In vivo characterization of Ca2+ dynamics in pancreatic β-cells of Zebrafish

Glucose homeostasis is fundamental for all living organisms. In vertebrates, the hormone insulin regulates the metabolism of carbohydrates, fats and proteins. In order to sustain the glucose homeostasis, the pancreatic β-cells, which produce and secrete insulin, must coordinate their efforts to secrete the right amounts of insulin required by the organism. In vitro studies, have suggested that a subpopulation of β-cells, referred to as “hub-cells”, coordinate islet Ca2+ dynamics during insulin secretion. However, it is unclear whether the hub-cell model pertains to an in vivo scenario, where the islet is densely vascularized and innervated. In this thesis, we employed the genetically-encoded calcium indicator GCaMP6, confocal imaging and optogenetics, to characterize the Ca2+ dynamics of the zebrafish β-cells in vivo. We found that pancreatic β-cells present endogenous Ca2+ spikes in vivo under basal conditions. These Ca2+ spikes are rapidly suppressed after lowering glucose levels via insulin administration. In addition, the temporal inhibition of blood flow decreases the Ca2+ spikes, suggesting that β-cells are systemically connected. Furthermore, β-cells show a synchronized response to a pericardial glucose injection. Specifically, we found that Ca2+ spikes originate and emanate from a subset of β-cells that are the first to respond to a glucose stimulus. We define these cells as “leader-cells”. We tested if these cells could coordinate the islet in vivo by employing 2-photon laser ablation. Whereas ablation of control cells had no significant effect on the amplitude and duration of the subsequent Ca2+ spikes responses, ablation of leader cells led to a reduction in the Ca2+ response. Furthermore, we developed systems for optogenetic interrogation of β-cells in vivo. We show that the light-gated Cl- ion pump halorhodopsin (NpHR) can be applied to inhibit β-cell depolarization in the zebrafish. We also present the optically orthogonal system of the red Ca2+ indicator K-GECO1 in combination with the blue-shifted channelrhodopsin CheRiff to activate individual β-cell in vivo. Using these new tools, we provide examples where the activation of individual β-cells showed heterogeneous potential to trigger influx of Ca2+ in the rest of the β-cells. Overall, our results led us to propose a hierarchical model of islet coordination. In contrast to the majority of β-cells, which occupy the bottom of the hierarchy since they present low capability to recruit other cells, the leader cells occupy the top levels, being capable to coordinate a majority of the islet’s β-cells.:List of figures xii List of Tables xiii 1. Introduction 1 1.1. Diabetes and insulin 1 1.2. The endocrine pancreas 2 1.3. The diabetes pandemic 4 1.4. β-cell development in zebrafish and mammals 4 1.5. β-cells function and heterogeneity 6 1.6. β-cell coordination 8 1.7. Genetically-encoded calcium indicators 10 1.8. Genetically-encoded optogenetic actuators 13 1.9. Models to study In vivo β-cell coordination 16 2. In vivo β-cell Ca2+ dynamics 19 2.1. β-cells present endogenous Ca2+ spikes in vivo, which are not present ex vivo 19 2.2. Insulin injection reduces endogenous β-cell Ca2+ activity 22 2.3. Pharmacological inhibition of β-cell Ca2+ spikes interferes with glucose control 24 2.4 Transient blood flow interruption decreases β-cell calcium spikes 26 2.5 Glucose bolus leads to a synchronous response of β-cells 29 3. Leader β-cells coordinates Ca2+ dynamics in vivo 32 3.1. High speed 2D and 3D imaging reveals “leader” β-cells 32 3.2. Pan-islet response to glucose is impaired after leader β-cells ablation 41 4. Optically orthogonal toolset for in vivo optogenetics and Ca2+ imaging 46 4.1. Development of optogenetics actuators systems in zebrafish β-cells 46 4.2. Red fluorescent calcium reporters in zebrafish β-cells 47 4.3. In vivo temporal optogenetic silencing of β-cells 50 4.4. In vivo temporal optogenetic silencing of a subset of β-cells can inhibit the islet response 52 4.5. In vivo temporal optogenetic activation of β-cells 55 5. Discussion and future directions 61 5.1. β-cell calcium spikes are systemically influenced 61 5.2. First responder β-cells are present in vivo 64 5.3. Leader β-cells coordinate Ca2+ influx in vivo 66 5.4. β-cell optogenetic interrogation shows heterogeneous potential of individual β-cells for islet coordination 68 6. Materials and methods 75 6.1. Zebrafish strains and husbandry 75 6.2. Transgenic lines generation 76 6.3. Glucose measurements 77 6.4. Pericardial injection of glucose and insulin 77 6.5. Live imaging 77 6.6. Fast whole islet live imaging 78 6.7. Selective two-photon laser ablation of leader cells in the zebrafish islet. 78 6.7. Selective one-photon optogenetic interrogation of β-cells in the zebrafish islet. 79 6.8. Islet blood flow imaging 80 6.9. Mechanical heart stop 80 6.10. Immunostaining 80 6.11. TUNEL assay 81 6.12 Image analysis of GCaMP6s fluorescence intensity from in vivo imaging. 82 6.13 Quantification of GCaMP6s fluorescence intensity 82 6.14 Spatial drift correction images. 