Influence of charge ratio of liposome/DNA complexes on their size after extrusion and transfection efficiency

Marija Brgles, Maja Šantak, Beata Halassy, Dubravko Forcic, Jelka TomašicInstitute of Immunology, Research and Development Department, Zagreb, CroatiaBackground: Physicochemical characteristics of liposome/DNA complexes influence transfection efficiency and affect each other in a very intricate way. The result of this is discrepancies in conclusions drawn about the individual influence of each one.Methods: Aiming to elucidate the influence of liposome/DNA charge ratio and size on transfection efficiency and on each other, we used liposome/DNA complexes with charge ratio (+/-) in the range of 1–50 and extruded through membranes of 400, 200, and 100 nm. Plasmid DNA encoding green fluorescent protein was used to measure transfection efficiency by flow cytometry. Sizes of liposome/DNA complexes were measured by dynamic light scattering.Results: Liposome size was reduced after extrusion but this was mainly driven by the charge ratio and not by the size of the membrane pores. Reduction of complex size at each charge ratio positively correlated with transfection efficiency. When the size of the complexes was approximately constant, increasing the charge ratio was found to promote transfection efficiency. Cationic lipid N-(1-(2,3-dioleoyloxy)propyl)N,N,N trimethylammonium chloride was used for modulation of positive charge and a cytotoxicity test showed that increasing its amount increases cytotoxicity.Conclusion: It can be concluded that charge ratio dictates the size of the complex whereas overall size reduction and higher charge ratios promote transfection efficiency in vitro.Keywords: transfection efficiency, liposome charge, liposome siz

Communicating Environmental Problems as a Basis for Creating Sustainable Family Habits. CRO Laudato Si’ Research Results

The paper presents the results of a research conducted among Catholic believers in Croatia during 2020 and 2021. The research was carried out as part of the CRO Laudato Si' project (N=1324).  In the second phase, four focus groups were conducted with 20 participants. The overall objective of the research is to determine whether families communicate about environmental problems and to describe the experience of communication and sustainable habits in the family. The results show that communication on environmental problems in the family is democratic and open. The vast majority of respondents (97%) mostly or completely agreed with the statement: “It is necessary to point out the importance of environmental problems in one's own family and society”. The results of the focus group provide a deeper insight into family relations and reveal the family as a social relationship within which environmental problems are not discussed regularly, but some habits are cultivated spontaneously. These are daily sustainable habits such as waste separation, water and energy saving, etc. However, the results show that younger family members pass on information on this topic to the elderly and therefore their role on micro-level is significant.  That role is related to meso level, and to educational institutions in the phase of secondary socialization.  We conclude that families from our sample have a non-economic exchange of information on environmental problems. Communication (non-verbal especially) about environmental problems in the family is important for both children and parents and can empower the willingness to change unsustainable family habits

Projekt CRO Laudato Si’: cele, działania i rezultaty społeczne

On the eve of 2020, and the fifth anniversary of the publication of the Encyclical Letter - Laudato Si’. On the care for the common home at the Catholic University of Croatia, the scientific-professional project CRO Laudato Si’ was launched. The project team was composed of scientists from different disciplines, and the project partners were the National Fraternity of the Franciscan Secular Order and the Franciscan Youth. As part of the project, scientific and practical activities were carried out. One of the main scientific activities was empirical scientific research, which was conducted, using a survey method over several months in 2020. The paper will present the results of the research, related to the role of the project in the knowledge of the encyclical Laudato Si’ and the awakening of interest in the encyclical, according to the socio-demographic characteristics of the respondents. The results show that men have heard of the encyclical Laudato Si’ to a somewhat greater extent than women. The largest number of respondents, who heard about the encyclical for the first time through the project, are those who belong to the youngest age group, followed by those with high school education. Arousing interest in Encyclical Letter topics is more encouraged among women, those over 65 years old, respondents with completed primary school, and those who are self-employed or retired.U progu 2020 roku, w piątą rocznicę opublikowania encykliki Laudato Si’. W trosce o wspólny dom na Katolickim Uniwersytecie Chorwacji zapoczątkowano projekt naukowo-zawodowy CRO Laudato Si’. Zespół projektowy składał się z naukowców z różnych dyscyplin, a partnerami projektu była Wspólnota Narodowa Świeckiego Zakonu Franciszkańskiego oraz Młodzież Franciszkańska. W ramach projektu prowadzono działania naukowe i praktyczne. Jednym z głównych działań naukowych były empiryczne badania, przeprowadzone metodą ankietową w okresie kilku miesięcy w 2020 roku. W artykule zostały przedstawione wyniki badań związanych z rolą projektu na temat znajomości encykliki Laudato Si’ oraz rozbudzanie zainteresowania encykliką uwzględniając społeczno-demograficzny charakter respondentów. Wyniki badań wskazują, że mężczyźni słyszeli o encyklice Laudato Si’ w nieco większym stopniu niż kobiety. Najwięcej respondentów, którzy w ramach projektu po raz pierwszy usłyszeli o encyklice, to ci, którzy należą do najmłodszej grupy wiekowej, a następnie osoby z wykształceniem średnim. Zainteresowanie tematyką encykliki jest wyższe wśród kobiet, osób powyżej 65 roku życia, respondentów z ukończoną szkołą podstawową oraz osób samozatrudnionych lub emerytów

