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1.
Angew Chem Int Ed Engl ; 62(43): e202311590, 2023 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-37675854

RESUMEN

The combination of gene therapy and immunotherapy concepts, along recent advances in DNA nanotechnology, have the potential to provide important tools for cancer therapies. We present the development of stimuli-responsive microcapsules, loaded with a viral immunogenetic agent, harnessing the immune response against the Coronavirus Disease 2019, COVID-19, to selectively attack liver cancer cells (hepatoma) or recognize breast cancer or hepatoma, by expression of green fluorescence protein, GFP. The pH-responsive microcapsules, modified with DNA-tetrahedra nanostructures, increased hepatoma permeation by 50 %. Incorporation of a GFP-encoding lentivirus vector inside the tumor-targeting pH-stimulated miRNA-triggered and Alpha-fetoprotein-dictated microcapsules enables the demonstration of neoplasm selectivity, with approximately 5,000-, 8,000- and 50,000-fold more expression in the cancerous cells, respectively. The incorporation of the SARS-CoV-2 spike protein in the gene vector promotes specific recognition of the immune-evading hepatoma by the COVID-19-analogous immune response, which leads to cytotoxic and inflammatory activity, mediated by serum components taken from vaccinated or recovered COVID-19 patients, resulting in effective elimination of the hepatoma (>85 % yield).

2.
Nat Biomed Eng ; 7(11): 1493-1513, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-37550423

RESUMEN

The study of cardiac physiology is hindered by physiological differences between humans and small-animal models. Here we report the generation of multi-chambered self-paced vascularized human cardiac organoids formed under anisotropic stress and their applicability to the study of cardiac arrhythmia. Sensors embedded in the cardiac organoids enabled the simultaneous measurement of oxygen uptake, extracellular field potentials and cardiac contraction at resolutions higher than 10 Hz. This microphysiological system revealed 1 Hz cardiac respiratory cycles that are coupled to the electrical rather than the mechanical activity of cardiomyocytes. This electro-mitochondrial coupling was driven by mitochondrial calcium oscillations driving respiration cycles. Pharmaceutical or genetic inhibition of this coupling results in arrhythmogenic behaviour. We show that the chemotherapeutic mitoxantrone induces arrhythmia through disruption of this pathway, a process that can be partially reversed by the co-administration of metformin. Our microphysiological cardiac systems may further facilitate the study of the mitochondrial dynamics of cardiac rhythms and advance our understanding of human cardiac physiology.


Asunto(s)
Fenómenos Bioquímicos , Miocitos Cardíacos , Animales , Humanos , Miocitos Cardíacos/metabolismo , Arritmias Cardíacas , Contracción Miocárdica/fisiología , Organoides
3.
Elife ; 122023 01 27.
Artículo en Inglés | MEDLINE | ID: mdl-36705566

RESUMEN

Background: Viral infection is associated with a significant rewire of the host metabolic pathways, presenting attractive metabolic targets for intervention. Methods: We chart the metabolic response of lung epithelial cells to SARS-CoV-2 infection in primary cultures and COVID-19 patient samples and perform in vitro metabolism-focused drug screen on primary lung epithelial cells infected with different strains of the virus. We perform observational analysis of Israeli patients hospitalized due to COVID-19 and comparative epidemiological analysis from cohorts in Italy and the Veteran's Health Administration in the United States. In addition, we perform a prospective non-randomized interventional open-label study in which 15 patients hospitalized with severe COVID-19 were given 145 mg/day of nanocrystallized fenofibrate added to the standard of care. Results: SARS-CoV-2 infection produced transcriptional changes associated with increased glycolysis and lipid accumulation. Metabolism-focused drug screen showed that fenofibrate reversed lipid accumulation and blocked SARS-CoV-2 replication through a PPARα-dependent mechanism in both alpha and delta variants. Analysis of 3233 Israeli patients hospitalized due to COVID-19 supported in vitro findings. Patients taking fibrates showed significantly lower markers of immunoinflammation and faster recovery. Additional corroboration was received by comparative epidemiological analysis from cohorts in Europe and the United States. A subsequent prospective non-randomized interventional open-label study was carried out on 15 patients hospitalized with severe COVID-19. The patients were treated with 145 mg/day of nanocrystallized fenofibrate in addition to standard-of-care. Patients receiving fenofibrate demonstrated a rapid reduction in inflammation and a significantly faster recovery compared to patients admitted during the same period. Conclusions: Taken together, our data suggest that pharmacological modulation of PPARα should be strongly considered as a potential therapeutic approach for SARS-CoV-2 infection and emphasizes the need to complete the study of fenofibrate in large randomized controlled clinical trials. Funding: Funding was provided by European Research Council Consolidator Grants OCLD (project no. 681870) and generous gifts from the Nikoh Foundation and the Sam and Rina Frankel Foundation (YN). The interventional study was supported by Abbott (project FENOC0003). Clinical trial number: NCT04661930.


