RESUMEN
A two-step strategy combining assisted benchmark testing (entry controls) and External Quality Assessments (EQAs) with blinded simulated clinical specimens to enhance and maintain the quality of nucleic acid amplification testing was developed. This strategy was successfully applied to 71 diagnostic laboratories in The Netherlands when upscaling the national diagnostic capacity during the SARS-CoV-2 pandemic. The availability of benchmark testing in combination with advice for improvement substantially enhanced the quality of the laboratory testing procedures for SARS-CoV-2 detection. The three subsequent EQA rounds demonstrated high quality testing with regard to specificity (99.6% correctly identified) and sensitivity (93.3% correctly identified). Even with the implementation of novel assays, changing workflows using diverse equipment and a high degree of assay heterogeneity, the overall high quality was maintained using this two-step strategy. We show that in contrast to the limited value of Cq value for absolute proxies of viral load, these Cq values can, in combination with metadata on strategies and techniques, provide valuable information for laboratories to improve their procedures. In conclusion, our two-step strategy (preparation phase followed by a series of EQAs) is a rapid and flexible system capable of scaling, improving, and maintaining high quality diagnostics even in a rapidly evolving (e.g. pandemic) situation.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/epidemiología , Laboratorios , Técnicas de Laboratorio Clínico/métodos , Prueba de COVID-19 , Benchmarking , Patología Molecular , Sensibilidad y EspecificidadRESUMEN
Respiratory syncytial virus (RSV) is a common pathogen causing mostly cold-like symptoms, but in very young infants and elderly individuals it can lead to severe disease and even death. There are currently promising developments both in vaccine development and in therapeutics that are expected to be approved soon. To get an impression within European countries of the laboratory diagnostics and surveillance activities, in anticipation of these developments, we queried the members of the European Respiratory Syncytial Virus Laboratory Network (RSV-LabNet, under the umbrella of the PROMISE project) via an online survey. The answers from the consortium members showed scattered monitoring and the application of a broad array of techniques in the laboratories. A majority of the members expressed strong interest in harmonization and collaboration for setting up surveillance programs and the need for sharing laboratory protocols. The additional value of RSV whole-genome sequencing is broadly appreciated, but implementation requires further development and closer collaboration. The RSV-LabNet can have an important responsibility in establishing contacts and exchange of expertise and providing a platform for communication to advance diagnostics, preparedness, and surveillance.
Asunto(s)
Laboratorios , Virus Sincitial Respiratorio Humano , Anciano , Lactante , Humanos , Virus Sincitial Respiratorio Humano/genética , Europa (Continente)/epidemiología , Secuenciación Completa del GenomaRESUMEN
We investigated a large outbreak of SARS-CoV-2 infections among passengers and crew members (60 cases in 132 persons) on a cruise ship sailing for 7 days on rivers in the Netherlands. Whole-genome analyses suggested a single or limited number of viral introductions consistent with the epidemiologic course of infections. Although some precautionary measures were taken, no social distancing was exercised, and air circulation and ventilation were suboptimal. The most plausible explanation for introduction of the virus is by persons (crew members and 2 passengers) infected during a previous cruise, in which a case of COVID-19 had occurred. The crew was insufficiently prepared on how to handle the situation, and efforts to contact public health authorities was inadequate. We recommend installing clear handling protocols, direct contacts with public health organizations, training of crew members to recognize outbreaks, and awareness of air quality on river-cruise ships, as is customary for most seafaring cruises.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/epidemiología , COVID-19/prevención & control , Países Bajos/epidemiología , Ríos , Brotes de Enfermedades/prevención & control , NavíosRESUMEN
Mutations in transcription factor p63 are associated with developmental disorders that manifest defects in stratified epithelia including the epidermis. The underlying cellular and molecular mechanism is however not yet understood. We established an epidermal commitment model using human induced pluripotent stem cells (iPSCs) and characterized differentiation defects of iPSCs derived from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients carrying p63 mutations. Transcriptome analyses revealed stepwise cell fate transitions during epidermal commitment: Specification from multipotent simple epithelium to basal stratified epithelia and ultimately to the mature epidermal fate. Differentiation defects of EEC iPSCs caused by p63 mutations occurred during the specification switch from the simple epithelium to the basal-stratified epithelial fate. Single-cell transcriptome and pseudotime analyses of cell states identified mesodermal activation that was associated with the deviated commitment route of EEC iPSCs. Integrated analyses of differentially regulated genes and p63-dependent dynamic genomic enhancers during epidermal commitment suggest that p63 directly controls epidermal gene activation at the specification switch and has an indirect effect on mesodermal gene repression. Importantly, inhibitors of mesodermal induction enhanced epidermal commitment of EEC iPSCs. Our findings demonstrate that p63 is required for specification of stratified epithelia, and that epidermal commitment defects caused by p63 mutations can be reversed by repressing mesodermal induction. This study provides insights into disease mechanisms underlying stratified epithelial defects caused by p63 mutations and suggests potential therapeutic strategies for the disease.
