Biology subjects

Salgado, D.

Publications and source records attributed to Salgado, D..

5 recordsLinked to original sources

bioRxiv2023

The Escherichia coli TolC efflux pump protein is immunogenic and elicits protective antibodies

Antimicrobial resistance is an increasing worldwide public health burden that threatens to make the existent antimicrobials obsolete. Among the mechanisms of antimicrobial resistance is the overexpression of efflux pumps, such as the AcrA-AcrB-TolC which extrude diverse compounds, reducing the intracellular concentration of antimicrobials. TolC is the outer membrane protein of this pump and has recently gained attention as a therapeutic target. However, little is known about the immune response generated against the TolC protein. Here we evaluated the cellular and humoral immune response against the TolC from Escherichia coli. An in silico epitope prediction of the E. coli TolC showed several residues could bind to human antibodies, and we showed that human plasma presented anti-TolC IgG and IgA antibodies. Gram-negative infected patients presented a slight increase in anti-TolC IgM amounts, compared to controls. Recombinant E. coli TolC protein stimulated macrophages in vitro to produce nitric oxide, as well as IL-6 and TNF-, assessed by Griess assay and ELISA, respectively. Immunization of mice with TolC intraperitoneally and an in vitro re-stimulation of lymph node cells led to increased percentage of T cell proliferation and IFN{gamma} production, evaluated by flow cytometry and ELISA, respectively. We observed that TolC mouse immunization stimulated anti-TolC IgM and IgG production, with a higher level of IgG1 and IgG2, amongst the IgG subclasses. Finally, TolC IgG from mouse immune serum could bind to live E. coli, increase bacterial uptake by macrophages in vitro. TolC immunized mice had a survival rate increased in 60% post infection with E. coli. Our results showed that TolC is immunogenic, activating macrophages, T and B cells, leading to the production of protective antibodies against E. coli.

immunology↗

bioRxiv2020

Hox-dependent coordination of cardiac progenitor cell patterning and differentiation

Perturbation of addition of second heart field (SHF) cardiac progenitor cells to the poles of the heart tube results in congenital heart defects (CHD). The transcriptional programs and upstream regulatory events operating in different subpopulations of the SHF remain unclear. Here, we profile the transcriptome and chromatin accessibility of anterior and posterior SHF sub-populations at genome-wide levels and demonstrate that Hoxb1 negatively regulates differentiation in the posterior SHF. Spatial mis-expression of Hoxb1 in the anterior SHF results in hypoplastic right ventricle. Activation of Hoxb1 in embryonic stem cells arrests cardiac differentiation, whereas Hoxb1-deficient embryos display premature cardiac differentiation. Moreover, ectopic differentiation in the posterior SHF of embryos lacking both Hoxb1 and its paralog Hoxa1 results in atrioventricular septal defects. Our results show that Hoxb1 plays a key role in patterning cardiac progenitor cells that contribute to both cardiac poles and provide new insights into the pathogenesis of CHD.

developmental biology↗

bioRxiv2019

Auto-inhibition of myoblast fusion by cyclic receptor signalling.

Fusion of nascent myoblasts to pre-existing myofibres is critical for skeletal muscle growth and repair. The vast majority of molecules known to regulate myoblast fusion are necessary in this process. Here we uncover, through high-throughput in vitro assays and in vivo studies in the chicken embryo, that TGF{beta} (SMAD2/3-dependent) signalling acts as a molecular brake on muscle fusion. While constitutive activation of the pathway arrests fusion, its inhibition leads to a striking over-fusion phenotype. This dynamic control of TGF{beta} signalling in the embryonic muscle relies on a unique receptor complementation mechanism, prompted by the merging of myoblasts with myofibres, each carrying one component of the heterodimer receptor complex. The competence of myofibres to fuse is restored through endocytic degradation of activated receptors. Altogether, this study shows that muscle fusion is a self-regulated process that relies on cyclic TGF{beta} signalling to regulate its pace.

developmental biology↗

bioRxiv2019

Piezo1 is required for outflow tract and aortic valve development.

Aims-During embryogenesis, the onset of circulatory blood flow generates a variety of hemodynamic forces which reciprocally induce changes in cardiovascular development and performance. It has been known for some time that these forces can be detected by as yet unknown mechanosensory systems which in turn promote cardiogenic events such as outflow tract and aortic valve development. PIEZO1 is a mechanosensitive ion channel present in endothelial cells where it serves to detect hemodynamic forces making it an ideal candidate to play a role during cardiac development. We sought to determine whether PIEZO1 is required for outflow tract and aortic valve development. Methods and results-By analysing heart development in zebrafish we have determined that piezo1 is expressed in the developing outflow tract where it serves to detect hemodynamic forces. In particular, we have found that mechanical forces generated during the cardiac cycle activate Piezo1 which triggers nitric oxide to be released in the outflow tract. Consequently, disrupting Piezo1 signalling leads to defective outflow tract and aortic valve development and indicates this gene may be involved in the etiology of congenital heart diseases. Based on these findings, we analysed genomic data generated from a cohort of bicuspid aortic valve patients and identified 3 probands who each harboured a novel variant in PIEZO1. Subsequent in vitro and in vivo assays indicates that these variants behave as dominant negatives leading to an inhibition of normal PIEZO1 mechanosensory activity and defective aortic valve development. Conclusion-These data indicate that the mechanosensitive ion channel piezo1 is required for OFT and aortic valve development and, furthermore, dominant negative variants of PIEZO1 appear to be associated with BAV in humans.

developmental biology↗

bioRxiv2018

A human cell model of cardiac pathophysiological valvulogenesis

Genetically modified mice have advanced our understanding of valve development and related pathologies. Yet, little is known regarding human valvulogenesis in health and diseases. Genuine human in vitro models that reproduce valvular (patho)biology are thus needed. We here developed a human pluripotent stem cell-derived model fit to decode the early steps of human valvulogenesis and to recapitulate valve disease traits in a dish.\n\nUsing cellular based, single cell omics-informed and in vivo-validated approaches, we derived a population of pre-valvular endocardial cells from a pluripotent stem cell source. These human prevalvular cells (HPVCs) expressed gene patterns conforming to the atrio-ventricular canal (AVC) endocardium signature originally established in E9.0 mouse embryos. In fact, HPVC treated with BMP2, cultured onto mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, underwent endothelial-to-mesenchymal transition and expressed markers of valve interstitial cells of different valvular layers demonstrating tissue functionality. HPVCs also differentiated into tendinous/chondrogenic cells in line with the valvular repertoire. Extending this valvulogenic model to patient specific iPS cells, we recapitulated features of mitral valve prolapse and uncovered that dysregulation of the SHH pathway is likely to be at the origin of the disease thus providing a putative therapeutic target.\n\nHuman pluripotent stem cells recapitulate early valvulogenesis and provide a powerful model to systematically decipher the origin and lineage contribution of different valvular cell types in humans as well as to study valve diseases in a dish.

developmental biology↗