83 6.15 Statistical analysis 84 7. References 85 8. Annexes 90 9. Acknowledgments 97Die Glukosehomöostase ist für alle lebenden Organismen von grundlegender Bedeutung. Bei Wirbeltieren reguliert das Hormon Insulin den Stoffwechsel von Kohlenhydraten, Fetten und Proteinen. Um die Glukosehomöostase aufrechtzuerhalten, müssen die β-Zellen der Bauchspeicheldrüse, welche Insulin produzieren und absondern, ihre Bemühungen koordinieren, um die richtigen Mengen an Insulin zu sekretieren, die der Organismus benötigt. In-vitro-Studien haben gezeigt, dass eine Subpopulation von β-Zellen, die als „Hub-Zellen“ bezeichnet werden, die Insulinsekretion der Inseln koordiniert. Es ist jedoch unklar, ob sich die Hub-Cell-Theorie auf ein in-vivo-Szenario bezieht, bei dem die Insel dicht vaskularisiert und von Neuronen innerviert ist. In dieser Arbeit verwendeten wir den genetisch kodierten Calcium-Indikator GCaMP6, konfokale Bildgebung und Optogenetik, um die Ca2+-Dynamik der Zebrafisch-β-Zellen in vivo zu charakterisieren. Wir fanden heraus, dass Pankreas-β-Zellen in vivo unter basalen Bedingungen endogene Ca2+-Spitzen aufweisen. Diese Ca2+-Spitzen werden nach Senkung des Glukosespiegels durch Insulinverabreichung schnell unterdrückt. Darüber hinaus verringert die zeitliche Hemmung des Blutflusses die Ca2+-Spitzen, was darauf hindeutet, dass β-Zellen systemisch verbunden sind. Darüber hinaus zeigen β-Zellen eine synchronisierte Reaktion auf die perdikale Glukoseinjektion. Insbesondere fanden wir heraus, dass Ca2+-Spitzen von den β-Zellen hervorgerufen werden, die zuerst auf den Glukosestimulus reagieren. Wir definieren diese Zellen als 'Leader-Zellen'. Wir haben in vivo durch den Einsatz einer 2-Photonen-Laserablation getestet, ob diese Zellen die Insel koordinieren können. Während die Ablation von Kontrollzellen keinen signifikanten Einfluss auf die Amplitude und Dauer der nachfolgenden Ca2+-Spitzenreaktionen hatte, führte die Ablation von Leader-Zellen zu einer signifikanten Verringerung der GCaMP-Reaktion. Darüber hinaus haben wir Systeme für die optogenetische Abfrage von β-Zellen in vivo entwickelt: Wir zeigen, dass die lichtgesteuerte Cl—Ionenpumpe Halorhodopsin (NpHR) angewendet werden kann, um die Depolarisation von β-Zellen in vivo zu hemmen. Wir präsentieren auch das optisch orthogonale System des roten Ca2+-Indikators K-GECO1 in Kombination mit dem blauverschobenen Channelrhodopsin CheRiff, um einzelne β-Zellen in vivo abzufragen. Unter Verwendung dieser neuen Werkzeuge liefern wir Beispiele, bei denen die Aktivierung einzelner β-Zellen ein heterogenes Potenzial für die Auslösung des Ca2+-Einstroms in die übrigen β-Zellen in vivo zeigte. Insgesamt bietet diese Studie Hinweise darauf, dass eine Untergruppe von β-Zellen ein hohes Potenzial zur Koordination der Ca2+-Dynamik der Insel in vivo aufweist.:List of figures xii List of Tables xiii 1. Introduction 1 1.1. Diabetes and insulin 1 1.2. The endocrine pancreas 2 1.3. The diabetes pandemic 4 1.4. β-cell development in zebrafish and mammals 4 1.5. β-cells function and heterogeneity 6 1.6. β-cell coordination 8 1.7. Genetically-encoded calcium indicators 10 1.8. Genetically-encoded optogenetic actuators 13 1.9. Models to study In vivo β-cell coordination 16 2. In vivo β-cell Ca2+ dynamics 19 2.1. β-cells present endogenous Ca2+ spikes in vivo, which are not present ex vivo 19 2.2. Insulin injection reduces endogenous β-cell Ca2+ activity 22 2.3. Pharmacological inhibition of β-cell Ca2+ spikes interferes with glucose control 24 2.4 Transient blood flow interruption decreases β-cell calcium spikes 26 2.5 Glucose bolus leads to a synchronous response of β-cells 29 3. Leader β-cells coordinates Ca2+ dynamics in vivo 32 3.1. High speed 2D and 3D imaging reveals “leader” β-cells 32 3.2. Pan-islet response to glucose is impaired after leader β-cells ablation 41 4. Optically orthogonal toolset for in vivo optogenetics and Ca2+ imaging 46 4.1. Development of optogenetics actuators systems in zebrafish β-cells 46 4.2. Red fluorescent calcium reporters in zebrafish β-cells 47 4.3. In vivo temporal optogenetic silencing of β-cells 50 4.4. In vivo temporal optogenetic silencing of a subset of β-cells can inhibit the islet response 52 4.5. In vivo temporal optogenetic activation of β-cells 55 5. Discussion and future directions 61 5.1. β-cell calcium spikes are systemically influenced 61 5.2. First responder β-cells are present in vivo 64 5.3. Leader β-cells coordinate Ca2+ influx in vivo 66 5.4. β-cell optogenetic interrogation shows heterogeneous potential of individual β-cells for islet coordination 68 6. Materials and methods 75 6.1. Zebrafish strains and husbandry 75 6.2. Transgenic lines generation 76 6.3. Glucose measurements 77 6.4. Pericardial injection of glucose and insulin 77 6.5. Live imaging 77 6.6. Fast whole islet live imaging 78 6.7. Selective two-photon laser ablation of leader cells in the zebrafish islet. 78 6.7. Selective one-photon optogenetic interrogation of β-cells in the zebrafish islet. 79 6.8. Islet blood flow imaging 80 6.9. Mechanical heart stop 80 6.10. Immunostaining 80 6.11. TUNEL assay 81 6.12 Image analysis of GCaMP6s fluorescence intensity from in vivo imaging. 82 6.13 Quantification of GCaMP6s fluorescence intensity 82 6.14 Spatial drift correction images. 83 6.15 Statistical analysis 84 7. References 85 8. Annexes 90 9. Acknowledgments 9

