Ligand-dependent Hedgehog pathway activation in Rhabdomyosarcoma: the oncogenic role of the ligands.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. The Hedgehog (HH) pathway is known to develop an oncogenic role in RMS. However, the molecular mechanism that drives activation of the pathway in RMS is not well understood. METHODS: The expression of HH ligands was studied by qPCR, western blot and immunohistochemistry. Functional and animal model studies were carried out with cells transduced with shRNAs against HH ligands or treated with HH-specific inhibitors (Vismodegib and MEDI-5304). Finally, the molecular characterisation of an off-target effect of Vismodegib was also made. RESULTS: The results showed a prominent expression of HH ligands supporting an autocrine ligand-dependent activation of the pathway. A comparison of pharmacologic Smoothened inhibition (Vismodegib) and HH ligand blocking (MEDI-5304) is also provided. Interestingly, a first description of pernicious off-target effect of Vismodegib is also reported. CONCLUSIONS: The clarification of the HH pathway activation mechanism in RMS opens a door for targeted therapies against HH ligands as a possible alternative in the future development of better treatment protocols. Moreover, the description of a pernicious off-target effect of Vismodegib, via unfolded protein response activation, may mechanistically explain its previously reported inefficiency in several ligand-dependent cancers.

Gender differences in the relationship between body mass index (BMI) changes and the prevalence and severity of wheezing and asthma in the first year of life

BACKGROUND: Rapid weight gain has been recently associated with asthma at school age, but its influence in respiratory symptoms during infancy is still unknown. METHODS: Answers from 6541 parents living in six different cities of Brazil to the International Study of Wheezing in Infants (EISL) questionnaire were analysed. Data from reported weight and height at birth and at one year were used to calculate BMI. Rapid body mass index (BMI) gain was defined by the difference in BMI superior to 1.0z and excessive by the difference superior to 2.0z. RESULTS: Rapid BMI gain was found in 45.8% infants and excessive in 24.4%. Boys showed a significantly higher BMI gain than girls. Girls with rapid BMI gain showed a significantly higher prevalence of hospitalisation for wheezing (8.8% vs. 6.4%; aOR: 1.4, 95%CI: 1.1-1.8), severe wheezing (18.1% vs. 15.0%; aOR: 1.3, 95%CI: 1.0-1.5) and medical diagnosis of asthma (7.5% vs. 5.7%; aOR: 1.3, 95%CI: 1.0-1.7). Girls with excessive BMI gain also had a significantly higher prevalence of hospitalisation for wheezing (9.8% vs. 6.7%; aOR: 1.5, 95%CI: 1.1-2.0) and severe wheezing (18.9% vs. 15.5%; aOR: 1.3, 95%CI: 1.0-1.6). No significant association was found among boys. CONCLUSIONS: The majority of the evaluated infants showed BMI gain above expected in the first year of life. Although more commonly found in boys, rapid and excessive BMI gain in the first year of life was significantly related to more severe patterns of wheezing in infancy among girls

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Quantum Monte Carlo estimation of complex-time correlations for the study of the ground-state dynamic structure function.

We present a method based on the path integral Monte Carlo formalism for the calculation of ground-state time correlation functions in quantum systems. The key point of the method is the consideration of time as a complex variable whose phase δ acts as an adjustable parameter. By using high-order approximations for the quantum propagator, it is possible to obtain Monte Carlo data all the way from purely imaginary time to δ values near the limit of real time. As a consequence, it is possible to infer accurately the spectral functions using simple inversion algorithms. We test this approach in the calculation of the dynamic structure function S(q, ω) of two one-dimensional model systems, harmonic and quartic oscillators, for which S(q, ω) can be exactly calculated. We notice a clear improvement in the calculation of the dynamic response with respect to the common approach based on the inverse Laplace transform of the imaginary-time correlation function.

Gender differences in the relationship between body mass index (BMI) changes and the prevalence and severity of wheezing and asthma in the first year of life.

