Suchergebnisse

Myocardial fibroblast activation coupled with extracellular matrix production is a pathological signature of myocardial fibrosis and is governed mainly by transforming growth factor TGFβ-Smad2/3 signaling. Targeting the ubiquitous TGFβ leads to cellular homeostasis deregulation with adverse consequences. We previously showed the anti-fibrotic effects upon downregulation of 90-kDa heat shock protein (Hsp90), a chaperone that associates to the TGFβ signaling cascade. In the present study, we use a fluorescent-labeled Hsp90 protein inhibitor (CTPR390–488) with specific Hsp90 binding properties to reduce myocardial pro-fibrotic events in vitro and in vivo. The mechanism of action involves the disruption of TGFβRI-Hsp90 complex, resulting in a decrease in TGFβ signaling and reduction in extracellular matrix collagen. In vivo, decreased myocardial collagen deposition was observed upon CTPR390–488 treatment in a pro-fibrotic mouse model. This is the first study demonstrating the ability of an engineered Hsp90 protein inhibitor to block collagen expression, reduce the motility of myocardial TGFβ-activated fibroblasts and ameliorate angiotensin-II induced cardiac myocardial fibrosis in vivo. ; This work was supported by the Spanish Ministerio de Economia, Industria y Competitividad [BIO2015-72124-EXP] and [BIO2016- 77367-R], and the European Research CouncilERC-2014-CoG-648071-ProNANO. The Instituto de Formación e Investigación Marqués de Valdecilla IDIVAL [InnVal 15/31] and [InnVal 17/22], and the FEDER European Union, SODERCAN "Proyecto Puente 2017".

© 2021 The Author(s). ; Revealing the intracellular location of novel therapeutic agents is paramount for the understanding of their effect at the cell ultrastructure level. Here, we apply a novel correlative cryo 3D imaging approach to determine the intracellular fate of a designed protein–nanomaterial hybrid with antifibrotic properties that shows great promise in mitigating myocardial fibrosis. Cryo 3D structured illumination microscopy (cryo-3D-SIM) pinpoints the location and cryo soft X-ray tomography (cryo-SXT) reveals the ultrastructural environment and subcellular localization of this nanomaterial with spatial correlation accuracy down to 70 nm in whole cells. This novel high resolution 3D cryo correlative approach unambiguously locates the nanomaterial after overnight treatment within multivesicular bodies which have been associated with endosomal trafficking events by confocal microscopy. Moreover, this approach allows assessing the cellular response towards the treatment by evaluating the morphological changes induced. This is especially relevant for the future usage of nanoformulations in clinical practices. This correlative super-resolution and X-ray imaging strategy joins high specificity, by the use of fluorescence, with high spatial resolution at 30 nm (half pitch) provided by cryo-SXT in whole cells, without the need of staining or fixation, and can be of particular benefit to locate specific molecules in the native cellular environment in bio-nanomedicine. ; This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 754397. This work was partially supported by the European Research Council ERC-CoG-648071-ProNANO, ERC-PoC-841063-NIMM; Spanish State Research Agency, Spain; (BIO2015-72124-EXP), (MDM-2017-0720), (PID2019-111649RB-I00) and (RTI2018-095214-B-I00); the Basque Government (Elkartek KK-2017/00008; RIS3-2019222005); and the Next Generation and Innovation Valdecilla (PREVAL19/05), (IDIVAL INNVAL17/22), (INNVAL20/34), (NVAL18/11) and (NextVal 18/14). This work was performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency – Grant No. MDM-2017-0720 (CIC biomaGUNE).

