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Haematopoiesis in the bone marrow (BM) maintains blood and immune cell production throughout postnatal life. Haematopoiesis first emerges in human BM at 11-12 weeks after conception1,2, yet almost nothing is known about how fetal BM (FBM) evolves to meet the highly specialized needs of the fetus and newborn. Here we detail the development of FBM, including stroma, using multi-omic assessment of mRNA and multiplexed protein epitope expression. We find that the full blood and immune cell repertoire is established in FBM in a short time window of 6-7 weeks early in the second trimester. FBM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell subsets emerging for the first time. The substantial expansion of B lymphocytes in FBM contrasts with fetal liver at the same gestational age. Haematopoietic progenitors from fetal liver, FBM and cord blood exhibit transcriptional and functional differences that contribute to tissue-specific identity and cellular diversification. Endothelial cell types form distinct vascular structures that we show are regionally compartmentalized within FBM. Finally, we reveal selective disruption of B lymphocyte, erythroid and myeloid development owing to a cell-intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in Down syndrome (trisomy 21).
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Células de la Médula Ósea/citología , Médula Ósea , Síndrome de Down/sangre , Síndrome de Down/inmunología , Feto/citología , Hematopoyesis , Sistema Inmunológico/citología , Linfocitos B/citología , Células Dendríticas/citología , Síndrome de Down/metabolismo , Síndrome de Down/patología , Células Endoteliales/patología , Eosinófilos/citología , Células Eritroides/citología , Granulocitos/citología , Humanos , Inmunidad , Células Mieloides/citología , Células del Estroma/citologíaRESUMEN
Developmental studies have shown that the evolutionarily conserved Wnt Planar Cell Polarity (PCP) pathway is essential for the development of a diverse range of tissues and organs including the brain, spinal cord, heart and sensory organs, as well as establishment of the left-right body axis. Germline mutations in the highly conserved PCP gene VANGL2 in humans have only been associated with central nervous system malformations, and functional testing to understand variant impact has not been performed. Here we report three new families with missense variants in VANGL2 associated with heterotaxy and congenital heart disease p.(Arg169His), non-syndromic hearing loss p.(Glu465Ala) and congenital heart disease with brain defects p.(Arg135Trp). To test the in vivo impact of these and previously described variants, we have established clinically-relevant assays using mRNA rescue of the vangl2 mutant zebrafish. We show that all variants disrupt Vangl2 function, although to different extents and depending on the developmental process. We also begin to identify that different VANGL2 missense variants may be haploinsufficient and discuss evidence in support of pathogenicity. Together, this study demonstrates that zebrafish present a suitable pipeline to investigate variants of unknown significance and suggests new avenues for investigation of the different developmental contexts of VANGL2 function that are clinically meaningful.
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Cardiopatías Congénitas , Pez Cebra , Animales , Humanos , Polaridad Celular/genética , Células Germinativas/metabolismo , Mutación de Línea Germinal/genética , Cardiopatías Congénitas/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Abnormalities of the arterial valves, including bicuspid aortic valve (BAV) are amongst the most common congenital defects and are a significant cause of morbidity as well as predisposition to disease in later life. Despite this, and compounded by their small size and relative inaccessibility, there is still much to understand about how the arterial valves form and remodel during embryogenesis, both at the morphological and genetic level. Here we set out to address this in human embryos, using Spatial Transcriptomics (ST). We show that ST can be used to investigate the transcriptome of the developing arterial valves, circumventing the problems of accurately dissecting out these tiny structures from the developing embryo. We show that the transcriptome of CS16 and CS19 arterial valves overlap considerably, despite being several days apart in terms of human gestation, and that expression data confirm that the great majority of the most differentially expressed genes are valve-specific. Moreover, we show that the transcriptome of the human arterial valves overlaps with that of mouse atrioventricular valves from a range of gestations, validating our dataset but also highlighting novel genes, including four that are not found in the mouse genome and have not previously been linked to valve development. Importantly, our data suggests that valve transcriptomes are under-represented when using commonly used databases to filter for genes important in cardiac development; this means that causative variants in valve-related genes may be excluded during filtering for genomic data analyses for, for example, BAV. Finally, we highlight "novel" pathways that likely play important roles in arterial valve development, showing that mouse knockouts of RBP1 have arterial valve defects. Thus, this study has confirmed the utility of ST for studies of the developing heart valves and broadens our knowledge of the genes and signalling pathways important in human valve development.