Asunto(s)
Labio Leporino/genética , Fisura del Paladar/genética , Displasia Ectodérmica/genética , Epitelio/metabolismo , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Epidermis/embriología , Epidermis/metabolismo , Epitelio/embriología , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/metabolismo , Queratinocitos/metabolismo , Mutación , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
We report on four families affected by a clinical presentation of complex hereditary spastic paraplegia (HSP) due to recessive mutations in DDHD2, encoding one of the three mammalian intracellular phospholipases A(1) (iPLA(1)). The core phenotype of this HSP syndrome consists of very early-onset (<2 years) spastic paraplegia, intellectual disability, and a specific pattern of brain abnormalities on cerebral imaging. An essential role for DDHD2 in the human CNS, and perhaps more specifically in synaptic functioning, is supported by a reduced number of active zones at synaptic terminals in Ddhd-knockdown Drosophila models. All identified mutations affect the protein's DDHD domain, which is vital for its phospholipase activity. In line with the function of DDHD2 in lipid metabolism and its role in the CNS, an abnormal lipid peak indicating accumulation of lipids was detected with cerebral magnetic resonance spectroscopy, which provides an applicable diagnostic biomarker that can distinguish the DDHD2 phenotype from other complex HSP phenotypes. We show that mutations in DDHD2 cause a specific complex HSP subtype (SPG54), thereby linking a member of the PLA(1) family to human neurologic disease.
Asunto(s)
Genes Recesivos , Mutación , Fosfolipasas/genética , Paraplejía Espástica Hereditaria/genética , Adolescente , Adulto , Secuencia de Bases , Sistema Nervioso Central/patología , Niño , Preescolar , Facies , Femenino , Orden Génico , Genotipo , Humanos , Imagen por Resonancia Magnética , Masculino , Neuroimagen , Linaje , Fenotipo , Paraplejía Espástica Hereditaria/diagnóstico , Adulto JovenRESUMEN
Intellectual disability (ID) disorders are genetically and phenotypically highly heterogeneous and present a major challenge in clinical genetics and medicine. Although many genes involved in ID have been identified, the etiology is unknown in most affected individuals. Moreover, the function of most genes associated with ID remains poorly characterized. Evidence is accumulating that the control of gene transcription through epigenetic modification of chromatin structure in neurons has an important role in cognitive processes and in the etiology of ID. However, our understanding of the key molecular players and mechanisms in this process is highly fragmentary. Here, we identify a chromatin-modification module that underlies a recognizable form of ID, the Kleefstra syndrome phenotypic spectrum (KSS). In a cohort of KSS individuals without mutations in EHMT1 (the only gene known to be disrupted in KSS until now), we identified de novo mutations in four genes, MBD5, MLL3, SMARCB1, and NR1I3, all of which encode epigenetic regulators. Using Drosophila, we demonstrate that MBD5, MLL3, and NR1I3 cooperate with EHMT1, whereas SMARCB1 is known to directly interact with MLL3. We propose a highly conserved epigenetic network that underlies cognition in health and disease. This network should allow the design of strategies to treat the growing group of ID pathologies that are caused by epigenetic defects.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/genética , Discapacidad Intelectual/genética , Animales , Cromatina , Proteínas Cromosómicas no Histona/genética , Receptor de Androstano Constitutivo , Proteínas de Unión al ADN/genética , Drosophila , Epigénesis Genética , Femenino , Humanos , Recién Nacido , Masculino , Mutación , Proteína SMARCB1 , Síndrome , Factores de Transcripción/genéticaRESUMEN
