Treatment of infantile-onset Pompe disease in a rat model with muscle-directed AAV gene therapy.

OBJECTIVE: Pompe disease (PD) is caused by deficiency of the lysosomal enzyme acid α-glucosidase (GAA), leading to progressive glycogen accumulation and severe myopathy with progressive muscle weakness. In the Infantile-Onset PD (IOPD), death generally occurs <1 year of age. There is no cure for IOPD. Mouse models of PD do not completely reproduce human IOPD severity. Our main objective was to generate the first IOPD rat model to assess an innovative muscle-directed adeno-associated viral (AAV) vector-mediated gene therapy. METHODS: PD rats were generated by CRISPR/Cas9 technology. The novel highly myotropic bioengineered capsid AAVMYO3 and an optimized muscle-specific promoter in conjunction with a transcriptional cis-regulatory element were used to achieve robust Gaa expression in the entire muscular system. Several metabolic, molecular, histopathological, and functional parameters were measured. RESULTS: PD rats showed early-onset widespread glycogen accumulation, hepato- and cardiomegaly, decreased body and tissue weight, severe impaired muscle function and decreased survival, closely resembling human IOPD. Treatment with AAVMYO3-Gaa vectors resulted in widespread expression of Gaa in muscle throughout the body, normalizing glycogen storage pathology, restoring muscle mass and strength, counteracting cardiomegaly and normalizing survival rate. CONCLUSIONS: This gene therapy holds great potential to treat glycogen metabolism alterations in IOPD. Moreover, the AAV-mediated approach may be exploited for other inherited muscle diseases, which also are limited by the inefficient widespread delivery of therapeutic transgenes throughout the muscular system.

The E1a Adenoviral Gene Upregulates the Yamanaka Factors to Induce Partial Cellular Reprogramming.

The induction of pluripotency by enforced expression of different sets of genes in somatic cells has been achieved with reprogramming technologies first described by Yamanaka's group. Methodologies for generating induced pluripotent stem cells are as varied as the combinations of genes used. It has previously been reported that the adenoviral E1a gene can induce the expression of two of the Yamanaka factors (c-Myc and Oct-4) and epigenetic changes. Here, we demonstrate that the E1a-12S over-expression is sufficient to induce pluripotent-like characteristics closely to epiblast stem cells in mouse embryonic fibroblasts through the activation of the pluripotency gene regulatory network. These findings provide not only empirical evidence that the expression of one single factor is sufficient for partial reprogramming but also a potential mechanistic explanation for how viral infection could lead to neoplasia if they are surrounded by the appropriate environment or the right medium, as happens with the tumorogenic niche.

Molecular characterization of epithelial-mesenchymal transition and medical treatment related-genes in non-functioning pituitary neuroendocrine tumors.

Introduction: Different medical therapies have been developed for pituitary adenomas. However, Non-Functioning Pituitary Neuroendocrine Tumors (NF-PitNET) have shown little response to them. Furthermore, epithelial-mesenchymal transition (EMT) has been linked to resistance to medical treatment in a significant number of tumors, including pituitary adenomas. Methods: We aimed to evaluate the expression of EMT-related markers in 72 NF-PitNET and 16 non-tumoral pituitaries. To further explore the potential usefulness of medical treatment for NF-PitNET we assessed the expression of somatostatin receptors and dopamine-associated genes. Results: We found that SNAI1, SNAI2, Vimentin, KLK10, PEBP1, Ki-67 and SSTR2 were associated with invasive NF-PitNET. Furthermore, we found that the EMT phenomenon was more common in NF-PitNET than in GH-secreting pituitary tumors. Interestingly, PEBP1 was overexpressed in recurrent NF-PitNET, and could predict growth recurrence with 100% sensitivity but only 43% specificity. In parallel with previously reported studies, SSTR3 is highly expressed in our NF-PitNET cohort. However, SSTR3 expression is highly heterogeneous among the different histological variants of NF-PitNET with very low levels in silent corticotroph adenomas. Conclusion: NF-PitNET showed an enhanced EMT phenomenon. SSTR3 targeting could be a good therapeutic candidate in NF-PitNET except for silent corticotroph adenomas, which express very low levels of this receptor. In addition, PEBP1 could be an informative biomarker of tumor regrowth, useful for predictive medicine in NF-PitNET.