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Enfermedad de la Válvula Aórtica Bicúspide , Enfermedades de las Válvulas Cardíacas , Humanos , Ratones , Animales , Enfermedades de las Válvulas Cardíacas/genética , Válvula Aórtica/anomalías , Enfermedad de la Válvula Aórtica Bicúspide/metabolismo , Perfilación de la Expresión Génica , Transcriptoma/genéticaRESUMEN
The establishment of the left-right axis is crucial for the placement, morphogenesis and function of internal organs. Left-right specification is proposed to be dependent on cilia-driven fluid flow in the embryonic node. Planar cell polarity (PCP) signalling is crucial for patterning of nodal cilia, yet downstream effectors driving this process remain elusive. We have examined the role of the JNK gene family, a proposed downstream component of PCP signalling, in the development and function of the zebrafish node. We show jnk1 and jnk2 specify length of nodal cilia, generate flow in the node and restrict southpaw to the left lateral plate mesoderm. Moreover, loss of asymmetric southpaw expression does not result in disturbances to asymmetric organ placement, supporting a model in which nodal flow may be dispensable for organ laterality. Later, jnk3 is required to restrict pitx2c expression to the left side and permit correct endodermal organ placement. This work uncovers multiple roles for the JNK gene family acting at different points during left-right axis establishment. It highlights extensive redundancy and indicates JNK activity is distinct from the PCP signalling pathway.
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Tipificación del Cuerpo , Pez Cebra , Animales , Tipificación del Cuerpo/genética , Cilios/metabolismo , Regulación del Desarrollo de la Expresión Génica , Mesodermo/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Much has been learned over the last half century regarding the molecular and genetic changes that take place during cardiac development. As yet, however, these advances have not been translated into knowledge regarding the marked changes that take place in the anatomical arrangements of the different cardiac components. As such, therefore, many aspects of cardiac development are still described on the basis of speculation rather than evidence. In this review, we show how controversial aspects of development can readily be arbitrated by the interested spectator by taking advantage of the material now gathered together in the Human Developmental Biology Resource; HDBR. We use the material to demonstrate the changes taking place during the formation of the ventricular loop, the expansion of the atrioventricular canal, the incorporation of the systemic venous sinus, the formation of the pulmonary vein, the process of atrial septation, the remodelling of the pharyngeal arches, the major changes occurring during formation of the outflow tract, the closure of the embryonic interventricular communication, and the formation of the ventricular walls. We suggest that access to the resource makes it possible for the interested observer to arbitrate, for themselves, the ongoing controversies that continue to plague the understanding of cardiac development.
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The separation of the outflow tract of the developing heart into the systemic and pulmonary arterial channels remains controversial and poorly understood. The definitive outflow tracts have three components. The developing outflow tract, in contrast, has usually been described in two parts. When the tract has exclusively myocardial walls, such bipartite description is justified, with an obvious dogleg bend separating proximal and distal components. With the addition of non-myocardial walls distally, it becomes possible to recognise three parts. The middle part, which initially still has myocardial walls, contains within its lumen a pair of intercalated valvar swellings. The swellings interdigitate with the distal ends of major outflow cushions, formed by the remodelling of cardiac jelly, to form the primordiums of the arterial roots. The proximal parts of the major cushions, occupying the proximal part of the outflow tract, which also has myocardial walls, themselves fuse and muscularise. The myocardial shelf thus formed remodels to become the free-standing subpulmonary infundibulum. Details of all these processes are currently lacking. In this account, we describe the anatomical changes seen during the overall remodelling. Our interpretations are based on the interrogation of serially sectioned histological and high-resolution episcopic microscopy datasets prepared from developing human and mouse embryos, with some of the datasets processed and reconstructed to reveal the specific nature of the tissues contributing to the separation of the outflow channels. Our findings confirm that the tripartite postnatal arrangement can be correlated with the changes occurring during development.