BACKGROUND: Rapid weight gain has been recently associated with asthma at school age, but its influence in respiratory symptoms during infancy is still unknown. METHODS: Answers from 6541 parents living in six different cities of Brazil to the International Study of Wheezing in Infants (EISL) questionnaire were analysed. Data from reported weight and height at birth and at one year were used to calculate BMI. Rapid body mass index (BMI) gain was defined by the difference in BMI superior to 1.0z and excessive by the difference superior to 2.0z. RESULTS: Rapid BMI gain was found in 45.8% infants and excessive in 24.4%. Boys showed a significantly higher BMI gain than girls. Girls with rapid BMI gain showed a significantly higher prevalence of hospitalisation for wheezing (8.8% vs. 6.4%; aOR: 1.4, 95%CI: 1.1-1.8), severe wheezing (18.1% vs. 15.0%; aOR: 1.3, 95%CI: 1.0-1.5) and medical diagnosis of asthma (7.5% vs. 5.7%; aOR: 1.3, 95%CI: 1.0-1.7). Girls with excessive BMI gain also had a significantly higher prevalence of hospitalisation for wheezing (9.8% vs. 6.7%; aOR: 1.5, 95%CI: 1.1-2.0) and severe wheezing (18.9% vs. 15.5%; aOR: 1.3, 95%CI: 1.0-1.6). No significant association was found among boys. CONCLUSIONS: The majority of the evaluated infants showed BMI gain above expected in the first year of life. Although more commonly found in boys, rapid and excessive BMI gain in the first year of life was significantly related to more severe patterns of wheezing in infancy among girls.

Prolyl isomerase Pin1 and protein kinase HIPK2 cooperate to promote cortical neurogenesis by suppressing Groucho/TLE:Hes1-mediated inhibition of neuronal differentiation.

The Groucho/transducin-like Enhancer of split 1 (Gro/TLE1):Hes1 transcriptional repression complex acts in cerebral cortical neural progenitor cells to inhibit neuronal differentiation. The molecular mechanisms that regulate the anti-neurogenic function of the Gro/TLE1:Hes1 complex during cortical neurogenesis remain to be defined. Here we show that prolyl isomerase Pin1 (peptidyl-prolyl cis-trans isomerase NIMA-interacting 1) and homeodomain-interacting protein kinase 2 (HIPK2) are expressed in cortical neural progenitor cells and form a complex that interacts with the Gro/TLE1:Hes1 complex. This association depends on the enzymatic activities of both HIPK2 and Pin1, as well as on the association of Gro/TLE1 with Hes1, but is independent of the previously described Hes1-activated phosphorylation of Gro/TLE1. Interaction with the Pin1:HIPK2 complex results in Gro/TLE1 hyperphosphorylation and weakens both the transcriptional repression activity and the anti-neurogenic function of the Gro/TLE1:Hes1 complex. These results provide evidence that HIPK2 and Pin1 work together to promote cortical neurogenesis, at least in part, by suppressing Gro/TLE1:Hes1-mediated inhibition of neuronal differentiation.

The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx).

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

Active application of liquid etching agent improves adhesion of fibre posts to intraradicular dentine.

