Automated Detection and Analysis of Depolarization Events in Human Cardiomyocytes using MaDEC

Optical imaging-based methods for assessing the membrane electrophysiology of in vitro human cardiac cells allow for non-invasive temporal assessment of the effect of drugs and other stimuli. Automated methods for detecting and analyzing the depolarization events (DEs) in image-based data allow quantitative assessment of these different treatments. In this study, we use 2-photon microscopy of fluorescent voltage-sensitive dyes (VSDs) to capture the membrane voltage of actively beating human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). We built a custom and freely available Matlab software, called MaDEC, to detect, quantify, and compare DEs of hiPS-CMs treated with the β-adrenergic drugs, propranolol and isoproterenol. The efficacy of our software is quantified by comparing detection results against manual DE detection by expert analysts, and comparing DE analysis results to known drug-induced electrophysiological effects. The software accurately detected DEs with true positive rates of 98–100% and false positive rates of 1–2%, at signal-to-noise ratios (SNRs) of 5 and above. The MaDEC software was also able to distinguish control DEs from drug-treated DEs both immediately as well as 10 min after drug administration

The potential role of stromal derived factor 1α in remote ischaemic conditioning

Background Alleviating the injury associated with ST-elevation myocardial infarction is central to improving the global burden of coronary heart disease. The chemokine stromal cell-derived factor 1α (SDF-1α) and its receptor, CXCR4, have dual potential benefit in this regard: acutely protecting the heart from lethal ischaemia-reperfusion injury (IRI) whilst mitigating adverse ventricular remodelling by recruiting progenitor cells to the site of injury. This project hypothesised that SDF-1α mediates the acute cardioprotection conferred by remote ischaemic conditioning (RIC), the phenomenon whereby brief cycles of non-lethal tissue ischaemia and reperfusion remote from the heart protects against myocardial IRI. Methods and Results This thesis defines a paradigm for evidencing a role in RIC that includes induction of cardioprotection by exogenous administration of SDF-1α at the time of reperfusion, abolition of cardioprotection by specific antagonism of CXCR4, increased production of SDF-1α as a direct effect of RIC, and absence of cardioprotection in CXCR4-deficient mice. A murine in vivo model of myocardial IRI and a novel ELISA for active SDF-1α were established and used to investigate this paradigm. This thesis provides the first description of cardioprotection against myocardial IRI as a result of exogenous SDF-1α administered prior to reperfusion. Moreover, AMD3100, a highly specific inhibitor of CXCR4, abolishes the beneficial effect of RIC in vivo. Next, SDF-1α cleavage and inactivation was unexpectedly demonstrated to increase after RIC, which may be attributable to up-regulation of dipeptidyl peptidase-4. Finally, inducible cardiomyocyte-specific CXCR4 deletion unexpectedly conferred protection against myocardial IRI. The protective mechanism was not established and, furthermore, it prohibited the use of these mice in experiments to validate the role of CXCR4 signalling in RIC. Conclusions The intrinsic role of SDF-1α in RIC remains equivocal. However, modulation of the SDF-1α-CXCR4 axis with other approaches, including exogenous SDF-1α, has potential utility in cardioprotection against myocardial IRI

Investigating the effects of stem cell therapies in experimental models of renal ischemia-reperfusion injury

