Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography.

Plus-strand RNA viruses are proficient at remodeling host cell membranes for optimal viral genome replication and the production of infectious progeny. These ultrastructural alterations result in the formation of viral membranous organelles and may be observed by different imaging techniques, providing nanometric resolution. Guided by confocal and electron microscopy, this study describes the generation of wide-field volumes using cryogenic soft-X-ray tomography (cryo-SXT) on SARS-CoV-2-infected human lung adenocarcinoma cells. Confocal microscopy showed accumulation of double-stranded RNA (dsRNA) and nucleocapsid (N) protein in compact perinuclear structures, preferentially found around centrosomes at late stages of the infection. Transmission electron microscopy (TEM) showed accumulation of membranous structures in the vicinity of the infected cell nucleus, forming a viral replication organelle containing characteristic double-membrane vesicles and virus-like particles within larger vesicular structures. Cryo-SXT revealed viral replication organelles very similar to those observed by TEM but indicated that the vesicular organelle observed in TEM sections is indeed a vesiculo-tubular network that is enlarged and elongated at late stages of the infection. Overall, our data provide additional insight into the molecular architecture of the SARS-CoV-2 replication organelle.

Differential Roles of Lipin1 and Lipin2 in the Hepatitis C Virus Replication Cycle.

Although their origin, nature and structure are not identical, a common feature of positive-strand RNA viruses is their ability to subvert host lipids and intracellular membranes to generate replication and assembly complexes. Recently, lipin1, a cellular enzyme that converts phosphatidic acid into diacylglycerol, has been implicated in the formation of the membranous web that hosts hepatitis C virus (HCV) replicase. In the liver, lipin1 cooperates with lipin2 to maintain glycerolipid homeostasis. We extended our previous study of the lipin family on HCV infection, by determining the impact of the lipin2 silencing on viral replication. Our data reveal that lipin2 silencing interferes with HCV virion secretion at late stages of the infection, without significantly affecting viral replication or assembly. Moreover, uninfected lipin2-, but not lipin1-deficient cells display alterations in mitochondrial and Golgi apparatus morphology, suggesting that lipin2 contributes to the maintenance of the overall organelle architecture. Finally, our data suggest a broader function of lipin2 for replication of HCV and other RNA viruses, in contrast with the specific impact of lipin1 silencing on HCV replication. Overall, this study reveals distinctive functions of lipin1 and lipin2 in cells of hepatic origin, a context in which they are often considered functionally redundant.

Creation of the TXP parenting questionnaire and study of its psychometric properties. / Creación y estudio de las propiedades psicométricas del cuestionario de socialización parental TXP.

Parenting is linked to conduct disorders (CD) and substance related disorders (SRD) in adolescents, but with differences according to cultural context. A questionnaire with two versions (parenting questionnaire TXP-A for adolescents and TXP-C  for primary caregivers) was designed using the Delphi method to evaluate parenting practices related to CD and SRD in a Spanish population. It was validated in a community sample of 631 adolescents aged between 14 and 16 and their caregivers. Results suggest a 29-item TXP-A questionnaire with bifactorial structure: affection-communication and control-structure, with high internal (Cronbach’s alpha=0.89) and test-retest (intraclass correlation coefficient=0.94) reliabilities. Both factors are related to SRD (r=0.273, p<0.001) and with most of the psychopathological dimensions studied. The total score and affection-communication are related to dissocial disorder (t=3.259, p=0.001) and its severity (r=-0,119; p=0.003). Inter-observer reliability between adolescents and caregivers is low, in part because the 16-item TXP-C has a different bifactorial structure: affection-communication and prosocial values. TXP-C’s internal (Cronbach’s alpha=0.87) and test-retest (intraclass correlation coefficient=0.94) reliabilities are high. The total score and affection-communication were related to dissocial disorder (t=2.586; p=0.010) but TXP-C did not discriminate according to SRD. In conclusion, the TXP-A questionnaire for adolescents seems to be a reliable, valid and unbiased instrument that evaluates the perception of parenting practices, relating higher affection-communication and control-structure to less psychopathology and alcohol and drug use. TXP-C also seems to be reliable and unbiased, but shows less evidence of validity regarding substance use and psychopathology. .

El estilo parental de socialización se relaciona con trastornos de conducta (TC) y trastornos relacionados con sustancias (TRS) en adolescentes, con diferencias según el contexto cultural. Se diseñó mediante método Delphi un cuestionario con dos versiones (Cuestionario de socialización parental TXP-A para adolescentes y TXP-C para cuidador principal) para evaluar en población española las prácticas de socialización parental relacionadas con TC y TRS. Se validó en una muestra comunitaria de 631 adolescentes entre 14 y 16 años y sus cuidadores. Los resultados recomiendan un cuestionario TXP-A de 29 ítems y estructura bifactorial: afecto-comunicación y control-estructura, mostrando alta fiabilidad interna (alfa de Cronbach=0,89) y test-retest (coeficiente de correlación intraclase=0,94). Ambos factores correlacionan con TRS (r=0,273; p<0,001) y con la mayoría de las dimensiones psicopatológicas estudiadas. La puntuación total y afecto-comunicación se relacionan con el trastorno disocial (t=3,259; p=0,001) y su gravedad (r=-0,119; p=0,003). La fiabilidad interjueces entre adolescentes y cuidadores es baja, en parte porque el TXP-C, de 16 ítems, presenta una estructura bifactorial diferente: afecto-comunicación y valores prosociales. La fiabilidad interna (alfa de Cronbach= 0,87) y test-retest (coeficiente de correlación intraclase=0,94) del TXP-C son altas. La puntuación total y afecto-comunicación se relacionan con el trastorno disocial (t=2,586; p=0,010) pero no discrimina según el TRS. En conclusión, el cuestionario TXP-A para adolescentes parece un instrumento fiable, válido y sin sesgos que evalúa la percepción de las prácticas de socialización parental, relacionando mayores puntuaciones en afecto-comunicación y control-estructura con menor psicopatología y consumo de alcohol y drogas. El TXP-C también parece fiable y sin sesgos, pero muestra menos evidencias de validez respecto al consumo de sustancias y la psicopatología.

