Application of the dual-luciferase reporter assay to the analysis of promoter activity in Zebrafish embryos

<p>Abstract</p> <p>Background</p> <p>The dual-luciferase assay has been widely used in cell lines to determine rapidly but accurately the activity of a given promoter. Although this strategy has proved very useful, it does not allow the promoter and gene function to be analyzed in the context of the whole organism.</p> <p>Results</p> <p>Here, we present a rapid and sensitive assay based on the classical dual-luciferase reporter technique which can be used as a new tool to characterize the minimum promoter region of a gene as well as the <it>in vivo </it>response of inducible promoters to different stimuli. We illustrate the usefulness of this system for studying both constitutive (telomerase) and inducible (NF-κB-dependent) promoters. The flexibility of this assay is demonstrated by induction of the NF-κB-dependent promoters using simultaneous microinjection of different pathogen-associated molecular patterns as well as with the use of morpholino-gene mediated knockdown.</p> <p>Conclusion</p> <p>This assay has several advantages compared with the classical <it>in vitro </it>(cell lines) and <it>in vivo </it>(transgenic mice) approaches. Among others, the assay allows a rapid and quantitative measurement of the effects of particular genes or drugs in a given promoter in the context of a whole organism and it can also be used in high throughput screening experiments.</p

Treatment of skeletal and non-skeletal alterations of Mucopolysaccharidosis type IVA by AAV-mediated gene therapy.

Mucopolysaccharidosis type IVA (MPSIVA) or Morquio A disease, a lysosomal storage disorder, is caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency, resulting in keratan sulfate (KS) and chondroitin-6-sulfate accumulation. Patients develop severe skeletal dysplasia, early cartilage deterioration and life-threatening heart and tracheal complications. There is no cure and enzyme replacement therapy cannot correct skeletal abnormalities. Here, using CRISPR/Cas9 technology, we generate the first MPSIVA rat model recapitulating all skeletal and non-skeletal alterations experienced by patients. Treatment of MPSIVA rats with adeno-associated viral vector serotype 9 encoding Galns (AAV9-Galns) results in widespread transduction of bones, cartilage and peripheral tissues. This led to long-term (1 year) increase of GALNS activity and whole-body correction of KS levels, thus preventing body size reduction and severe alterations of bones, teeth, joints, trachea and heart. This study demonstrates the potential of AAV9-Galns gene therapy to correct the disabling MPSIVA pathology, providing strong rationale for future clinical translation to MPSIVA patients.We thank A. Arbos, S. Turon, M. Morro, J. Barrero, L. Hernandez, a. Vazquez, V. Melgarejo, and L. Noya for technical assistance. This work was supported by grants from the Ministerio de Ciencia, Innovacion y Universidades, Plan Nacional I+D+I and the European Union through Regional Development Funds (ERDF) (SAF2017-86166-R), Generalitat de Catalunya (2017SGR1508 and ICREA Academia Award to F.B.) and MPS Espana Foundation. This work is part of a public-private partnership on gene therapy between UAB and ESTEVE Pharmaceuticals, Spain. V.S. and G.E. received a predoctoral fellowship from the Generalitat de Catalunya, Spain.S

Beneficial effects of manually assisted chiropractic adjusting instrument in a rabbit model of osteoarthritis.

