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

Common position of indels that cause deviations from canonical genome organization in different measles virus strains

BACKGROUND: The canonical genome organization of measles virus (MV) is characterized by total size of 15 894 nucleotides (nts) and defined length of every genomic region, both coding and non-coding. Only rarely have reports of strains possessing non-canonical genomic properties (possessing indels, with or without the change of total genome length) been published. The observed mutations are mutually compensatory in a sense that the total genome length remains polyhexameric. Although programmed and highly precise pseudo-templated nucleotide additions during transcription are inherent to polymerases of all viruses belonging to family Paramyxoviridae, a similar mechanism that would serve to non-randomly correct genome length, if an indel has occurred during replication, has so far not been described in the context of a complete virus genome. ----- METHODS: We compiled all complete MV genomic sequences (64 in total) available in open access sequence databases. Multiple sequence comparisons and phylogenetic analyses were performed with the aim of exploring whether non-recombinant and non-evolutionary linked measles strains that show deviations from canonical genome organization possess a common genetic characteristic. ----- RESULTS: In 11 MV sequences we detected deviations from canonical genome organization due to short indels located within homopolymeric stretches or next to them. In nine out of 11 identified non-canonical MV sequences, a common feature was observed: one mutation, either an insertion or a deletion, was located in a 28 nts long region in F gene 5' untranslated region (positions 5051-5078 in genomic cDNA of canonical strains). This segment is composed of five tandemly linked homopolymeric stretches, its consensus sequence is G6-7C7-8A6-7G1-3C5-6. Although none of the mononucleotide repeats within this segment has fixed length, the total number of nts in canonical strains is always 28. These nine non-canonical strains, as well as the tenth (not mutated in 5051-5078 segment), can be grouped in three clusters, based on their passage histories/epidemiological data/genetic similarities. There are no indications that the 3 clusters are evolutionary linked, other than the fact that they all belong to clade D. ----- CONCLUSIONS: A common narrow genomic region was found to be mutated in different, non-related, wild type strains suggesting that this region might have a function in non-random genome length corrections occurring during MV replication

Genetic heterogeneity of L-Zagreb mumps virus vaccine strain

<p>Abstract</p> <p>Background</p> <p>The most often used mumps vaccine strains Jeryl Lynn (JL), RIT4385, Urabe-AM9, L-Zagreb and L-3 differ in immunogenicity and reactogenicity. Previous analyses showed that JL, Urabe-AM9 and L-3 are genetically heterogeneous.</p> <p>Results</p> <p>We identified the heterogeneity of L-Zagreb throughout the entire genome. Two major variants were defined: variant A being identical to the consensus sequence of viral seeds and vaccine(s) and variant B which differs from variant A in three nucleotide positions. The difference between viral variants in L-Zagreb strain is insufficient for distinct viral strains to be defined. We demonstrated that proportion of variants in L-Zagreb viral population depends on cell substrate used for viral replication in vitro and in vivo.</p> <p>Conclusion</p> <p>L-Zagreb strain should be considered as a single strain composed of at least two variant viral genomes.</p

Population Variability Generated during Rescue Process and Passaging of Recombinant Mumps Viruses

Recombinant mumps viruses (MuVs) based on established vaccine strains represent attractive vector candidates as they have known track records for high efficacy and the viral genome does not integrate in the host cells. We developed a rescue system based on the consensus sequence of the L- Zagreb vaccine and generated seven different recombinant MuVs by (a) insertion of one or two additional transcription units (ATUs), (b) lengthening of a noncoding region to the extent that the longest noncoding region in MuV genome is created, or (c) replacement of original L-Zagreb sequences with sequences rich in CG and AT dinucleotides. All viruses were successfully rescued and faithfully matched sequences of input plasmids. In primary rescued stocks, low percentages of heterogeneous positions were found (maximum 0.12%) and substitutions were predominantly obtained in minor variants, with maximally four substitutions seen in consensus. ATUs did not accumulate more mutations than the natural MuV genes. Six substitutions characteristic for recombinant viruses generated in our system were defined, as they repetitively occurred during rescue processes. In subsequent passaging of primary rescue stocks in Vero cells, different inconsistencies within quasispecies structures were observed. In order to assure that unwanted mutations did not emerge and accumulate, sub-consensus variability should be closely monitored. As we show for Pro408Leu mutation in L gene and a stop codon in one of ATUs, positively selected variants can rise to frequencies over 85% in only few passages

