RESUMEN
Understanding mechanisms underlying various physiological and pathological processes often requires accurate and fully automated analysis of dense cell populations that collectively migrate. In such multicellular systems, there is a rising interest in the relations between biophysical and cell cycle progression aspects. A seminal tool that led to a leap in real-time study of cell cycle is the fluorescent ubiquitination-based cell cycle indicator (FUCCI). Here, we introduce ConfluentFUCCI, an open-source graphical user interface-based framework that is designed, unlike previous tools, for fully automated analysis of cell cycle progression, cellular dynamics, and cellular morphology, in highly dense migrating cell collectives. We integrated into ConfluentFUCCI's pipeline state-of-the-art tools such as Cellpose, TrackMate, and Napari, some of which incorporate deep learning, and we wrap the entire tool into an isolated computational environment termed container. This provides an easy installation and workflow that is independent of any specific operation system. ConfluentFUCCI offers accurate nuclear segmentation and tracking using FUCCI tags, enabling comprehensive investigation of cell cycle progression at both the tissue and single-cell levels. We compare ConfluentFUCCI to the most recent relevant tool, showcasing its accuracy and efficiency in handling large datasets. Furthermore, we demonstrate the ability of ConfluentFUCCI to monitor cell cycle transitions, dynamics, and morphology within densely packed epithelial cell populations, enabling insights into mechanotransductive regulation of cell cycle progression. The presented tool provides a robust approach for investigating cell cycle-related phenomena in complex biological systems, offering potential applications in cancer research and other fields.
Asunto(s)
Ciclo Celular , Movimiento Celular , Ciclo Celular/fisiología , Movimiento Celular/fisiología , Humanos , Programas Informáticos , Ubiquitinación , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Age is a significant risk factor for the coronavirus disease 2019 (COVID-19) severity due to immunosenescence and certain age-dependent medical conditions (e.g., obesity, cardiovascular disorder, and chronic respiratory disease). However, despite the well-known influence of age on autoantibody biology in health and disease, its impact on the risk of developing severe COVID-19 remains poorly explored. Here, we performed a cross-sectional study of autoantibodies directed against 58 targets associated with autoimmune diseases in 159 individuals with different COVID-19 severity (71 mild, 61 moderate, and 27 with severe symptoms) and 73 healthy controls. We found that the natural production of autoantibodies increases with age and is exacerbated by SARS-CoV-2 infection, mostly in severe COVID-19 patients. Multiple linear regression analysis showed that severe COVID-19 patients have a significant age-associated increase of autoantibody levels against 16 targets (e.g., amyloid ß peptide, ß catenin, cardiolipin, claudin, enteric nerve, fibulin, insulin receptor a, and platelet glycoprotein). Principal component analysis with spectrum decomposition and hierarchical clustering analysis based on these autoantibodies indicated an age-dependent stratification of severe COVID-19 patients. Random forest analysis ranked autoantibodies targeting cardiolipin, claudin, and platelet glycoprotein as the three most crucial autoantibodies for the stratification of severe COVID-19 patients ≥50 years of age. Follow-up analysis using binomial logistic regression found that anti-cardiolipin and anti-platelet glycoprotein autoantibodies significantly increased the likelihood of developing a severe COVID-19 phenotype with aging. These findings provide key insights to explain why aging increases the chance of developing more severe COVID-19 phenotypes.
RESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with increased levels of autoantibodies targeting immunological proteins such as cytokines and chemokines. Reports further indicate that COVID-19 patients may develop a broad spectrum of autoimmune diseases due to reasons not fully understood. Even so, the landscape of autoantibodies induced by SARS-CoV-2 infection remains uncharted territory. To gain more insight, we carried out a comprehensive assessment of autoantibodies known to be linked to diverse autoimmune diseases observed in COVID-19 patients in a cohort of 231 individuals, of which 161 were COVID-19 patients (72 with mild, 61 moderate, and 28 with severe disease) and 70 were healthy controls. Dysregulated IgG and IgA autoantibody signatures, characterized mainly by elevated concentrations, occurred predominantly in patients with moderate or severe COVID-19 infection. Autoantibody levels often accompanied anti-SARS-CoV-2 antibody concentrations while stratifying COVID-19 severity as indicated by random forest and principal component analyses. Furthermore, while young versus elderly COVID-19 patients showed only slight differences in autoantibody levels, elderly patients with severe disease presented higher IgG autoantibody concentrations than young individuals with severe COVID-19. This work maps the intersection of COVID-19 and autoimmunity by demonstrating the dysregulation of multiple autoantibodies triggered during SARS-CoV-2 infection. Thus, this cross-sectional study suggests that SARS-CoV-2 infection induces autoantibody signatures associated with COVID-19 severity and several autoantibodies that can be used as biomarkers of COVID-19 severity, indicating autoantibodies as potential therapeutical targets for these patients.
