One Size Does Not Fit All: The Effect of Chain Length and Charge Density of Poly(ethylene imine) Based Copolymers on Delivery of pDNA, mRNA, and RepRNA Polyplexes

Nucleic acid delivery systems are commonly translated between different modalities, such as DNA and RNA of varying length and structure, despite physical differences in these molecules that yield disparate delivery efficiency with the same system. Here, we synthesized a library of poly­(2-ethyl-2-oxazoline)/poly­(ethylene imine) copolymers with varying molar mass and charge densities in order to probe how pDNA, mRNA, and RepRNA polyplex characteristics affect transfection efficiency. The library was utilized in a full factorial design of experiment (DoE) screening, with outputs of luciferase expression, particle size, surface charge, and particle concentration. The optimal copolymer molar mass and charge density was found as 83 kDa/100%, 72 kDa/100%, and 45 kDa/80% for pDNA, RepRNA, and mRNA, respectively. While 10 of the synthesized copolymers enhanced the transfection efficiency of pDNA and mRNA, only 2 copolymers enhanced RepRNA transfection efficiency, indicating a narrow and more stringent design space for RepRNA. These findings suggest that there is not a “one size fits all” polymer for different nucleic acid species

IN and DNA + IN regimens induce the highest serum IgG and IgA responses after three vaccinations.

<p>Mice were vaccinated three times with the indicated vaccine modalities, serum samples taken 1 week after the last vaccination (week 8) were tested for antigen-specific IgG (a) and IgA (b) by ELISA. Data were analysed using one-way ANOVA with Tukey’s post test, * P <0.05, ** P < 0.01, *** P < 0.001.</p

Adaptivity based on error estimation for viscoplastic softening materials

The definitive version is available at http://www3.interscience.wiley.com/journal/70002928/abstract?CRETRY=1&SRETRY=0This paper focuses on the numerical simulation of strain softening mechanical problems. Two problems arise: (1) the constitutive model has to be regular and (2) the numerical technique must be able to capture the two scales of the problem (the macroscopic geometrical representation and the microscopic behaviour in the localization bands). The Perzyna viscoplastic model is used in order to obtain a regularized softening model allowing to simulate strain localization phenomena. This model is applied to quasistatic examples. The viscous regularization of quasistatic processes is also discussed: in quasistatics, the internal length associated with the obtained band width is no longer only a function of the material parameters but also depends on the boundary value problem (geometry and loads, specially loading velocity). An adaptive computation is applied to softening viscoplastic materials showing strain localization. As the key ingredient of the adaptive strategy, a residual-type error estimator is generalized to deal with such highly non-linear material model. In several numerical examples the adaptive process is able to detect complex collapse modes that are not captured by a first, even if fine, mesh. Consequently, adaptive strategies are found to be essential to detect the collapse mechanism and to assess the optimal location of the elements in the mesh.Peer ReviewedPostprint (author’s final draft

DNA vaccination induces Th1 skewed responses.

<p>Levels of IgG1 and IgG2a were assessed in serum one week after the last vaccination (week 8) by ELISA (a) and IgG1:IgG2a ratios were calculated as a surrogate of Th1/Th2 bias (b).</p

Detection of mucosal antigen-specific IgG and IgA responses in the vagina.

<p>Mice were vaccinated three times and vaginal lavage samples were taken 1 week after the last vaccination and tested for antigen-specific IgG (a) and IgA (b) by ELISA. Results are expressed as specific antibody (ng)/total antibody (μg). One-way ANOVA with Tukey’s post test was used to analyse data. * P <0.05, ** P < 0.01, *** P < 0.001.</p

IN vaccination induces potent IL-2 responses in splenocytes after stimulation with HIV-1 Env peptides.

<p>Animals were vaccinated three times by the routes indicated. One week after the final vaccination splenocytes were cultured with two HIV Env-specific peptide pools, and IL-2-producing cells were measured using ELIspot kits. Negative responses were assigned a value of 1 to allowing plotting on a log scale. One-way ANOVA with Tukey’s post test was used to analyse data * P <0.05, ** P < 0.01, *** P < 0.001.</p

Vaccination schedule.

<p>Animals were vaccinated at three time points with the indicated vaccine combinations. DNA = intramuscular injection with HIV-1 gp140-encoding plasmid with electroporation, IM = Intramuscular injection with HIV-1 CN54 gp140 Env protein with MPLA, IN = Intranasal delivery of HIV-1 CN54 gp140 Env protein with MPLA.</p><p>Vaccination schedule.</p

Determination of paternity

The primary theme of the thesis is "paternity determination", an interesting part of private law. The goal of the thesis was to find juridical legislation that could be applied by legal institutions in the Czech Republic, and have been neither legally controlled nor properly spoken through. Also the thesis deals with surrogate motherhood institutes, same-sex parenthood, baby hatches and paternity determination of children, made by assisted reproduction. The thesis is divided into five chapters, which are divided further into sub-chapters. Following the introduction, the primary terms of parental difficulties, paternity determination and the term family are explored. The second chapter discusses motherhood institutes. It speaks primarily of surrogate motherhood's challenges, including other countries' attitude to this topic. The following chapter is about fatherhood determination, containing the juridical legislative analysis of the problem in the Czech law. The fourth chapter expands on the issues of same sex couple parenthood, and the forms of family coexistence. These issues are described in great detail from both the view of Czech law, and also as a global issue. The last chapter discusses the issues of baby hatches, and legislation regarding anonymity for those who utilize them in the Czech..

Mean humoral responses for each vaccination protocol.

<p>Mean humoral responses for each vaccination protocol.</p

DNA and IN vaccinations induces a distinct cytokine profile compared to IM vaccinations.

<p>Cytokines were detected using multiplex bead assay capable of measuring 23 murine cytokines. Differences in cytokine production between groups were observed with selected cytokines IL-2 (a), IL-4 (b), IL-13 (c), IL-17 (d), IFNγ (e), MIP-1α (f), MIP-1β (g) and MCP1 (h). Kruskal-Wallis analysis with Dunn’s multiple comparison test was used to analyse data, * P <0.05, ** P < 0.01, *** P < 0.001.</p