Immortalization of Rat Embryo Fibroblasts by a 3′-Untranslated Region

We have exploited a cross-species expression screen to search for cellular immortalizing activities. A newt blastemal cDNA expression library was transfected into rat embryo fibroblasts and immortal cell lines were selected. This identified a 1-kb cDNA fragment which has a low representation in the cDNA library and is derived from the 3′-UTR of an α-glucosidase-related mRNA. Expression of this sequence in rat embryo fibroblasts has shown that it is active in promoting colony formation and immortalization. It is also able to cooperate with an immortalization-defective deletion mutant of SV40 T antigen, indicating that it can exert its growth-stimulatory activity in the pathway activated by a viral immortalizing oncogene. This is the first example of an immortalizing activity mediated by an RNA sequence, and further analysis of its mechanism should provide new insights into senescence and immortalization.     

MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells

Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30–40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation are not sufficient to predict a positive response of NSCLC cells to a cocktail of MET and EGFR inhibitors and highlights the importance of identifying more reliable biomarkers to predict the efficacy of treatments in NSCLC patients resistant to EGFR TKI.     

Antimicrobial activity of copper and silver nanofilms on nosocomial bacterial species

Contaminated surfaces are possible vehicles in infection transmission. It is known that both Copper (Cu) and Silver (Ag) efficiently inactivate microbes by direct contact. Aiming at using these metals for benefitting from their antimicrobial effect, but to avoid subsequent toxic effects, we evaluated the antimicrobial activity of nanometric thin Silver and Copper films covering less expensive materials. Using a modified version of the Japan Industrial Standard JIS Z 2801:2000, we demonstrated the antimicrobial activity of the surfaces covered with metal ions nanofilms on microorganisms possibly involved in nosocomial infections and on Bacillus anthracis, bacteria with possible implication in bioterrorist attacks. Copper covered surfaces proved to have better antimicrobial activity than Silver surfaces. Silver covered surfaces showed better activity on Gram negative bacteria than on Gram positive cocci. Going deeper with studies on antimicrobial effects using new methods with better direct and/or functional discriminatory capacity is needed in order to provide additional information on the mechanisms of Silver and Copper nanofilms antimicrobial activity.     

Protection against Pseudomonas aeruginosa lung infection in mice by recombinant OprF-pulsed dendritic cell immunization

Background

Porcine reproductive and respiratory syndrome (PRRS) has now been widely recognized as an economically important disease. The objective of this study was to compare the molecular and biological characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) field isolates in China to those of the modified live virus (MLV) PRRS vaccine and its parent strain (ATCC VR2332).

Results

Five genes (GP2, GP3, GP4, GP5 and NSP2) of seven isolates of PRRSV from China, designated LS-4, HM-1, HQ-5, HQ-6, GC-2, GCH-3 and ST-7/2008, were sequenced and analyzed. Phylogenetic analyses based on the nucleotide sequence of the ORF2-5 and NSP2 showed that the seven Chinese isolates belonged to the same genetic subgroup and were related to the North American PRRSV genotype. Comparative analysis with the relevant sequences of another Chinese isolate (BJ-4) and North American (VR2332 and MLV) viruses revealed that these isolates have 80.8-92.9% homology with VR-2332, and 81.3-98.8% identity with MLV and 80.7-92.9% with BJ-4. All Nsp2 nonstructural protein of these seven isolates exhibited variations (a 29 amino acids deletion) in comparison with other North American PRRSV isolates. Therefore, these isolates were novel strain with unique amino acid composition. However, they all share more than 97% identity with other highly pathogenic Chinese PRRSV strains. Additionally, there are extensive amino acid (aa) mutations in the GP5 protein and the Nsp2 protein when compared with the previous isolates.

Conclusions

These results might be useful to study the genetic diversity of PRRSV in China and to track the infection sources as well as for vaccines development.     

Polyunsaturated fatty acid pattern in liver and erythrocyte phospholipids from obese patients

