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Posts Taged confocal-microscopy

Experimental & molecular medicine: a new article with NANBIOSIS U17


The UAH research groupTranslational research of chronic diseases associated with aging and kidney disease has recently published an article in which unit NANBIOSIS unit 17 and Leica SP5 confocal microscope has had a great contribution

Located in the Support Center for Research in Medicine and Biology, Faculty of Medicine and Healthe Sciences, University of Alcalá (UAH). The Unit is equipped with a Leica TCS-SP5 confocal microscope. The confocal module is equipped with three spectral detection channels, AOBS (Acousto-optical beam splitter) and a resonant scanner system that allows analysis at high speed and resolution and makes possible the analysis of dynamic in vivo physiological processes in short periods, significantly improving the survival of living biological samples by shortening the exposure times to lasers. Includes an argon laser, a He/Ne laser, a DPSS laser diode and a violet excitation laser. The microscope is coupled to a cell incubation kit that allows multi-position time-lapse experiments. The equipment includes a workstation and four software for acquisition and analysis, which allow 3D visualizations, co-location studies, FRAP (Fluorescent Recovery after Photo-bleaching), FLIP (Fluorescent Loss in Photobleaching) and FRET (Fluorescence Resonant Energy Transfer). The equipment allows 3D characterization in detail of living cells and tissues through the use of different fluorochromes, expression and localization of molecules in 2/3D, colocalization and interaction of proteins or other types of molecules; endocytosis and intracellular transport, in situ hybridization with fluorescent probes, interaction studies between cells and materials, etc.

The unit provides researchers with a wide array of routine and specialized services as well as the latest advances in microscopy, including technical and scientific support to scientists for the study of cell/tissue biology, physiology and pathogenesis of diseases.

Article of reference

Campillo, S., Bohorquez, L., Gutiérrez-Calabrés, E., García-Ayuso, D., Miguel, V., Griera, M., Calle, Y., de Frutos, S., Rodríguez-Puyol, M., Rodríguez-Puyol, D., & Calleros, L. Indoxyl sulfate- and P-cresol-induced monocyte adhesion and migration is mediated by integrin-linked kinase-dependent podosome formation Experimental & molecular medicine, 2022, 54(3): 226–238. https://doi.org/10.1038/s12276-022-00738-8


Cardiovascular disease is an important cause of death in patients with chronic kidney disease (CKD). Protein-bound uremic toxins, such as p-cresyl and indoxyl sulfate (IS), are poorly removed during hemodialysis, leading to vascular endothelial dysfunction and leukocyte extravasation. These processes can be related to dynamic adhesion structures called podosomes. Several studies have  indicated the role of integrin-linked kinase (ILK) in the accumulation of integrin-associated proteins in podosomes. Here, we investigated the involvement of ILK and podosome formation in the adhesion and extravasation of monocytes under p-cresol (pc) and IS exposure. Incubation of THP-1 human monocyte cells with these toxins upregulated ILK kinase activity. Together, both toxins increased cell adhesion, podosome formation, extracellular matrix degradation, and migration of THP-1 cells, whereas ILK depletion with specific small interfering RNAs suppressed these processes. Interestingly, F-actin colocalized with cortactin in podosome cores, while ILK was colocalized in podosome rings under toxin stimulation. Podosome Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) and AKT protein depletion demonstrated that monocyte adhesion depends on podosome formation and that the ILK/AKT signaling pathway is involved in these processes. Ex vivo experiments showed that both toxins induced adhesion and podosome formation in leukocytes from wild-type mice, whereas these effects were not observed in leukocytes of conditional ILK-knockdown animals. In summary, under pc and IS stimulation, monocytes increase podosome formation and transmigratory capacity through an ILK/AKT signaling pathway-dependent mechanism, which could lead to vascular injury. Therefore, ILK could be a potential therapeutic target for the treatment of vascular damage associated with CKD.

