Complexation of lipofectamine and cholesterol-modified DNA sequences studied by single-molecule fluorescence techniques

Lipoplex formation for normal and cholesterol-modified oligonucleotides is investigated by fluorescence correlation spectroscopy (FCS). To overcome the problems related to the fitting of autocorrelation curves when fluorescence bursts are present, the baseline fluorescence levels and the fluorescence bursts in the same trace were separately analyzed. This approach was not previously used in FCS studies of lipoplexes and allowed a more detailed characterization of this heterogeneous system. From the baseline levels, the number of free/bound DNA molecules and the presence of tens to hundreds of nanometer-sized lipoplexes were estimated using various mathematical models. Analysis of the fluorescent bursts provided an indication about the sizes of the lipoplexes, the number of DNA molecules in these aggregates, and the relative amount of lipids in each aggregate. An explanation for the higher transfection efficiency previously reported for one of the cholesterol-modified oligonucleotide compounds was found in relation to the formation of large size lipoplexes.     

Foam cell‐derived 4‐hydroxynonenal induces endothelial cell senescence in a TXNIP‐dependent manner

Vascular endothelial cell (VEC) senescence is considered an early event in the development of atherosclerotic lesions. Stressful stimuli, in particular oxidative stress, have been linked to premature senescence in the vasculature. Foam cells are a major source of reactive oxygen species and may play a role in the induction of VEC senescence; hence, we investigated their involvement in the induction of VEC senescence in a co‐culture transwell system. Primary bovine aortic endothelial cells, exposed to the secretome of THP‐1 monocyte‐derived foam cells, were analysed for the induction of senescence. Senescence associated β‐galactosidase activity and the expression of p16 and p21 were increased, whereas phosphorylated retinoblastoma protein was reduced. This senescent phenotype was mediated by 4‐hydroxnonenal (4‐HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4‐HNE in the co‐culture medium blunted this effect. Furthermore, both foam cells and 4‐HNE increased the expression of the pro‐oxidant thioredoxin‐interacting protein (TXNIP). Molecular manipulation of TXNIP expression confirmed its involvement in foam cell‐induced senescence. Previous studies showed that peroxisome proliferator‐activated receptor (PPAR)δ was activated by 4‐hydroalkenals, such as 4‐HNE. Pharmacological interventions supported the involvement of the 4‐HNE‐PPARδ axis in the induction of TXNIP and VEC senescence. The association of TXNIP with VEC senescence was further supported by immunofluorescent staining of human carotid plaques in which the expression of both TXNIP and p21 was augmented in endothelial cells. Collectively, these findings suggest that foam cell‐released 4‐HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence.     

ORF7 of varicella-zoster virus is a neurotropic factor

Varicella-zoster virus (VZV) is the causative agent of chickenpox and herpes zoster (shingles). After the primary infection, the virus remains latent in sensory ganglia and reactivates upon weakening of the cellular immune system due to various conditions, erupting from sensory neurons and infecting the corresponding skin tissue. The current varicella vaccine is highly attenuated in the skin and yet retains its neurovirulence and may reactivate and damage sensory neurons. The factors involved in neuronal invasion and establishment of latency are still elusive. Previously, we constructed a library of whole-gene deletion mutants carrying a bacterial artificial chromosome sequence and a luciferase marker in order to perform a comprehensive VZV genome functional analysis. Here, screening of dispensable gene deletion mutants in differentiated neuronal cells led to the identification of ORF7 as the first known, likely a main, VZV neurotropic factor. ORF7 is a virion component localized to the Golgi compartment in infected cells, whose deletion causes loss of polykaryon formation in epithelial cell culture. Interestingly, ORF7 deletion completely abolishes viral spread in human nervous tissue ex vivo and in an in vivo mouse model. This finding adds to our previous report that ORF7 is also a skin-tropic factor. The results of our investigation will not only lead to a better understanding of VZV neurotropism but could also contribute to the development of a neuroattenuated vaccine candidate against shingles or a vector for delivery of other antigens.     

Immunohistochemical expression of androgen receptor and prostate-specific antigen in breast cancer

AR (androgen receptor) and PSA (prostate-specific antigen) are involved in the pathogenesis of breast cancer, but their role is not clearly defined. The purpose of this study was to analyze by immunohistochemistry the AR and PSA (prostate-specific antigen) expression in 156 female breast carcinomas and to correlate the results with some histopathological parameters, like ER (estrogen receptor), PR (progesterone receptor), HER2/neu, nodal and metastasis status, histological type and grade. ARs and PSA were expressed in 112/156 (72%) and respectively in 61/156 (39%) of cases and we found a positive correlation between AR and PSA expression in breast carcinomas (p<0.0002). We also found an association between the histological type of the tumor and AR (p<0.001), respectively PSA (p=0.01) and between AR and the grade of differentiation (p=0.007) and the nodal status (p=0.02). No correlations were found between the metastasis status and AR or PSA. 47.3% (53/112) of AR-positive cases and 46% (28/61) of PSA-positive cases were ER-negative. High frequency of AR (87.5%) and PSA (75%) expression was found in medullary carcinomas and 53% of lobular invasive carcinomas co-expressed AR and PSA. We found an inverse correlation between HER2/neu and PSA (p=0.05). Although most of the PSA-positive carcinomas were lymph node-negative, well and moderately differentiated, we did not find any statistically significant correlations between these parameters and PSA expression. Our study confirms that ARs are commonly expressed in breast cancer and the expression of PSA and AR are highly correlated. Moreover, all the lobular carcinomas and the majority of medullary carcinomas co-expressed AR and PSA, the majority of AR-positive carcinomas were lymph node-negative, well and moderately differentiated, and large number of ER-negative carcinomas expressed AR and PSA.     

