SERPINA1 long transcripts produce non-secretory alpha1-antitrypsin isoform: In vitro translation in living cells.

SERPINA1 is a well-studied serpin gene due to its dramatic impact on human health. Translation initiation at the main SERPINA1 start codon produces the only known alpha1-antitrypsin (AAT) isoform intended for secretion. AAT performs essential functions by inhibiting proteases and modulating immunity. However, SERPINA1 expression at the level of translation is not sufficiently studied. Here we hypothesize that the main SERPINA1 ORF can be alternatively translated, producing a non-secretory AAT isoform by either masking or excluding a signal peptide. We defined SERPINA1 long mRNA isoforms specific for prostate (DU145) and liver (HepG2) cell lines and studied their individual expression by in vitro assay. We found that all long transcripts produce both glycosylated secretory AAT-eGFP fusion protein and non-glycosylated intracellular AAT-eGFP (initiated from an alternative AUG-2 start codon), with the proportion regulated by the SERPINA1 5'-UTR. Both fusion proteins localize to distinct cellular compartments: in contrast to a fusion with the secretory AAT accumulating in the ER, the intracellular one exhibits nuclear-cytoplasmic shuttling. We detected putative endogenous AAT isoform enriching the nuclear speckles. CONCLUSION: Alternative translation initiation might be a mechanism through which SERPINA1 expands the biological diversity of its protein products. Our findings open up new prospects for the study of SERPINA1 gene expression.

Detonation nanodiamonds as enhancers of E. coli photodynamic inactivation by phthalocyanines in a high molarity buffer solution.

Antimicrobial therapy, especially inactivation of multi-antibiotic-resistant strains, requires creating new approaches for drug action and targeted delivery in different environmental conditions. In this work, detonation nanodiamonds (DNDs) were used to deliver polycationic zinc phthalocyanines to E. coli cells. It is shown that in aqueous solutions, zinc phthalocyanines with cholinyl peripheral substituents form complexes with negatively charged DND based on electrostatic interactions. About 40-70 phthalocyanine molecules can bind to a single DND particle, depending on the number of charged groups of the dye molecule. During the complex formation, quenching of phthalocyanine fluorescence and a decrease in its ability to generate reactive oxygen species were observed. In the presence of bacterial cells, phthalocyanine left the complex and induced a photodynamic effect, the magnitude of which depended on the phthalocyanine charge, the molarity of the buffer solution, and the stoichiometry of the phthalocyanine-DND complex. It was found that at physiological values of the ionic strength of the solution, the photodynamic effect of phthalocyanine with a charge of 8+ in combination with a DND is higher than that of the initial phthalocyanine. Thus, nanodiamonds are a promising platform for the delivery of photosensitizers in antimicrobial therapy.

Fibroin-Gelatin Composite Stimulates the Regeneration of a Splinted Full-Thickness Skin Wound in Mice.

The effects of composite fibroin-gelatin microparticles (100-250 µ) on the rate of wound healing and regeneration under conditions of contraction prevention were studied on the model of splinted full-thickness skin wound in a mouse. Subcutaneous injection of these particles into the defect area accelerated wound healing and promoted re-epithelialization and recovery of normal structure of the epidermis. In addition, the composite microparticles promoted the formation of connective tissue of characteristic structure, replacing the derma over the entire defect, and stimulated regeneration of subcutaneous muscle (panniculus carnosus) and skin appendages (sebaceous glands and hair follicles).

Osteogenic differentiation of mouse bone marrow stromal cells on fibroin microcarriers.

We investigated the proliferation and osteogenic differentiation of mesenchymal stem cells cultured on fibroin microcarriers. Effective cell proliferation on the surface of the microcarriers, determined by the large surface area, and the contribution of microcarrier mineralization to the stimulation of the osteogenic differentiation of mesenchymal stem cells was revealed.

Effects of Fibroin Microcarriers on Inflammation and Regeneration of Deep Skin Wounds in Mice.

The process of tissue regeneration following damage takes place with direct participation of the immune system. The use of biomaterials as scaffolds to facilitate healing of skin wounds is a new and interesting area of regenerative medicine and biomedical research. In many ways, the regenerative potential of biological material is related to its ability to modulate the inflammatory response. At the same time, all foreign materials, once implanted into a living tissue, to varying degree cause an immune reaction. The modern approach to the development of bioengineered structures for applications in regenerative medicine should be directed toward using the properties of the inflammatory response that improve healing, but do not lead to negative chronic manifestations. In this work, we studied the effect of microcarriers comprised of either fibroin or fibroin supplemented with gelatin on the dynamics of the healing, as well as inflammation, during regeneration of deep skin wounds in mice. We found that subcutaneous administration of microcarriers to the wound area resulted in uniform contraction of the wounds in mice in our experimental model, and microcarrier particles induced the infiltration of immune cells. This was associated with increased expression of proinflammatory cytokines TNF, IL-6, IL-1ß, and chemokines CXCL1 and CXCL2, which contributed to full functional recovery of the injured area and the absence of fibrosis as compared to the control group.

Changes in morphology of actin filaments and expression of alkaline phosphatase at 3D cultivation of MG-63 osteoblast-like cells on mineralized fibroin scaffolds.

3D cultivation of MG-63 osteoblast-like cells on mineralized fibroin scaffolds leads to an increase in the expression of alkaline phosphatase, an early marker of bone formation. Increased expression is associated with the actin cytoskeleton reorganization under the influence of 3D cultivation and osteogenic calcium phosphate component of the microcarrier.