Genetic causes for autosomal recessive forms of dilated cardiomyopathy (DCM) are only rarely identified, although they are thought to contribute considerably to sudden cardiac death and heart failure, especially in young children. Here, we describe 11 young patients (5-13 years) with a predominant presentation of dilated cardiomyopathy (DCM). Metabolic investigations showed deficient protein N-glycosylation, leading to a diagnosis of Congenital Disorders of Glycosylation (CDG). Homozygosity mapping in the consanguineous families showed a locus with two known genes in the N-glycosylation pathway. In all individuals, pathogenic mutations were identified in DOLK, encoding the dolichol kinase responsible for formation of dolichol-phosphate. Enzyme analysis in patients' fibroblasts confirmed a dolichol kinase deficiency in all families. In comparison with the generally multisystem presentation in CDG, the nonsyndromic DCM in several individuals was remarkable. Investigation of other dolichol-phosphate dependent glycosylation pathways in biopsied heart tissue indicated reduced O-mannosylation of alpha-dystroglycan with concomitant functional loss of its laminin-binding capacity, which has been linked to DCM. We thus identified a combined deficiency of protein N-glycosylation and alpha-dystroglycan O-mannosylation in patients with nonsyndromic DCM due to autosomal recessive DOLK mutations.
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Cardiomiopatía Dilatada/genética , Distroglicanos/metabolismo , Genes Recesivos , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Adolescente , Cardiomiopatía Dilatada/metabolismo , Niño , Preescolar , Fosfatos de Dolicol/metabolismo , Femenino , Expresión Génica , Glicosilación , Haplotipos , Homocigoto , Humanos , Masculino , Linaje , Saccharomyces cerevisiae/genética , Sarcolema/metabolismoRESUMEN
Hox proteins are homeobox containing transcription factors that play important roles in patterning the presumptive central nervous system and the axial mesoderm in the early vertebrate embryo. Hox genes are first expressed during gastrula stages and recent studies suggest that their function goes beyond their role as patterning determinants. To improve our understanding of the role of Hox proteins during early vertebrate development, we designed a strategy to identify target genes of the zebrafish hoxb1b using overexpression and whole-genome microarray analysis. We directly compared the hoxb1b microarray data with those resulting from heterologous over-expression of the Xenopus XhoxD1 gene in zebrafish embryos. Both genes are the first expressed hox genes in their respective native embryos and display similar spatial expression patterns. The zebrafish transcriptome was analysed prior to the start of the expression of the endogenous hoxb1b gene and we observed extensive overlap between the hoxb1b and XhoxD1 putative downstream genes suggesting evolutionary functional conservation between these hox genes. Furthermore, genes encoding transcription factors and proteins that are known to be involved in cell adhesion and movement were over-represented among the candidate downstream genes, indicating the involvement of the developmentally earliest expressed hox genes in transcriptional networks and cell movement processes.