Lung Volume Calculation in Preclinical MicroCT: A Fast Geometrical Approach.

In this study, we present a time-efficient protocol for thoracic volume calculation as a proxy for total lung volume. We hypothesize that lung volume can be calculated indirectly from this thoracic volume. We compared the measured thoracic volume with manually segmented and automatically thresholded lung volumes, with manual segmentation as the gold standard. A linear regression formula was obtained and used for calculating the theoretical lung volume. This volume was compared with the gold standard volumes. In healthy animals, thoracic volume was 887.45 mm3, manually delineated lung volume 554.33 mm3 and thresholded aerated lung volume 495.38 mm3 on average. Theoretical lung volume was 554.30 mm3. Finally, the protocol was applied to three animal models of lung pathology (lung metastasis and transgenic primary lung tumor and fungal infection). In confirmed pathologic animals, thoracic volumes were: 893.20 mm3, 860.12 and 1027.28 mm3. Manually delineated volumes were 640.58, 503.91 and 882.42 mm3, respectively. Thresholded lung volumes were 315.92 mm3, 408.72 and 236 mm3, respectively. Theoretical lung volume resulted in 635.28, 524.30 and 863.10.42 mm3. No significant differences were observed between volumes. This confirmed the potential use of this protocol for lung volume calculation in pathologic models.

Non-Anthocyanin Compounds in Minority Red Grapevine Varieties Traditionally Cultivated in Galicia (Northwest Iberian Peninsula), Analysis of Flavanols, Flavonols, and Phenolic Acids.

Non-anthocyanin compounds (NAN) such as flavonol, flavanol, and phenolic acids should be considered in the characterization of minority red grapevine varieties because these compounds are involved in copigmentation reactions and are potent antioxidants. Sixteen NAN were extracted, identified, and quantified by High Performance Liquid Chromatography (HPLC) from grapes of 28 red genotypes of Vitis vinifera L. grown in Galicia (Northwest of Spain) in 2018 and 2019 vintages. The percentage of total NAN with respect to the total polyphenol content (TPC) values was calculated for each sample and established into three categories: high percentage NAN varieties (NANV), those varieties showing low percentages of NAN (ANV), and finally those varieties showing medium percentages of NAN (NANAV). 'Xafardán' and 'Zamarrica', classified as NANAV, had high values of TPC and showed good percentages of flavonol and flavanol compounds. Principal component analyses (PCA) were performed with flavonol, flavanol, and phenolic acid profiles. The flavonol and flavanol profiles allowed a good discrimination of samples by variety and year, respectively. The flavonol profile should therefore be considered as a potential varietal marker. The results could help in the selection of varieties to be disseminated and in the identification of the most appropriate agronomic and oenological techniques that should be performed on them.

Treatment of skeletal and non-skeletal alterations of Mucopolysaccharidosis type IVA by AAV-mediated gene therapy.

Mucopolysaccharidosis type IVA (MPSIVA) or Morquio A disease, a lysosomal storage disorder, is caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency, resulting in keratan sulfate (KS) and chondroitin-6-sulfate accumulation. Patients develop severe skeletal dysplasia, early cartilage deterioration and life-threatening heart and tracheal complications. There is no cure and enzyme replacement therapy cannot correct skeletal abnormalities. Here, using CRISPR/Cas9 technology, we generate the first MPSIVA rat model recapitulating all skeletal and non-skeletal alterations experienced by patients. Treatment of MPSIVA rats with adeno-associated viral vector serotype 9 encoding Galns (AAV9-Galns) results in widespread transduction of bones, cartilage and peripheral tissues. This led to long-term (1 year) increase of GALNS activity and whole-body correction of KS levels, thus preventing body size reduction and severe alterations of bones, teeth, joints, trachea and heart. This study demonstrates the potential of AAV9-Galns gene therapy to correct the disabling MPSIVA pathology, providing strong rationale for future clinical translation to MPSIVA patients.

