Pt(II)-dendrimers as bio-imaging marker for bacteria in two-photon excitation microscopy

The use of luminescent markers based on metal complexes in two-photon excitation microscopy techniques are of great interest in the field of bioimaging. However, despite the excellent luminescent properties of Pt(II) complexes, their application in this field is still limited, due to their poor solubility and quenching problems in aqueous media [1]. The insertion of a Pt(II) complex into a dendritic structure, gives as a result an unique luminescent marker soluble in biological media. Dendrimers provides excellent properties to the metal complex such as solubility in aqueous media, protection against quenching processes and binding to bacterial surfaces. The new probe can be used as bacteria cells marker in luminescent microscopy, operating under one or two-photon excitation (OPE/TPE) conditions, as well as in electron microscopy, thus providing a powerful tool in the field of bioimaging.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Tailoring two-photon fluorescent probes for pH bioimaging in living cells

Fluorescence biosensors are indispensable basic tools in modern biology. These type of molecules allow real-time visualization of biological events inside living cells. Especially important in many of these processes (proliferation, apoptosis or defense tasks) is the control of the cellular pH. In consequence, a great variety of structural models have been developed for pH bioimaging in fluorescence microscopy. Nonetheless, these efforts have been mainly focused on the development of one-photon (OP) probes. Recently, we described a biosensor with excellent photophysical properties and appropriate two-photon absorption (TPA) behavior. This sensor allows selective and specific detection of hydroxyl radicals solely inside lysosomes.Based on this scaffold, we have synthesized and characterized new TPA fluorescent probes. These molecules have an “off-on” response to different pH environments with a strong selectivity and sensitivity toward H+. These naphthalene-indolenine derivatives have a high synthetic versatility through affordable and efficient synthesis. The synthetic modification of this model allows tuning subcellular targets through minor modifications and without affecting their emission properties. The effectiveness of these probes and their structural modifications for different pH-related applications has been probed in mouse embrionary fibroblast (MEF) cells.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Cardiac electrical defects in progeroid mice and Hutchinson-Gilford progeria syndrome patients with nuclear lamina alterations

Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disease caused by defective prelamin A processing, leading to nuclear lamina alterations, severe cardiovascular pathology, and premature death. Prelamin A alterations also occur in physiological aging. It remains unknown how defective prelamin A processing affects the cardiac rhythm. We show age-dependent cardiac repolarization abnormalities in HGPS patients that are also present in the Zmpste24-/- mouse model of HGPS. Challenge of Zmpste24-/- mice with the ß-adrenergic agonist isoproterenol did not trigger ventricular arrhythmia but caused bradycardia-related premature ventricular complexes and slow-rate polymorphic ventricular rhythms during recovery. Patch-clamping in Zmpste24-/- cardiomyocytes revealed prolonged calcium-transient duration and reduced sarcoplasmic reticulum calcium loading and release, consistent with the absence of isoproterenol-induced ventricular arrhythmia. Zmpste24-/- progeroid mice also developed severe fibrosis-unrelated bradycardia and PQ interval and QRS complex prolongation. These conduction defects were accompanied by overt mislocalization of the gap junction protein connexin43 (Cx43). Remarkably, Cx43 mislocalization was also evident in autopsied left ventricle tissue from HGPS patients, suggesting intercellular connectivity alterations at late stages of the disease. The similarities between HGPS patients and progeroid mice reported here strongly suggest that defective cardiac repolarization and cardiomyocyte connectivity are important abnormalities in the HGPS pathogenesis that increase the risk of arrhythmia and premature death.Peer ReviewedPostprint (published version

V-shaped pyranylidene/triphenylamine-based chromophores with enhanced photophysical, electrochemical and nonlinear optical properties