Asunto(s)
Movimiento Celular/fisiología , Embrión no Mamífero/metabolismo , Gastrulación , Perfilación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Células Cultivadas , Embrión no Mamífero/citología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Hibridación in Situ , Análisis de Secuencia por Matrices de Oligonucleótidos , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Knockout of the Nkx2.1 (Titf-1) homeobox gene in the mouse leads to severe malformation and size reduction of the basal telencephalon/preoptic area and basal hypothalamus, indicating an important role of this gene in forebrain patterning. Here we show that abrogation of the orthologous gene in the frog Xenopus laevis by way of morpholino knockdown also affects the relative size of major regions in both the telencephalon (subpallium versus pallium) and diencephalon (hypothalamus versus thalamus). Remarkably, while a similar effect on the telencephalon was noted previously in Nkx2.1-knockout mice, the effect on the diencephalon seems to be specific for Xenopus. This difference may be explained by the partially dissimilar expression of the orthologous genes in the forebrain of Xenopus and mouse. In both species Nkx2.1 is expressed in the basal telencephalon/preoptic area and basal hypothalamus, but in Xenopus this gene is additionally expressed in the alar hypothalamus. Phylogenetic comparison of Nkx2.1 expression in the forebrain suggests that the expression in the basal telencephalon-preoptic region and alar hypothalamus appeared in the transition from jawless to jawed vertebrates, but the alar hypothalamic expression was later dramatically reduced during evolution to birds and mammals. Our study suggests that changes in the regulation of Nkx2.1 expression have played an important role on the evolution of forebrain development, and emphasizes the potential of the combined analysis of expression and function of master control genes in different vertebrates for unraveling the origin of brain complexity and diversity.
Asunto(s)
Evolución Biológica , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/fisiología , Prosencéfalo/embriología , Prosencéfalo/metabolismo , Proteínas de Xenopus/genética , Animales , Animales Modificados Genéticamente , Embrión no Mamífero , Proteínas Nucleares , Factor Nuclear Tiroideo 1 , Factores de Transcripción , Proteínas de Xenopus/deficiencia , Proteínas de Xenopus/fisiología , Xenopus laevisRESUMEN
The specification of neuronal cell types in the developing neural tube is orchestrated by signaling centers. However, how patterned territories of the central nervous system (CNS) are organized into structures with appropriate size and shape is still unclear. We report that in the absence of the mouse transcription factor mBtd/Sp8, a posterior shift of the isthmic organizer (IsO) occurs, suggesting a crucial role for Sp8 in this process. In addition, large patches of cells ectopically expressing Fgf8, Otx2 and/or Wnt1 in the rostral hindbrain are detected in Sp8 mutant embryos. In this context, midbrain dopaminergic neurons are found posterior to the IsO. Furthermore, we provide evidence that cell proliferation in the mid- and hindbrain is tightly controlled by Sp8 activity. Our observations are consistent with a role for Sp8 in restricting Fgf8 expression at the IsO.
Asunto(s)
Tipificación del Cuerpo , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Mesencéfalo/embriología , Rombencéfalo/embriología , Factores de Transcripción/metabolismo , Animales , Tipificación del Cuerpo/genética , Proteínas de Unión al ADN/genética , Mesencéfalo/metabolismo , Ratones , Ratones Noqueados , Rombencéfalo/metabolismo , Factores de Transcripción/genéticaRESUMEN
The Hox paralogous group 1 (PG1) genes are the first and initially most anterior Hox genes expressed in the embryo. In Xenopus, the three PG1 genes, Hoxa1, Hoxb1 and Hoxd1, are expressed in a widely overlapping domain, which includes the region of the future hindbrain and its associated neural crest. We used morpholinos to achieve a complete knockdown of PG1 function. When Hoxa1, Hoxb1 and Hoxd1 are knocked down in combination, the hindbrain patterning phenotype is more severe than in the single or double knockdowns, indicating a degree of redundancy for these genes. In the triple PG1 knockdown embryos the hindbrain is reduced and lacks segmentation. The patterning of rhombomeres 2 to 7 is lost, with a concurrent posterior expansion of the rhombomere 1 marker, Gbx2. This effect could be via the downregulation of other Hox genes, as we show that PG1 function is necessary for the hindbrain expression of Hox genes from paralogous groups 2 to 4. Furthermore, in the absence of PG1 function, the cranial neural crest is correctly specified but does not migrate into the pharyngeal arches. Embryos with no active PG1 genes have defects in derivatives of the pharyngeal arches and, most strikingly, the gill cartilages are completely missing. These results show that the complete abrogation of PG1 function in Xenopus has a much wider scope of effect than would be predicted from the single and double PG1 knockouts in other organisms.