Osteoarthritis (OA) is a degenerative disease characterized by injury of all joint tissues. Our previous study showed that in experimental osteoporosis, chiropractic manipulation (CM) exerts protective effects on bone. We here assessed whether CM might ameliorate OA by improving subchondral bone sclerosis, cartilage integrity and synovitis. Male New-Zealand rabbits underwent knee surgery to induce OA by anterior cruciate ligament injury. CM was performed using the chiropractic instrument ActivatorV 3 times/week for 8 weeks as follows: force 2 setting was applied to the tibial tubercle of the rabbit right hind limb (TM-OA), whereas the corresponding left hind limb received a false manipulation (FM-OA) consisting of ActivatorV firing in the air and slightly touching the tibial tubercle. After sacrifice, subchondral bone integrity was assessed in the tibiae by microCT and histology. Cartilage damage and synovitis were estimated by Mankin's and Krenn's scores, respectively, and histological techniques. Bone mineral density and content in both cortical and trabecular compartments of subchondral bone decreased in OA rabbits compared to controls, but partially reversed in the TM-OA group. Trabecular bone parameters in the latter group also showed a significant improvement compared to FM-OA group. Moreover RANKL, OPG, ALP and TRAP protein expression in subchondral bone significantly decreased in TM-OA rabbits with respect to FM-OA group. CM was associated with lower Mankin's and Krenn's scores and macrophage infiltrate together with a decreased protein expression of pro-inflammatory, fibrotic and angiogenic factors, in TM-OA rabbits with respect to FM-OA. Our results suggest that CM may mitigate OA progression by improving subchondral bone as well as cartilage and synovial membrane status.AODM was supported by grants from the Spanish Chiropractic Association (AEQ). AM was supported by grants from Spanish Ministry of Economy and Competitiveness and Carlos III Institute of Health (CP15/00053 and PI16/00991). We thank Dr. Carlos Guillén-Viejo (School of Pharmacy, Universidad Complutense de Madrid) for his help in advising in molecular biology methods. The authors are also grateful to Mark S. Davis for his assistance with editing and proofreading the article.S

A new antibiotic-loaded sol-gel can prevent bacterial prosthetic joint infection: From in vitro studies to an in vivo model

The aim of this study was to evaluate the effect of a moxifloxacin-loaded organicinorganic sol-gel with different antibiotic concentration in the in vitro biofilm development and treatment against Staphylococcus aureus, S. epidermidis, and Escherichia coli, cytotoxicity and cell proliferation of MC3T3-E1 osteoblasts; and its efficacy in preventing the prosthetic joint infection (PJI) caused by clinical strains of S. aureus and E. coli using an in vivo murine model. Three bacterial strains, S. epidermidis ATCC 35984, S. aureus 15981, and, E. coli ATCC 25922, were used for microbiological studies. Biofilm formation was induced using tryptic-soy supplemented with glucose for 24 h, and then, adhered and planktonic bacteria were estimated using drop plate method and absorbance, respectively. A 24-h-mature biofilm of each species growth in a 96- well plate was treated for 24 h using a MBECTM biofilm Incubator lid with pegs coated with the different types of sol-gel, after incubation, biofilm viability was estimated using alamrBlue. MC3T3-E1 cellular cytotoxicity and proliferation were evaluated using CytoTox 96 Non-Radioactive Cytotoxicity Assay and alamarBlue, respectively. The microbiological studies showed that sol-gel coatings inhibited the biofilm development and treated to a mature biofilm of three evaluated bacterial species. The cell studies showed that the sol-gel both with and without moxifloxacin were non-cytotoxic and that cell proliferation was inversely proportional to the antibiotic concentration containing by sol-gel. In the in vivo study, mice weight increased over time, except in the E. coliinfected group without coating. The most frequent symptoms associated with infection were limping and piloerection; these symptoms were more frequent in infected groups with non-coated implants than infected groups with coated implants. The response of moxifloxacin-loaded sol-gel to infection was either total or completely absent. No differences in bone mineral density were observed between groups with coated and non-coated implants and macrophage presence lightly increased in the bone grown directly in contact with the antibiotic-loaded sol-gel. In conclusion, moxifloxacinloaded sol-gel coating is capable of preventing PJI caused by both Gram-positive and Gram-negative speciesThe authors acknowledge the financial support from the Regional Government of Madrid through the program MULTIMAT-CHALLENGE (S2013/MIT-2862), and from the Mutua Madrileña Foundation (04078/001). JA-C was funded by an FPI grant from the Spanish Ministry of Economics and Competitiveness (BES-2014-069007). AM was funded by grants from Instituto de Salud Carlos III through the “Miguel Servet” program (CP15/00053). DR was funded by a grant from the Fundación Conchita Rábag

