Insulin receptor membrane retention by a traceable chimeric mutant

Background: The insulin receptor (IR) regulates glucose homeostasis, cell growth and differentiation. It has been hypothesized that the specific signaling characteristics of IR are in part determined by ligand-receptor complexes localization. Downstream signaling could be triggered from the plasma membrane or from endosomes. Regulation of activated receptor's internalization has been proposed as the mechanism responsible for the differential isoform and ligand-specific signaling. Results: We generated a traceable IR chimera that allows the labeling of the receptor at the cell surface. This mutant binds insulin but fails to get activated and internalized. However, the mutant heterodimerizes with wild type IR inhibiting its auto-phosphorylation and blocking its internalization. IR membrane retention attenuates AP-1 transcriptional activation favoring Akt activation. Conclusions: These results suggest that the mutant acts as a selective dominant negative blocking IR internalization-mediated signaling.Fil: Giudice, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica; ArgentinaFil: Jares, Elizabeth Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Coluccio Leskow, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica; Argentina. Universidad Nacional de Luján. Departamento de Ciencias Básicas; Argentin

Insulin and insulin like growth factor II endocytosis and signaling via insulin receptor B

Insulin and insulin-like growth factors (IGFs) act on tetrameric tyrosine kinase receptors controlling essential functions including growth, metabolism, reproduction and longevity. The insulin receptor (IR) binds insulin and IGFs with different affinities triggering different cell responses. RESULTS: We showed that IGF-II induces cell proliferation and gene transcription when IR-B is over-expressed. We combined biotinylated ligands with streptavidin conjugated quantum dots and visible fluorescent proteins to visualize the binding of IGF-II and insulin to IR-B and their ensuing internalization. By confocal microscopy and flow cytometry in living cells, we studied the internalization kinetic through the IR-B of both IGF-II, known to elicit proliferative responses, and insulin, a regulator of metabolism. CONCLUSIONS: IGF-II promotes a faster internalization of IR-B than insulin. We propose that IGF-II differentially activates mitogenic responses through endosomes, while insulin-activated IR-B remains at the plasma membrane. This fact could facilitate the interaction with key effector molecules involved in metabolism regulation.Fil: Giudice, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Barcos, Lucía Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Guaimas, Francisco Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús. Instituto de Investigaciones Biotecnológicas (sede Chascomús); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Penas Steinhardt, Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R .A. Margni"; ArgentinaFil: Giordano, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina. Max Planck Institute for Biophysical. Laboratory of Cellular Dynamics; AlemaniaFil: Jares, Elizabeth Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Coluccio Leskow, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad Nacional de Luján. Departamento de Ciencias Básicas; Argentin

ESIPT and FRET probes for monitoring nanoparticle polymer coating stability

Coating strategies of inorganic nanoparticles (NPs) can provide properties unavailable to the NP core alone, such as targeting, specific sensing, and increased biocompatibility. Non-covalent amphiphilic NP capping polymers function via hydrophobic interactions with surface ligands and are extensively used to transfer NPs to aqueous media. For applications of coated NPs as actuators (sensors, markers, or for drug delivery) in a complex environment, such as biological systems, it is important to achieve a deep understanding of the factors affecting coating stability and behavior. We have designed a system that tests the coating stability of amphiphilic polymers through a simple fluorescent readout using either polarity sensing ESIPT (excited state intramolecular proton transfer) dyes or NP FRET (Förster resonance energy transfer). The stability of the coating was determined in response to changes in polarity, pH and ionic strength in the medium. Using the ESIPT system we observed linear changes in signal up to ∼20-25% v/v of co-solvent addition, constituting a break point. Based on such data, we propose a model for coating instability and the important adjustable parameters, such as the electrical charge distribution. FRET data provided confirmatory evidence for the model. The ESIPT dyes and FRET based methods represent new, simple tools for testing NP coating stability in complex environments.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