Compact analytical flow system for the simultaneous determination of L-lactic and L-malic in red wines.

During the malolactic fermentation of red wines, L-malic acid is mainly converted to L-lactic acid. Both acids should be precisely measured during the entire process to guarantee the quality of the final wine, thus making real-time monitoring approaches of great importance in the winemaking industry. Traditional analytical methods based on laboratory procedures are currently applied and cannot be deployed on-site. In this work, we report on the design and development of a bi-parametric compact analytical flow system integrating two electrochemical biosensors that could be potentially applied in this scenario. The developed flow-system will allow for the first time the simultaneous measurement of both acids in real scenarios at the real-time and in remote way. Miniaturized thin-film platinum four-electrode chips are fabricated on silicon substrates by standard photolithographic techniques and further implemented in a polymeric fluidic structure. This includes a 15 µL flow cell together with the required fluidic channels for sample and reagent fluid management. The four-electrode chip includes counter and pseudo-reference electrodes together with two working electrodes. These are sequentially modified with electropolymerized polypyrrole membranes that entrap the specific receptors for selectively detecting both target analytes. The analytical performance of both biosensors is studied by chronoamperometry, showing a linear range from 5 × 10-6 to 1 × 10-4 M (LOD of 3.2 ± 0.3 × 10-6 M) and from 1 × 10-7 to 1 × 10-6 M (LOD of 6.7 ± 0.2 × 10-8 M) for the L-lactate and the L-malate, respectively. Both biosensors show long-term stability, retaining more than the 90% of their initial sensitivity after more than 30 days, this being a prerequisite for monitoring the whole process of the malolactic fermentation of the red wines (time between 20 and 40 days). The flow system performance is assessed with several wine samples collected during the malolactic fermentation process of three red wines, showing an excellent agreement with the results obtained with the standard method.

Disease correction by AAV-mediated gene therapy in a new mouse model of mucopolysaccharidosis type IIID.

Gene therapy is a promising therapeutic alternative for Lysosomal Storage Disorders (LSD), as it is not necessary to correct the genetic defect in all cells of an organ to achieve therapeutically significant levels of enzyme in body fluids, from which non-transduced cells can uptake the protein correcting their enzymatic deficiency. Animal models are instrumental in the development of new treatments for LSD. Here we report the generation of the first mouse model of the LSD Muccopolysaccharidosis Type IIID (MPSIIID), also known as Sanfilippo syndrome type D. This autosomic recessive, heparan sulphate storage disease is caused by deficiency in N-acetylglucosamine 6-sulfatase (GNS). Mice deficient in GNS showed lysosomal storage pathology and loss of lysosomal homeostasis in the CNS and peripheral tissues, chronic widespread neuroinflammation, reduced locomotor and exploratory activity and shortened lifespan, a phenotype that closely resembled human MPSIIID. Moreover, treatment of the GNS-deficient animals with GNS-encoding adeno-associated viral (AAV) vectors of serotype 9 delivered to the cerebrospinal fluid completely corrected pathological storage, improved lysosomal functionality in the CNS and somatic tissues, resolved neuroinflammation, restored normal behaviour and extended lifespan of treated mice. Hence, this work represents the first step towards the development of a treatment for MPSIIID.

Analysis of free and total sulfur dioxide in wine by using a gas-diffusion analytical system with pH detection.

The use of sulfur dioxide as preservative in winemaking industry has a direct impact on wine quality. The standard methods to analyze this parameter require several processes and are time consuming. In this paper a simple and rapid analytical method for free and total sulfur dioxide detection is proposed. This method is based on the separation of the analyte from the sample with a permeable gas diffusion membrane and its indirect detection with a pH sensor. The system has been validated and optimized for free sulfur dioxide detection in the range of 1-60mgL-1 and for total sulfur dioxide in the range of 30-300mgL-1 with a limit of detection of 0.5mgL-1. Validation of the system has been carried out using a total of 70 samples of white and red wines and two standard methods, the Ripper and the Paul method. The obtained values have demonstrated a good agreement for both methods.