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Estructuras Embrionarias , Matriz Extracelular , Cardiopatías Congénitas , Corazón , Ratones , Animales , Humanos , Ventrículos Cardíacos , Arteria PulmonarRESUMEN
The planar cell polarity pathway is required for heart development and whilst the functions of most pathway members are known, the roles of the jnk genes in cardiac morphogenesis remain unknown as mouse mutants exhibit functional redundancy, with early embryonic lethality of compound mutants. In this study zebrafish were used to overcome early embryonic lethality in mouse models and establish the requirement for Jnk in heart development. Whole mount in-situ hybridisation and RT-PCR demonstrated that evolutionarily conserved alternative spliced jnk1a and jnk1b transcripts were expressed in the early developing heart. Maternal zygotic null mutant zebrafish lines for jnk1a and jnk1b, generated using CRISPR-Cas9, revealed a requirement for jnk1a in formation of the proximal, first heart field (FHF)-derived portion of the cardiac ventricular chamber. Rescue of the jnk1a mutant cardiac phenotype was only possible by injection of the jnk1a EX7 Lg alternatively spliced transcript. Analysis of mutants indicated that there was a reduction in the size of the hand2 expression field in jnk1a mutants which led to a specific reduction in FHF ventricular cardiomyocytes within the anterior lateral plate mesoderm. Moreover, the jnk1a mutant ventricular defect could be rescued by injection of hand2 mRNA. This study reveals a novel and critical requirement for Jnk1 in heart development and highlights the importance of alternative splicing in vertebrate cardiac morphogenesis. Genetic pathways functioning through jnk1 may be important in human heart malformations with left ventricular hypoplasia.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Ventrículos Cardíacos/citología , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Empalme Alternativo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Recuento de Células , Células Cultivadas , Exones , Regulación del Desarrollo de la Expresión Génica , Ventrículos Cardíacos/embriología , Ventrículos Cardíacos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Although scholars have noted the detrimental nature of the various changes in higher education prompted by neoliberalism, its impact on the experiences of international Higher Degree by Research (HDR) students has yet to be adequately studied. Informed by Bourdieu's concepts of doxa, field, habitus, and capital, this paper examines the ways in which neoliberalism as doxa in the Australian higher education field has colonised the perception and practice of Chinese international HDR students whilst some students were able to demonstrate resilience to the pervasive neoliberal practices. The paper draws on a larger qualitative research project including interviews with 18 Chinese HDR students from four Australian universities. Data suggest that Chinese HDR research students gradually developed intensified dispositions of self-reliance and self-exploitation in response to neoliberal academic practices whilst others were enculturated into a floating habitus (or vulnerable position) in relation to academic publishing as they attempted to negotiate the tensions across fields and over time. Data further reveal that some participants demonstrated resilience to neoliberalism when empowered by their supervisors with less utilitarian and more critically reflexive supervisory practices. The paper argues that the embrace of neoliberalism in the Australian higher education field has become widespread yet controversial, and that thinking and enacting resilience sociologically may de-neoliberalise the higher education field in Australia and beyond.
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During axonal ensheathment, noncompact myelin channels formed at lateral edges of the myelinating process become arranged into tight paranodal spirals that resemble loops when cut in cross section. These adhere to the axon, concentrating voltage-dependent sodium channels at nodes of Ranvier and patterning the surrounding axon into distinct molecular domains. The signals responsible for forming and maintaining the complex structure of paranodal myelin are poorly understood. Here, we test the hypothesis that the planar cell polarity determinant Vangl2 organizes paranodal myelin. We show that Vangl2 is concentrated at paranodes and that, following conditional knockout of Vangl2 in oligodendrocytes, the paranodal spiral loosens, accompanied by disruption to the microtubule cytoskeleton and mislocalization of autotypic adhesion molecules between loops within the spiral. Adhesion of the spiral to the axon is unaffected. This results in disruptions to axonal patterning at nodes of Ranvier, paranodal axon diameter and conduction velocity. When taken together with our previous work showing that loss of the apico-basal polarity protein Scribble has the opposite phenotype-loss of axonal adhesion but no effect on loop-loop autotypic adhesion-our results identify a novel mechanism by which polarity proteins control the shape of nodes of Ranvier and regulate conduction in the CNS.