We report the synthesis and comprehensive study of two chromophores based on 4H-pyranylidene moiety as a part of the π-conjugated spacer. Triphenylamine (TPA) acts as donor and tricarbonitrile-based electron-accepting groups complete these V-shaped D–A–D architectures (A, acceptor; D, donor). Their electrochemical, photophysical and nonlinear optical properties are analyzed in detail by using a joint experimental and theoretical approach. The two chromophores exhibit near-infrared fluorescence, large Stokes shift, enhanced emission in tetrahydrofuran/water mixtures and good photostability. Additionally, the dimerization of triphenylamine groups to tetraphenylbenzidine (TPB) takes place upon electrochemical and chemical oxidation showing their peculiar electrochemical behavior and film formation capabilities. Interestingly, high molecular first hyperpolarizabilities and two-photon absorption cross-sections were found, highlighting their potential applications in electro-optical devices. Overall, our work demonstrates that these near-infrared (NIR) fluorescent chromophores are versatile materials with a myriad of applications ranging from optoelectronics to biological applications.The work at the University of Málaga was funded by the MICINN (PID2019-110305GB-I00, PID2019-104293GB-I00, RTI2018-095410-B-I00, EuroNanoMed 2019 PCI2019-111825-2), the Institute of Health Carlos III (ISCIII) RETIC ARADYAL (RD16/0006/0012) and by the Junta de Andalucıa (P09-FQM-4708, UMA18-FEDERJA-080, UMA18-FEDERJA-007, PIER-0084-2019). The work at the University of Zaragoza was funded by the MICINN (PID2019-104307GB-I00/AEI/10.13039/501100011033) and Gobierno de Aragón (E47_20R). The work at the University of Stuttgart was funded by the German Science Foundation (DFG) through the project “LU 1445/7-1, project number 416982273” on electrooptical hybrid modulators, C. Malacrida is acknowledged for discussions. S. G.-V. thanks the MINECO for a FPU predoctoral fellowship (FPU17/04908) and CB-M for FPU fellowship (FPU16/02516). Computer resources, technical expertise and assistance provided by the SCBI (Supercomputing and Bioinformatics) centre of the University of Málaga are gratefully acknowledged. We thank the Vibrational spectroscopy lab (EVI) of the Research Central Services (SCAI) of the University of Málaga and John Pearson (BIONAND) for help with laser confocal microscopy analysis. We gratefully acknowledge the ICTS “NANBIOSIS” facilities, more specifically the U28 Unit of the Andalusian Centre for Nanomedicine & Biotechnology (BIONAND), for their help with the 2PA characterization and the microscopy studies.Peer reviewe

Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin

Epicardial epithelial-mesenchymal transition (EMT) is hypothesized to generate cardiovascular progenitor cells that differentiate into various cell types, including coronary smooth muscle and endothelial cells, perivascular and cardiac interstitial fibroblasts and cardiomyocytes. Here we show that an epicardial-specific knockout of Wt1 leads to a reduction of mesenchymal progenitor cells and their derivatives. We demonstrate that Wt1 is essential for repression of the epithelial phenotype in epicardial cells and during Embryonic Stem (ES) cell differentiation, through direct transcriptional regulation of Snail (Snai1) and E-cadherin (Cdh1), two of the major mediators of EMT. Some mesodermal lineages fail to form in Wt1 null embryoid bodies but this effect is rescued by the expression of Snai1, underlining the importance of EMT in generating these differentiated cells. These new insights into the molecular mechanisms regulating cardiovascular progenitor cells and EMT will shed light on the pathogenesis of heart diseases and may help the development of cell based therapies

Cardiac electrical defects in progeroid mice and Hutchinson-Gilford progeria syndrome patients with nuclear lamina alterations

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease caused by defective prelamin A processing, leading to nuclear lamina alterations, severe cardiovascular pathology, and premature death. Prelamin A alterations also occur in physiological aging. It remains unknown how defective prelamin A processing affects the cardiac rhythm. We show age-dependent cardiac repolarization abnormalities in HGPS patients that are also present in the Zmpste24−/− mouse model of HGPS. Challenge of Zmpste24−/− mice with the β-adrenergic agonist isoproterenol did not trigger ventricular arrhythmia but caused bradycardia-related premature ventricular complexes and slow-rate polymorphic ventricular rhythms during recovery. Patch-clamping in Zmpste24−/− cardiomyocytes revealed prolonged calcium-transient duration and reduced sarcoplasmic reticulum calcium loading and release, consistent with the absence of isoproterenol-induced ventricular arrhythmia. Zmpste24−/− progeroid mice also developed severe fibrosis-unrelated bradycardia and PQ interval and QRS complex prolongation. These conduction defects were accompanied by overt mislocalization of the gap junction protein connexin43 (Cx43). Remarkably, Cx43 mislocalization was also evident in autopsied left ventricle tissue from HGPS patients, suggesting intercellular connectivity alterations at late stages of the disease. The similarities between HGPS patients and progeroid mice reported here strongly suggest that defective cardiac repolarization and cardiomyocyte connectivity are important abnormalities in the HGPS pathogenesis that increase the risk of arrhythmia and premature death

Development of the Myocardial Interstitium.

