RESUMO
Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications.
Assuntos
Álcalis/química , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vírion/imunologia , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Terapia Genética/métodos , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação , Homologia de Sequência de Aminoácidos , Espectrofotometria Ultravioleta , Vírion/genética , Vírion/metabolismo , Montagem de Vírus/genética , Montagem de Vírus/imunologiaRESUMO
The heritability of high-density lipoprotein cholesterol (HDL-C) level is estimated at approximately 50%. Recent genome-wide association studies have identified genes involved in regulation of high-density lipoprotein cholesterol (HDL-C) levels. The precise genetic profile determining heritability of HDL-C however are far from complete and there is substantial room for further characterization of genetic profiles influencing blood lipid levels. Here we report an association study comparing the distribution of 139 SNPs from more than 30 genes between groups that represent extreme ends of HDL-C distribution. We genotyped 704 individuals that were selected from Genome Database of Latvian Population. 10 SNPs from CETP gene showed convincing association with low HDL-C levels (rs1800775, rs3764261, rs173539, rs9939224, rs711752, rs708272, rs7203984, rs7205804, rs11076175 and rs9929488) while 34 SNPs from 10 genes were nominally associated (p<0.05) with HDL-C levels. We have also identified haplotypes from CETP with distinct effects on determination of HDL-C levels. Our conclusion: So far the SNPs in CETP gene are identified as the most common genetic factor influencing HDL-C levels in the representative sample from Latvian population.
Assuntos
Proteínas de Transferência de Ésteres de Colesterol/genética , HDL-Colesterol/genética , Haplótipos , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , HDL-Colesterol/sangue , LDL-Colesterol/sangue , LDL-Colesterol/genética , Bases de Dados Genéticas , Feminino , Estudo de Associação Genômica Ampla , Hereditariedade , Humanos , Letônia , Masculino , Pessoa de Meia-Idade , Fatores de RiscoAssuntos
Tremor Essencial/genética , Testes Genéticos , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Polimorfismo de Nucleotídeo Único , Estudos de Casos e Controles , Tremor Essencial/epidemiologia , Estudo de Associação Genômica Ampla/métodos , Humanos , Letônia/epidemiologiaRESUMO
We have previously documented that transient polyploidy is a potential cell survival strategy underlying the clonogenic re-growth of tumour cells after genotoxic treatment. In an attempt to better define this mechanism, we recently documented the key role of meiotic genes in regulating the DNA repair and return of the endopolyploid tumour cells (ETC) to diploidy through reduction divisions after irradiation. Here, we studied the role of the pluripotency and self-renewal stem cell genes NANOG, OCT4 and SOX2 in this polyploidy-dependent survival mechanism. In irradiation-resistant p53-mutated lymphoma cell-lines (Namalwa and WI-L2-NS) but not sensitive p53 wild-type counterparts (TK6), low background expression of OCT4 and NANOG was up-regulated by ionising radiation with protein accumulation evident in ETC as detected by OCT4/DNA flow cytometry and immunofluorescence (IF). IF analysis also showed that the ETC generate PML bodies that appear to concentrate OCT4, NANOG and SOX2 proteins, which extend into complex nuclear networks. These polyploid tumour cells resist apoptosis, overcome cellular senescence and undergo bi- and multi-polar divisions transmitting the up-regulated OCT4, NANOG and SOX2 self-renewal cassette to their descendents. Altogether, our observations indicate that irradiation-induced ETC up-regulate key components of germ-line cells, which potentially facilitate survival and propagation of the tumour cell population.