Publications

Understanding the dopaminergic system is a priority in neurobiology and neuropharmacology. Dopamine receptors are involved in the modulation of fundamental physiological functions, and dysregulation of dopaminergic transmission is associated with major neurological disorders. However, the available tools to dissect the endogenous dopaminergic circuits have limited specificity, reversibility, resolution, or require genetic manipulation. Here, we introduce azodopa, a novel photoswitchable ligand that enables reversible spatiotemporal control of dopaminergic transmission. We demonstrate that azodopa activates D1-like receptors in vitro in a light-dependent manner. Moreover, it enables reversibly photocontrolling zebrafish motility on a timescale of seconds and allows separating the retinal component of dopaminergic neurotransmission. Azodopa increases the overall neural activity in the cortex of anesthetized mice and displays illumination-dependent activity in individual cells. Azodopa is the first photoswitchable dopamine agonist with demonstrated efficacy in wild-type animals and opens the way to remotely controlling dopaminergic neurotransmission for fundamental and therapeutic purposes.

JTD Keywords: azobenzene, behavior, brainwave, d-1, dopamine, gpcr, in vivo electrophysiology, inhibitors, optogenetics, optopharmacology, photochromism, photopharmacology, photoswitch, stimulation, zebrafish, Azobenzene, Receptors, Zebrafish

Primary ventricular fibrillation (PVF) is a major driver of cardiac arrest in the acute phase of ST-segment elevation myocardial infarction (STEMI). Enrichment of cardiomyocyte plasma membranes with dietary polyunsaturated fatty acids (PUFA) reduces vulnerability to PVF experimentally, but clinical data are scarce. PUFA status in serum phospholipids is a valid surrogate biomarker of PUFA status in cardiomyocytes within a wide range of dietary PUFA. In this nested case-control study (n = 58 cases of STEMI-driven PVF, n = 116 control non-PVF STEMI patients matched for age, sex, smoking status, dyslipidemia, diabetes mellitus and hypertension) we determined fatty acids in serum phospholipids by gas-chromatography, and assessed differences between cases and controls, applying the Benjamini-Hochberg procedure on nominal P-values to control the false discovery rate (FDR). Significant differences between cases and controls were restricted to linoleic acid (LA), with PVF patients showing a lower level (nominal P = 0.002; FDR-corrected P = 0.027). In a conditional logistic regression model, each one standard deviation increase in the proportion of LA was related to a 42% lower prevalence of PVF (odds ratio = 0.58; 95% confidence interval, 0.37, 0.90; P = 0.02). The association lasted after the inclusion of confounders. Thus, regular consumption of LA-rich foods (nuts, oils from seeds) may protect against ischemia-driven malignant arrhythmias.

JTD Keywords: Arrhythmias, Fish-oil, Omega-3-fatty-acids, Sudden cardiac death

The identification of key components relevant to sensory perception of quality from commercial chocolate samples was accomplished after chemometric processing of GC×GC-MS (Comprehensive Two-dimensional Gas Chromatography with Mass Spectrometric Detection) profiles corresponding to HS-SPME (Headspace Solid Phase Microextraction) extracts of the samples. Descriptive sensory evaluation of samples was carried out using Optimized Descriptive Profile (ODP) procedures, where sensory attributes of 24 commercial chocolate samples were used to classify them in two classes (low and high chocolate flavor). 2D Fisher Ratio analysis was applied to four-way chromatographic data tensors (1st dimension retention time 1tR × 2nd dimension retention time 2tR × m/z × sample), to identify the crucial areas on the chromatograms that resulted on ODP class separation on Principal Component Analysis (PCA) scores plot. Comparing the relevant sections of the chromatograms to the analysis of the corresponding mass spectra, it was possible to assess that most of the information regarding the sample main sensory attributes can be related to only 14 compounds (2,5-dimethylpyrazine, 2,6-dimethyl-4-heptanol, 1-octen-3-ol, trimethylpyrazine, β-pinene, o-cimene, 2-ethyl-3,5-dimethylpyrazine, tetramethylpyrazine, benzaldehyde, 1,3,5-trimethylbenzene, 6-methyl-5-hepten-2-one, limonene, benzeneethanol and 1,1-dimethylbutylbenzene) among the complex blend of volatiles found on these extremely complex samples.