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Cresta Neural/metabolismo , Rombencéfalo/anomalías , Rombencéfalo/metabolismo , Factores de Transcripción/deficiencia , Proteínas de Xenopus/deficiencia , Xenopus laevis/embriología , Animales , Tipificación del Cuerpo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Cresta Neural/anomalías , Cresta Neural/embriología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rombencéfalo/embriología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/anomalías , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
The facultative intracellular pathogen Bartonella henselae is responsible for a broad range of clinical manifestations, including the formation of vascular tumors as a result of increased proliferation and survival of colonized endothelial cells. This remarkable interaction with endotoxin-sensitive endothelial cells and the apparent lack of septic shock are considered to be due to a reduced endotoxic activity of the B. henselae lipopolysaccharide. Here, we show that B. henselae ATCC 49882(T) produces a deep-rough-type lipopolysaccharide devoid of O-chain and report on its complete structure and Toll-like receptor-dependent biological activity. The major short-chain lipopolysaccharide was studied by chemical analyses, electrospray ionization, and matrix-assisted laser desorption/ionization mass spectrometry, as well as by NMR spectroscopy after alkaline deacylation. The carbohydrate portion of the lipopolysaccharide consists of a branched trisaccharide containing a glucose residue attached to position 5 of an alpha-(2-->4)-linked 3-deoxy-d-manno-oct-2-ulosonic acid disaccharide. Lipid A is a pentaacylated beta-(1'-->6)-linked 2,3-diamino-2,3-dideoxy-glucose disaccharide 1,4'-bisphosphate with two amide-linked residues each of 3-hydroxydodecanoic and 3-hydroxyhexadecanoic acids and one residue of either 25-hydroxyhexacosanoic or 27-hydroxyoctacosanoic acid that is O-linked to the acyl group at position 2'. The lipopolysaccharide studied activated Toll-like receptor 4 signaling only to a low extent (1,000-10,000-fold lower compared with that of Salmonella enterica sv. Friedenau) and did not activate Toll-like receptor 2. Some unusual structural features of the B. henselae lipopolysaccharide, including the presence of a long-chain fatty acid, which are shared by the lipopolysaccharides of other bacteria causing chronic intracellular infections (e.g. Legionella and Chlamydia), may provide the molecular basis for low endotoxic potency.
Asunto(s)
Bartonella henselae/química , Lipopolisacáridos/química , Infecciones por Bartonella , Bartonella henselae/inmunología , Bartonella henselae/aislamiento & purificación , Conformación de Carbohidratos , Secuencia de Carbohidratos , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/metabolismo , Metilación , Datos de Secuencia Molecular , Espectrometría de Masa por Ionización de Electrospray , TransfecciónRESUMEN
LPSs play an important role in bacterial pathogenesis. In this study, the LPS expression of the seven known Bordetella species and its dependency on growth temperature was analysed by oxidative silver staining of proteinase-K-treated whole bacteria separated by Tricine-SDS-PAGE. The bordetellae were found to have extensively variable LPS in a species-specific way. In addition, the human and ovine Bordetella parapertussis strains exhibited host-specific LPS expression. LPSs from human B. parapertussis strains grown at 37 and 25 degrees C were distinct. Growth temperature also affected LPS production by several Bordetella bronchiseptica strains. In some of these cases, BvgAS, the global regulator of virulence factors, was involved in this regulation of LPS biosynthesis. In contrast, no evidence was found for the involvement of the Bordetella pertussis BvgAS system in regulation of LPS synthesis. The obligate human pathogens B. pertussis and Bordetella holmesii are closely related but were shown to produce immunologically distinct LPSs. These species are isolated from the upper respiratory tract and blood, respectively. This raises several interesting questions concerning the potential role of LPS as a virulence factor in the infection processes.