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Vaina de Mielina , Nódulos de Ranvier , Axones , Polaridad Celular , OligodendroglíaRESUMEN
DNA from archived organs is presumed unsuitable for genomic studies because of excessive formalin-fixation. As next generation sequencing (NGS) requires short DNA fragments, and Uracil-N-glycosylase (UNG) can be used to overcome deamination, there has been renewed interest in the possibility of genomic studies using these collections. We describe a novel method of DNA extraction capable of providing PCR amplicons of at least 400 bp length from such excessively formalin-fixed human tissues. When compared with a leading commercial formalin-fixed DNA extraction kit, our method produced greater yields of DNA and reduced sequence variations. Analysis of PCR products using bacterial sub-cloning and Sanger sequencing from UNG-treated DNA unexpectedly revealed increased sequence variations, compared with untreated samples. Finally, whole exome NGS was performed on a myocardial sample fixed in formalin for 2 years and compared with lymphocyte-derived DNA (as a gold standard) from the same patient. Despite the reduction in the number and quality of reads in the formalin-fixed DNA, we were able to show that bioinformatic processing by joint calling and variant quality score recalibration (VQSR) increased the sensitivity four-fold to 56% and doubled specificity to 68% when compared with a standard hard-filtering approach. Thus, high-quality DNA can be extracted from excessively formalin-fixed tissues and bioinformatic processing can optimise sensitivity and specificity of results. Sequencing of several sub-cloned amplicons is an important methodological step in assessing DNA quality.
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ADN/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Fijación del Tejido , Formaldehído , HumanosRESUMEN
Planar cell polarity (PCP) pathways control the orientation and alignment of epithelial cells within tissues. Van Gogh-like 2 (Vangl2) is a key PCP protein that is required for the normal differentiation of kidney glomeruli and tubules. Vangl2 has also been implicated in modifying the course of acquired glomerular disease, and here, we further explored how Vangl2 impacts on glomerular pathobiology in this context. Targeted genetic deletion of Vangl2 in mouse glomerular epithelial podocytes enhanced the severity of not only irreversible accelerated nephrotoxic nephritis but also lipopolysaccharide-induced reversible glomerular damage. In each proteinuric model, genetic deletion of Vangl2 in podocytes was associated with an increased ratio of active-MMP9 to inactive MMP9, an enzyme involved in tissue remodelling. In addition, by interrogating microarray data from two cohorts of renal patients, we report increased VANGL2 transcript levels in the glomeruli of individuals with focal segmental glomerulosclerosis, suggesting that the molecule may also be involved in certain human glomerular diseases. These observations support the conclusion that Vangl2 modulates glomerular injury, at least in part by acting as a brake on MMP9, a potentially harmful endogenous enzyme. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Polaridad Celular , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Glomérulos Renales/metabolismo , Proteínas de la Membrana/metabolismo , Nefrosis Lipoidea/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Podocitos/metabolismo , Adulto , Animales , Estudios de Casos y Controles , Células Cultivadas , Modelos Animales de Enfermedad , Activación Enzimática , Femenino , Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/patología , Glomeruloesclerosis Focal y Segmentaria/fisiopatología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Glomérulos Renales/patología , Glomérulos Renales/fisiopatología , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Nefrosis Lipoidea/genética , Nefrosis Lipoidea/patología , Nefrosis Lipoidea/fisiopatología , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Podocitos/patología , Transducción de Señal , Adulto JovenRESUMEN
The arterial roots are important transitional regions of the heart, connecting the intrapericardial components of the aortic and pulmonary trunks with their ventricular outlets. They house the arterial (semilunar) valves and, in the case of the aorta, are the points of coronary arterial attachment. Moreover, because of the semilunar attachments of the valve leaflets, the arterial roots span the anatomic ventriculo-arterial junction. By virtue of this arrangement, the interleaflet triangles, despite being fibrous, are found on the ventricular aspect of the root and located within the left ventricular cavity. Malformations and diseases of the aortic root are common and serious. Despite the mouse being the animal model of choice for studying cardiac development, few studies have examined the structure of their arterial roots. As a consequence, our understanding of their formation and maturation is incomplete. We set out to clarify the anatomical and histological features of the mouse arterial roots, particularly focusing on their walls and the points of attachment of the valve leaflets. We then sought to determine the embryonic lineage relationships between these tissues, as a forerunner to understanding how they form and mature over time. Using histological stains and immunohistochemistry, we show that the walls of the mouse arterial roots show a gradual transition, with smooth muscle cells (SMC) forming the bulk of wall at the most distal points of attachments of the valve leaflets, while being entirely fibrous at their base. Although the interleaflet triangles lie within the ventricular chambers, we show that they are histologically indistinguishable from the arterial sinus walls until the end of gestation. Differences become apparent after birth, and are only completed by postnatal day 21. Using Cre-lox-based lineage tracing technology to label progenitor populations, we show that the SMC and fibrous tissue within the walls of the mature arterial roots share a common origin from the second heart field (SHF) and exclude trans-differentiation of myocardium as a source for the interleaflet triangle fibrous tissues. Moreover, we show that the attachment points of the leaflets to the walls, like the leaflets themselves, are derived from the outflow cushions, having contributions from both SHF-derived endothelial cells and neural crest cells. Our data thus show that the arterial roots in the mouse heart are similar to the features described in the human heart. They provide a framework for understanding complex lesions and diseases affecting the aortic root.