JTD Keywords: classification, cocoa, dark chocolate, feature-selection, fisher ratio, gcxgc-ms, impact, olfactometry, principal component analysis, sensorial analysis, Chocolate flavor, Fisher ratio, Flight mass-spectrometry, Gc×gc-ms, Principal component analysis, Sensorial analysis

Diamine oxydase and peroxidase have been co-immobilized onto layered double hydroxide (LDH) thin films for the development of real-time histamine biosensors. The chosen LDH materials are Mg2AlCO3, Mg4FeCl and Ca2AlCl. Prepared bi-enzymatic hybrid nanomaterials are capable of detecting histamine through the electrochemical oxidation of H(2)O(2)and are used as the sensitive membrane for potentiometric microelectrode. Histamine biosensors developed in this work have fast response of less than 20 s, are sensitive and selective, with a large dynamic range of 10(-8)-10(-3) M and a limit of detection of less than 10(-8) M. The detection limit of the developed bi-enzymatic biosensors is relatively higher than those corresponding with gas and liquid chromatography, which are still considered as the reference methods. Finally, the reproducibility, the specificity and the storage stability of the biosensors were studied.

JTD Keywords: Biogenic-amines, Biosensor, Diamine oxidase, Film, Fish, Histamine, Hybrid nanomaterial, Immobilization, Layer double hydroxide, Potentiometric biosensor, Specificity

The principles underlying the biomechanics of morphogenesis are largely unknown. Epiboly is an essential embryonic event in which three tissues coordinate to direct the expansion of the blastoderm. How and where forces are generated during epiboly, and how these are globally coupled remains elusive. Here we developed a method, hydrodynamic regression (HR), to infer 3D pressure fields, mechanical power, and cortical surface tension profiles. HR is based on velocity measurements retrieved from 2D+T microscopy and their hydrodynamic modeling. We applied HR to identify biomechanically active structures and changes in cortex local tension during epiboly in zebrafish. Based on our results, we propose a novel physical description for epiboly, where tissue movements are directed by a polarized gradient of cortical tension. We found that this gradient relies on local contractile forces at the cortex, differences in elastic properties between cortex components and the passive transmission of forces within the yolk cell. All in all, our work identifies a novel way to physically regulate concerted cellular movements that might be instrumental for the mechanical control of many morphogenetic processes.

JTD Keywords: Epiboly, Hydrodynamics, Mechanics, Morphogenesis, Zebrafish

exThe CFDP1 proteins have been linked to craniofacial development and osteogenesis in vertebrates, though specific human syndromes have not yet been identified. Alterations of craniofacial development represent the main cause of infant disability and mortality in humans. For this reason, it is crucial to understand the cellular functions and mechanism of action of the CFDP1 protein in model vertebrate organisms. Using a combination of genomic, molecular and cell biology approaches, we have performed a functional analysis of the cfdp1 gene and its encoded protein, zCFDP1, in the zebrafish model system. We found that zCFDP1 is present in the zygote, is rapidly produced after MTZ transition and is highly abundant in the head structures. Depletion of zCFDP1, induced by an ATG-blocking morpholino, produces considerable defects in craniofacial structures and bone mineralization. Together, our results show that zCFDP1 is an essential protein required for proper development and provide the first experimental evidence showing that in vertebrates it actively participates to the morphogenesis of craniofacial territories.

JTD Keywords: Craniofacial development, BCNT protein family, Zebrafish, Morpholino

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

JTD Keywords: Developmental Biology, Zebrafish, Yolk, Atomic Force Microscopy, Cortical Tension, Microrheology, Nanoparticle tracking

Genetic labeling techniques allow for noninvasive lineage tracing of cells in vivo. Two-photon inducible activators provide spatial resolution for superficial cells, but labeling cells located deep within tissues is precluded by scattering of the far-red illumination required for two-photon photolysis. Three-photon illumination has been shown to overcome the limitations of two-photon microscopy for in vivo imaging of deep structures, but whether it can be used for photoactivation remains to be tested. Here we show, both theoretically and experimentally, that three-photon illumination overcomes scattering problems by combining longer wavelength excitation with high uncaging three-photon cross-section molecules. We prospectively labeled heart muscle cells in zebrafish embryos and found permanent labeling in their progeny in adult animals with negligible tissue damage. This technique allows for a noninvasive genetic manipulation in vivo with spatial, temporal and cell-type specificity, and may have wide applicability in experimental biology.

JTD Keywords: Multi-photon microscopy, Photoactivation, Three-photon microscopy, Zebrafish