Democracia en el Estado de México: fortalezas y debilidades

En toda interacción humana puede estar presente el poder. Por tanto, todas las formas de interacción humana son susceptibles de catalogarse como democráticas o como autoritarias (e incluso híbridas, cuando conjugan elementos democráticos y autoritarios). En la interacción democrática o bien está ausente el poder o bien éste es ejercido con tolerancia y con apego a normas preestablecidas. En la interacción autoritaria prevalece la intolerancia, la arbitrariedad y la violencia

Vocabulario de la sociedad civil, la ruralidad y los movimientos sociales en América Latina

El Vocabulario de la Sociedad Civil, la Ruralidad y los Movimientos Sociales en América Latina tiene como objetivo desarrollar vocablos relacionados con temas de gran trascendencia para la vida colectiva de la población Latinoamericana; pretende introducir a estudiantes, personas del ámbito académico y activistas en la comprensión de estas categorías de análisis. A través de la mirada de 70 especialistas que participaron en este vocabulario, es posible comprender muchos de los términos que se utilizan dentro de la investigación social y áreas relacionadas con las ciencias políticas, ambientales y rurales, a partir de una mayor explicación y detalle. Es por ello que se inserta este trabajo desde una mirada colectiva y amplia de los conceptos que se exponen. En este libro podrá encontrar las ideas de varios autores y autoras de distintas universidades, con una visión multi, inter y transdisciplinaria. El esfuerzo que se realizó para conjuntar varios términos y analizar su compleja red de interpretaciones, permitirá que este manuscrito pueda ser consultado por estudiantes, personas del ámbito científico-académico, y ciudadanía; porque contiene el estado del arte, la historia del paulatino avance de múltiples conceptos y su vigencia en el contexto actual

c-Abl Tyrosine Kinase Is Regulated Downstream of the Cytoskeletal Protein Synemin in Head and Neck Squamous Cell Carcinoma Radioresistance and DNA Repair