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Válvula Aórtica/anomalías , Válvula Aórtica/crecimiento & desarrollo , Cardiopatías Congénitas/embriología , Corazón/crecimiento & desarrollo , Válvula Pulmonar/anomalías , Válvula Pulmonar/crecimiento & desarrollo , Animales , Estenosis de la Válvula Aórtica/etiología , Estenosis de la Válvula Aórtica/patología , Técnica del Anticuerpo Fluorescente , Síndrome del Corazón Izquierdo Hipoplásico/etiología , Síndrome del Corazón Izquierdo Hipoplásico/patología , Ratones , Ratones Mutantes , Miocitos del Músculo Liso/fisiología , Cresta Neural/crecimiento & desarrolloRESUMEN
Planar cell polarity (PCP) is the mechanism by which cells orient themselves in the plane of an epithelium or during directed cell migration, and is regulated by a highly conserved signalling pathway. Mutations in the PCP gene Vangl2, as well as in other key components of the pathway, cause a spectrum of cardiac outflow tract defects. However, it is unclear why cells within the mesodermal heart tissue require PCP signalling. Using a new conditionally floxed allele we show that Vangl2 is required solely within the second heart field (SHF) to direct normal outflow tract lengthening, a process that is required for septation and normal alignment of the aorta and pulmonary trunk with the ventricular chambers. Analysis of a range of markers of polarised epithelial tissues showed that in the normal heart, undifferentiated SHF cells move from the dorsal pericardial wall into the distal outflow tract where they acquire an epithelial phenotype, before moving proximally where they differentiate into cardiomyocytes. Thus there is a transition zone in the distal outflow tract where SHF cells become more polarised, turn off progenitor markers and start to differentiate to cardiomyocytes. Membrane-bound Vangl2 marks the proximal extent of this transition zone and in the absence of Vangl2, the SHF-derived cells are abnormally polarised and disorganised. The consequent thickening, rather than lengthening, of the outflow wall leads to a shortened outflow tract. Premature down regulation of the SHF-progenitor marker Isl1 in the mutants, and accompanied premature differentiation to cardiomyocytes, suggests that the organisation of the cells within the transition zone is important for maintaining the undifferentiated phenotype. Thus, Vangl2-regulated polarisation and subsequent acquisition of an epithelial phenotype is essential to lengthen the tubular outflow vessel, a process that is essential for on-going cardiac morphogenesis.
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Ventrículos Cardíacos/embriología , Proteínas del Tejido Nervioso/fisiología , Animales , Diferenciación Celular , Polaridad Celular , Células Madre Embrionarias/fisiología , Epitelio/embriología , Ventrículos Cardíacos/citología , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfogénesis , Pericardio/embriología , FenotipoRESUMEN
At present, there is significant interest in the morphology of the coronary arteries, not least due to the increasingly well-recognised association between anomalous origin of the arteries and sudden cardiac death. Much has also been learnt over the last decade regarding the embryology of the arteries. In this review, therefore, we provide a brief introduction into the recent findings regarding their development. In particular, we emphasise that new evidence, derived using the developing murine heart, points to the arterial stems growing out from the adjacent sinuses of the aortic root, rather than the arteries growing in, as is currently assumed. As we show, the concept of outgrowth provides an excellent explanation for several of the abnormal arrangements encountered in the clinical setting. Before summarising these abnormal features, we draw attention to the need to describe the heart in an attitudinally appropriate manner, following the basic rule of human anatomy, rather than describing the cardiac components with the heart in the "Valentine" orientation. We then show how the major abnormalities involving the coronary arteries in humans can be summarised in terms of abnormal origin from the pulmonary circulation, abnormal aortic origin, or fistulous communications between the coronary arteries and the cardiac cavities. In the case of abnormal aortic origin, we highlight those malformations known to be associated with sudden cardiac death.