Asunto(s)
Enfermedades Autoinmunes , COVID-19 , Anciano , Humanos , Autoanticuerpos , Estudios Transversales , SARS-CoV-2 , Inmunoglobulina GRESUMEN
BACKGROUND: COVID-19 is a heterogenous disease resulting in long-term sequela in predisposed individuals. It is not uncommon that recovering patients endure non-respiratory ill-defined manifestations, including anosmia, and neurological and cognitive deficit persisting beyond recovery-a constellation of conditions that are grouped under the umbrella of long-term COVID-19 syndrome. Association between COVID-19 and autoimmune responses in predisposed individuals was shown in several studies. AIM AND METHODS: To investigate autoimmune responses against neuronal and CNS autoantigens in SARS-CoV-2-infected patients, we performed a cross-sectional study with 246 participants, including 169 COVID-19 patients and 77 controls. Levels of antibodies against the acetylcholine receptor, glutamate receptor, amyloid ß peptide, alpha-synucleins, dopamine 1 receptor, dopamine 2 receptor, tau protein, GAD-65, N-methyl D-aspartate (NMDA) receptor, BDNF, cerebellar, ganglioside, myelin basic protein, myelin oligodendrocyte glycoprotein, S100-B, glial fibrillary acidic protein, and enteric nerve were measured using an Enzyme-Linked Immunosorbent Assay (ELISA). Circulating levels of autoantibodies were compared between healthy controls and COVID-19 patients and then classified by disease severity (mild [n = 74], severe [n = 65], and requiring supplemental oxygen [n = 32]). RESULTS: COVID-19 patients were found to have dysregulated autoantibody levels correlating with the disease severity, e.g., IgG to dopamine 1 receptor, NMDA receptors, brain-derived neurotrophic factor, and myelin oligodendrocyte glycoprotein. Elevated levels of IgA autoantibodies against amyloid ß peptide, acetylcholine receptor, dopamine 2 receptor, myelin basic protein, and α-synuclein were detected in COVID-19 patients compared with healthy controls. Lower IgA autoantibody levels against NMDA receptors, and IgG autoantibodies against glutamic acid decarboxylase 65, amyloid ß peptide, tau protein, enteric nerve, and S100-B were detected in COVID-19 patients versus healthy controls. Some of these antibodies have known clinical correlations with symptoms commonly reported in the long COVID-19 syndrome. CONCLUSIONS: Overall, our study shows a widespread dysregulation in the titer of various autoantibodies against neuronal and CNS-related autoantigens in convalescent COVID-19 patients. Further research is needed to provide insight into the association between these neuronal autoantibodies and the enigmatic neurological and psychological symptoms reported in COVID-19 patients.
RESUMEN
COVID-19 shares the feature of autoantibody production with systemic autoimmune diseases. In order to understand the role of these immune globulins in the pathogenesis of the disease, it is important to explore the autoantibody spectra. Here we show, by a cross-sectional study of 246 individuals, that autoantibodies targeting G protein-coupled receptors (GPCR) and RAS-related molecules associate with the clinical severity of COVID-19. Patients with moderate and severe disease are characterized by higher autoantibody levels than healthy controls and those with mild COVID-19 disease. Among the anti-GPCR autoantibodies, machine learning classification identifies the chemokine receptor CXCR3 and the RAS-related molecule AGTR1 as targets for antibodies with the strongest association to disease severity. Besides antibody levels, autoantibody network signatures are also changing in patients with intermediate or high disease severity. Although our current and previous studies identify anti-GPCR antibodies as natural components of human biology, their production is deregulated in COVID-19 and their level and pattern alterations might predict COVID-19 disease severity.