The intermediate filament synemin has been previously identified as novel regulator of cancer cell therapy resistance and DNA double strand break (DSB) repair. c-Abl tyrosine kinase is involved in both of these processes. Using PamGene technology, we performed a broad-spectrum kinase activity profiling in three-dimensionally, extracellular matrix grown head and neck cancer cell cultures. Upon synemin silencing, we identified 86 deactivated tyrosine kinases, including c-Abl, in irradiated HNSCC cells. Upon irradiation and synemin inhibition, c-Abl hyperphosphorylation on tyrosine (Y) 412 and threonine (T) 735 was significantly reduced, prompting us to hypothesize that c-Abl tyrosine kinase is an important signaling component of the synemin-mediated radioresistance pathway. Simultaneous targeting of synemin and c-Abl resulted in similar radiosensitization and DSB repair compared with single synemin depletion, suggesting synemin as an upstream regulator of c-Abl. Immunoprecipitation assays revealed a protein complex formation between synemin and c-Abl pre- and post-irradiation. Upon pharmacological inhibition of ATM, synemin/c-Abl protein-protein interactions were disrupted implying synemin function to depend on ATM kinase activity. Moreover, deletion of the SH2 domain of c-Abl demonstrated a decrease in interaction, indicating the dependency of the protein-protein interaction on this domain. Mechanistically, radiosensitization upon synemin knockdown seems to be associated with an impairment of DNA repair via regulation of non-homologous end joining independent of c-Abl function. Our data generated in more physiological 3D cancer cell culture models suggest c-Abl as further key determinant of radioresistance downstream of synemin

Modelling pancreatic β-cell inflammation in zebrafish identifies the natural product wedelolactone for human islet protection

Islet inflammation and cytokine production are implicated in pancreatic β-cell dysfunction and diabetes pathogenesis. However, we lack therapeutics to protect the insulin-producing β-cells from inflammatory damage. Closing this clinical gap requires the establishment of new disease models of islet inflammation to facilitate screening efforts aimed at identifying new protective agents. Here, we have developed a genetic model of Interleukin-1β (Il-1β)-driven islet inflammation in zebrafish, a vertebrate that allows for non-invasive imaging of β-cells and in vivo drug discovery. Live imaging of immune cells and β-cells in our model revealed dynamic migration, increased visitation and prolonged macrophage retention in the islet, together with robust activation of NF-κB signalling in β-cells. We find that Il-1β-mediated inflammation does not cause β-cell destruction but, rather, it impairs β-cell function and identity. In vivo, β-cells exhibit impaired glucose-stimulated calcium influx and reduced expression of genes involved in function and maturity. These defects are accompanied by α-cell expansion, glucose intolerance and hyperglycemia following a glucose challenge. Notably, we show that a medicinal plant derivative (wedelolactone) is capable of reducing the immune-cell infiltration while also ameliorating the hyperglycemic phenotype of our model. Importantly, these anti-diabetic properties in zebrafish are predictive of wedelolactone's efficacy in protecting rodent and human islets from cytokine-induced apoptosis. In summary, this new zebrafish model of diabetes opens a window to study the interactions between immune and β-cells in vivo, while also allowing the identification of therapeutic agents for protecting β-cells from inflammation

Management of coronary disease in patients with advanced kidney disease

BACKGROUND Clinical trials that have assessed the effect of revascularization in patients with stable coronary disease have routinely excluded those with advanced chronic kidney disease. METHODS We randomly assigned 777 patients with advanced kidney disease and moderate or severe ischemia on stress testing to be treated with an initial invasive strategy consisting of coronary angiography and revascularization (if appropriate) added to medical therapy or an initial conservative strategy consisting of medical therapy alone and angiography reserved for those in whom medical therapy had failed. The primary outcome was a composite of death or nonfatal myocardial infarction. A key secondary outcome was a composite of death, nonfatal myocardial infarction, or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. RESULTS At a median follow-up of 2.2 years, a primary outcome event had occurred in 123 patients in the invasive-strategy group and in 129 patients in the conservative-strategy group (estimated 3-year event rate, 36.4% vs. 36.7%; adjusted hazard ratio, 1.01; 95% confidence interval [CI], 0.79 to 1.29; P=0.95). Results for the key secondary outcome were similar (38.5% vs. 39.7%; hazard ratio, 1.01; 95% CI, 0.79 to 1.29). The invasive strategy was associated with a higher incidence of stroke than the conservative strategy (hazard ratio, 3.76; 95% CI, 1.52 to 9.32; P=0.004) and with a higher incidence of death or initiation of dialysis (hazard ratio, 1.48; 95% CI, 1.04 to 2.11; P=0.03). CONCLUSIONS Among patients with stable coronary disease, advanced chronic kidney disease, and moderate or severe ischemia, we did not find evidence that an initial invasive strategy, as compared with an initial conservative strategy, reduced the risk of death or nonfatal myocardial infarction