Production- and Purification-Relevant Properties of Human and Murine Cytomegalovirus

There is a large unmet need for a prophylactic vaccine against human cytomegalovirus (HCMV) to combat the ubiquitous infection that is ongoing with this pathogen. A vaccination against HCMV could protect immunocompromised patients and prevent birth defects caused by congenital HCMV infections. Moreover, cytomegalovirus (CMV) has a number of features that make it a very interesting vector platform for gene therapy. In both cases, preparation of a highly purified virus is a prerequisite for safe and effective application. Murine CMV (MCMV) is by far the most studied model for HCMV infections with regard to the principles that govern the immune surveillance of CMVs. Knowledge transfer from MCMV and mice to HCMV and humans could be facilitated by better understanding and characterization of the biological and biophysical properties of both viruses. We carried out a detailed investigation of HCMV and MCMV growth kinetics as well as stability under the influence of clarification and different storage conditions. Further, we investigated the possibilities to concentrate and purify both viruses by ultracentrifugation and ion-exchange chromatography. Defective enveloped particles were not separately analyzed; however, the behavior of exosomes was examined during all experiments. The effectiveness of procedures was monitored using CCID50 assay, Nanoparticle tracking analysis, ELISA for host cell proteins, and quantitative PCR for host cell DNA. MCMV generally proved to be more robust in handling. Despite its greater sensitivity, HCMV was efficiently (100% recovery) purified and concentrated by anion-exchange chromatography using QA monolithic support. The majority of the host genomic DNA as well as most of the host cell proteins were removed by this procedure

Mass spectrometry-based investigation of measles and mumps virus proteome

Abstract Background Measles (MEV) and mumps virus (MUV) are enveloped, non-segmented, negative single stranded RNA viruses of the family Paramyxoviridae, and are the cause of measles and mumps, respectively, both preventable by vaccination. Aside from proteins coded by the viral genome, viruses are considered to contain host cell proteins (HCPs). The presence of extracellular vesicles (ECVs), which are often co-purified with viruses due to their similarity in size, density and composition, also contributes to HCPs detected in virus preparations, and this has often been neglected. The aim was to identify which virus-coded proteins are present in MEV and MUV virions, and to try to detect which HCPs, if any, are incorporated inside the virions or adsorbed on their outer surface, and which are more likely to be a contamination from co-purified ECVs. Methods MUV, MEV and ECVs were purified by ultracentrifugation, hydrophobic interaction chromatography and immunoaffinity chromatography, proteins in the samples were resolved by SDS-PAGE and subjected to identification by MALDI-TOF/TOF-MS. A comparative analysis of HCPs present in all samples was carried out. Results By proteomics approach, it was verified that almost all virus-coded proteins are present in MEV and MUV particles. Protein C in MEV which was until now considered to be non-structural viral protein, was found to be present inside the MeV virions. Results on the presence of HCPs in differently purified virus preparations imply that actin, annexins, cyclophilin A, moesin and integrin β1 are part of the virions. Conclusions All HCPs detected in the viruses are present in ECVs as well, indicating their possible function in vesicle formation, or that most of them are only present in ECVs. Only five HCPs were constantly present in purified virus preparations, regardless of the purification method used, implying they are likely the integral part of the virions. The approach described here is helpful for further investigation of HCPs in other virus preparations