AIM: To assess the effectiveness of an active application of liquid etching, compared with the standard gel formulation on smear layer removal from post space walls and push-out bond strength of luted fibre posts. METHODOLOGY: Human extracted teeth were collected and root filled. After post space preparation and cleaning with 10% ethylenediaminetetraacetic acid for 30 s, teeth were assigned to four groups (n = 11) according to etching procedure: (i) 37% phosphoric acid (H3 PO4 ) gel; (ii) 37% H3 PO4 liquid applied with an endodontic needle; (iii) 37% H3 PO4 liquid applied with an Endovac; (iv) no etching procedure (control group). Three teeth per group were sectioned longitudinally and prepared for SEM examination to evaluate the presence of smear layer, debris, sealer/gutta-percha remnants, and the number of open tubules. Eight teeth per group were bonded with an etch-and-rinse adhesive, and fibre posts were luted with a resin-based cement. After cutting, specimens were prepared for a push-out test. Data were analysed by anova and post hoc tests (P < 0.05). RESULTS: Improved smear layer removal was obtained in Group 2, followed by Group 1, Group 3, and the control group (P < 0.05). The mean values for the bond strength of the push-out test were: Group 1, 8.3 ± 2.9 MPa (coronal); 7.7 ± 3.0 (middle); 3.3 ±1.9 MPa (apical); Group 2, 7.8 ± 2.1 MPa (coronal); 6.9 ± 3.9 MPa (middle); 3.7 ± 1.3 MPa (apical); Group 3, 9.7 ± 2.8 MPa (coronal); 8.6 ± 2.1 MPa (middle); 6.9 ± 2.3 MPa (apical); and Group 4, 2.9 ± 3.0 MPa (coronal); 2.6 ± 2.0 MPa (middle); 1.1 ± 2.0 MPa (apical). CONCLUSIONS: Liquid phosphoric acid applied with an endodontic needle yielded better canal wall smear layer removal and higher bond strength values when an etch-and-rinse system was used.

Onset temperature of Bose-Einstein condensation in incommensurate solid (4)He.

The temperature dependence of the one-body density matrix in (4)He crystals presenting vacancies is computed with path integral Monte Carlo simulations. The main purpose of this study is to estimate the onset temperature T(0) of Bose-Einstein condensation in these systems. We see that T(0) depends on the vacancy concentration X(v) of the simulated system, but not following the law T(0) ~ X(v)(2/3) obtained assuming noninteracting vacancies. For the lowest X(v) we study, that is X(v)= 1/256, we get T(0) = 0.15 ± 0.05 K, close to the temperatures at which a finite fraction of nonclassical rotational inertia is experimentally observed. Below T(0), vacancies do not act as classical point defects becoming completely delocalized entities.

Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21(Cip1) expression and hampers tumour cell growth in vitro and in vivo.

Rhabdomyosarcoma (RMS) is a paediatric soft-tissue sarcoma arising from skeletal muscle precursors coexpressing markers of proliferation and differentiation. Inducers of myogenic differentiation suppress RMS tumourigenic phenotype. The Notch target gene HES1 is upregulated in RMS and prevents tumour cell differentiation in a Notch-dependent manner. However, Notch receptors regulating this phenomenon are unknown. In agreement with data in RMS primary tumours, we show here that the Notch3 receptor is overexpressed in RMS cell lines versus normal myoblasts. Notch3-targeted downregulation in RMS cells induces hyper-phosphorylation of p38 and Akt essential for myogenesis, resulting in the differentiation of tumour cells into multinucleated myotubes expressing Myosin Heavy Chain. These phenomena are associated to a marked decrease in HES1 expression, an increase in p21(Cip1) level and the accumulation of RMS cells in the G1 phase. HES1-forced overexpression in RMS cells reverses, at least in part, the pro-differentiative effects of Notch3 downregulation. Notch3 depletion also reduces the tumourigenic potential of RMS cells both in vitro and in vivo. These results indicate that downregulation of Notch3 is sufficient to force RMS cells into completing a correct full myogenic program providing evidence that it contributes, partially through HES1 sustained expression, to their malignant phenotype. Moreover, they suggest Notch3 as a novel potential target in human RMS.

Emulsion polymerization of vinyl acetate: safe optimization of a hazardous complex process.

