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1.
Int J Mol Sci ; 22(2)2021 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-33430070

RESUMEN

The nosocomial opportunistic Gram-negative bacterial pathogen Acinetobacter baumannii is resistant to multiple antimicrobial agents and an emerging global health problem. The polymyxin antibiotic colistin, targeting the negatively charged lipid A component of the lipopolysaccharide on the bacterial cell surface, is often considered as the last-resort treatment, but resistance to colistin is unfortunately increasing worldwide. Notably, colistin-susceptible A. baumannii can also develop a colistin dependence after exposure to this drug in vitro. Colistin dependence might represent a stepping stone to resistance also in vivo. However, the mechanisms are far from clear. To address this issue, we combined proteogenomics, high-resolution microscopy, and lipid profiling to characterize and compare A. baumannii colistin-susceptible clinical isolate (Ab-S) of to its colistin-dependent subpopulation (Ab-D) obtained after subsequent passages in moderate colistin concentrations. Incidentally, in the colistin-dependent subpopulation the lpxA gene was disrupted by insertion of ISAjo2, the lipid A biosynthesis terminated, and Ab-D cells displayed a lipooligosaccharide (LOS)-deficient phenotype. Moreover, both mlaD and pldA genes were perturbed by insertions of ISAjo2 and ISAba13, and LOS-deficient bacteria displayed a capsule with decreased thickness as well as other surface imperfections. The major changes in relative protein abundance levels were detected in type 6 secretion system (T6SS) components, the resistance-nodulation-division (RND)-type efflux pumps, and in proteins involved in maintenance of outer membrane asymmetry. These findings suggest that colistin dependence in A. baumannii involves an ensemble of mechanisms seen in resistance development and accompanied by complex cellular events related to insertional sequences (ISs)-triggered LOS-deficiency. To our knowledge, this is the first study demonstrating the involvement of ISAjo2 and ISAba13 IS elements in the modulation of the lipid A biosynthesis and associated development of dependence on colistin.

2.
Front Microbiol ; 11: 1069, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32523583

RESUMEN

Mitochondria play crucial roles in cellular metabolism, signaling, longevity, and immune defense. Recent evidences have revealed that the host microbiota, including bacterial pathogens, impact mitochondrial behaviors and activities in the host. The pathogenicity of Pseudomonas aeruginosa requires quorum sensing (QS) cell-cell communication allowing the bacteria to sense population density and collectively control biofilm development, virulence traits, adaptation and interactions with the host. QS molecules, like N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C12-HSL), can also modulate the behavior of host cells, e.g., epithelial barrier properties and innate immune responses. Here, in two types of cells, fibroblasts and intestinal epithelial cells, we investigated whether and how P. aeruginosa 3O-C12-HSL impacts the morphology of mitochondrial networks and their energetic characteristics, using high-resolution transmission electron microscopy, fluorescence live-cell imaging, assay for mitochondrial bioenergetics, and quantitative mass spectrometry for mitoproteomics and bioinformatics. We found that 3O-C12-HSL induced fragmentation of mitochondria, disruption of cristae and inner membrane ultrastructure, altered major characteristics of respiration and energetics, and decreased mitochondrial membrane potential, and that there are distinct cell-type specific details of these effects. Moreover, this was mechanistically accompanied by differential expression of both common and cell-type specific arrays of components in the mitochondrial proteome involved in their structural organization, electron transport chain complexes and response to stress. We suggest that this effect of 3O-C12-HSL on mitochondria may represent one of the events in the interaction between P. aeruginosa and host mitochondria and may have an impact on the pathogens strategy to hijack host cell activities to support their own survival and spreading.

3.
Reprod Domest Anim ; 55(3): 293-300, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31894881

RESUMEN

The avian seminal fluid (SF) is a protein-rich fluid, derived from the testis, the rudimentary epididymis and, finally, from the cloacal gland. The SF interacts with spermatozoa and the inner cell lining of the female genital tract, to modulate sperm functions and female immune responsiveness. Its complex proteome might either be free or linked to extracellular vesicles (EVs) as it is the case in mammals, where EVs depict the tetraspanin CD9; and where those EVs derived from the epididymis (epididymosomes) also present the receptor CD44. In the present study, sperm-free SF from Red Jungle Fowl, White Leghorn and an advanced intercross (AIL, 12th generation) were studied using flow cytometry of the membrane marker tetraspanin CD9, Western blotting of the membrane receptor CD44 and electron microscopy in non-enriched (whole SF) or enriched fractions obtained by precipitation using a commercial kit (Total Exosome Precipitation Solution). Neither CD9- nor CD44 could be detected, and the ultrastructure confirmed the relative absence of EVs, raising the possibility that avian SF interacts differently with the female genitalia as compared to the seminal plasma of mammals.


