RESUMEN
The cell wall is a distinctive feature of filamentous fungi, providing them with structural integrity and protection from both biotic and abiotic factors. Unlike plant cell walls, fungi rely on structurally strong hydrophobic chitin core for mechanical strength together with alpha- and beta-glucans, galactomannans and glycoproteins. Cell wall stress conditions are known to alter the cell wall through the signaling cascade of the cell wall integrity (CWI) pathway and can result in increased cell wall chitin deposition. A previously isolated set of Aspergillus niger cell wall mutants was screened for increased cell wall chitin deposition. UV-mutant RD15.8#16 was found to contain approximately 60% more cell wall chitin than the wild type. In addition to the chitin phenotype, RD15.8#16 exhibits a compact colony morphology and increased sensitivity towards SDS. RD15.8#16 was subjected to classical genetic approach for identification of the underlying causative mutation, using co-segregation analysis and SNP genotyping. Genome sequencing of RD15.8#16 revealed eight SNPs in open reading frames (ORF) which were individually checked for co-segregation with the associated phenotypes, and showed the potential relevance of two genes located on chromosome IV. In situ re-creation of these ORF-located SNPs in a wild type background, using CRISPR/Cas9 genome editing, showed the importance Rab GTPase dissociation inhibitor A (gdiA) for the phenotypes of RD15.8#16. An alteration in the 5' donor splice site of gdiA reduced pre-mRNA splicing efficiency, causing aberrant cell wall assembly and increased chitin levels, whereas gene disruption attempts showed that a full gene deletion of gdiA is lethal.
Asunto(s)
Aspergillus niger/genética , Quitina/metabolismo , Proteínas Fúngicas/genética , Genes Esenciales , Inhibidores de Disociación de Guanina Nucleótido/genética , Aspergillus niger/metabolismo , Sistemas CRISPR-Cas , Pared Celular/metabolismo , Eliminación de Gen , Edición Génica , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Técnicas de Genotipaje , Polimorfismo de Nucleótido Simple , Empalme del ARN/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
OBJECTIVE: Family history (FH) in Preventive Primary Pediatric Care is to identify children at risk for complex diseases and provide personal preventive strategies. This study was to assess parents' opinion on FH collection. METHODS: Semi-structured interviews were conducted. Among issues addressed were: former experiences with FH, knowledge about FH, family definition and sharing information about FH. RESULTS: The importance of FH for participants depended on their knowledge, perceived family health status and former experiences. After insight into FH, parents shift to believing it to be important, but certain barriers exist in reporting FH. CONCLUSION: Parents suggest that the importance of FH should be more emphasized and more trusting relationship with Preventive Primary Pediatric Care should be invested in.