Aislamiento de núcleos para citometría de flujo en plantas con alto contenido de compuestos mucilaginosos: un ejemplo en el género Viola L. (Violaceae)

Flow cytometry analysis has been widely applied in the determination of nuclear DNA content and ploidy level in many organisms. Despite being the most appropriate method for DNA content measurement, flow cytometry also presents some limitations. A fairly common, but little-studied problem is the effect on measurements of the presence of secondary metabolites. A good example is the genus Viola, which is composed of 525-600 species distributed worldwide. These species have proved to be problematic for flow cytometric analyses due to the release of extremely mucilaginous compounds into the nuclear suspension. In this work, the genome size of 13 species of Viola using flow cytometry are presented for the first time. Despite obtaining histograms with high coefficients of variation, we here present an optimized protocol to remove cytoplasmic compounds, particularly mucilaginous ones, from plant nuclei that pave the way for its application to estimate the genome size of other species exhibiting similar problems. Statistical analyses revealed significant differences between sections Viola and Melanium, and within each section (P < 0.001). Furthermore, statistically significant differences were not detected among samples of the same species.El análisis mediante citometría de flujo ha sido aplicado de modo general para determinar el contenido de ADN nuclear y el nivel de ploidía en muchos organismos. A pesar de ser el método más adecuado para medir la cantidad de ADN, esta técnica también presenta algunas limitaciones. Un problema bastante común, aunque poco estudiado, es el efecto de los metabolitos secundarios en los resultados obtenidos. Un ejemplo respecto a la presencia de estos compuestos se encuentra en el género Viola, compuesto por 525-600 especies distribuidas por todo el mundo. Las especies de este género ya han sido previamente descritas como problemáticas en los análisis de citometría de flujo debido a la presencia de compuestos extremadamente mucilaginosos en las suspensiones de núcleos. En el presente trabajo se analiza por primera vez el tamaño genómico de 13 especies del genero Viola mediante el empleo de citometría de flujo. A pesar de los altos valores mostrados en los coeficientes de variación de los histogramas, se presenta un protocolo optimizado para eliminar compuestos citoplasmáticos, y más concretamente mucilaginosos de las suspensiones nucleares, siendo de aplicación en la estimación del tamaño genómico de plantas con problemas similares. Los análisis estadísticos mostraron diferencias significativas entre las secciones Viola y Melanium, así como dentro de cada sección (P < 0,001). Además, no se encontraron diferencias significativas entre aquellas muestras pertenecientes a la misma especie

A Phase I-II multicenter trial with Avelumab plus autologous dendritic cell vaccine in pre-treated mismatch repair-proficient (MSS) metastatic colorectal cancer patients; GEMCAD 1602 study

[Background]: Immune check-point blockade (ICB) has shown clinical beneft in mismatch repair-defcient/microsatellite instability high metastatic colorectal cancer (mCRC) but not in mismatch repair-profcient/microsatellite stable patients. Cancer vaccines with autologous dendritic cells (ADC) could be a complementary therapeutic approach to ICB as this combination has the potential to achieve synergistic efects. [Methods]: This was a Phase I/II multicentric study with translational sub-studies, to evaluate the safety, pharmacodynamics and anti-tumor efects of Avelumab plus ADC vaccine in heavily pre-treated MSS mCRC patients. Primary objective was to determine the maximum tolerated dose and the efcacy of the combination. The primary end-point was 40% progressionfree survival at 6 months with a 2 Simon Stage. [Results]: A total of 28 patients were screened and 19 pts were included. Combined therapy was safe and well tolerated. An interim analysis (Simon design frst-stage) recommended early termination because only 2/19 (11%) patients were disease free at 6 months. Median PFS was 3.1 months [2.1–5.3 months] and overall survival was 12.2 months [3.2–23.2 months]. Stimulation of immune system was observed in vitro but not clinically. The evaluation of basal RNA-seq noted signifcant changes between pre and post-therapy liver biopsies related to lipid metabolism and transport, infammation and oxidative stress pathways. [Conclusions]: The combination of Avelumab plus ADC vaccine is safe and well tolerated but exhibited modest clinical activity. Our study describes, for the frst-time, a de novo post-therapy metabolic rewiring, that could represent novel immunotherapyinduced tumor vulnerabilities.The study was funded by grants from the FIS PI17/00732 from Instituto de Salud Carlos III, Premi Fi de Residència Emili Letang from Hospital Clínic Barcelona, Plan Nacional de I + D (PID-107139RB-C21 to DB-R and PID2020-115051RB-I00 to MC) and Grupo Español Multidisciplinar en Cáncer Digestivo (GEMCAD). The study was funded with Grants from Catalan Agency for Management of University and Research Grants (AGAUR) (2014-SGR-474, 2017-SGR-1174 and 2017-SGR-1033), Fundació la Marató de TV3 (201330.10), Instituto de Salud Carlos III (PI13/01728 and PI19/00740) and Fundacion Olga Torres (Modalitat A. 2019/2020) to JM. IMMETCOLS signature is under patent protection (EP21382772.8.) This research was financially supported by GEMCAD and (OR Avelumab was provided) by Merck, S.L.U., Madrid, Spain, an affiliate of Merck KGaA, Darmstadt, Germany, as part of an alliance between the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: https://doi.org/10.13039/100009945) and Pfizer