Asunto(s)
Vesículas Extracelulares , Semen , Animales , Western Blotting/veterinaria , Pollos/fisiología , Vesículas Extracelulares/ultraestructura , Citometría de Flujo/veterinaria , Receptores de Hialuranos/análisis , Masculino , Microscopía Electrónica , Especificidad de la Especie , Tetraspanina 29/análisis
4.
Angiogenesis ; 22(4): 553-567, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31486010

RESUMEN

Inhibiting pathologic angiogenesis can halt disease progression, but such inhibition may offer only a temporary benefit, followed by tissue revascularization after treatment stoppage. This revascularization, however, occurs by largely unknown phenotypic changes in pathologic vessels. To investigate the dynamics of vessel reconfiguration during revascularization, we developed a model of reversible murine corneal angiogenesis permitting longitudinal examination of the same vasculature. Following 30 days of angiogenesis inhibition, two types of vascular structure were evident: partially regressed persistent vessels that were degenerate and barely functional, and fully regressed, non-functional empty basement membrane sleeves (ebms). While persistent vessels maintained a limited flow and retained collagen IV+ basement membrane, CD31+ endothelial cells (EC), and α-SMA+ pericytes, ebms were acellular and expressed only collagen IV. Upon terminating angiogenesis inhibition, transmission electron microscopy and live imaging revealed that revascularization ensued by a rapid reversal of EC degeneracy in persistent vessels, facilitating their phenotypic normalization, vasodilation, increased flow, and subsequent new angiogenic sprouting. Conversely, ebms were irreversibly sealed from the circulation by excess collagen IV deposition that inhibited EC migration and prevented their reuse. Fully and partially regressed vessels therefore have opposing roles during revascularization, where fully regressed vessels inhibit new sprouting while partially regressed persistent vessels rapidly reactivate and serve as the source of continued pathologic angiogenesis.


Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Movimiento Celular/efectos de los fármacos , Neovascularización de la Córnea , Células Endoteliales , Pericitos , Animales , Membrana Basal/metabolismo , Membrana Basal/patología , Neovascularización de la Córnea/tratamiento farmacológico , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Masculino , Pericitos/metabolismo , Pericitos/patología , Ratas , Ratas Wistar
5.
Arterioscler Thromb Vasc Biol ; 39(7): 1402-1418, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31242036

RESUMEN

Objective- Pathological neovascularization is crucial for progression and morbidity of serious diseases such as cancer, diabetic retinopathy, and age-related macular degeneration. While mechanisms of ongoing pathological neovascularization have been extensively studied, the initiating pathological vascular remodeling (PVR) events, which precede neovascularization remains poorly understood. Here, we identify novel molecular and cellular mechanisms of preneovascular PVR, by using the adult choriocapillaris as a model. Approach and Results- Using hypoxia or forced overexpression of VEGF (vascular endothelial growth factor) in the subretinal space to induce PVR in zebrafish and rats respectively, and by analyzing choriocapillaris membranes adjacent to choroidal neovascular lesions from age-related macular degeneration patients, we show that the choriocapillaris undergo robust induction of vascular intussusception and permeability at preneovascular stages of PVR. This PVR response included endothelial cell proliferation, formation of endothelial luminal processes, extensive vesiculation and thickening of the endothelium, degradation of collagen fibers, and splitting of existing extravascular columns. RNA-sequencing established a role for endothelial tight junction disruption, cytoskeletal remodeling, vesicle- and cilium biogenesis in this process. Mechanistically, using genetic gain- and loss-of-function zebrafish models and analysis of primary human choriocapillaris endothelial cells, we determined that HIF (hypoxia-induced factor)-1α-VEGF-A-VEGFR2 signaling was important for hypoxia-induced PVR. Conclusions- Our findings reveal that PVR involving intussusception and splitting of extravascular columns, endothelial proliferation, vesiculation, fenestration, and thickening is induced before neovascularization, suggesting that identifying and targeting these processes may prevent development of advanced neovascular disease in the future. Visual Overview- An online visual overview is available for this article.


Asunto(s)
Neovascularización Patológica/etiología , Remodelación Vascular/fisiología , Adulto , Animales , Humanos , Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Degeneración Macular/etiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiología , Pez Cebra
6.
Haematologica ; 104(7): 1482-1492, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-30630981

RESUMEN

As platelet activation is an irreversible and potentially harmful event, platelet stimulatory signaling must be tightly regulated to ensure the filtering-out of inconsequential fluctuations of agonist concentrations in the vascular milieu. Herein, we show that platelet activation via G protein-coupled receptors is gradient-dependent, i.e., determined not only by agonist concentrations per se but also by how rapidly concentrations change over time. We demonstrate that gradient-dependent inhibition is a common feature of all major platelet stimulatory G protein-coupled receptors, while platelet activation via the non-G protein-coupled receptor glycoprotein VI is strictly concentration-dependent. By systematically characterizing the effects of variations in temporal agonist concentration gradients on different aspects of platelet activation, we demonstrate that gradient-dependent inhibition of protease-activated receptors exhibits different kinetics, with platelet activation occurring at lower agonist gradients for protease-activated receptor 4 than for protease-activated receptor 1, but shares a characteristic bimodal effect distribution, as gradient-dependent inhibition increases over a narrow range of gradients, below which aggregation and granule secretion is effectively shut off. In contrast, the effects of gradient-dependent inhibition on platelet activation via adenosine diphosphate and thromboxane receptors increase incrementally over a large range of gradients. Furthermore, depending on the affected activation pathway, gradient-dependent inhibition results in different degrees of refractoriness to subsequent autologous agonist stimulation. Mechanistically, our study identifies an important role for the cyclic adenosine monophosphate-dependent pathway in gradient-dependent inhibition. Together, our findings suggest that gradient-dependent inhibition may represent a new general mechanism for hemostatic regulation in platelets.