ESIPT and FRET probes for monitoring nanoparticle polymer coating stability

Coating strategies of inorganic nanoparticles (NPs) can provide properties unavailable to the NP core alone, such as targeting, specific sensing, and increased biocompatibility. Non-covalent amphiphilic NP capping polymers function via hydrophobic interactions with surface ligands and are extensively used to transfer NPs to aqueous media. For applications of coated NPs as actuators (sensors, markers, or for drug delivery) in a complex environment, such as biological systems, it is important to achieve a deep understanding of the factors affecting coating stability and behavior. We have designed a system that tests the coating stability of amphiphilic polymers through a simple fluorescent readout using either polarity sensing ESIPT (excited state intramolecular proton transfer) dyes or NP FRET (Förster resonance energy transfer). The stability of the coating was determined in response to changes in polarity, pH and ionic strength in the medium. Using the ESIPT system we observed linear changes in signal up to ∼20-25% v/v of co-solvent addition, constituting a break point. Based on such data, we propose a model for coating instability and the important adjustable parameters, such as the electrical charge distribution. FRET data provided confirmatory evidence for the model. The ESIPT dyes and FRET based methods represent new, simple tools for testing NP coating stability in complex environments.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

New Algorithm to Determine True Colocalization in Combination with Image Restoration and Time-Lapse Confocal Microscopy to Map Kinases in Mitochondria

The subcellular localization and physiological functions of biomolecules are closely related and thus it is crucial to precisely determine the distribution of different molecules inside the intracellular structures. This is frequently accomplished by fluorescence microscopy with well-characterized markers and posterior evaluation of the signal colocalization. Rigorous study of colocalization requires statistical analysis of the data, albeit yet no single technique has been established as a standard method. Indeed, the few methods currently available are only accurate in images with particular characteristics. Here, we introduce a new algorithm to automatically obtain the true colocalization between images that is suitable for a wide variety of biological situations. To proceed, the algorithm contemplates the individual contribution of each pixel's fluorescence intensity in a pair of images to the overall Pearsońs correlation and Manders' overlap coefficients. The accuracy and reliability of the algorithm was validated on both simulated and real images that reflected the characteristics of a range of biological samples. We used this algorithm in combination with image restoration by deconvolution and time-lapse confocal microscopy to address the localization of MEK1 in the mitochondria of different cell lines. Appraising the previously described behavior of Akt1 corroborated the reliability of the combined use of these techniques. Together, the present work provides a novel statistical approach to accurately and reliably determine the colocalization in a variety of biological images

Cabbage and fermented vegetables : From death rate heterogeneity in countries to candidates for mitigation strategies of severe COVID-19

Large differences in COVID-19 death rates exist between countries and between regions of the same country. Some very low death rate countries such as Eastern Asia, Central Europe, or the Balkans have a common feature of eating large quantities of fermented foods. Although biases exist when examining ecological studies, fermented vegetables or cabbage have been associated with low death rates in European countries. SARS-CoV-2 binds to its receptor, the angiotensin-converting enzyme 2 (ACE2). As a result of SARS-CoV-2 binding, ACE2 downregulation enhances the angiotensin II receptor type 1 (AT(1)R) axis associated with oxidative stress. This leads to insulin resistance as well as lung and endothelial damage, two severe outcomes of COVID-19. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is the most potent antioxidant in humans and can block in particular the AT(1)R axis. Cabbage contains precursors of sulforaphane, the most active natural activator of Nrf2. Fermented vegetables contain many lactobacilli, which are also potent Nrf2 activators. Three examples are: kimchi in Korea, westernized foods, and the slum paradox. It is proposed that fermented cabbage is a proof-of-concept of dietary manipulations that may enhance Nrf2-associated antioxidant effects, helpful in mitigating COVID-19 severity.Peer reviewe

Nrf2-interacting nutrients and COVID-19 : time for research to develop adaptation strategies