Robust l-malate bienzymatic biosensor to enable the on-site monitoring of malolactic fermentation of red wines.

Monitoring the malolactic fermentation process is strictly required to guarantee the sensorial quality and freshness of red wines. This could be achieved by in-field and real-time continuous measurements of l-malate concentration in the fermentation tanks. The potential of a miniaturized amperometric bienzymatic biosensor as an analytical tool to be applied in such scenario is described in this paper. The biosensor comprises a thin-film gold electrode as transducer, malate dehydrogenase (MDH) and diaphorase (DP) enzymes together with nicotinamide adenine dinucleotide (NAD+) cofactor as the selective receptor and an adequate redox mediator to record the corresponding amperometric signal. Three different biosensor architectures are studied, whose main differences lie in the immobilization of the different chemical components onto the electrode surface. In all cases a fast-electrosynthethized polypyrrole (PPy) membrane is generated for this purpose. The experimental conditions are optimized and the best architecture shows a sensitivity of 1365 ± 110 mA M-1 cm-2 and a detection limit of 6.3 × 10-8 M in a concentration range of 1 × 10-7 M - 1 × 10-6 M. The biosensor presents an excellent working stability as it retains above 90% of its sensitivity after 37 days, thus enabling the monitoring of the malolactic fermentation of three red wines. The obtained results show excellent agreement with the standard colorimetric method.

Monitoring of malolactic fermentation in wine using an electrochemical bienzymatic biosensor for L-lactate with long term stability.

L-lactic acid is monitored during malolactic fermentation process of wine and its evolution is strongly related with the quality of the final product. The analysis of L-lactic acid is carried out off-line in a laboratory. Therefore, there is a clear demand for analytical tools that enabled real-time monitoring of this process in field and biosensors have positioned as a feasible alternative in this regard. The development of an amperometric biosensor for L-lactate determination showing long-term stability is reported in this work. The biosensor architecture includes a thin-film gold electrochemical transducer selectively modified with an enzymatic membrane, based on a three-dimensional matrix of polypyrrole (PPy) entrapping lactate oxidase (LOX) and horseradish peroxidase (HRP) enzymes. The experimental conditions of the biosensor fabrication regarding the pyrrole polymerization and the enzymes entrapment are optimized. The biosensor response to L-lactate is linear in a concentration range of 1 × 10(-6)-1 × 10(-4) M, with a detection limit of 5.2 × 10(-7) M and a sensitivity of - (13500 ± 600) µA M(-1) cm(-2). The biosensor shows an excellent working stability, retaining more than 90% of its original sensitivity after 40 days. This is the determining factor that allowed for the application of this biosensor to monitor the malolactic fermentation of three red wines, showing a good agreement with the standard colorimetric method.

Protein kinase Cδ regulates nuclear export of FOXO1 through phosphorylation of the chaperone 14-3-3ζ.

AIMS/HYPOTHESIS: Forkhead box protein O1 (FOXO1) is a transcription factor essential for beta cell fate. Protein kinase B-dependent phosphorylation of FOXO1 at S256 (P-FOXO1) enables its binding to 14-3-3 dimers and nuclear export. Dephosphorylated FOXO1 enters nuclei and activates pro-apoptotic genes. Since our previous observations suggest that protein kinase C delta (PKCδ) induces nuclear accumulation of FOXO1, the underlying mechanism was examined. METHODS: In human islets, genetically modified mice and INS-1E cells apoptosis was assessed by TUNEL staining. Subcellular translocation of proteins was examined by confocal microscopy and signalling pathways were analysed by western blotting and overlay assay. RESULTS: In PKCδ-overexpressing (PKCδ-tg) mouse islet cells and INS-1E cells FOXO1 accumulated in nuclei, surprisingly, as P-FOXO1. PKCδ-tg decelerated IGF-1-dependent stimulation of nuclear export, indicating that changes in export caused nuclear retention of P-FOXO1. Nuclear accumulation of P-FOXO1 was accompanied by increased phosphorylation of 14-3-3ζ at S58 and reduced dimerisation of 14-3-3ζ. Palmitic acid further augmented phosphorylation of 14-3-3ζ and triggered nuclear accumulation of FOXO1 in both INS-1E and human islet cells. Furthermore, the overexpression of a phosphomimicking mutant of 14-3-3ζ (S58D) enhanced nuclear FOXO1. In accordance with the nuclear accumulation of P-FOXO1, PKCδ overexpression alone did not increase apoptotic cell death. Additionally, insulin secretion and glucose homeostasis in PKCδ-overexpressing mice remained unaffected. CONCLUSIONS/INTERPRETATION: These results suggest that PKCδ-mediated phosphorylation of 14-3-3ζ contributes to the nuclear retention of FOXO1, even when FOXO1 is phosphorylated as under non-stress conditions. P-FOXO1 does not induce pro-apoptotic genes, but may rather exert beneficial effects on beta cells.