Asunto(s)
COVID-19 , Fenofibrato , Humanos , Fenofibrato/uso terapéutico , Lípidos , PPAR alfa , Estudios Prospectivos , SARS-CoV-2 , Resultado del Tratamiento
4.
Lab Chip ; 22(23): 4469-4480, 2022 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-36281785

RESUMEN

Aminoglycosides are an important class of antibiotics that play a critical role in the treatment of life-threatening infections, but their use is limited by their toxicity. In fact, gentamicin causes severe nephrotoxicity in 17% of hospitalized patients. The kidney proximal tubule is particularly vulnerable to drug-induced nephrotoxicity due to its role in drug transport. In this work, we developed a perfused vascularized model of human kidney tubuloids integrated with tissue-embedded microsensors that track the metabolic dynamics of aminoglycoside-induced renal toxicity in real time. Our model shows that gentamicin disrupts proximal tubule polarity at concentrations 20-fold below its TC50, leading to a 3.2-fold increase in glucose uptake, and reverse TCA cycle flux culminating in a 40-fold increase in lipid accumulation. Blocking glucose reabsorption using the SGLT2 inhibitor empagliflozin significantly reduced gentamicin toxicity by 10-fold. These results demonstrate the utility of sensor-integrated kidney-on-chip platforms to rapidly identify new metabolic mechanisms that may underly adverse drug reactions. The results should improve our ability to modulate the toxicity of novel aminoglycosides.


Asunto(s)
Aminoglicósidos , Antibacterianos , Humanos , Aminoglicósidos/toxicidad , Aminoglicósidos/metabolismo , Antibacterianos/toxicidad , Gentamicinas/toxicidad , Riñón/metabolismo , Túbulos Renales Proximales/metabolismo
5.
Elife ; 102021 10 25.
Artículo en Inglés | MEDLINE | ID: mdl-34694226

RESUMEN

Severe acute respiratory syndrome (SARS)-CoV-2 infection leads to severe disease associated with cytokine storm, vascular dysfunction, coagulation, and progressive lung damage. It affects several vital organs, seemingly through a pathological effect on endothelial cells. The SARS-CoV-2 genome encodes 29 proteins, whose contribution to the disease manifestations, and especially endothelial complications, is unknown. We cloned and expressed 26 of these proteins in human cells and characterized the endothelial response to overexpression of each, individually. Whereas most proteins induced significant changes in endothelial permeability, nsp2, nsp5_c145a (catalytic dead mutant of nsp5), and nsp7 also reduced CD31, and increased von Willebrand factor expression and IL-6, suggesting endothelial dysfunction. Using propagation-based analysis of a protein-protein interaction (PPI) network, we predicted the endothelial proteins affected by the viral proteins that potentially mediate these effects. We further applied our PPI model to identify the role of each SARS-CoV-2 protein in other tissues affected by coronavirus disease (COVID-19). While validating the PPI network model, we found that the tight junction (TJ) proteins cadherin-5, ZO-1, and ß-catenin are affected by nsp2, nsp5_c145a, and nsp7 consistent with the model prediction. Overall, this work identifies the SARS-CoV-2 proteins that might be most detrimental in terms of endothelial dysfunction, thereby shedding light on vascular aspects of COVID-19.


