Caracterización del patrón de isoformas de splicing de SMS1 en la proliferación tumorigénica

[spa] La esfingomielina sintasa (SGMS) cataliza la transferencia reversible de un residuo de fosforilcolina de la fosfatidilcolina al hidroxilo primario de la ceramida dando lugar a la producción de la esfingomielina y diacilglicerol (DAG). Se ha establecido una posible relación entre la acción de la SGMS y la proliferación tumorogénica. La actividad de la SGMS puede servir para regular los niveles celulares de ceramida, diacilglicerol y esfingomielina, que son importantes en la dinámica celular. Existen dos isoformas de SGMS en mamíferos (SGMS1 y SGMS2), las cuales presentan seis dominios transmembrana, y cuyo sitio catalítico está constituido por tres residuos en dos de los bucles comprendidos entre los dominios transmembrana. Además, se ha visto que, en humanos, para la SGMS1, existen diferentes isoformas de splicing es decir cuyo patrón de exones (tanto codificantes como no traducidos -5’- y 3’-UTR) incluye diferentes combinaciones. La proporción de estas isoformas en niveles de mRNA es característica de cada tejido estudiado. Sin embargo, no se ha explorado la presencia de estas isoformas en las células tumorales. Recientemente, se están desarrollando fármacos para el tratamiento del cáncer desde un nuevo enfoque denominado Terapia Lipídica de Membrana (TLM). Esta terapia se basa en la modificación de los componentes y la estructura de la membrana que permiten un cambio en las cascadas de señalización celulares y por tanto dan lugar a un cambio en el comportamiento celular. Este enfoque ha conducido al diseño del agente antitumoral ácido 2-hidroxioleico (2OHOA). En este trabajo se han utilizado muestras de cDNA procedentes de líneas tumorales de distinto origen (glioma, colon pulmón y mama), tratadas o no (controles) con 2OHOA, que fueron analizadas mediante técnicas de PCR (convencional y a tiempo real) y electroforesis en geles de agarosa y capilar. De esta manera se ha: (1) explorado la presencia de las diferentes isoformas de SGMS-1 y -2 (si las había) en líneas tumorales de distinto origen (glioma, colon, pulmón y mama); y (2) determinado si dicho patrón de isoformas es relevante en la respuesta al tratamiento con 2OHOA.[eng] Sphingomyelin synthase catalyses the reversible transfer of a phosphorylcholine residue from phosphatidylcholine to ceramide producing sphingomyelin and diacylglycerol. It has been established a possible connection between the action of SGMS and tumorigenic proliferation. SGMS activity may serve to regulate the cellular levels of ceramide, diacylglycerol and sphingomyelin, which are important in cell dynamics. There are two isoforms of SGMS in mammals (SGMS1 and SGMS2), which have six transmembrane domains, and whose catalytic site consists of three residues in two of the loops comprised between the transmembrane domains. In addition, different splicing isoforms have been found in humans for SGMS1, that is to say, the pattern of exons (both coding and untranslated -5'- and 3'-UTR) includes different combinations. The proportion of these isoforms at mRNA levels is characteristic of each tissue studied. However, the presence of these isoforms in tumor cells has not been explored. Recently, drugs for the treatment of cancer have been developed from a new approach called Membrane Lipid Therapy (TLM). The basis of this therapy is the modification of cell membrane components and structure allowing a change in the cellular signaling cascades which results in a change in cellular behavior. This approach has led to the design of the antitumor agent 2-hydroxyoleic acid (2OHOA). In this work, cDNA samples from different tumor cell lines (glioma, colon, lung and breast), treated or not (controls) with 2OHOA, were analyzed using PCR (conventional and real time) and electrophoresis, both capillary and in agarose gels. In this way, (1) has been explored the presence of different SGMS- 1 and -2 isoforms (if present) in tumor cell lines of different origin (glioma, colon, lung and breast); and (2) determined if this isoform pattern is relevant in the response to treatment with 2OHOA

Ca2+-modulated photoactivatable imaging reveals neuron-astrocyte glutamatergic circuitries within the nucleus accumbens

We thank Alfonso Araque, Eduardo Martín, Juliana M Rosa, and Gertrudis Perea for expert advice and critical reading of the manuscript. This work was supported by grants from the Spanish Ministry of Science and Innovation (Ramón y Cajal RYC-2016-20414, RTI2018-094887-B-I00, and PID2021-122586NB-I00) to M.N. and Fondo Europeo de Desarrollo Regional (FEDER) and PID2020-115091RB-10 to R.T. The professional editing service NB Revisions was used for technical preparation of the text prior to submission