Biological Activities and Proteomic Profile of the Venom of Vipera ursinii ssp., a very Rare Karst Viper from Croatia

The karst viper (Vipera ursinii ssp.) favours high-mountain dry grasslands in southern and south-eastern Croatia. It is medically less important than other Vipera species, because of its remote habitat and the very small amount of venom that it injects by its relatively short fangs. The scientific literature on Vipera ursinii deals mostly with the morphology, ecology and distribution range of this snake, due to the species’ conservation issues, while the toxinological aspects of its venom have not so far been investigated. Here we report on the composition and biological activity of the Vipera ursinii ssp. venom. Using a proteomics approach, we have identified 25 proteins in the venom that belong to seven protein families: snake venom metalloproteinase, serine protease, secreted phospholipase A2, cysteine-rich secretory protein, snake C-type lectin-like protein, serine protease inhibitor and nerve growth factor. The Vipera ursinii ssp. venom was found to be distinctively insecticidal. Its lethal toxicity towards crickets was more than five times greater than that of Vipera ammodytes ammodytes venom, while the opposite held in mice. Interestingly, the mode of dying after injecting a mouse with Vipera ursinii ssp. venom may suggest the presence of a neurotoxic component. Neurotoxic effects of European vipers have so far been ascribed exclusively to ammodytoxins and ammodytoxin-like basic secreted phospholipases A2. Structural and immunological analyses of the Vipera ursinii ssp. venom, however, confirmed that ammodytoxin-like proteins are not present in this venom

Refinement strategy for antivenom preparation of high yield and quality.

Antivenoms from hyperimmune animal plasma are the only specific pharmaceuticals against snakebites. The improvement of downstream processing strategies is of great interest, not only in terms of purity profile, but also from yield-to-cost perspective and rational use of plasma of animal origin. We report on development of an efficient refinement strategy for F(ab')2-based antivenom preparation. Process design was driven by the imperative to keep the active principle constantly in solution as a precautionary measure to preserve stability of its conformation (precipitation of active principle or its adsorption to chromatographic stationary phase has been completely avoided). IgG was extracted from hyperimmune horse plasma by 2% (V/V) caprylic acid, depleted from traces of precipitating agent and digested by pepsin. Balance between incomplete IgG fraction breakdown, F(ab')2 over-digestion and loss of the active principle's protective efficacy was achieved by adjusting pepsin to substrate ratio at the value of 4:300 (w/w), setting pH to 3.2 and incubation period to 1.5 h. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. Developed manufacturing strategy gave 100% pure and aggregate-free F(ab')2 preparation, as shown by size-exclusion HPLC and confirmed by MS/MS. The overall yield of 75% or higher compares favorably to others so far reported. This optimised procedure looks also promising for large-scale production of therapeutic antivenoms, since high yield of the active drug and fulfillment of the regulatory demand considering purity was achieved. The recovery of the active substance was precisely determined in each purification step enabling accurate estimation of the process cost-effectiveness

Optimization of tetanus toxoid ammonium sulfate precipitation process using response surface methodology

<p>Tetanus toxoid (TTd) is a highly immunogenic, detoxified form of tetanus toxin, a causative agent of tetanus disease, produced by <i>Clostridium tetani</i>. Since tetanus disease cannot be eradicated but is easily prevented by vaccination, the need for the tetanus vaccine is permanent. The aim of this work was to investigate the possibility of optimizing TTd purification, i.e., ammonium sulfate precipitation process. The influence of the percentage of ammonium sulfate, starting amount of TTd, buffer type, pH, temperature, and starting purity of TTd on the purification process were investigated using optimal design for response surface models. Responses measured for evaluation of the ammonium sulfate precipitation process were TTd amount (Lf/mL) and total protein content. These two parameters were used to calculate purity (Lf/mgPN) and the yield of the process. Results indicate that citrate buffer, lower temperature, and lower starting amount of TTd result in higher purities of precipitates. Gel electrophoresis combined with matrix-assisted laser desorption ionization–mass spectrometric analysis of precipitates revealed that there are no inter-protein cross-links and that all contaminating proteins have pIs similar to TTd, so this is most probably the reason for the limited success of purification by precipitation.</p