Asunto(s)
Permeabilidad Capilar , Endotelio Vascular/metabolismo , Interacciones Huésped-Patógeno , SARS-CoV-2/metabolismo , Proteínas Virales/metabolismo , Animales , COVID-19/virología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Mapas de Interacción de Proteínas , Proteínas de Uniones Estrechas/metabolismo
6.
Sci Transl Med ; 13(582)2021 02 24.
Artículo en Inglés | MEDLINE | ID: mdl-33627489

RESUMEN

The kidney plays a critical role in fluid homeostasis, glucose control, and drug excretion. Loss of kidney function due to drug-induced nephrotoxicity affects over 20% of the adult population. The kidney proximal tubule is a complex vascularized structure that is particularly vulnerable to drug-induced nephrotoxicity. Here, we introduce a model of vascularized human kidney spheroids with integrated tissue-embedded microsensors for oxygen, glucose, lactate, and glutamine, providing real-time assessment of cellular metabolism. Our model shows that both the immunosuppressive drug cyclosporine and the anticancer drug cisplatin disrupt proximal tubule polarity at subtoxic concentrations, leading to glucose accumulation and lipotoxicity. Impeding glucose reabsorption using glucose transport inhibitors blocked cyclosporine and cisplatin toxicity by 1000- to 3-fold, respectively. Retrospective study of 247 patients who were diagnosed with kidney damage receiving cyclosporine or cisplatin in combination with the sodium-glucose cotransporter-2 (SGLT2) inhibitor empagliflozin showed significant (P < 0.001) improvement of kidney function, as well as reduction in creatinine and uric acid, markers of kidney damage. These results demonstrate the potential of sensor-integrated kidney-on-chip platforms to elucidate mechanisms of action and rapidly reformulate effective therapeutic solutions, increasing drug safety and reducing the cost of clinical and commercial failures.


Asunto(s)
Preparaciones Farmacéuticas , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Humanos , Riñón , Dispositivos Laboratorio en un Chip , Estudios Retrospectivos , Transportador 1 de Sodio-Glucosa
7.
J Am Chem Soc ; 142(51): 21460-21468, 2020 12 23.
Artículo en Inglés | MEDLINE | ID: mdl-33290051

RESUMEN

Inspired by nature, where dynamic networks control the levels of gene expression and the activities of transcribed/translated proteins, we introduce nucleic acid-based constitutional dynamic networks (CDNs) as functional modules mimicking native circuits by demonstrating CDNs-guided programmed synthesis of genes, controlled transcription of RNAs, and dictated transcription/translation synthesis of proteins. An auxiliary CDN consisting of four dynamically equilibrated constituents AA', AB', BA', and BB' is orthogonally triggered by two different inputs yielding two different compositionally reconfigured CDNs. Subjecting the parent auxiliary CDN to two hairpins, HA and HB, and two templates TA and TB and a nicking/replication machinery leads to the cleavage of the hairpins and to the activation of the nicking/replication machineries that synthesize two "genes", e.g., the histidine-dependent DNAzyme g1 and the Zn2+-ion-dependent DNAzyme g2. The triggered orthogonal reconfiguration of the parent CDN to the respective CDNs leads to the programmed preferred CDN-guided synthesis of g1 or g2. Similarly, the triggered reconfigured CDNs are subjected to two hairpins HC and HD, the templates I'/I and J'/J, and the RNA polymerase (RNAp)/NTPs machinery. While the cleavage of the hairpins by the constituents associated with the parent CDN leads to the transcription of the broccoli aptamer recognizing the DFHBI ligand and of the aptamer recognizing the malachite green (MG) ligand, the orthogonally triggered CDNs lead to the CDNs-guided enhanced transcription of either the DFHBI aptamer or the MG aptamer. In addition, subjecting the triggered reconfigured CDNs to predesigned hairpins HE and HF, the templates M'/M and N'/N, the RNAp/NTPs machinery, and the cell-free ribosome t-RNA machinery leads to the CDNs-guided transcription/translation of the green fluorescence protein (GFP) or red fluorescence protein (RFP).