Computed tomography assesment in the characterization of mouse model for Costello Syndrome

Proceeding of: 2008 World Molecular Imaging Congress (WMIC 2008), 10-13 september 2008. Nice, Franc

Clickable albumin nanoparticles for pretargeted drug delivery toward PD-L1 overexpressing tumors in combination immunotherapy

We present a simple methodology to design a pretargeted drug delivery system, based on clickable anti-programmed death ligand 1 (anti-PD-L1) antibodies (Abs) and clickable bovine serum albumin (BSA) nanoparticles (NPs). Pretargeted drug delivery is based on the decoupling of a targeting moiety and a drug-delivering vector which can then react in vivo after separate injections. This may be key to achieve active targeting of drug-delivering NPs toward cancerous tissue. In pretargeted approaches, drug-delivering NPs were observed to accumulate in a higher amount in the targeted tissue due to shielding-related enhanced blood circulation and size-related enhanced tissue penetration. In this work, BSA NPs were produced using the solvent precipitation methodology that renders colloidally stable NPs, which were subsequently functionalized with a clickable moiety based on chlorosydnone (Cl-Syd). Those reactive groups are able to specifically react with dibenzocyclooctyne (DBCO) groups in a click-type fashion, reaching second-order reaction rate constants as high as 1.9 M-1·s-1, which makes this reaction highly suitable for in vivo applications. The presence of reactive Cl-Syd was demonstrated by reacting the functionalized NPs with a DBCO-modified sulfo-cyanine-5 dye. With this reaction, it was possible to infer the number of reactive moieties per NPs. Finally, and with the aim of demonstrating the suitability of this system to be used in pretargeted strategies, functionalized fluorescent NPs were used to label H358 cells with a clickable anti-PD-L1 Ab, applying the reaction between Cl-Syd and DBCO as corresponding clickable groups. The results of these experiments demonstrate the bio-orthogonality of the system to perform the reaction in vitro, in a period as short as 15 mi

Monitoring vascular normalization induced by antiangiogenic treatment with (18)F-fluoromisonidazole-PET

This work was supported by the following sources: Fondo de Investigacion Sanitaria (Ministry of Health, Spain; numbers FIS PI10/0288, FIS PI13/00430, FIS PI 11/00616, CPII14/00005 and FIS PI14/00860; the first two awarded to MQF and the last three to MD), and "Fondo Europeo de Desarrollo Regional (FEDER) - Una manera de hacer Europa". MQF is a recipient of a 2010 Beca-Retorno from the AECC Scientific Foundation. Rosae Foundation and AVON Espana S.A.U. contributed to this work with unrestricted donations. Dovitinib was kindly provided by Novartis.BACKGROUND: Rationalization of antiangiogenics requires biomarkers. Vascular re-normalization is one widely accepted mechanism of action for this drug class. The interstitium of tumors with abnormal vasculature is hypoxic. We sought to track vascular normalization with (18)F-misonidazole ([18F]-FMISO, a probe that detects hypoxia) PET, in response to window-of-opportunity (WoO) treatment with the antiangiogenic dovitinib. METHODS: Two patient-derived pancreas xenografts (PDXs; Panc215 and Panc286) and the spontaneous breast cancer model MMTV-PyMT were used. Animals were treated during 1 week of WoO treatment with vehicle or dovitinib, preceded and followed by [18F]-FMISO-PET, [18F]-FDG-PET, and histologic assessment (dextran extravasation, hypoxia and microvessel staining, and necrosis, cleaved caspase-3 and Ki67 measurements). After WoO treatment, gemcitabine (pancreas)/adriamycin (breast) or vehicle was added and animals were treated until the humane endpoint. Tumor growth inhibition (TGI) and survival were the parameters studied. RESULTS: [18F]-FMISO SUV did not change after dovitinib-WoO treatment compared to vehicle-WoO (0.54 vs. 0.6) treatment in Panc215, but it decreased significantly in Panc286 (0.58 vs. 1.18; P < 0.05). In parallel, 10-KDa perivascular dextran extravasation was not reduced with dovitinib or vehicle-WoO treatment in Panc215, but it was reduced in Panc286. Whereas the addition of dovitinib to gemcitabine was indifferent in Panc215, it increased TGI in Panc286 (TGI switched from -59% to +49%). [18F]-FMISO SUV changes were accompanied by an almost 100% increase in interstitial gemcitabine delivery (665-1260 ng/mL). The results were validated in the PyMT model. CONCLUSIONS: [18F]-FMISO accurately monitored vascular re-normalization and improved interstitial chemotherapy delivery.This work was supported by the following sources: Fondo de Investigacion Sanitaria (Ministry of Health, Spain; numbers FIS PI10/0288, FIS PI13/00430, FIS PI 11/00616, CPII14/00005 and FIS PI14/00860; the first two awarded to MQF and the last three to MD), and "Fondo Europeo de Desarrollo Regional (FEDER) - Una manera de hacer Europa". MQF is a recipient of a 2010 Beca-Retorno from the AECC Scientific Foundation. Rosae Foundation and AVON Espana S.A.U. contributed to this work with unrestricted donations. Dovitinib was kindly provided by Novartis.S