Asunto(s)
Adenosina Difosfato/farmacología , Plaquetas/metabolismo , AMP Cíclico/farmacología , Activación Plaquetaria/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Adulto , Plaquetas/efectos de los fármacos , Epoprostenol/farmacología , Humanos , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , Trombina/metabolismo , Tromboxano A2/metabolismo
7.
Electromagn Biol Med ; 32(1): 95-120, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23320614

RESUMEN

This study was designed to investigate the transient and cumulative impairments in spatial and non-spatial memory of C57Bl/6J mice exposed to GSM 1.8 GHz signal for 90 min daily by a typical cellular (mobile) phone at a specific absorption rate value of 0.11 W/kg. Free-moving male mice 2 months old were irradiated in two experimental protocols, lasting for 66 and for 148 days respectively. Each protocol used three groups of animals (n = 8 each for exposed, sham exposed and controls) in combination with two behavioural paradigms, the object recognition task and the object location task sequentially applied at different time points. One-way analysis of variance revealed statistically significant impairments of both types of memory gradually accumulating, with more pronounced effects on the spatial memory. The impairments persisted even 2 weeks after interruption of the 8 weeks daily exposure, whereas the memory of mice as detected by both tasks showed a full recovery approximately 1 month later. Intermittent every other day exposure for 1 month had no effect on both types of memory. The data suggest that visual information processing mechanisms in hippocampus, perirhinal and entorhinal cortex are gradually malfunctioning upon long-term daily exposure, a phenotype that persists for at least 2 weeks after interruption of radiation, returning to normal memory performance levels 4 weeks later. It is postulated that cellular repair mechanisms are operating to eliminate the memory affecting molecules. The overall contribution of several possible mechanisms to the observed cumulative and transient impairments in spatial and non-spatial memory is discussed.


Asunto(s)
Teléfono Celular , Memoria/fisiología , Memoria/efectos de la radiación , Percepción Espacial/fisiología , Percepción Espacial/efectos de la radiación , Absorción , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Reconocimiento en Psicología/efectos de la radiación , Factores de Tiempo
8.
J Neurosci ; 30(20): 6838-51, 2010 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-20484626

RESUMEN

alpha-Synuclein is central in Parkinson's disease pathogenesis. Although initially alpha-synuclein was considered a purely intracellular protein, recent data suggest that it can be detected in the plasma and CSF of humans and in the culture media of neuronal cells. To address a role of secreted alpha-synuclein in neuronal homeostasis, we have generated wild-type alpha-synuclein and beta-galactosidase inducible SH-SY5Y cells. Soluble oligomeric and monomeric species of alpha-synuclein are readily detected in the conditioned media (CM) of these cells at concentrations similar to those observed in human CSF. We have found that, in this model, alpha-synuclein is secreted by externalized vesicles in a calcium-dependent manner. Electron microscopy and liquid chromatography-mass spectrometry proteomic analysis demonstrate that these vesicles have the characteristic hallmarks of exosomes, secreted intraluminar vesicles of multivesicular bodies. Application of CM containing secreted alpha-synuclein causes cell death of recipient neuronal cells, which can be reversed after alpha-synuclein immunodepletion from the CM. High- and low-molecular-weight alpha-synuclein species, isolated from this CM, significantly decrease cell viability. Importantly, treatment of the CM with oligomer-interfering compounds before application rescues the recipient neuronal cells from the observed toxicity. Our results show for the first time that cell-produced alpha-synuclein is secreted via an exosomal, calcium-dependent mechanism and suggest that alpha-synuclein secretion serves to amplify and propagate Parkinson's disease-related pathology.


Asunto(s)
Calcio/metabolismo , Exosomas/fisiología , Cuerpos Multivesiculares/patología , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Análisis de Varianza , Animales , Brefeldino A/farmacología , Calcio/farmacología , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Corteza Cerebral/citología , Cromatografía Líquida de Alta Presión/métodos , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Pruebas Inmunológicas de Citotoxicidad/métodos , Relación Dosis-Respuesta a Droga , Endocitosis/efectos de los fármacos , Exosomas/ultraestructura , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Inmunoprecipitación/métodos , Espectrometría de Masas/métodos , Microscopía Confocal/métodos , Microscopía Electrónica de Transmisión/métodos , Peso Molecular , Cuerpos Multivesiculares/efectos de los fármacos , Cuerpos Multivesiculares/ultraestructura , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma/patología , Neuroblastoma/ultraestructura , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/ultraestructura , Péptidos/farmacología , Piperidinas/farmacología , Presenilina-1/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Pirazoles/farmacología , Ratas , Receptores de Transferrina/metabolismo , Suero/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructura , Temperatura , Transfección , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo
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