 1

Carrot torradovirus 1 (CaTV1) is a new member of the genus Torradovirus within the family Secoviridae. CaTV1 genome sequences were obtained from a previous next-generation sequencing (NGS) study and were compared to other members and tentative new members of the genus. The virus has a bipartite genome, and RACE was used to amplify and sequence each end of RNA1 and RNA2. As a result, RNA1 and RNA2 are estimated to contain 6944 and 4995 nucleotides, respectively, with RNA1 encoding the proteins involved in virus replication, and RNA2 encoding the encapsidation and movement proteins. Sequence comparisons showed that CaTV1 clustered within the non-tomato-infecting torradoviruses and is most similar to motherwort yellow mottle virus (MYMoV). The nucleotide sequence identities of the Pro-Pol and coat protein regions were below the criteria established by the ICTV for demarcating species, confirming that CaTV1 should be classified as a member of a new species within the genus Torradovirus

Detection and transmission of Carrot torrado virus, a novel putative member of the Torradovirus genus

A new Torradovirus tentatively named Carrot torrado virus (CaTV) was an incidental finding following a next generation sequencing study investigating internal vascular necrosis in carrot. The closest related viruses are Lettuce necrotic leaf curl virus (LNLCV) found in the Netherlands in 2011 and Motherwort yellow mottle virus (MYMoV) found in Korea in 2014. Primers for reverse transcriptase-PCR (RT-PCR) and RT-qPCR were designed with the aim of testing for the presence of virus in plant samples collected from the field. Both methods successfully amplified the target from infected samples but not from healthy control samples. The specificity of the CaTV assay was also checked against other known carrot viruses and no cross-reaction was seen. A comparative study between methods showed RT-qPCR was the most reliable method, giving positive results in samples where RT-PCR fails. Evaluation of the Ct values following RT-qPCR and a direct comparison demonstrated this was due to improved sensitivity. The previous published Torradovirus genus specific RT-PCR primers were tested and shown to detect CaTV. Also, virus transmission experiments carried out suggest that unlike other species of the same genus, Carrot torrado virus could be aphid-transmitted

Transcriptome sequencing identifies novel persistent viruses in herbicide resistant wild-grasses

Herbicide resistance in wild grasses is widespread in the UK, with non-target site resistance (NTSR) to multiple chemistries being particularly problematic in weed control. As a complex trait, NTSR is driven by complex evolutionary pressures and the growing awareness of the role of the phytobiome in plant abiotic stress tolerance, led us to sequence the transcriptomes of herbicide resistant and susceptible populations of black-grass and annual rye-grass for the presence of endophytes. Black-grass (Alopecurus myosuroides; Am) populations, displaying no overt disease symptoms, contained three previously undescribed viruses belonging to the Partititiviridae (AMPV1 and AMPV2) and Rhabdoviridae (AMVV1) families. These infections were widespread in UK black-grass populations and evidence was obtained for similar viruses being present in annual rye grass (Lolium rigidum), perennial rye-grass (Lolium perenne) and meadow fescue (Festuca pratensis). In black-grass, while no direct causative link was established linking viral infection to herbicide resistance, transcriptome sequencing showed a high incidence of infection in the NTSR Peldon population. The widespread infection of these weeds by little characterised and persistent viruses and their potential evolutionary role in enhancing plant stress tolerance mechanisms including NTSR warrants further investigation