Intensification of Antiretroviral Therapy with a CCR5 Antagonist in Patients with Chronic HIV-1 Infection: Effect on T Cells Latently Infected

Objective: The primary objective was to assess the effect of MVC intensification on latently infected CD4+ T cells in chronically HIV-1-infected patients receiving antiretroviral therapy. Methods: We performed an open-label pilot phase II clinical trial involving chronically HIV-1-infected patients receiving stable antiretroviral therapy whose regimen was intensified with 48 weeks of maraviroc therapy. We analyzed the latent reservoir, the residual viremia and episomal 2LTR DNA to examine the relationship between these measures and the HIV-1 latent reservoir, immune activation, lymphocyte subsets (including effector and central memory T cells), and markers associated with bacterial translocation. Results: Overall a non significant reduction in the size of the latent reservoir was found (p = 0.068). A mean reduction of 1.82 IUPM was observed in 4 patients with detectable latent reservoir at baseline after 48 weeks of intensification. No effect on plasma residual viremia was observed. Unexpectedly, all the patients had detectable 2LTR DNA circles at week 24, while none of them showed those circles at the end of the study. No changes were detected in CD4+ or CD8+ counts, although a significant decrease was found in the proportion of HLA-DR+/CD38+ CD4+ and CD8+ T-cells. LPS and sCD14 levels increased. Conclusions: Intensification with MVC was associated with a trend to a decrease in the size of the latent HIV-1 reservoir in memory T cells. No impact on residual viremia was detected. Additional studies with larger samples are needed to confirm the results

Red “Universidad, género, docencia e igualdad”

La Red de investigación en docencia universitaria “Universidad, docencia, genero e igualdad” persigue avanzar en la calidad e innovación de las enseñanzas universitarias a partir de la inclusión de la perspectiva de género. Se busca dar cumplimiento a las directrices generales de los nuevos planes de estudio respecto del principio de igualdad de oportunidades entre hombres y mujeres en la formación universitaria (Real Decreto 1393/2007. BOE nº 260, 30 de octubre de 2007). En la quinta edición de la Red, y dada su composición multidisciplinar, se ha trabajado en tres líneas de investigación: 1) mantenimiento del “Portal web con recursos docentes con perspectiva de género”, proyecto financiado por el Instituto de la Mujer (PACUI, 2012) e iniciado en el curso 2012-2013; 2) desarrollo de la primera versión de “iLengUA”, una herramienta informática para un discurso inclusivo e igualitario; y 3) diseño de una Guía de recomendaciones para la inclusión de la perspectiva de género en la docencia universitaria

Spatiotemporal Characteristics of the Largest HIV-1 CRF02_AG Outbreak in Spain: Evidence for Onward Transmissions

Background and Aim: The circulating recombinant form 02_AG (CRF02_AG) is the predominant clade among the human immunodeficiency virus type-1 (HIV-1) non-Bs with a prevalence of 5.97% (95% Confidence Interval-CI: 5.41–6.57%) across Spain. Our aim was to estimate the levels of regional clustering for CRF02_AG and the spatiotemporal characteristics of the largest CRF02_AG subepidemic in Spain.Methods: We studied 396 CRF02_AG sequences obtained from HIV-1 diagnosed patients during 2000–2014 from 10 autonomous communities of Spain. Phylogenetic analysis was performed on the 391 CRF02_AG sequences along with all globally sampled CRF02_AG sequences (N = 3,302) as references. Phylodynamic and phylogeographic analysis was performed to the largest CRF02_AG monophyletic cluster by a Bayesian method in BEAST v1.8.0 and by reconstructing ancestral states using the criterion of parsimony in Mesquite v3.4, respectively.Results: The HIV-1 CRF02_AG prevalence differed across Spanish autonomous communities we sampled from (p &lt; 0.001). Phylogenetic analysis revealed that 52.7% of the CRF02_AG sequences formed 56 monophyletic clusters, with a range of 2–79 sequences. The CRF02_AG regional dispersal differed across Spain (p = 0.003), as suggested by monophyletic clustering. For the largest monophyletic cluster (subepidemic) (N = 79), 49.4% of the clustered sequences originated from Madrid, while most sequences (51.9%) had been obtained from men having sex with men (MSM). Molecular clock analysis suggested that the origin (tMRCA) of the CRF02_AG subepidemic was in 2002 (median estimate; 95% Highest Posterior Density-HPD interval: 1999–2004). Additionally, we found significant clustering within the CRF02_AG subepidemic according to the ethnic origin.Conclusion: CRF02_AG has been introduced as a result of multiple introductions in Spain, following regional dispersal in several cases. We showed that CRF02_AG transmissions were mostly due to regional dispersal in Spain. The hot-spot for the largest CRF02_AG regional subepidemic in Spain was in Madrid associated with MSM transmission risk group. The existence of subepidemics suggest that several spillovers occurred from Madrid to other areas. CRF02_AG sequences from Hispanics were clustered in a separate subclade suggesting no linkage between the local and Hispanic subepidemics