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Aorta Torácica/embriología , Anomalías de los Vasos Coronarios/embriología , Vasos Coronarios/embriología , Arteria Pulmonar/embriología , Animales , Aorta Torácica/anatomía & histología , Anomalías de los Vasos Coronarios/patología , Vasos Coronarios/anatomía & histología , Humanos , Ratones , Arteria Pulmonar/anatomía & histología , Circulación PulmonarRESUMEN
The evolutionarily conserved planar cell polarity (PCP) pathway (or noncanonical Wnt pathway) drives several important cellular processes, including epithelial cell polarization, cell migration and mitotic spindle orientation. In vertebrates, PCP genes have a vital role in polarized convergent extension movements during gastrulation and neurulation. Here we show that mice with mutations in genes involved in Bardet-Biedl syndrome (BBS), a disorder associated with ciliary dysfunction, share phenotypes with PCP mutants including open eyelids, neural tube defects and disrupted cochlear stereociliary bundles. Furthermore, we identify genetic interactions between BBS genes and a PCP gene in both mouse (Ltap, also called Vangl2) and zebrafish (vangl2). In zebrafish, the augmented phenotype results from enhanced defective convergent extension movements. We also show that Vangl2 localizes to the basal body and axoneme of ciliated cells, a pattern reminiscent of that of the BBS proteins. These data suggest that cilia are intrinsically involved in PCP processes.
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Síndrome de Bardet-Biedl/patología , Proteínas Asociadas a Microtúbulos/genética , Chaperonas Moleculares/genética , Proteínas del Tejido Nervioso/metabolismo , Animales , Síndrome de Bardet-Biedl/genética , Polaridad Celular/genética , Cilios/química , Cóclea/patología , Células Epiteliales/química , Párpados/fisiopatología , Chaperoninas del Grupo II , Ratones , Ratones Mutantes , Mutación , Proteínas del Tejido Nervioso/análisis , Defectos del Tubo Neural/patología , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Monk fruit (Siraitia grosvenorii) is an herbaceous perennial vine of the Cucurbitaceae family cultivated commercially mainly in southern China. There is very little information available about the fungal endophytes in monk fruit. In this study, monk fruit plants were grown from seeds in a research greenhouse at Kwantlen Polytechnic University in British Columbia, Canada to explore the abundance and diversity of their fungal endophytes. Fungal endophytes were isolated from seeds, seedlings, mature monk fruit plants, and fruits, and cultured on potato dextrose agar and water agar media. Isolates were identified by microscopic examination and BLAST comparison of ITS sequences to published sequences in GenBank. At least 150 species of fungal endophytes representing 60 genera and 20 orders were recovered from monk fruit tissues. Non-metric multidimensional scaling (NMDS) was carried out to explore the similarity of fungal communities among roots, stems, leaves, flowers, fruits, and seeds based on fungal orders. Our study showed that monk fruit plants are a rich source of fungal endophytes with the greatest abundance and diversity in leaves. This work has deepened our understanding of the intricate interactions between plants and fungi that sustain ecosystems and underpin plant health and resilience.