Objective: Our aim was to study the fatty acid (FA) composition of liver phospholipids and its relation to that in erythrocyte membranes from patients with obese nonalcoholic fatty liver disease (NAFLD), as an indication of lipid metabolism alterations leading to steatosis. Research Methods and Procedures: Eight control subjects who underwent antireflux surgery and 12 obese patients with NAFLD who underwent subtotal gastrectomy with a gastro‐jejunal anastomosis in Roux‐en‐Y were studied. The oxidative stress status of patients was assessed by serum F₂‐isoprostanes levels (gas chromatography/negative ion chemical ionization tandem mass spectrometry). Analysis of FA composition of liver and erythrocyte phospholipids was carried out by gas‐liquid chromatography. Results: Patients with NAFLD showed serum F₂‐isoprostanes levels 84% higher than controls. Compared with controls, liver phospholipids from obese patients exhibited significantly 1) lower levels of 20:4n‐6, 22:5n‐3, 22:6n‐3 [docosahexaenoic acid (DHA)], total long‐chain polyunsaturated FA (LCPUFA), and total n‐3 LCPUFA, 2) higher 22:5n‐6 [docosapentaenoic acid (DPAn‐6)] levels and n‐6/n‐3 LCPUFA ratios, and 3) comparable levels of n‐6 LCPUFA. Levels of DHA and DPAn‐6 in liver were positively correlated with those in erythrocytes (r = 0.77 and r = 0.90, respectively; p < 0.0001), whereas DHA and DPAn‐6 showed a negative association in both tissues (r = ?0.79, p < 0.0001 and r = ?0.58, p < 0.01, respectively), associated with lower DHA/DPAn‐6 ratios. Discussion: Obese patients with NAFLD showed marked alterations in the polyunsaturated fatty acid pattern of the liver. These changes are significantly correlated with those found in erythrocytes, thus suggesting that erythrocyte FA composition could be a reliable indicator of derangements in liver lipid metabolism in obese patients.     

The use of GMOs (genetically modified organisms): agricultural biotechnology or agricultural biopolitics?

Agricultural biotechnologies embrace a large array of conventional and modern technologies, spanning from composting organic by-products of agriculture to innovative improvement of quality traits of about twenty out of the mostly cultivated plants. In EU a rather restrictive legislative framework has been installed for GMOs, requiring a risk assessment disproportionate with respect to conventional agriculture and organic farming products. The latter are far from being proved safe for human and animal health, and for the environment.Biotechnology of GMOs has been overtaken by biopolitics. On one side there are biotechnological challenges to be tackled, on another side there is plenty of ground for biopolitical decisions about GMOs. Perhaps the era of harsh confrontation could be fruitfully replaced by sensible cooperation, in order to get a sustainable agricultural development.     

Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not

The temporal and spatial regulation of cytokinesis requires an interaction between the anaphase mitotic spindle and the cell cortex. However, the relative roles of the spindle asters or the central spindle bundle are not clear in mammalian cells. The central spindle normally serves as a platform to localize key regulators of cell cleavage, including passenger proteins. Using time-lapse and immunofluorescence analysis, we have addressed the consequences of eliminating the central spindle by ablation of PRC1, a microtubule bundling protein that is critical to the formation of the central spindle. Without a central spindle, the asters guide the equatorial cortical accumulation of anillin and actin, and of the passenger proteins, which organize into a subcortical ring in anaphase. Furrowing goes to completion, but abscission to create two daughter cells fails. We conclude the central spindle bundle is required for abscission but not for furrowing in mammalian cells.     

Mutation analysis of oxisterol-binding-protein gene in patients with age-related macular degeneration

Allelic variants of several genes are increasingly recognized as susceptibility factors in age-related macular degeneration (AMD). Because of its metabolic characteristics the macula is sensitive to oxidative damage, and supplementation with antioxidants has been shown to be effective in slowing the progression of disease in AMD patients. The oxisterol-binding-protein (OSBP2) gene is expressed mainly in the retinal pigmented epithelium underlying the macular region. Its product specifically binds and transports oxisterols, the cytotoxic effects of which may be involved in macular damage. The aim of this study was to search for allelic variants of OSBP2 gene, as well as to evaluate several risk factors in 24 patients with AMD; 17 with nonexudative (NE) and 7 with neovascular (NV) form. Total cholesterol was elevated in 66% of the patients, high-density lipoprotein (HDL) cholesterol was reduced in 12%; vitamin A or vitamin E deficiency was not observed. OSBP2 gene analysis was performed in AMD patients and in 110 control subjects by single-stranded conformational polymorphism (SSCP) analysis followed by direct sequencing. Six allelic variants were detected: 2 nonpolymorphic unique exonic variants in 2 AMD subjects and 4 polymorphic variants (2 exonic and 2 intronic). These data indicate a possible role of OSBP2 gene in the pathogenesis of oxidative damage to the macula induced by oxysterols in AMD patients.     

Twenty-four novel mutations in Wilson disease patients of predominantly Italian origin

Herein we report the results of mutation analysis of the ATP7B gene in a group of 134 Wilson disease (WD) families (268 chromosomes) prevalently of Italian origin. Using the SSCP and sequencing methods we identified 71 disease-causing mutations. Twenty-four were novel, while 19 more mutations already described, were identified in new populations in this study. A known mutation G591D showed a regional distribution, since it was only detected in 38.5% of the analyzed chromosomes in WD patients originating from Apulia, a region of South Italy. Detection of new mutations in the ATP7B gene increases our capability of molecular analysis that is essential for early diagnosis and treatment of WD.