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Autoanticuerpos/inmunología , COVID-19/inmunología , Receptores Acoplados a Proteínas G/inmunología , Sistema Renina-Angiotensina/inmunología , Autoanticuerpos/sangre , Autoinmunidad , Biomarcadores/sangre , COVID-19/sangre , COVID-19/clasificación , Estudios Transversales , Femenino , Humanos , Aprendizaje Automático , Masculino , Análisis Multivariante , Receptor de Angiotensina Tipo 1/inmunología , Receptores CXCR3/inmunología , SARS-CoV-2 , Índice de Severidad de la EnfermedadRESUMEN
Biomaterials folded into nanoparticles (NPs) can be utilized as targeted drug delivery systems for cancer therapy. NPs may provide a vehicle for the anticancer drug lonidamine (LND), which inhibits glycolysis but was suspended from use at the clinical trial stage because of its hepatotoxicity due to poor solubility and pharmacokinetic properties. The NPs prepared by coassembly of the anionic polypeptide poly gamma glutamic acid (γ-PGA) and a designed amphiphilic and positively charged peptide (designated as mPoP-NPs) delivered LND to the mitochondria in cell cultures. In this study, we demonstrate that LND-mPoP-NP effective drug concentrations can be increased to reach therapeutically relevant concentrations. The self-assembled NP solution was subjected to snap-freezing and lyophilization and the resultant powder was redissolved in a tenth of the original volume. The NP size and their ability to target the proximity of the mitochondria of breast cancer cells were both maintained in this new formulation, C-LND-mPoP-NPs. Furthermore, these NPs exhibited 40% better cytotoxicity, relative to the nonlyophilized LND-mPoP-NPs and led to tumor growth inhibition with no adverse side effects upon intravenous administration in a xenograft breast cancer murine model.
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Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Indazoles/uso terapéutico , Nanopartículas/uso terapéutico , Péptidos/uso terapéutico , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Indazoles/farmacología , Ratones Endogámicos BALB C , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Nanopartículas/ultraestructura , Péptidos/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lateral heterogeneity of cell membranes has been demonstrated in numerous studies showing anomalous diffusion of membrane proteins; it has been explained by models and experiments suggesting dynamic barriers to free diffusion, that temporarily confine membrane proteins into microscopic patches. This picture, however, comes short of explaining a steady-state patchy distribution of proteins, in face of the transient opening of the barriers. In our previous work we directly imaged persistent clusters of MHC-I, a type I transmembrane protein, and proposed a model of a dynamic equilibrium between proteins newly delivered to the cell surface by vesicle traffic, temporary confinement by dynamic barriers to lateral diffusion, and dispersion of the clusters by diffusion over the dynamic barriers. Our model predicted that the clusters are dynamic, appearing when an exocytic vesicle fuses with the plasma membrane and dispersing with a typical lifetime that depends on lateral diffusion and the dynamics of barriers. In a subsequent work, we showed this to be the case. Here we test another prediction of the model, and show that changing the stability of actin barriers to lateral diffusion changes cluster lifetimes. We also develop a model for the distribution of cluster lifetimes, consistent with the function of barriers to lateral diffusion in maintaining MHC-I clusters.
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Citoesqueleto de Actina/metabolismo , Antígenos HLA/química , Antígenos HLA/metabolismo , Modelos Moleculares , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Ratones , Faloidina/metabolismo , Estabilidad Proteica , Tiazolidinas/metabolismoRESUMEN
We test here a previously proposed hypothesis about lateral heterogeneity of cell membranes, a model predicting that heterogeneity is maintained by a combination of delivery and intake of molecules with barriers to lateral free diffusion. To test the validity of the model, we observed green fluorescent protein tagged major histocompatibility complex class I patches on the plasma membrane of mouse fibroblasts, using total internal reflection fluorescence microscopy in real time. The dynamic characterization revealed the life course of these patches comprises delivery of molecules at a short instant, followed by a slow, exponential decay, corresponding to diffusion of the molecules over dynamic barriers to free lateral diffusion. The characteristic lifetime of the patches, extracted from the measurements, is approximately 30 s, in excellent agreement with the predictions of the model.