Creación y estudio de las propiedades psicométricas del cuestionario de socialización parental TXP / Creation of the TXP parenting questionnaire and study of its psychometric properties

El estilo parental de socialización se relaciona con trastornos de conducta (TC) y trastornos relacionados con sustancias (TRS) en adolescentes, con diferencias según el contexto cultural. Se diseñó mediante método Delphi un cuestionario con dos versiones (Cuestionario de socialización parental TXP-A para adolescentes y TXP-C para cuidador principal) para evaluar en población española las prácticas de socialización parental relacionadas con TC y TRS. Se validó en una muestra comunitaria de 631 adolescentes entre 14 y 16 años y sus cuidadores. Los resultados recomiendan un cuestionario TXP-A de 29 ítems y estructura bifactorial: afecto-comunicación y controlestructura, mostrando alta fiabilidad interna (alfa de Cronbach = 0,89) y test-retest (coeficiente de correlación intraclase = 0,94). Ambos factores correlacionan con TRS (r = 0,273; p < 0,001) y con la mayoría de las dimensiones psicopatológicas estudiadas. La puntuación total y afectocomunicación se relacionan con el trastorno disocial (t = 3,259; p = 0,001) y su gravedad (r = -0,119; p = 0,003). La fiabilidad interjueces entre adolescentes y cuidadores es baja, en parte porque el TXP-C, de 16 ítems, presenta una estructura bifactorial diferente: afecto-comunicación y valores prosociales. La fiabilidad interna (alfa de Cronbach = 0,87) y test-retest (coeficiente de correlación intraclase = 0,94) del TXP-C son altas. La puntuación total y afecto-comunicación se relacionan con el trastorno disocial (t = 2,586; p = 0,010) pero no discrimina según el TRS. En conclusión, el cuestionario TXP-A para adolescentes parece un instrumento fiable, válido y sin sesgos que evalúa la percepción de las prácticas de socialización parental, relacionando mayores puntuaciones en afecto-comunicación y control-estructura con menor psicopatología y consumo de alcohol y drogas. El TXP-C también parece fiable y sin sesgos, pero muestra menos evidencias de validez respecto al consumo de sustancias y la psicopatología

Parenting is linked to conduct disorders (CD) and substance related disorders (SRD) in adolescents, but with differences according to cultural context. A questionnaire with two versions (parenting questionnaire TXP-A for adolescents and TXP-C for primary caregivers) was designed using the Delphi method to evaluate parenting practices related to CD and SRD in a Spanish population. It was validated in a community sample of 631 adolescents aged between 14 and 16 and their caregivers. Results suggest a 29-item TXP-A questionnaire with bifactorial structure: affection-communication and control-structure, with high internal (Cronbach's alpha = 0.89) and test-retest (intraclass correlation coefficient = 0.94) reliabilities. Both factors are related to SRD (r = 0.273, p < 0.001) and with most of the psychopathological dimensions studied. The total score and affection-communication are related to dissocial disorder (t = 3.259, p = 0.001) and its severity (r = -0,119; p = 0.003). Inter-observer reliability between adolescents and caregivers is low, in part because the 16-item TXP-C has a different bifactorial structure: affection-communication and prosocial values. TXP-C's internal (Cronbach’s alpha=0.87) and test-retest (intraclass correlation coefficient=0.94) reliabilities are high. The total score and affection-communication were related to dissocial disorder (t = 2.586; p = 0.010) but TXP-C did not discriminate according to SRD. In conclusion, the TXP-A questionnaire for adolescents seems to be a reliable, valid and unbiased instrument that evaluates the perception of parenting practices, relating higher affection-communication and control-structure to less psychopathology and alcohol and drug use. TXP-C also seems to be reliable and unbiased, but shows less evidence of validity regarding substance use and psychopathology

Host phosphatidic acid phosphatase lipin1 is rate limiting for functional hepatitis C virus replicase complex formation.

Hepatitis C virus (HCV) infection constitutes a significant health burden worldwide, because it is a major etiologic agent of chronic liver disease, cirrhosis and hepatocellular carcinoma. HCV replication cycle is closely tied to lipid metabolism and infection by this virus causes profound changes in host lipid homeostasis. We focused our attention on a phosphatidate phosphate (PAP) enzyme family (the lipin family), which mediate the conversion of phosphatidate to diacylglycerol in the cytoplasm, playing a key role in triglyceride biosynthesis and in phospholipid homeostasis. Lipins may also translocate to the nucleus to act as transcriptional regulators of genes involved in lipid metabolism. The best-characterized member of this family is lipin1, which cooperates with lipin2 to maintain glycerophospholipid homeostasis in the liver. Lipin1-deficient cell lines were generated by RNAi to study the role of this protein in different steps of HCV replication cycle. Using surrogate models that recapitulate different aspects of HCV infection, we concluded that lipin1 is rate limiting for the generation of functional replicase complexes, in a step downstream primary translation that leads to early HCV RNA replication. Infection studies in lipin1-deficient cells overexpressing wild type or phosphatase-defective lipin1 proteins suggest that lipin1 phosphatase activity is required to support HCV infection. Finally, ultrastructural and biochemical analyses in replication-independent models suggest that lipin1 may facilitate the generation of the membranous compartment that contains functional HCV replicase complexes.