The incidence of end-stage renal disease is increasing in Western Society. Renal transplantation is known to be the optimal treatment for ESRD, being associated with significant reduction in morbidity, mortality for patients and cost for wider society when compared to remaining on dialysis. Unfortunately, the growing number of patients listed for renal transplantation has occurred without a matched supply in the number of suitable organs. This has led to longer average waiting times for increased numbers of patients, who consequently suffer adverse outcomes at considerable cost to the National Health Service as a result of organ shortage. One strategy employed by clinicians to meet demand for organs has been to transplant ‘suboptimal’ kidneys’ historically rejected as unsuitable for transplantation, which are usually retrieved from older and less fit donors. Sometimes referred to as ‘extended criteria’ or ‘marginal kidneys’, such allografts are more prone to damage in the peri-transplantation period, with the major pathological process recognised to be ischemia-reperfusion injury (IRI). Although functioning ‘marginal’ allografts have been shown to confer benefit to recipients, early transplant failure is associated with negative outcomes. Consequently, there is a real need to develop treatments to mitigate renal IRI, especially since the use of ‘marginal’ kidneys is likely to increase. Stem cell therapy has been shown to protect solid organs from IRI in a number of different animal models. Consequently, there is great interest in researching the ability of stem cell-based therapies to ameliorate solid organ damage and perhaps to encourage organ regeneration. However, debate exists regarding the exact mechanism by which stem cells produce their effects. Some researchers suggest that stem cells directly differentiate to replace specialised cell types in damage organs. Other investigators conclude that stem cells produce their effects in a paracrine fashion via the release of extracellular vesicles with the horizontal transfer of genetic material between cells. Unfortunately, no therapies are currently in widespread use to reduce damage to allografts in the peri-transplant period. In part, this reflects the lack of robust small animal models for screening potential renal IRI therapies before testing in large animal models. Furthermore, clinical application has been limited by safety concerns, and particularly by the risk of stem cells undergoing malignant transformation and subsequent tumour formation in recipients. However, investigators hypothesise that the use of stem cell-derived, extracellular vesicles may confer similar beneficial therapeutic efficacy, but lack many of the side effects associated with stem cells themselves. This thesis describes experiments in which stem cell-based therapies are tested in conventional and novel animal models of renal IRI and renal transplantation. In Chapter 3, initial experiments unexpectedly demonstrated the potential of ex vivo expanded stem cells to undergo malignant change and induce tumour formation in recipient animals. Therefore, the subsequent research investigated the effects of freshly isolated stem cells or those of novel extracellular vesicle preparations. In Chapter 4, experiments unexpectedly demonstrated the shortcomings of a conventional rat model of renal IRI. Therefore, Chapter 5 describes the development of a novel rat of model of renal IRI, in which stem cell-based therapies may be tested. Using this animal model, Chapters 6 and Chapter 7 describe the investigation of novel stem cell-based therapies and their effects on renal IRI. Some of these treatments were found to protect kidneys from IRI damage with preservation of renal function and structure in the medium to long-term. Chapter 8 describes a rat model of renal transplantation, in which therapies were investigated after being screened for efficacy in the novel rat IRI model. Although no functional difference was demonstrated, renal histology was preserved by treatment, although the mechanisms by which this effect occurred remain unclear. These findings suggest that stem cells and their extracellular vesicles have the potential to reduce peri-transplantation renal IRI and hence improve long-term outcomes of ‘marginal’ allografts. However, clinical translation requires the long-term efficacy and safety of these novel therapies to be investigated in large animal models of renal transplantation, before further testing in pilot studies

Automated detection and analysis of depolarization events in human cardiomyocytes using MaDEC.

Optical imaging-based methods for assessing the membrane electrophysiology of in vitro human cardiac cells allow for non-invasive temporal assessment of the effect of drugs and other stimuli. Automated methods for detecting and analyzing the depolarization events (DEs) in image-based data allow quantitative assessment of these different treatments. In this study, we use 2-photon microscopy of fluorescent voltage-sensitive dyes (VSDs) to capture the membrane voltage of actively beating human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). We built a custom and freely available Matlab software, called MaDEC, to detect, quantify, and compare DEs of hiPS-CMs treated with the β-adrenergic drugs, propranolol and isoproterenol. The efficacy of our software is quantified by comparing detection results against manual DE detection by expert analysts, and comparing DE analysis results to known drug-induced electrophysiological effects. The software accurately detected DEs with true positive rates of 98-100% and false positive rates of 1-2%, at signal-to-noise ratios (SNRs) of 5 and above. The MaDEC software was also able to distinguish control DEs from drug-treated DEs both immediately as well as 10min after drug administration

Toxicological Study in vitro, in vivo and Transcriptomic of Mycotoxins. Estudio Toxicológico in vitro, in vivo y Transcriptómico de Micotoxinas