Ecology of Antibiotic Resistance Genes: Characterization of Enterococci from Houseflies Collected in Food Settings

In this project, enterococci from the digestive tracts of 260 houseflies (Musca domestica L.) collected from five restaurants were characterized. Houseflies frequently (97% of the flies were positive) carried enterococci (mean, 3.1 × 10³ CFU/fly). Using multiplex PCR, 205 of 355 randomly selected enterococcal isolates were identified and characterized. The majority of these isolates were Enterococcus faecalis (88.2%); in addition, 6.8% were E. faecium, and 4.9% were E. casseliflavus. E. faecalis isolates were phenotypically resistant to tetracycline (66.3%), erythromycin (23.8%), streptomycin (11.6%), ciprofloxacin (9.9%), and kanamycin (8.3%). Tetracycline resistance in E. faecalis was encoded by tet(M) (65.8%), tet(O) (1.7%), and tet(W) (0.8%). The majority (78.3%) of the erythromycin-resistant E. faecalis isolates carried erm(B). The conjugative transposon Tn916 and members of the Tn916/Tn1545 family were detected in 30.2% and 34.6% of the identified isolates, respectively. E. faecalis carried virulence genes, including a gelatinase gene (gelE; 70.7%), an aggregation substance gene (asa1; 33.2%), an enterococcus surface protein gene (esp; 8.8%), and a cytolysin gene (cylA; 8.8%). Phenotypic assays showed that 91.4% of the isolates with the gelE gene were gelatinolytic and that 46.7% of the isolates with the asa1 gene aggregated. All isolates with the cylA gene were hemolytic on human blood. This study showed that houseflies in food-handling and -serving facilities carry antibiotic-resistant and potentially virulent enterococci that have the capacity for horizontal transfer of antibiotic resistance genes to other bacteria.     

Vascular endothelium in atherosclerosis

Their strategic location between blood and tissue and their constitutive properties allow endothelial cells (EC) to monitor the transport of plasma molecules, by employing bidirectional receptor-mediated and receptor-independent transcytosis and endocytosis, and to regulate vascular tone, cellular cholesterol and lipid homeostasis. These cells are also involved in signal transduction, immunity, inflammation and haemostasis. Cardiovascular risk factors, such as hyperlipaemia/dyslipidaemia trigger the molecular machinery of EC to respond to insults by modulation of their constitutive functions followed by dysfunction and ultimately by injury and apoptosis. The gradual activation of EC consists initially in the modulation of two constitutive functions: (1) permeability, i.e. increased transcytosis of lipoproteins, and (2) biosynthetic activity, i.e. enhanced synthesis of the basement membrane and extracellular matrix. The increased transcytosis and the reduced efflux of β-lipoproteins (βLp) lead to their retention within the endothelial hyperplasic basal lamina as modified lipoproteins (MLp) and to their subsequent alteration (oxidation, glycation, enzymatic modifications). MLp generate chemoattractant and inflammatory molecules, triggering EC dysfunction (appearance of new adhesion molecules, secretion of chemokines, cytokines), characterised by monocyte recruitment, adhesion, diapedesis and residence within the subendothelium. In time, EC in the athero-prone areas alter their net negative surface charge, losing their non-thrombogenic ability, become loaded with lipid droplets and turn into foam cells. Prolonged and/or repeated exposure to cardiovascular risk factors can ultimately exhaust the protective effect of the endogenous anti-inflammatory system within EC. As a consequence, EC may progress to senescence, lose their integrity and detach into the circulation.     

p21 functions in a post-mitotic block checkpoint in the apoptotic response to vinblastine

We have shown previously that in KB-3 (HeLa) cells vinblastine causes downregulation of the CDK inhibitor p21 through a c-Jun regulated pathway. To test the hypothesis that p21 downregulation is necessary to alleviate a protective function, we transfected p21 in KB-3 cells and examined the apoptotic response to vinblastine. The results showed that cells overexpressing p21 were apoptosis-resistant, not through an ability of p21 to cause cell cycle arrest prior to mitotic arrest, but through altering the fate of mitotically arrested cells after drug treatment. Moreover, p21 null HCT116 cells were more prone to vinblastine-induced apoptosis relative to wild-type cells. The results provide support for a model whereby p21 downregulation promotes vinblastine-induced apoptosis by alleviating its protective function following mitotic arrest.     

Phenotypic and molecular methods used for identification of oral streptococci and related microorganisms

The oral cavity contains the greatest biodiversity, over 70 species being isolated from mouth mucosa, saliva, denture surfaces and/or dental-plaque. The oral streptococci, representing over 80% of the mouth micro flora, are able to synthesize glucosyl-transferases, enzymes involved in glucans production. Glucans are involved in production of an extracellular slime layer promoting adhesion and formation of a dental plaque biofilm. The 43 isolates studied obtained from partially and/or totally edentulous, were identified by VITEK system using gram-positive identification cards. Species-specific regions within the genes coding for glucosyl-transferases (gtf genes) were targeted for PCR identification of isolates. Sequencing of 16S rRNA was used as gold standard for strain confirmation. VITEK system identified a number of 11 strains as S. mitis/oralis, 12 strains as S. anginosus/gordonii, 12 strains as S. sanguinis/parasanguinis, 3 strains as S. salivarius, 3 strains as S. plurianimalium, 1 strain as S. cristatus and 1 strain as S. alactolyticus, respectively. The PCR system targeting gtf genes was able to identify S. oralis, S. salivarius and S. gordonii strains. Sequence of 16S rRNA discriminated among streptococci species and revealed 16 strains of Leuconostoc mesenteroides. Many studies are needed in order to select the most reliable phenotypic and genotypic methods in order to improve the identification algorithm for oral streptococci used by clinical laboratories. Their accurate identification is mandatory for better understanding their role in human infections.