Absence of p.R50X Pygm read-through in McArdle disease cellular models

International audienceMcArdle disease is an autosomal recessive disorder caused by the absence of muscle glycogen phosphorylase, which leads to blocked muscle glycogen breakdown. We used three different cellular models to evaluate the efficiency of different read-through agents (including amlexanox, Ataluren, RTC13 and G418) in McArdle disease. The first model consisted of HeLa cells transfected with two different GFP-PYGM constructs presenting the Pygm p.R50X mutation (GFP-PYGM p.R50X and PYGM Ex1-GFP p.R50X). The second cellular model was based on the creation of HEK293T cell lines stably expressing the PYGM Ex1-GFP p.R50X construct. As these plasmids encode murine Pygm cDNA without any intron sequence, their transfection in cells would allow for analysis of the efficacy of read-through agents with no concomitant nonsense-mediated decay interference. The third model consisted of skeletal muscle cultures derived from the McArdle mouse model (knock-in for the p.R50X mutation in the Pygm gene). We found no evidence of read-through at detectable levels in any of the models evaluated. We performed a literature search and compared the premature termination codon context sequences with reported positive and negative read-through induction, identifying a potential role for nucleotide positions -9, -8, -3, -2, +13 and +14 (the first nucleotide of the stop codon is assigned as +1). The Pygm p.R50X mutation presents TGA as a stop codon, G nucleotides at positions -1 and -9, and a C nucleotide at -3, which potentially generate a good context for read-through induction, counteracted by the presence of C at -2 and its absence at +4

Subcutaneous anti-COVID-19 hyperimmune immunoglobulin for prevention of disease in asymptomatic individuals with SARS-CoV-2 infection: a double-blind, placebo-controlled, randomised clinical trialResearch in context

Summary: Background: Anti-COVID-19 hyperimmune immunoglobulin (hIG) can provide standardized and controlled antibody content. Data from controlled clinical trials using hIG for the prevention or treatment of COVID-19 outpatients have not been reported. We assessed the safety and efficacy of subcutaneous anti-COVID-19 hyperimmune immunoglobulin 20% (C19-IG20%) compared to placebo in preventing development of symptomatic COVID-19 in asymptomatic individuals with SARS-CoV-2 infection. Methods: We did a multicentre, randomized, double-blind, placebo-controlled trial, in asymptomatic unvaccinated adults (≥18 years of age) with confirmed SARS-CoV-2 infection within 5 days between April 28 and December 27, 2021. Participants were randomly assigned (1:1:1) to receive a blinded subcutaneous infusion of 10 mL with 1 g or 2 g of C19-IG20%, or an equivalent volume of saline as placebo. The primary endpoint was the proportion of participants who remained asymptomatic through day 14 after infusion. Secondary endpoints included the proportion of individuals who required oxygen supplementation, any medically attended visit, hospitalisation, or ICU, and viral load reduction and viral clearance in nasopharyngeal swabs. Safety was assessed as the proportion of patients with adverse events. The trial was terminated early due to a lack of potential benefit in the target population in a planned interim analysis conducted in December 2021. ClinicalTrials.gov registry: NCT04847141. Findings: 461 individuals (mean age 39.6 years [SD 12.8]) were randomized and received the intervention within a mean of 3.1 (SD 1.27) days from a positive SARS-CoV-2 test. In the prespecified modified intention-to-treat analysis that included only participants who received a subcutaneous infusion, the primary outcome occurred in 59.9% (91/152) of participants receiving 1 g C19-IG20%, 64.7% (99/153) receiving 2 g, and 63.5% (99/156) receiving placebo (difference in proportions 1 g C19-IG20% vs. placebo, −3.6%; 95% CI -14.6% to 7.3%, p = 0.53; 2 g C19-IG20% vs placebo, 1.1%; −9.6% to 11.9%, p = 0.85). None of the secondary clinical efficacy endpoints or virological endpoints were significantly different between study groups. Adverse event rate was similar between groups, and no severe or life-threatening adverse events related to investigational product infusion were reported. Interpretation: Our findings suggested that administration of subcutaneous human hyperimmune immunoglobulin C19-IG20% to asymptomatic individuals with SARS-CoV-2 infection was safe but did not prevent development of symptomatic COVID-19. Funding: Grifols