© 2021 The Author(s). ; Protein-based hybrid nanomaterials have recently emerged as promising platforms to fabricate tailored multifunctional biologics for biotechnological and biomedical applications. This work shows a simple, modular, and versatile strategy to design custom protein hybrid nanomaterials. This approach combines for the first time the engineering of a therapeutic protein module with the engineering of a nanomaterial-stabilizing module within the same molecule, resulting in a multifunctional hybrid nanocomposite unachievable through conventional material synthesis methodologies. As the first proof of concept, a multifunctional system was designed ad hoc for the therapeutic intervention and monitoring of myocardial fibrosis. This hybrid nanomaterial combines a designed Hsp90 inhibitory domain and a metal nanocluster stabilizing module resulting in a biologic drug labelled with a metal nanocluster. The engineered nanomaterial actively reduced myocardial fibrosis and heart hypertrophy in an animal model of cardiac remodeling. In addition to the therapeutic effect, the metal nanocluster allowed for in vitro, ex vivo, and in vivo detection and imaging of the fibrotic disease under study. This study evidences the potential of combining protein engineering and protein-directed nanomaterial engineering approaches to design custom nanomaterials as theranostic tools, opening up unexplored routes to date for the next generation of advanced nanomaterials in medicine. ; This work was partially supported by the European Research Council ERC-CoG-648071-ProNANO and ERC-PoC-841063-NIMM; Agencia Estatal de Investigación, Spain (PID2019-111649RB-I00; RTI2018-095214-B-I00; BIO2016-77367-C2-1-R; and BIO2015-72124-EXP); the Basque Government (Elkartek KK-2017/00008; RIS3-2019222005); and IDIVAL InnVal 17/22. C. S. C. thanks Gipuzkoa Foru Aldundia (Gipuzkoa Fellows program; grant number 2019-FELL-000018-01/62/2019) for financial support. E.L.-M. thanks the Spanish Ministry of Science and Innovation for the FPI grant (BES-2017-079646). This work was performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency – Grant No. MDM-2017-0720 (CIC biomaGUNE). SXRF maps were acquired at the I18 beamline in the frame of the Diamond proposals SP20603.

Altres ajuts: Fondo Investigaciones Sanitarias Grants 07/0601 And PI11/00726, by CIBER de Diabetes y Enfermedades Metabólicas Relacionadas (CIBERDEM); CIBERDEM is an initiative by Carlos III Health Institute in Madrid and the Spanish Health Ministry, by PROMETEO/2009/029, AP-091/11, and ACOMP/2013/039 from the Valencian Government. ; BACKGROUND: The role of genetic variations within the ghrelin gene on cardiometabolic profile and nutritional status is still not clear in humans, particularly in elderly people. OBJECTIVES: We investigated six SNPs of the ghrelin gene and their relationship with metabolic syndrome (MS) components. SUBJECTS AND METHODS: 824 subjects (413 men/411 women, age 77.31±5.04) participating in the Mataró aging study (n = 310) and the Hortega study (n = 514) were analyzed. Anthropometric variables, ghrelin, lipids, glucose and blood pressure levels were measured, and distribution of SNPs -994CT (rs26312), -604GA (rs27647), -501AC (rs26802), R51Q (rs34911341), M72L (rs696217) and L90G (rs4684677) of the ghrelin gene evaluated. Genotypes were determined by multiplex PCR and SNaPshot minisequencing. MS (IDF criteria) was found in 54.9%.RESULTS:No association between any of the SNPs and levels of total fasting circulating ghrelin levels was found. C/A-A/A genotype of M72L was associated with increased risk of central obesity according to IDF criteria, while G/A-G/G genotypes of -604GA with reduced risk. A/A genotype of -501AC polymorphism was associated to decreased BMI. In relation to lipid profile, the same genotypes of -604GA were associated with increased total cholesterol and LDL-cholesterol and -501AC with reduced triglycerides. There were no associations with systolic or diastolic blood pressure levels or with hypertension, glucose levels or diabetes and ghrelin polymorphisms. However, G/G genotype of -604GA was associated with glucose >100 mg/dL. Haplotype analysis showed that only one haplotype is associated with increased risk of waist circumference and central obesity. The analysis ...

Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly, open-source Python control software. Therefore, cryoSIM democratizes the ability to detect molecules using super-resolution fluorescence imaging of cryopreserved specimens for correlation with their cellular ultrastructure.

Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly, open-source Python control software. Therefore, cryoSIM democratizes the ability to detect molecules using super-resolution fluorescence imaging of cryopreserved specimens for correlation with their cellular ultrastructure.