Las micotoxinas, producidas por el metabolismo secundario de hongos filamentosos, se consideran contaminantes comunes de alimentos y piensos y constituyen una preocupación en el ámbito de la seguridad alimentaria. Se han estudiado los efectos tóxicos y mecanismos de acción de las micotoxinas emergentes Beauvericina (BEA) y Eniatinas (ENs) mediante ensayos in vitro en células Jurkat, in vivo en rata Wistar y transcriptómicos por secuenciación del ARN; así como la presencia de multi-micotoxinas en alimentos, piensos y muestras biológicas. Las revisiones realizadas ponen de manifiesto que (i) la investigación in vivo de fusarotoxinas se basa principalmente en estudios de toxicidad subaguda y subcrónica con cerdos y ratas expuestos por vía oral a micotoxinas como Fumonisina B1, Deoxinivalenol (DON) y Zearalenona (ZEN); (ii) los objetivos principales del análisis de micotoxinas en muestras biológicas son el desarrollo de métodos analíticos, estudios de biomonitorización y evaluación de la exposición, siendo la orina humana la muestra biológica más analizada y destacando Ocratoxina A (OTA), ZEN y DON como las micotoxinas más estudiadas. A pesar de la elevada frecuencia de micotoxinas detectadas en muestras biológicas, los bajos niveles cuantificados indican que las ingestas diarias probables son generalmente inferiores a las ingestas tolerables permitidas; (iii) las micotoxinas de Alternaria más analizadas en alimentos y piensos son Alternariol, Alternariol Metil Éter, Tentoxinona y Ácido Tenuazónico, detectando las mayores concentraciones en legumbres, tomates y cereales. Los resultados del análisis de piensos de rata de laboratorio demuestran una elevada incidencia de contaminación por multi-micotoxinas, principalmente ZEN, ENs y DON. Los ensayos in vitro muestran disminución de la viabilidad celular, arresto del ciclo celular y activación de la apoptosis con aumento de la actividad de las caspasas tras la exposición de células Jurkat a EN B y BEA, demostrando esta última efectos in vitro más pronunciados, así como potencial genotóxico. La revisón bibliográfica de la Quercetina revela actividad protectora in vitro frente al efecto citotóxico de micotoxinas como OTA, DON, ZEN, BEA y ENs, sugiriendo su ingesta en alimentos como una estrategia potencial para prevenir la toxicidad inducida por micotoxinas. Los estudios in vivo muestran ausencia de efectos adversos observables tras la administración subaguda de EN A y administración única vía oral de ENs en rata Wistar, a pesar de detectar las micotoxinas en suero, órganos y tejidos, confirmando así su absorción intestinal tras administración oral. El estudio transcriptómico revela alteración del genoma humano tras la exposición de células Jurkat a BEA y EN B, indicando daño mitocondrial con alteración de las vías de fosforilación oxidativa y cadena de transporte de electrones. Se necesitan nuevos estudios que permitan la evaluación del riesgo potencial de la exposición a estos compuestos para proteger así la salud humana y animal.Mycotoxins, produced by secondary metabolism of filamentous fungi, are common food and feed contaminants, and they constitute a concern of food safety. The present work studied the toxic effects and mechanisms of action of the emerging mycotoxins Beauvericin (BEA) and Enniatins (ENs) through in vitro tests in Jurkat cells, in vivo assays in Wistar rats, and transcriptomic approach by RNA-seq; as well as multi-mycotoxin presence in food, feed, and biological samples. The reviews indicated that (i) the in vivo research of fusarotoxins were mainly based on subacute and subchronic toxicity studies with pigs and rats orally exposed to mycotoxins such as Fumonisin B1, Deoxynivalenol (DON) and Zearalenone (ZEN), as well as mycotoxin combinations generally including DON; (ii) the main objectives of mycotoxins analysis in biological samples were method development, biomonitoring studies, and exposure assessment, with human urine as the most analyzed biological sample and Ochratoxin A (OTA), ZEN and DON as the most studied mycotoxins. Despite the high frequency of mycotoxins detected in biological samples, the low quantified levels suggested probable daily intakes generally lower than the permited tolerable intakes; (iii) the most analyzed Alternaria mycotoxins in food and feed were Alternariol, Alternariol Methyl Ether, Tentoxinone and Tenuazonic Acid, detecting the highest concentrations in legumes, tomatoes and cereals. The results of the laboratory rat feed analysis demonstrated high incidence of multi-mycotoxin contamination mainly with ZEN, ENs and DON. The in vitro assays indicated decrease in cell viability, cell cycle arrest, and apoptosis activation with increase of caspase activity after EN B and BEA exposure to Jurkat cells. BEA demonstrated more pronounced in vitro effects, as well as, genotoxic potential. The review about Quercetin showed in vitro protective activity against the cytotoxic effect of mycotoxins such as OTA, DON, ZEN, BEA and ENs, suggesting their intake in food as a potential strategy to prevent mycotoxin-induced toxicity. The in vivo studies showed absence of observed adverse effects after subacute administration of EN A and single oral dose administration of ENs in Wistar rats, despite their detection in serum, organs and tissues, thus confirming their intestinal absorption after oral administration. The transcriptomic study revealed human genome preturbation after Jurkat cells exposure to BEA and EN B, indicating mitochondrial damage with alteration of the oxidative phosphorylation and electron transport chain pathways. Further studies are needed to evaluate the potential risk of the exposure to these compounds and thus, to protect human and animal health