The Role of Nucleoprotein in Immunity to Human Negative-Stranded RNA Viruses&mdash;Not Just Another Brick in the Viral Nucleocapsid

Negative-stranded RNA viruses (NSVs) are important human pathogens, including emerging and reemerging viruses that cause respiratory, hemorrhagic and other severe illnesses. Vaccine design traditionally relies on the viral surface glycoproteins. However, surface glycoproteins rarely elicit effective long-term immunity due to high variability. Therefore, an alternative approach is to include conserved structural proteins such as nucleoprotein (NP). NP is engaged in myriad processes in the viral life cycle: coating and protection of viral RNA, regulation of transcription/replication processes and induction of immunosuppression of the host. A broad heterosubtypic T-cellular protection was ascribed very early to this protein. In contrast, the understanding of the humoral immunity to NP is very limited in spite of the high titer of non-neutralizing NP-specific antibodies raised upon natural infection or immunization. In this review, the data with important implications for the understanding of the role of NP in the immune response to human NSVs are revisited. Major implications of the elicited T-cell immune responses to NP are evaluated, and the possible multiple mechanisms of the neglected humoral response to NP are discussed. The intention of this review is to remind that NP is a very promising target for the development of future vaccines

The difference between interpersonal and hyperpersonal communication in the business environment

Namjera ovog rada je prikazati usporedbu interpersonalne komunikacije ("licem u lice") s komunikacijom posredstvom tehničkih pomagala poput računala i mobitela u poslovnom okruženju. Detaljnom analizom stručne akademske literature, u ovome radu prikazana je interpersonalna komunikacija kao i njezine osnovne značajke. Isto tako je analizirana hiperpersonalna komunikacija i njezine temeljne značajke. U radu je provedena usporedba komunikacije „licem u lice“ s hiperpersonalnom komunikacijom. Nadalje, provedeno je istraživanje putem aknetnog upitnika u kojem je sudjelovalo 105 ispitanika. Na temelju rezultata istraživanja prva hipoteza sa podhipotezama je negirana, dok je druga potvrđena. Na kraju rada je izložen zaključak u kojem se ističe kako je interpersonalna komunikacija i dalje dominantna u poslovnom okruženju u odnosu na hiperpersonalnu.The purpose of this thesis is to show the comparison of interpersonal communication ( „face to face“) with communication through technical solutions such as computer and mobile phone in a business environment. In a detailed analysis of academic literature, this paper presents interpersonal communication as well as its basic features. Hyperpersonal communication and its core features were also analyzed as well as a comparison of “face to face” communication with hyperpersonal communication. Furthermore, a survey was conducted through a questionnaire involving 105 respondents. Based on the results of the survey, the first hypothesis with sub-hypotheses was denied, while the second was confirmed. At the end of the paper, a conclusion is drawn that emphasizes that interpersonal communication remains dominant in the business environment in relation to hyperpersonal

Old and new ways to combat human influenza virus

Influenza viruses have been with mankind for at least 300 years, with epidemics occurring every few years and pandemics every few decades. They replicate extremely rapidly in the host therefore demanding a fast and effective antiviral response. Despite the availability of seasonal trivalent vaccines and antivirals, which are effective for most recipients, influenza remains serious disease. The reason for that is a grand capacity of the influenza virus to adapt to new environmental conditions and evolutionary pressure. Vaccination remains the main protective measure against influenza for general population. The first vaccine was administered in the 1940s and ever since the influenza vaccine has provided tremendous relief against influenza infections. However, time has revealed the ultimate limit of the vaccine and the call for its reinvention has now come. The purpose of this review is to give a brief but comprehensive overview of the currently used prophylactic and therapeutic approaches against influenza and the new most promising developments in this field