HMGA1 overexpression in adipose tissue impairs adipogenesis and prevents diet-induced obesity and insulin resistance.

High-Mobility-Group-A1 (HMGA1) proteins are non-histone proteins that regulate chromatin structure and gene expression during embryogenesis, tumourigenesis and immune responses. In vitro studies suggest that HMGA1 proteins may be required to regulate adipogenesis. To examine the role of HMGA1 in vivo, we generated transgenic mice overexpressing HMGA1 in adipose tissues. HMGA1 transgenic mice showed a marked reduction in white and brown adipose tissue mass that was associated with downregulation of genes involved in adipogenesis and concomitant upregulation of preadipocyte markers. Reduced adipogenesis and decreased fat mass were not associated with altered glucose homeostasis since HMGA1 transgenic mice fed a regular-chow diet exhibited normal glucose tolerance and insulin sensitivity. However, when fed a high-fat diet, overexpression of HMGA1 resulted in decreased body-weight gain, reduced fat mass, but improved insulin sensitivity and glucose tolerance. Although HMGA1 transgenic mice exhibited impaired glucose uptake in adipose tissue due to impaired adipogenesis, the increased glucose uptake observed in skeletal muscle may account for the improved glucose homeostasis. Our results indicate that HMGA1 plays an important function in the regulation of white and brown adipogenesis in vivo and suggests that impaired adipocyte differentiation and decreased fat mass is not always associated with impaired whole-body glucose homeostasis.

Tobacco smoking, exposure to second-hand smoke, and asthma and wheezing in schoolchildren: a cross-sectional study.

AIM: To analyse the association between tobacco smoking, exposure to second-hand smoke (SHS) and reports of wheezing and asthma in a sample of schoolchildren. METHODS: A structured questionnaire was administered to 1766 students (7th grade, aged 12-13 years) at 25 schools in Terrassa, Spain (2006). We determined the prevalence of active smoking, exposure to SHS and reports of wheezing and asthma, and their association by means of prevalence odds ratios (OR) and 95% confidence intervals (CI). RESULTS: 97.5% of children were nonsmokers, 1.5% were experimental smokers and 1% were regular smokers. 41.1% of children reported exposure to SHS at home, 40.0% at school, 53.9% in their leisure time and 33.2% while using private or public transportation. Wheezing was reported by 9.2% of children, and 9.2% reported asthma. A significant association was found between smoking tobacco and wheezing: OR in experimental smokers = 3.0 (95% CI 1.2-7.7), and OR in active smokers = 4.2 (95% CI 1.4-12.5). Exposure to SHS while using transportation was associated with wheezing (OR = 1.4; 95% CI 1.0-2.0). Tobacco smoking and exposure to SHS were not associated with asthma. CONCLUSION: Active and experimental smokers, and those who reported exposure to SHS while using public or private transportation, had higher likelihood of reporting wheezing. No association between active or passive smoking and asthma was observed.

Vascular endothelial growth factor-mediated islet hypervascularization and inflammation contribute to progressive reduction of ß-cell mass.