Asunto(s)
Redes Reguladoras de Genes , Biosíntesis de Proteínas/genética , Animales , Aptámeros de Nucleótidos/genética , Proteínas Fluorescentes Verdes/genética , ARN Mensajero/genética
8.
Chem Sci ; 11(17): 4516-4524, 2020 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-34122910

RESUMEN

The enzymes glucose oxidase (GOx), acetylcholine esterase (AchE) and urease that drive biocatalytic transformations to alter pH, are integrated into pH-responsive DNA-based hydrogels. A two-enzyme-loaded hydrogel composed of GOx/urease or AchE/urease and a three-enzyme-loaded hydrogel composed of GOx/AchE/urease are presented. The biocatalytic transformations within the hydrogels lead to the dictated reconfiguration of nucleic acid bridges and the switchable control over the stiffness of the respective hydrogels. The switchable stiffness features are used to develop biocatalytically guided shape-memory and self-healing matrices. In addition, loading of GOx/insulin in a pH-responsive DNA-based hydrogel yields a glucose-triggered matrix for the controlled release of insulin, acting as an artificial pancreas. The release of insulin is controlled by the concentrations of glucose, hence, the biocatalytic insulin-loaded hydrogel provides an interesting sense-and-treat carrier for controlling diabetes.

9.
Annu Rev Biomed Eng ; 21: 219-239, 2019 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-31167098

RESUMEN

The liver is the central hub of xenobiotic metabolism and consequently the organ most prone to cosmetic- and drug-induced toxicity. Failure to detect liver toxicity or to assess compound clearance during product development is a major cause of postmarketing product withdrawal, with disastrous clinical and financial consequences. While small animals are still the preferred model in drug development, the recent ban on animal use in the European Union created a pressing need to develop precise and efficient tools to detect human liver toxicity during cosmetic development. This article includes a brief review of liver development, organization, and function and focuses on the state of the art of long-term cell culture, including hepatocyte cell sources, heterotypic cell-cell interactions, oxygen demands, and culture medium formulation. Finally, the article reviews emerging liver-on-chip devices and discusses the advantages and pitfalls of individual designs. The goal of this review is to provide a framework to design liver-on-chip devices and criteria with which to evaluate this emerging technology.


Asunto(s)
Técnicas de Cultivo de Célula , Hepatocitos/patología , Dispositivos Laboratorio en un Chip/tendencias , Hígado/metabolismo , Hígado/patología , Ingeniería de Tejidos/tendencias , Células 3T3 , Animales , Reactores Biológicos , Medios de Cultivo/química , Medios de Cultivo/farmacología , Diseño de Fármacos , Descubrimiento de Drogas , Células Endoteliales/citología , Unión Europea , Células Estrelladas Hepáticas/citología , Humanos , Macrófagos del Hígado/citología , Ratones , Microfluídica , Oxígeno/química , Distribución Tisular , Ingeniería de Tejidos/métodos
10.
Lab Chip ; 18(17): 2510-2522, 2018 08 21.
Artículo en Inglés | MEDLINE | ID: mdl-29992215

RESUMEN

Drug development is currently hampered by the inability of animal experiments to accurately predict human response. While emerging organ on chip technology offers to reduce risk using microfluidic models of human tissues, the technology still mostly relies on end-point assays and biomarker measurements to assess tissue damage resulting in limited mechanistic information and difficulties to detect adverse effects occurring below the threshold of cellular damage. Here we present a sensor-integrated liver on chip array in which oxygen is monitored using two-frequency phase modulation of tissue-embedded microprobes, while glucose, lactate and temperature are measured in real time using microfluidic electrochemical sensors. Our microphysiological platform permits the calculation of dynamic changes in metabolic fluxes around central carbon metabolism, producing a unique metabolic fingerprint of the liver's response to stimuli. Using our platform, we studied the dynamics of human liver response to the epilepsy drug Valproate (Depakine™) and the antiretroviral medication Stavudine (Zerit™). Using E6/E7LOW hepatocytes, we show TC50 of 2.5 and 0.8 mM, respectively, coupled with a significant induction of steatosis in 2D and 3D cultures. Time to onset analysis showed slow progressive damage starting only 15-20 hours post-exposure. However, flux analysis showed a rapid disruption of metabolic homeostasis occurring below the threshold of cellular damage. While Valproate exposure led to a sustained 15% increase in lipogenesis followed by mitochondrial stress, Stavudine exposure showed only a transient increase in lipogenesis suggesting disruption of ß-oxidation. Our data demonstrates the importance of tracking metabolic stress as a predictor of clinical outcome.


Asunto(s)
Dispositivos Laboratorio en un Chip , Análisis de Flujos Metabólicos/instrumentación , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Línea Celular , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Estavudina/efectos adversos , Ácido Valproico/efectos adversos
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