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eNOS (NOS3) is the enzyme that generates nitric oxide, a signalling molecule and regulator of vascular tone. Loss of eNOS function is associated with increased susceptibility to atherosclerosis, hypertension, thrombosis and stroke. Aortopathy and cardiac hypertrophy have also been found in eNOS null mice, but their aetiology is unclear. We evaluated eNOS nulls before and around birth for cardiac defects, revealing severe abnormalities in the ventricular myocardium and pharyngeal arch arteries. Moreover, in the aortic arch, there were fewer baroreceptors, which sense changes in blood pressure. Adult eNOS null survivors showed evidence of cardiac hypertrophy, aortopathy and cartilaginous metaplasia in the periductal region of the aortic arch. Notch1 and neuregulin were dysregulated in the forming pharyngeal arch arteries and ventricles, suggesting that these pathways may be relevant to the defects observed. Dysregulation of eNOS leads to embryonic and perinatal death, suggesting mutations in eNOS are candidates for causing congenital heart defects in humans. Surviving eNOS mutants have a deficiency of baroreceptors that likely contributes to high blood pressure and may have relevance to human patients who suffer from hypertension associated with aortic arch abnormalities.
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Embrión de Mamíferos , Cardiopatías Congénitas , Hipertensión , Ratones , Animales , Humanos , Corazón , Óxido Nítrico Sintasa de Tipo III/metabolismo , Aorta/metabolismo , Ratones Noqueados , CardiomegaliaRESUMEN
BACKGROUND: Epidemiological studies indicate a substantial excess familial recurrence of non-syndromic Tetralogy of Fallot (TOF), implicating genetic factors that remain largely unknown. The Rho induced kinase 1 gene (ROCK1) is a key component of the planar cell polarity signalling pathway, which plays an important role in normal cardiac development. The aim of this study was to investigate the role of genetic variation in ROCK1 on the risk of TOF. RESULTS: ROCK1 was sequenced in a discovery cohort of 93 non-syndromic TOF probands to identify rare variants. TagSNPs were selected to capture commoner variation in ROCK1. Novel variants and TagSNPs were genotyped in a discovery cohort of 458 TOF cases and 1331 healthy controls, and positive findings were replicated in a further 209 TOF cases and 1290 healthy controls. Association between genotypes and TOF was assessed using LAMP.A rare SNP (c.807C > T; rs56085230) discovered by sequencing was associated with TOF risk (p = 0.006) in the discovery cohort. The variant was also significantly associated with the risk of TOF in the replication cohort (p = 0.018). In the combined cohorts the odds ratio for TOF was 2.61 (95% CI 1.58-4.30); p < 0.0001. The minor allele frequency of rs56085230 in the cases was 0.02, and in the controls it was 0.007. The variant accounted for 1% of the population attributable risk (PAR) of TOF. We also found significant association with TOF for an uncommon TagSNP in ROCK1, rs288979 (OR 1.64 [95% CI 1.15-2.30]; p = 1.5x10â»5). The minor allele frequency of rs288979 in the controls was 0.043, and the variant accounted for 11% of the PAR of TOF. These association signals were independent of each other, providing additional internal validation of our result. CONCLUSIONS: Low frequency intermediate penetrance (LFIP) variants in the ROCK1 gene predispose to the risk of TOF.
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Predisposición Genética a la Enfermedad , Tetralogía de Fallot/genética , Quinasas Asociadas a rho/genética , Estudios de Casos y Controles , Estudios de Cohortes , Haplotipos , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
BACKGROUND AND PURPOSE: In the UK, there is a continuing effort within the National Health Service to reduce patient waiting times in emergency departments (EDs). This audit aimed to evaluate whether a reporting radiographer-led discharge system could reduce waiting times from x-ray to discharge with no detrimental effect on patient outcomes. METHODS: A prospective audit over 2 years was conducted. Patients were considered for discharge by a reporting radiographer-led service if they were >5 years old, attended the hospital ED between 9:00 and 17:00, Monday to Friday, had an injury below the elbow in the upper limb or below the knee in the lower limb that required an x-ray, and were able to be discharged home without further medical intervention. Outcomes of interest were overall waiting times, accuracy of diagnosis and re-attendance at the ED within 28 days. RESULTS: Between July 2006 and June 2008, 497 patients met the inclusion criteria and were discharged home by the radiographer-led service, and 2632 were discharged home using standard practices. Overall waiting times were >20 min quicker for the radiographer-led service at 100.9 min. The false negative rate was reduced from 2.09% to 0.2%, and re-attendance at the ED within 28 days for the same injury was reduced from 3.27% to only 0.4% for radiographer-led discharge. CONCLUSIONS: The service reduced waiting times and re-attendance rates while improving the accuracy of diagnosis. The efficacy of such services should be further studied in relation to more complex patient groups.