Type 2 diabetes (T2D) results from insulin resistance and inadequate insulin secretion. Insulin resistance initially causes compensatory islet hyperplasia that progresses to islet disorganization and altered vascularization, inflammation, and, finally, decreased functional ß-cell mass and hyperglycemia. The precise mechanism(s) underlying ß-cell failure remain to be elucidated. In this study, we show that in insulin-resistant high-fat diet-fed mice, the enhanced islet vascularization and inflammation was parallel to an increased expression of vascular endothelial growth factor A (VEGF). To elucidate the role of VEGF in these processes, we have genetically engineered ß-cells to overexpress VEGF (in transgenic mice or after adeno-associated viral vector-mediated gene transfer). We found that sustained increases in ß-cell VEGF levels led to disorganized, hypervascularized, and fibrotic islets, progressive macrophage infiltration, and proinflammatory cytokine production, including tumor necrosis factor-α and interleukin-1ß. This resulted in impaired insulin secretion, decreased ß-cell mass, and hyperglycemia with age. These results indicate that sustained VEGF upregulation may participate in the initiation of a process leading to ß-cell failure and further suggest that compensatory islet hyperplasia and hypervascularization may contribute to progressive inflammation and ß-cell mass loss during T2D.

Adipose tissue overexpression of vascular endothelial growth factor protects against diet-induced obesity and insulin resistance.

During the expansion of fat mass in obesity, vascularization of adipose tissue is insufficient to maintain tissue normoxia. Local hypoxia develops and may result in altered adipokine expression, proinflammatory macrophage recruitment, and insulin resistance. We investigated whether an increase in adipose tissue angiogenesis could protect against obesity-induced hypoxia and, consequently, insulin resistance. Transgenic mice overexpressing vascular endothelial growth factor (VEGF) in brown adipose tissue (BAT) and white adipose tissue (WAT) were generated. Vessel formation, metabolism, and inflammation were studied in VEGF transgenic mice and wild-type littermates fed chow or a high-fat diet. Overexpression of VEGF resulted in increased blood vessel number and size in both WAT and BAT and protection against high-fat diet-induced hypoxia and obesity, with no differences in food intake. This was associated with increased thermogenesis and energy expenditure. Moreover, whole-body insulin sensitivity and glucose tolerance were improved. Transgenic mice presented increased macrophage infiltration, with a higher number of M2 anti-inflammatory and fewer M1 proinflammatory macrophages than wild-type littermates, thus maintaining an anti-inflammatory milieu that could avoid insulin resistance. These studies suggest that overexpression of VEGF in adipose tissue is a potential therapeutic strategy for the prevention of obesity and insulin resistance.

Methylthioadenosine (MTA) inhibits melanoma cell proliferation and in vivo tumor growth.

BACKGROUND: Melanoma is the most deadly form of skin cancer without effective treatment. Methylthioadenosine (MTA) is a naturally occurring nucleoside with differential effects on normal and transformed cells. MTA has been widely demonstrated to promote anti-proliferative and pro-apoptotic responses in different cell types. In this study we have assessed the therapeutic potential of MTA in melanoma treatment. METHODS: To investigate the therapeutic potential of MTA we performed in vitro proliferation and viability assays using six different mouse and human melanoma cell lines wild type for RAS and BRAF or harboring different mutations in RAS pathway. We also have tested its therapeutic capabilities in vivo in a xenograft mouse melanoma model and using variety of molecular techniques and tissue culture we investigated its anti-proliferative and pro-apoptotic properties. RESULTS: In vitro experiments showed that MTA treatment inhibited melanoma cell proliferation and viability in a dose dependent manner, where BRAF mutant melanoma cell lines appear to be more sensitive. Importantly, MTA was effective inhibiting in vivo tumor growth. The molecular analysis of tumor samples and in vitro experiments indicated that MTA induces cytostatic rather than pro-apoptotic effects inhibiting the phosphorylation of Akt and S6 ribosomal protein and inducing the down-regulation of cyclin D1. CONCLUSIONS: MTA inhibits melanoma cell proliferation and in vivo tumor growth particularly in BRAF mutant melanoma cells. These data reveal a naturally occurring drug potentially useful for melanoma treatment.