Health status after invasive or conservative care in coronary and advanced kidney disease

BACKGROUND In the ISCHEMIA-CKD trial, the primary analysis showed no significant difference in the risk of death or myocardial infarction with initial angiography and revascularization plus guideline-based medical therapy (invasive strategy) as compared with guideline-based medical therapy alone (conservative strategy) in participants with stable ischemic heart disease, moderate or severe ischemia, and advanced chronic kidney disease (an estimated glomerular filtration rate of <30 ml per minute per 1.73 m2 or receipt of dialysis). A secondary objective of the trial was to assess angina-related health status. METHODS We assessed health status with the Seattle Angina Questionnaire (SAQ) before randomization and at 1.5, 3, and 6 months and every 6 months thereafter. The primary outcome of this analysis was the SAQ Summary score (ranging from 0 to 100, with higher scores indicating less frequent angina and better function and quality of life). Mixed-effects cumulative probability models within a Bayesian framework were used to estimate the treatment effect with the invasive strategy. RESULTS Health status was assessed in 705 of 777 participants. Nearly half the participants (49%) had had no angina during the month before randomization. At 3 months, the estimated mean difference between the invasive-strategy group and the conservative-strategy group in the SAQ Summary score was 2.1 points (95% credible interval, 120.4 to 4.6), a result that favored the invasive strategy. The mean difference in score at 3 months was largest among participants with daily or weekly angina at baseline (10.1 points; 95% credible interval, 0.0 to 19.9), smaller among those with monthly angina at baseline (2.2 points; 95% credible interval, 122.0 to 6.2), and nearly absent among those without angina at baseline (0.6 points; 95% credible interval, 121.9 to 3.3). By 6 months, the between-group difference in the overall trial population was attenuated (0.5 points; 95% credible interval, 122.2 to 3.4). CONCLUSIONS Participants with stable ischemic heart disease, moderate or severe ischemia, and advanced chronic kidney disease did not have substantial or sustained benefits with regard to angina-related health status with an initially invasive strategy as compared with a conservative strategy

Delayed colorectal cancer care during covid-19 pandemic (decor-19). Global perspective from an international survey

Background The widespread nature of coronavirus disease 2019 (COVID-19) has been unprecedented. We sought to analyze its global impact with a survey on colorectal cancer (CRC) care during the pandemic. Methods The impact of COVID-19 on preoperative assessment, elective surgery, and postoperative management of CRC patients was explored by a 35-item survey, which was distributed worldwide to members of surgical societies with an interest in CRC care. Respondents were divided into two comparator groups: 1) ‘delay’ group: CRC care affected by the pandemic; 2) ‘no delay’ group: unaltered CRC practice. Results A total of 1,051 respondents from 84 countries completed the survey. No substantial differences in demographics were found between the ‘delay’ (745, 70.9%) and ‘no delay’ (306, 29.1%) groups. Suspension of multidisciplinary team meetings, staff members quarantined or relocated to COVID-19 units, units fully dedicated to COVID-19 care, personal protective equipment not readily available were factors significantly associated to delays in endoscopy, radiology, surgery, histopathology and prolonged chemoradiation therapy-to-surgery intervals. In the ‘delay’ group, 48.9% of respondents reported a change in the initial surgical plan and 26.3% reported a shift from elective to urgent operations. Recovery of CRC care was associated with the status of the outbreak. Practicing in COVID-free units, no change in operative slots and staff members not relocated to COVID-19 units were statistically associated with unaltered CRC care in the ‘no delay’ group, while the geographical distribution was not. Conclusions Global changes in diagnostic and therapeutic CRC practices were evident. Changes were associated with differences in health-care delivery systems, hospital’s preparedness, resources availability, and local COVID-19 prevalence rather than geographical factors. Strategic planning is required to optimize CRC care