Corneal Reconstruction with EGFP-Labelled Limbal Mesenchymal Stem Cells in a Rabbit Model of Limbal Stem Cell Deficiency.

Ocular surface reconstruction is essential for treating corneal epithelial defects and vision recovery. Stem cell-based therapy demonstrates promising results but requires further research to elucidate stem cell survival, growth, and differentiation after transplantation in vivo. This study examined the corneal reconstruction promoted by EGFP-labeled limbal mesenchymal stem cells (L-MSCs-EGFP) and their fate after transplantation. EGFP labeling allowed us to evaluate the migration and survival rates of the transferred cells. L-MSCs-EGFP seeded onto decellularized human amniotic membrane (dHAM) were transplanted into rabbits with a modeled limbal stem cell deficiency. The localization and viability of the transplanted cells in animal tissue were analyzed using histology, immunohistochemistry, and confocal microscopy up to 3 months after transplantation. EGFP-labeled cells remained viable for the first 14 days after transplantation. By the 90th day, epithelialization of the rabbit corneas reached 90%, but the presence of viable labeled cells was not observed within the newly formed epithelium. Although labeled cells demonstrated low survivability in host tissue, the squamous corneal-like epithelium was partially restored by the 30th day after transplantation of the tissue-engineered graft. Overall, this study paves the way for further optimization of transplantation conditions and studying the mechanisms of corneal tissue restoration.

The Aß42 Peptide and IAPP Physically Interact in a Yeast-Based Assay.

Numerous studies have demonstrated that people with type 2 diabetes mellitus (associated with IAPP peptide aggregation) show an increased incidence of Alzheimer's disease (associated with Aß aggregation), but the mechanism responsible for this correlation is presently unknown. Here, we applied a yeast-based model to study the interactions of IAPP with PrP (associated with TSEs) and with the Aß42 peptide. We demonstrated that fluorescently tagged IAPP forms detergent-resistant aggregates in yeast cells. Using the FRET approach, we showed that IAPP and Aß aggregates co-localize and physically interact in yeast cells. We also showed that this interaction is specific and that there is no interaction between IAPP and PrP in the yeast system. Our data confirmed a direct physical interaction between IAPP and Aß42 aggregates in a living cell. Based on these findings, we hypothesize that this interaction may play a crucial role in seeding Aß42 aggregation in T2DM patients, thereby promoting the development of AD.

Human RAD51 Protein Forms Amyloid-like Aggregates In Vitro.

RAD51 is a central protein of homologous recombination and DNA repair processes that maintains genome stability and ensures the accurate repair of double-stranded breaks (DSBs). In this work, we assessed amyloid properties of RAD51 in vitro and in the bacterial curli-dependent amyloid generator (C-DAG) system. Resistance to ionic detergents, staining with amyloid-specific dyes, polarized microscopy, transmission electron microscopy (TEM), X-ray diffraction and other methods were used to evaluate the properties and structure of RAD51 aggregates. The purified human RAD51 protein formed detergent-resistant aggregates in vitro that had an unbranched cross-ß fibrillar structure, which is typical for amyloids, and were stained with amyloid-specific dyes. Congo-red-stained RAD51 aggregates demonstrated birefringence under polarized light. RAD51 fibrils produced sharp circular X-ray reflections at 4.7 Å and 10 Å, demonstrating that they had a cross-ß structure. Cytoplasmic aggregates of RAD51 were observed in cell cultures overexpressing RAD51. We demonstrated that a key protein that maintains genome stability, RAD51, has amyloid properties in vitro and in the C-DAG system and discussed the possible biological relevance of this observation.

Biocompatible Ir(III) Complexes as Oxygen Sensors for Phosphorescence Lifetime Imaging.

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.

Protein Misfolding during Pregnancy: New Approaches to Preeclampsia Diagnostics.

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.

Derivation and Characterization of EGFP-Labeled Rabbit Limbal Mesenchymal Stem Cells and Their Potential for Research in Regenerative Ophthalmology.

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.

An Efficient Method for Isolation of Plasmid DNA for Transfection of Mammalian Cell Cultures.

In this article, we present several protocols that describe the steps from cloning and obtaining a large amount of pure plasmid DNA to generation of lentiviruses based on these constructs. The protocols have been worked out on human cell culture HEK293T but can be adapted for other cell cultures. This protocol was designed to be simple to execute and cheap since it requires only materials and consumables widely available in molecular laboratories, such as salts, alcohols, etc., and no complicated laboratory equipment. These protocols are highly effective and can be performed in any standard molecular biology laboratory.

RNA-binding protein FXR1 is presented in rat brain in amyloid form.

Amyloids are ß-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.