There are large between- and within-country variations in COVID-19 death rates. Some very low death rate settings such as Eastern Asia, Central Europe, the Balkans and Africa have a common feature of eating large quantities of fermented foods whose intake is associated with the activation of the Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) anti-oxidant transcription factor. There are many Nrf2-interacting nutrients (berberine, curcumin, epigallocatechin gallate, genistein, quercetin, resveratrol, sulforaphane) that all act similarly to reduce insulin resistance, endothelial damage, lung injury and cytokine storm. They also act on the same mechanisms (mTOR: Mammalian target of rapamycin, PPAR gamma:Peroxisome proliferator-activated receptor, NF kappa B: Nuclear factor kappa B, ERK: Extracellular signal-regulated kinases and eIF2 alpha:Elongation initiation factor 2 alpha). They may as a result be important in mitigating the severity of COVID-19, acting through the endoplasmic reticulum stress or ACE-Angiotensin-II-AT(1)R axis (AT(1)R) pathway. Many Nrf2-interacting nutrients are also interacting with TRPA1 and/or TRPV1. Interestingly, geographical areas with very low COVID-19 mortality are those with the lowest prevalence of obesity (Sub-Saharan Africa and Asia). It is tempting to propose that Nrf2-interacting foods and nutrients can re-balance insulin resistance and have a significant effect on COVID-19 severity. It is therefore possible that the intake of these foods may restore an optimal natural balance for the Nrf2 pathway and may be of interest in the mitigation of COVID-19 severity

A versatile Near-Infrared Asymmetric Tricarbocyanine for Zinc Ion Sensing in Water

We have synthesized a near infrared emissive asymmetric tricarbocyanine conveniently functionalized to improve bioconjugation. The leading structure contains a versatile derivatization point at the meso position for facile radical-nucleophilic aromatic substitution. We have evaluated a DPEN (N,N-di(2-picolyl)ethylendiamine) derivative of this dye as a highly selective sensor for zinc (II) in aqueous medium, which performs in an appropriate sensitivity range for biological studies. The probe was successfully conjugated to a protein-ligand model with high affinity and specificity (biotin-streptavidin technology) rendering an excellent performance of sensing. In a general strategy to obtain sensitive probes combining fluorescent nanoparticles and molecular fluorophores, a preliminary design of a supramolecular assembly derived from the conjugation of the molecular sensor to quantum dots (QDs) was also investigated. The advantages and problems of FRET-based sensors are also discussed.Fil: Menéndez, Guillermo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentina;Fil: Samaniego Lopez, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentina;Fil: Jares, Elizabeth Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentina;Fil: Spagnuolo, Carla Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentina

NIR fluorescent biotinylated cyanine dye: optical properties and combination with quantum dots as a potential sensing device

We present a water soluble and fluorescent biotinylated probe derived from a carbocyanine dye. A high efficiency of energy transfer was measured when the dyes were placed on the surface of streptavidin conjugated Quantum dots. The system is a model platform for potential application as a FRET-based fluorescent sensor.Fil: Menéndez, Guillermo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Pichel, María Eva. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; ArgentinaFil: Spagnuolo, Carla Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Jares, Elizabeth Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentin

Quantum Dots as Templates for Self-Assembly of Photoswitchable Polymers: Small, Dual-Color Nanoparticles Capable of Facile Photomodulation

A photomodulatable amphiphilic polymer has been synthesized with a backbone of poly[isobutylene-alt-maleic anhydride] and pendant dodecyl alkyl chains, Lucifer Yellow (LY) fluorescent probes, and diheteroarylethenes photochromic (PC) groups. The latter serve as reversible UV-activated FRET acceptors for the LY donors. We characterized the spectral and switching properties of the polymer in an organic solvent (CHCl3). In an aqueous medium the polymer forms polymersomes, constituting fluorescence probes ~75 nm in diameter. Self-assembly of the polymer on the surface of a quantum dot (QD) serving as a template creates a dual-color photoswitchable nanoparticle (psNP) with improved properties due to the increase in polymer density and efficiency of PC photoconversion. The psNP exhibits a second QD red emission band that functions as an internal standard requiring only a single excitation wavelength, and is much reduced in size (<20 nm diameter) compared to the polymersomes. The QD template also greatly increases the depth of modulation by photochromic FRET of the LY emission monitored by both steady-state and time-resolved (lifetime) fluorescence (from 20% to 70%, and from 12% to 55%, respectively).Fil: Diaz, Sebastian Andres. Max Planck Institute for Biophysical Chemistry. Laboratory of Cellular Dynamics; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Giordano, Luciana. Max Planck Institute for Biophysical Chemistry. Laboratory of Cellular Dynamics; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Azcárate, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Jovin, Tomás. Max Planck Institute for Biophysical Chemistry. Laboratory of Cellular Dynamics; AlemaniaFil: Jares, Elizabeth Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentin