Amyloids are β-sheets-rich protein fibrils that cause neurodegenerative and other incurable human diseases affecting millions of people worldwide. However, a number of proteins is functional in the amyloid state in various organisms from bacteria to humans. Using an original proteomic approach, we identified a set of proteins forming amyloid-like aggregates in the brain of young healthy rats. One of them is the FXR1 protein, which is known to regulate memory and emotions. We showed that FXR1 clearly colocalizes in cortical neurons with amyloid-specific dyes Congo-Red, Thioflavines S and T. FXR1 extracted from brain by immunoprecipitation shows yellow-green birefringence after staining with Congo red. This protein forms in brain detergent-resistant amyloid oligomers and insoluble aggregates. RNA molecules that are colocalized with FXR1 in cortical neurons are insensitive to treatment with RNase A. All these data suggest that FXR1 functions in rat brain in amyloid form. The N-terminal amyloid-forming fragment of FXR1 is highly conserved across mammals. We assume that the FXR1 protein may be presented in amyloid form in brain of different species of mammals, including humans.
Synthesis of biocompatible near infrared phosphorescent complexes and their application in bioimaging as triplet oxygen sensors in live systems are still challenging areas of organometallic chemistry. We have designed and synthetized four novel iridium [Ir(N^C)2(N^N)]+ complexes (N^C–benzothienyl-phenanthridine based cyclometalated ligand; N^N–pyridin-phenanthroimidazol diimine chelate), decorated with oligo(ethylene glycol) groups to impart these emitters’ solubility in aqueous media, biocompatibility, and to shield them from interaction with bio-environment. These substances were fully characterized using NMR spectroscopy and ESI mass-spectrometry. The complexes exhibited excitation close to the biological “window of transparency”, NIR emission at 730 nm, and quantum yields up to 12% in water. The compounds with higher degree of the chromophore shielding possess low toxicity, bleaching stability, absence of sensitivity to variations of pH, serum, and complex concentrations. The properties of these probes as oxygen sensors for biological systems have been studied by using phosphorescence lifetime imaging experiments in different cell cultures. The results showed essential lifetime response onto variations in oxygen concentration (2.0–2.3 μs under normoxia and 2.8–3.0 μs under hypoxia conditions) in complete agreement with the calibration curves obtained “in cuvette”. The data obtained indicate that these emitters can be used as semi-quantitative oxygen sensors in biological systems.
Preeclampsia (PE) is a multisystem heterogeneous complication of pregnancy remaining a leading cause of maternal and perinatal morbidity and mortality over the world. PE has a large spectrum of clinical features and symptoms, which make diagnosis challenging. Despite a long period of studying, PE etiology is still unclear and there are no reliable rapid tests for early diagnosis of this disease. During the last decade, it was shown that proteins misfolding and aggregation are associated with PE. Several proteins, including amyloid beta peptide, transthyretin, alpha-1 antitrypsin, albumin, IgG k-free light chains, and ceruloplasmin are dysregulated in PE, resulting in toxic deposition of amyloid-like aggregates in the placenta and body fluids. It is also possible that aggregated proteins induce defective trophoblast invasion, placental ischemia, ER stress, and promote PE manifestation. The fact that protein aggregation is an emerging biomarker of PE provides an opportunity to develop new diagnostic approaches based on amyloids special features, such as Congo red (CR) staining and thioflavin T (ThT) enhanced fluorescence.
The development of cell-based approaches to the treatment of various cornea pathologies, including limbal stem cell deficiency (LSCD), is an area of current interest in regenerative biomedicine. In this context, the shortage of donor material is urgent, and limbal mesenchymal stem cells (L-MSCs) may become a promising cell source for the development of these novel approaches, being established mainly within the rabbit model. In this study, we obtained and characterized rabbit L-MSCs and modified them with lentiviral transduction to express the green fluorescent protein EGFP (L-MSCs-EGFP). L-MSCs and L-MSCs-EGFP express not only stem cell markers specific for mesenchymal stem cells but also ABCG2, ABCB5, ALDH3A1, PAX6, and p63a specific for limbal epithelial stem cells (LESCs), as well as various cytokeratins (3/12, 15, 19). L-MSCs-EGFP have been proven to differentiate into adipogenic, osteogenic, and chondrogenic directions, as well as to transdifferentiate into epithelial cells. The possibility of using L-MSCs-EGFP to study the biocompatibility of various scaffolds developed to treat corneal pathologies was demonstrated. L-MSCs-EGFP may become a useful tool for studying regenerative processes occurring during the treatment of various corneal pathologies, including LSCD, with the use of cell-based technologies.
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