Factors mediating lipofection potency of a series of cationic phosphonolipids in human cell lines

A series of cationic liposomes known as cationic phosphonolipids (CPs) were evaluated as vehicles for in vitro gene transfer in K562 erythroleukemia cells and 5637 epithelial carcinoma cells. For each CP and target cell type examined, detailed analyses were performed to determine optimal transfection conditions (lipid/ DNA (+/-) charge ratio, amount of complexed episomal DNA, liposomal and lipoplex size, complexation medium and duration of complex-cell exposure time). Lipofection conditions were determined to be both cell- and lipid-type specific. Complexation medium critically affected transfection competence. The initial size of the liposome was not always predictive of lipofection potency. The lipid chemical composition had a strong impact upon lipofection efficiency; DOPE inclusion in the liposome formulations was found to affect the levels of transgene expression in a cell-dependent way. Notably, effective transgene expression was characterized by prominent plasmid nuclear incorporation. Human Aγ- and ε-globin transgene nuclear incorporation and expression in 5637 cells post GLB.391-mediated lipofection lends credence to its use as a vehicle of therapeutic transgene delivery. © 2006 Elsevier B.V. All rights reserved

Hepatitis B virus basal core promoter mutations show lower replication fitness associated with cccDNA acetylation status

In chronic hepatitis B virus (HBV) infection, variants with mutations in the basal core promoter (BCP) and precore region predominate and associate with more severe disease forms. Studies on their effect on viral replication remain controversial. Increasing evidence shows that epigenetic modifications of cccDNA regulate HBV replication and disease outcome. Here we determined the transcription and viral replication efficiency of well-defined BCP and precore mutations and their effect on cccDNA epigenetic control. HBV monomers bearing BCP mutations A1762T/G1764A and A1762T/G1764A/C1766T, and precore mutations G1896A, G1899A and G1896A/G1899A, were transfected into HepG2 cells using a plasmid-free approach. Viral RNA transcripts were detected by Northern blot hybridization and RT PCR, DNA replicative intermediates by Southern blotting and RT PCR, and viral release was measured by ELISA. Acetylation of cccDNA-bound histones was assessed by Chromatin ImmunoPrecipitation (ChIP) assay and methylation of cccDNA by bisulfite sequencing. BCP mutations resulted in low viral release, mRNA transcription and pgRNA/cccDNA ratios that paralleled the acetylation of cccDNA-bound H4 histone and inversely correlated with the HDAC1 recruitment onto cccDNA. Independently of the mutations, cccDNA was a target for methylation, accompanied by the upregulation of DNMT1 expression and DNMT1 recruitment onto cccDNA. Our results suggest that BCP mutations decrease viral replication capacity possibly by modulating the acetylation and deacetylation of cccDNA-bound histones while precore mutations do not have a significant effect on viral replication. These data provide evidence that epigenetic factors contribute to the regulation of HBV viral replication

Factors mediating lipofection potency of a series of cationic phosphonolipids in human cell lines

A series of cationic liposomes known as cationic phosphonolipids (CPs) were evaluated as vehicles for in vitro gene transfer in K562 erythroleukemia cells and 5637 epithelial carcinoma cells. For each CP and target cell type examined, detailed analyses were performed to determine optimal transfection conditions (lipid/ DNA (+/-) charge ratio, amount of complexed episomal DNA, liposomal and lipoplex size, complexation medium and duration of complex-cell exposure time). Lipofection conditions were determined to be both cell- and lipid-type specific. Complexation medium critically affected transfection competence. The initial size of the liposome was not always predictive of lipofection potency. The lipid chemical composition had a strong impact upon lipofection efficiency; DOPE inclusion in the liposome formulations was found to affect the levels of transgene expression in a cell-dependent way. Notably, effective transgene expression was characterized by prominent plasmid nuclear incorporation. Human Aγ- and ε-globin transgene nuclear incorporation and expression in 5637 cells post GLB.391-mediated lipofection lends credence to its use as a vehicle of therapeutic transgene delivery. © 2006 Elsevier B.V. All rights reserved

Deleted in Azoospermia-Like (DAZL) gene-expressing cells in human amniotic fluid: a new source for germ cells research?

Objective: To evaluate whether amniotic fluid cells contain a germ-like cell subpopulation. Design: Experimental study. Setting: University hospital. Patient(s): None. Intervention(s): Cells from human amniotic fluid samples were analyzed for messenger RNA expression of Deleted in Azoospermia-Like gene (DAZL) and Oct-4 by reverse transcriptase polymerase chain reaction. DAZL and C-kit protein expression was assessed by flow cytometry. Immunocytochemistry also was performed to determine DAZL-, stage-specific embryonic antigen 4 (SSEA-4)-, and Oct-4-positive cells. Main Outcome Measure(s): DAZL gene expression in amniotic fluid cells. Result(s): Reverse transcriptase polymerase chain reaction, flow cytometric, and immunocytochemical analyses revealed that human amniotic fluid consists of a distinct cell population that expresses DAZL, C-kit, SSEA-4, and Oct-4. Conclusion(s): Our results suggest that human amniotic fluid represents a new source for the isolation of human DAZL-, C-kit-, SSEA-4-, and Oct-4-positive stem cells without raising the ethical issues associated with human embryonic research. © 2008 American Society for Reproductive Medicine

Combined GSTP1 and NQO1 germline polymorphisms in the susceptibility to Multiple Sclerosis

Germline polymorphisms of detoxification genes could influence susceptibility to Multiple Sclerosis (MS). Glutathione-S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase 1 (NQO1) are detoxifying enzymes involved in biotransformation of metabolites preventing cells from oxidative damage. In order to evaluate the possible contribution of the A313G GSTP1 inactivating polymorphism, alone and in combination with the C609T NQO1 genetic variant in MS susceptibility, we performed a case-control study consisting of 254 MS patients and 370 healthy donors. Genotypes were investigated using a new Real-Time PCR and PCR-RFLP assays. The GSTP1 polymorphism was evaluated in relation to patients' characteristics (clinical subtypes, age and gender) and the NQO1 gene status. GSTP1 genotype distribution was similar between cases and controls. Higher frequency of GSTP1 heterozygotes was observed in patients with relapsing remitting disease (RRMS) (p = 0.019), especially in those presenting a benign form (EDSS ≤2 after 10-15 years from the disease onset). Interestingly, genotype distribution analysis of combined GSTP1 and NQO1 polymorphisms revealed significantly higher frequency of GSTP1 heterozygous (A/G) and NQO1 variant genotypes (C/T and T/T) in patients as compared to the controls (p = 0.031). The increased incidence of combined GSTP1 and NQO1 variant genotypes in MS patients may suggest that defective function of detoxification enzymes might be an important determinant of susceptibility and clinical manifestation of the disease. Moreover, the results suggest a possible role for the GSTP1 heterozygous background in the development of RRMS. © 2015 Informa Healthcare USA, Inc