Parathyroid hormone-related protein (107-111) improves the bone regeneration potential of gelatin-glutaraldehyde biopolymer-coated hydroxyapatite

Biopolymer-coated nanocrystalline hydroxyapatite (HA) made as macroporous foams which are degradable and flexible are promising candidates as orthopaedic implants. The C-terminal (107-111) epitope of parathyroid hormone-related protein (PTHrP) exhibits osteogenic properties. The main aim of this study was to evaluate whether PTHrP (107-111) loading into gelatin-glutaraldehyde biopolymer-coated HA (HA(GIu)) scaffolds would produce an optimal biomaterial for tissue engineering applications. HA(GIu) scaffolds with and without PTHrP (107-111) were implanted into a cavitary defect performed in both distal tibial metaphysis of adult rats. Animals were sacrificed after 4 weeks for histological, microcomputerized tomography and gene expression analysis of the callus. At this time, bone healing occurred only in the presence of PTHrP (107-111)-containing HA(Giu) implant, related to an increase in bone volume/tissue volume and trabecular thickness, cortical thickness and gene expression of osteocalcin and vascular cell adhesion molecule I, but a decreased gene expression of Wnt inhibitors, SOST and dickkopf homolog 1. The autonomous osteogenic effect of the PTHrP (107-111)-loaded HA(Giu) scaffolds was confirmed in mouse and human osteoblastic cell cultures. Our findings demonstrate the advantage of loading PTHrP (107-111) into degradable HA(GIu) scaffolds for achieving an optimal biomaterial that is promising for low load bearing clinical applications. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved

Chrononutrition and Polyphenols: Roles and Diseases

Biological rhythms can influence the activity of bioactive compounds, and at the same time, the intake of these compounds can modulate biological rhythms. In this context, chrononutrition has appeared as a research field centered on the study of the interactions among biological rhythms, nutrition, and metabolism. This review summarizes the role of phenolic compounds in the modulation of biological rhythms, focusing on their effects in the treatment or prevention of chronic diseases. Heterotrophs are able to sense chemical cues mediated by phytochemicals such as phenolic compounds, promoting their adaptation to environmental conditions. This is called xenohormesis. Hence, the consumption of fruits and vegetables rich in phenolic compounds exerts several health benefits, mainly attributed to the product of their metabolism. However, the profile of phenolic compounds present in plants differs among species and is highly variable depending on agricultural and technological factors. In this sense, the seasonal consumption of polyphenol-rich fruits could induce important changes in the regulation of physiology and metabolism due to the particular phenolic profile that the fruits contain. This fact highlights the need for studies that evaluate the impact of these specific phenolic profiles on health to establish more accurate dietary recommendations.España MINISTERIO DE ECONOMÍA, INDUSTRIA Y COMPETITIVIDAD (AGL2016-77105-R