The Green\u27s Function Method for Solutions of Fourth Order Nonlinear Boundary Value Problem.

This thesis has demonstrated that Green’s functions have a wide range of applications with regard to boundary value problems. In particular, existence and uniqueness of solutions of a large class of fourth order boundary value problems has been established. In fact, given any fourth order ODE with homogeneous boundary conditions, as long as the corresponding Green’s function exists and f satisfies an appropriate Lipschitz condition, Theorem 2.1 guarantees such a solution under equally mild conditions. Similarly, Theorem 2.2 also guarantees such a solution under equally mild conditions. These theorems are contrasted with classical ODE existence theorems in that they get around the use of classical convergence analysis by assuming the existence of the Green’s function. Banach techniques are still used, but the existence of the Green’s function is the primary tool in showing existence and uniqueness. This requires, of course, that the Green’s function exists for particular problem, but the examples in Section 4 show that this s usually not a severe restriction. However, as mild as the restrictions seem to be, one should pay particular detail to the range of values on the Lipschitz constant(s). The Lipschitz constants corresponding to f must satisfy an inequality involving bounds on integrals of G and its derivatives, which, if G is badly behaved, may be a severe restriction. The examples of Section 4 illustrate these ideas. For example, Theorems 4.1-4.2 are specific cases in which Theorem 2.2 is applicable

Silencing of neurotropic flavivirus replication in the central nervous system by combining multiple microRNA target insertions in two distinct viral genome regions

AbstractIn recent years, microRNA-targeting has become an effective strategy for selective control of tissue-tropism and pathogenesis of both DNA and RNA viruses. Here, using a neurotropic flavivirus as a model, we demonstrate that simultaneous miRNA targeting of the viral genome in the open reading frame and 3′-noncoding regions for brain-expressed miRNAs had an additive effect and produced a more potent attenuation of the virus compared to separate targeting of those regions. Multiple miRNA co-targeting of these two distantly located regions completely abolished the virus neurotropism as no viral replication was detected in the developing brain of neonatal mice. Furthermore, no viral antigens were detected in neurons, and neuronal integrity in the brain of mice was well preserved. This miRNA co-targeting approach can be adapted for other viruses in order to minimize their replication in a cell- or tissue-type specific manner, but most importantly, to prevent virus escape from miRNA-mediated silencing

Stable and Highly Immunogenic MicroRNA-Targeted Single-Dose Live Attenuated Vaccine Candidate against Tick-Borne Encephalitis Constructed Using Genetic Backbone of Langat Virus

Tick-borne encephalitis virus (TBEV) is one of the most medically important tick-borne pathogens of the Old World. Despite decades of active research, efforts to develop of TBEV live attenuated virus (LAV) vaccines with acceptable safety and immunogenicity characteristics have not been successful. Here we report the development and evaluation of a highly attenuated and immunogenic microRNA-targeted TBEV LAV.</jats:p

Stable and Highly Immunogenic MicroRNA-Targeted Single-Dose Live Attenuated Vaccine Candidate against Tick-Borne Encephalitis Constructed Using Genetic Backbone of Langat Virus

Tick-borne encephalitis virus (TBEV) is one of the most medically important tick-borne pathogens of the Old World. Despite decades of active research, efforts to develop of TBEV live attenuated virus (LAV) vaccines with acceptable safety and immunogenicity characteristics have not been successful. Here we report the development and evaluation of a highly attenuated and immunogenic microRNA-targeted TBEV LAV.Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is one of the most medically important tick-borne pathogens of the Old World. Despite decades of active research, attempts to develop of a live attenuated virus (LAV) vaccine against TBEV with acceptable safety and immunogenicity characteristics have not been successful. To overcome this impasse, we generated a chimeric TBEV that was highly immunogenic in nonhuman primates (NHPs). The chimeric virus contains the prM/E genes of TBEV, which are expressed in the genetic background of an antigenically closely related, but less pathogenic member of the TBEV complex—Langat virus (LGTV), strain T-1674. The neurovirulence of this chimeric virus was subsequently controlled by robust targeting of the viral genome with multiple copies of central nervous system-enriched microRNAs (miRNAs). This miRNA-targeted T/1674-mirV2 virus was highly stable in Vero cells and was not pathogenic in various mouse models of infection or in NHPs. Importantly, in NHPs, a single dose of the T/1674-mirV2 virus induced TBEV-specific neutralizing antibody (NA) levels comparable to those seen with a three-dose regimen of an inactivated TBEV vaccine, currently available in Europe. Moreover, our vaccine candidate provided complete protection against a stringent wild-type TBEV challenge in mice and against challenge with a parental (not miRNA-targeted) chimeric TBEV/LGTV in NHPs. Thus, this highly attenuated and immunogenic T/1674-mirV2 virus is a promising LAV vaccine candidate against TBEV and warrants further preclinical evaluation of its neurovirulence in NHPs prior to entering clinical trials in humans