The dual PI3K/mTOR inhibitor PI-103 promotes immunosuppression, in vivo tumor growth and increases survival of sorafenib-treated melanoma cells.

Melanoma is the most lethal human skin cancer. If metastatic, it becomes very aggressive and resistant to standard modalities of anticancer treatment. During the last 10 years, several therapeutic strategies have been tested including the use of single and combined small drugs. Experimental results indicate that RAS and PI3K pathways are important for the development and maintenance of melanoma. In this study, we assessed the in vitro and in vivo inhibition potential of PI-103, a PI3K (p110alpha)/mTOR inhibitor and sorafenib, a BRAF inhibitor, as single agents and in combination in primary melanoma cell lines. Although PI-103 and sorafenib inhibited melanoma in vitro cell proliferation and viability, the inhibition of RAS pathway appeared to be more effective. The combination of the two agents in in vitro showed a synergistic effect inhibiting RAS and PI3K pathways in a cell line dependent manner. However, no cooperative effect was observed in blocking in vivo tumor growth in immunocompetent mice. In contrary to the expected, the data indicate that PI-103 induced immunosuppression promoting in vivo tumor growth and inhibiting apoptosis. Furthermore, in vitro studies examining the effects of the PI3K/mTOR inhibitor in tumor derived cell lines indicated that PI-103 induced the anti-apoptotic BH3 family proteins Mcl1, Bcl2 and Bcl(xL) favoring, the in vitro survival of sorafenib treated melanoma cells. These data certainly makes an argument for investigating unexpected effects of rational drug combinations on immunocompetent animal models prior to conducting clinical studies.

Secondhand smoke in hospitals of Catalonia (Spain) before and after a comprehensive ban on smoking at the national level.

OBJECTIVE: To assess changes in secondhand smoke exposure by means of airborne nicotine concentrations in public hospitals of Catalonia (Spain) before and after a comprehensive national smoking ban. METHODS: We monitored vapor-phase nicotine concentrations in 44 public hospitals in Catalonia (Spain) before the smoking ban (September-December 2005) and one year after (September-December 2006). We installed 5-7 sampling devices per hospital for 7 days in different places (228 pairs of samples), and 198 pairs of samples were available for the final analysis. RESULTS: The median nicotine concentration declined from 0.23 microg/m(3) (interquartile range: 0.13-0.63) before the law to 0.10 microg/m(3) (interquartile range: 0.02-0.19) after the law (% decline=56.5, p<0.01). We observed significant reductions in the median nicotine concentrations in all hospital locations, although secondhand smoke exposure was still present in some places (main hospital entrance, emergency department waiting rooms, fire escapes, and cafeterias). CONCLUSIONS: Secondhand smoke in hospitals has decreased after the ban. Assessment of airborne nicotine concentrations appears to be an objective and feasible system to monitor and reinforce the compliance of smoke-free legislations in this setting.

Overexpression of ubiquitous 6-phosphofructo-2-kinase in the liver of transgenic mice results in weight gain.

Fructose 2,6-bisphosphate (Fru-2,6-P2) is an important metabolite that controls glycolytic and gluconeogenic pathways in several cell types. Its synthesis and degradation are catalyzed by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2). Four genes, designated Pfkfb1-4, codify the different PFK-2 isozymes. The Pfkfb3 gene product, ubiquitous PFK-2 (uPFK-2), has the highest kinase/bisphosphatase activity ratio and is associated with proliferation and tumor metabolism. A transgenic mouse model that overexpresses uPFK-2 under the control of the phosphoenolpyruvate carboxykinase promoter was designed to promote sustained and elevated Fru-2,6-P2 levels in the liver. Our results demonstrate that in diet-induced obesity, high Fru-2,6-P2 levels in transgenic livers caused changes in hepatic gene expression profiles for key gluconeogenic and lipogenic enzymes, as well as an accumulation of lipids in periportal cells, and weight gain.