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Hydrogels structured with dual stimuli responsive for biomedical applications

Researchers of NANBIOSIS Unit 17 Confocal Microscopy Service have participated in the research carried out structuring hydrophobic domains in Poly(N-isopropylacrylamide-co-Methacrylic acid) hydrogels for biomedical aplplications.

Hydrogels are cross-linked polymeric networks, which have the ability to hold a large amount of water in their structure. Hydrogels can be designed to respond to a specific stimulus such as temperature, pH, ionic strength, light, etc., making making them suitable for biomedical applications, as drug delivery.

The most popular responsive polymeric hydrogel is made of poly(N-isopropylacrylamide) (PNIPAM). The copolymerization of NIPAM with an acrylic/methacrylic acid monomer permits the development of a hydrogel with a dual stimuli response: temperature and medium pH. Additionally, the acid groups can electrostatically interact with positively charged drugs, the interaction being sensitive to pH. Therefore, these hydrogel systems have great potential for drug delivery applications.

At it seemed that the structuring of dual stimuli responsive hydrogels had not been reported, the authors deat with the structuring of poly(N-isopropylacrylamide-co-methacrylic acid) hydrogels to create hydrophobic domains by means of copolymerization of NIPAM with methacrylic acid and a small percentage of a nitrocatechol monomer in an aqueous medium that contained SDS. This structured hydrogel allows is capable of loading hydrophobic molecules as well as charged drugs. The hydrogel permitted cell adhesion and growth as well as its detachment when the temperature fell below the LCST.

As reported in the article, fluorescence images of cells were obtained with a laser scanning confocal microscope (LSCM) (Leica TCS-SP5) through the Confocal Microscopy Service of ICTS ‘NANBIOSIS’ U17 of the Biomedical Research Networking Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN at the University of Alcalá, Madrid, Spain).
equipped with a Diode 405 nm and a continuous Ar ion laser (488, 514,
561 and 633 nm).

Article of refrence:

Structuring hydrophobic domains in Poly(N-isopropylacrylamide-co-
Methacrylic acid) hydrogels. Mar López-González, M. Melia Rodrigo, Mercedes Valiente, Isabel Trabado, Francisco Mendicutib, Gema Marcelo. European Polymer Journal. April 2020 https://doi.org/10.1016/j.eurpolymj.2020.109695

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New Antimicrobial surfaces with self-cleaning properties and NANBIOSIS Unit 17

In the last decades, increased resistance to conventional antibiotics has led to important research in the development of alternative strategies for preventing pathogen dissemination. Antimicrobial surfaces containing a biocidal agent inhibit or reduce microorganisms growth capacity on the surface of materials. In addition to this microbial proliferation inhibition, antimicrobial coatings may also confer additional properties.

Researchers from the University of Alcalá and the CSIC Institute of Catalysis and Petrochemistry, have carried out a research about the self-cleaning properties of antimicrobial surfaces functionalized by photocatalytic ZnO electrosprayed coatings.

The authors prepared electrosprayed photoactive coatings of sol-gel ZnO nanoparticles tested as dual action self-cleaning antimicrobial surfaces. The materials showed excellent photocatalytic and photodisinfection properties due to the release of bioavailable zinc and photogenerated oxidative species. The surfaces were free from bacterial colonization and biofilm formation.

Confocal microscopy of NANBIOSIS U17 was used to reveal biofilm matrix using FilmTracer SYPRO Ruby stain and to assess cell viability by means of Live/Dead BacLight Bacterial Viability Kit.

Article of reference:

Laura Valenzuela, Ana Iglesias, Marisol Faraldos, Ana Bahamonde, Roberto Rosal, Antimicrobial surfaces with self-cleaning properties functionalized by photocatalytic ZnO electrosprayed coatings, Journal of Hazardous Materials, Volume 369, 2019, Pages 665-673, https://doi.org/10.1016/j.jhazmat.2019.02.073.

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