The space between cardiac myocytes is commonly referred-to as the cardiac interstitium (CI). The CI is a unique, complex and dynamic microenvironment in which multiple cell types, extracellular matrix molecules, and instructive signals interact to crucially support heart homeostasis and promote cardiac responses to normal and pathologic stimuli. Despite the biomedical and clinical relevance of the CI, its detailed cellular structure remains to be elucidated. In this review, we will dissect the organization of the cardiac interstitium by following its changing cellular and molecular composition from embryonic developmental stages to adulthood, providing a systematic analysis of the biological components of the CI. The main goal of this review is to contribute to our understanding of the CI roles in health and disease. Anat Rec, 302:58-68, 2019. © 2018 Wiley Periodicals, Inc

Platinum-Doped Dendritic Structure as a Phosphorescent Label for Bacteria in Two-Photon Excitation Microscopy.

Herein, we present a water-soluble dendritric Pt(II) complex as a phosphorescent label for bacterial cells. The dendritic moiety endows the Pt(II) complex with unique properties such as water solubility, shielding from quenching by dioxygen, and binding to bacterial surfaces. The new biosensor was employed for two-photon excitation microscopy, and the binding was confirmed by electron microscopy, which demonstrates that such hybrid arrays can provide orthogonal yet complementary readouts

A New Versatile Platform for Assessment of Improved Cardiac Performance in Human-Engineered Heart Tissues

Cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) hold a great potential as human in vitro models for studying heart disease and for drug safety screening. Nevertheless, their associated immaturity relative to the adult myocardium limits their utility in cardiac research. In this study, we describe the development of a platform for generating three-dimensional engineered heart tissues (EHTs) from hPSC-CMs for the measurement of force while under mechanical and electrical stimulation. The modular and versatile EHT platform presented here allows for the formation of three tissues per well in a 12-well plate format, resulting in 36 tissues per plate. We compared the functional performance of EHTs and their histology in three different media and demonstrated that tissues cultured and maintained in maturation medium, containing triiodothyronine (T3), dexamethasone, and insulin-like growth factor-1 (TDI), resulted in a higher force of contraction, sarcomeric organization and alignment, and a higher and lower inotropic response to isoproterenol and nifedipine, respectively. Moreover, in this study, we highlight the importance of integrating a serum-free maturation medium in the EHT platform, making it a suitable tool for cardiovascular research, disease modeling, and preclinical drug testin

Cardiac electrical defects in progeroid mice and Hutchinson-Gilford progeria syndrome patients with nuclear lamina alterations

Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disease caused by defective prelamin A processing, leading to nuclear lamina alterations, severe cardiovascular pathology, and premature death. Prelamin A alterations also occur in physiological aging. It remains unknown how defective prelamin A processing affects the cardiac rhythm. We show age-dependent cardiac repolarization abnormalities in HGPS patients that are also present in the Zmpste24-/- mouse model of HGPS. Challenge of Zmpste24-/- mice with the ß-adrenergic agonist isoproterenol did not trigger ventricular arrhythmia but caused bradycardia-related premature ventricular complexes and slow-rate polymorphic ventricular rhythms during recovery. Patch-clamping in Zmpste24-/- cardiomyocytes revealed prolonged calcium-transient duration and reduced sarcoplasmic reticulum calcium loading and release, consistent with the absence of isoproterenol-induced ventricular arrhythmia. Zmpste24-/- progeroid mice also developed severe fibrosis-unrelated bradycardia and PQ interval and QRS complex prolongation. These conduction defects were accompanied by overt mislocalization of the gap junction protein connexin43 (Cx43). Remarkably, Cx43 mislocalization was also evident in autopsied left ventricle tissue from HGPS patients, suggesting intercellular connectivity alterations at late stages of the disease. The similarities between HGPS patients and progeroid mice reported here strongly suggest that defective cardiac repolarization and cardiomyocyte connectivity are important abnormalities in the HGPS pathogenesis that increase the risk of arrhythmia and premature death.Peer Reviewe