Sodium alginate-based composites as a collagen substitute for skin bioengineering

Abstract The skin is a combination of two different types of tissue—epithelial and connective (mesenchymal). The outer protective layer of the skin, the epidermis, consists of multiple layers of keratinocytes residing on the basement membrane that separates them from the underlying dermis, which consists of a well-vascularized fibrous extracellular matrix seeded mainly by fibroblasts and mesenchymal stromal cells. These skin features suggest that the development of a fibroblast-friendly porous scaffold covered with a flat dense sheath mimicking the basement membrane, and sufficient to support keratinocyte attachment, would be a reasonable approach in the generation of clinically-relevant skin substitutes useful for reconstructive dermatology and burn treatment. Therefore, we developed a procedure to obtain biocompatible composite bilayer scaffolds comprising a spongy dermis-like body (supporting vascularization and appropriate fibroblast and multipotent stromal cell activity) fused with a film-like cover (supporting keratinocyte attachment, growth and differentiation). The sodium alginate (SA), an algae-derived biopolymer, has been used as a base component for these scaffolds while collagen (CL) and fibrinogen (FG) were used as minor additives in variable concentrations. The slow rates of composite SA-based scaffold biodegradation were achieved by using Ba2+ as cross-linking cations. By manipulating the SA/CL/FG ratio we managed to obtain sponge scaffolds with highly interconnected porous structures, with an average pore size ranging from 60 to 300 μm, and sufficient tensile strength (3.12–5.26 MPa). The scaffolds biocompatibility with the major human skin cell types was confirmed by seeding the scaffold sponge compartment with primary skin fibroblasts and subcutaneous adipose-derived stromal cells while the film side biocompatibility was tested using primary human keratinocytes. The obtained results have shown that bilayer alginate-based scaffolds have biological and mechanical properties comparable with CL scaffolds but surpass them in cost efficiency and vascularization ability in the subcutaneous implantation model in laboratory mice.</jats:p

The Effect of Humic Aerosol Treatment on Grain Seeds Quality

Introduction. According to experts, in the coming years, the world market of organic products will continue to grow at a rate of 15-16% per year. The growth will equal about 20% of the world market of all agricultural products by 2025. In this connection, of a particular interest is the development of elements of agricultural technologies for the use of soil-improving substances and growth regulators of natural origin, including humic preparations, which provide for sufficiently high yields of agricultural products. Materials and Methods. The methods of using humic preparations were investigated on the example of spring barley seeds, treated with the traditional semi-dry method and using hot mist. Germination of seeds in the laboratory was carried out according to the Russian State Standard GOST 12038-84. The laboratory data were subjected to mathematical processing by the method of variance analysis. Results. The treatment of seeds with humic preparations led to an increase in sowing qualities, regardless of the method of seed treatment. However, the comparative efficiency analysis of the pre-sowing treatment of the test crop seeds with the aerosol and using the semi-dry method showed that the use of the developed equipment made it possible to increase the biological activity of humic preparations, which brought about an increase in the growth and biometric parameters of seedlings. Discussion and Conclusion. The treatment of spring barley seeds with humic preparations has some positive effect on their sowing and yield qualities. The strongest positive effect was obtained when treating seeds with reduced sowing qualities, which was quite often observed when harvesting seed crops in years with unfavorable conditions: heavy rainfall, lack of heat, as well as in violation of the technology of harvesting and preparing seeds. Our experimental studies showed that the use of the developed device for treating seeds with hot mist reduces energy costs and increases processing efficiency.</jats:p