Fast and exothermic discontinuous emulsion polymerization processes are particularly difficult to optimize from both safety and productivity point of view because of the occurrence of side undesired reactions (e.g. chain transfer to monomer, backbiting, propagation of tertiary radicals, termination by disproportion, etc.) and the hazards of boiling phenomena and stable foam formation under atmospheric pressure. Moreover, the relevant number of loading, heating and cooling steps, required before starting the monomer addition (that is, the desired reaction), makes a strict product quality reproducibility very difficult to obtain. Under these operating conditions, it is necessary to employ a suitable combined theoretical and experimental procedure able to detect the optimum process dosing time at both the laboratory and the industrial scale. In this work, it is shown how to use the topological criterion theory together with proper adiabatic calorimeter and RC1 experimental data to safely optimize the synthesis of polyvinyl acetate through the radical emulsion polymerization of vinyl acetate by the means of an indirectly cooled isoperibolic semibatch reactor.

Expression and purification of Z protein from Junín virus.

Arenaviridae comprises 23 recognized virus species with a bipartite ssRNA genome and an ambisense coding strategy. The virions are enveloped and include nonequimolar amounts of each genomic RNA species, designated L and S, coding for four ORFs (N, GPC, L, and Z). The arenavirus Junín (JUNV) is the etiological agent of Argentine Hemorrhagic Fever, an acute disease with high mortality rate. It has been proposed that Z is the functional counterpart of the matrix proteins found in other negative-stranded enveloped RNA viruses. Here we report the optimized expression of a synthetic gene of Z protein, using three expression systems (two bacterial and a baculoviral one). One of these recombinant proteins was used to generate antibodies. A bioinformatic analysis was made where Z was subdivided into three domains. The data presented contributes methodologies for Z recombinant production and provides the basis for the development of new experiments to test its function.

High-order time expansion path integral ground state.

The feasibility of path integral Monte Carlo ground state calculations with very few beads using a high-order short-time Green's function expansion is discussed. An explicit expression of the evolution operator which provides dramatic enhancements in the quality of ground-state wave functions is examined. The efficiency of the method makes possible to remove the trial wave function and thus obtain completely model-independent results still with a very small number of beads. If a single iteration of the method is used to improve a given model wave function, the result is invariably a shadow-type wave function, whose precise content is provided by the high-order algorithm employed.

Hazardous gas releases in urban areas: assessment of consequences through CFD modelling.

Release of hazardous materials in urban areas is a major concern in industrial risk assessment. The presence of high population density in such areas multiplies the magnitude of the consequences. In urban areas, many buildings with complex geometries are involved leading to 3D flow fields that strongly influence gas dispersion. Representing such complex geometries simply but realistically in detailed simulation models can be cumbersome and often limit their utility. In this work, a methodology for the construction of 3D urban models and their importation into CFD models was developed through the access to spatial geodatabases, leading to a relatively fast and simple domain design technique. Moreover, since the magnitude of consequences depends on the absorbed dose which in turn depends on both concentration and exposure time, a simple methodology for dose evaluation was developed and implemented in a CFD code that enables the estimation of regions with a given death probability. The approach was developed and applied to a case study with different atmospheric stratification conditions. The results were then compared with those obtained using integral models. It was found that integral models can both overestimate and underestimate the magnitude of consequences related to hazardous material releases in urban areas.

Usefulness of reagent strips for checking cure in spontaneous bacterial peritonitis after short-course treatment.

BACKGROUND: The usefulness of reagent strips to check cure of spontaneous bacterial peritonitis have not been evaluated to date. AIM: To assess the usefulness of ascitic fluid analysis by means of reagent strips to check cure after a 5-day antibiotic course. METHODS: We prospectively included all cirrhotic patients diagnosed with spontaneous bacterial peritonitis. On day 5, conventional and reagent strip ascitic fluid analyses were performed. RESULTS: Fifty-three episodes of spontaneous bacterial peritonitis in 51 cirrhotic patients were included. Five patients died before the fifth day and in two patients, the control paracentesis yielded no ascitic fluid. In nine out of 46 cases (19.6%), spontaneous bacterial peritonitis had not resolved by day 5. In 32 out of 33 cases in which the ascitic fluid polymorphonuclear count was <250/microL at day five, the reagent strips was negative. The negative predictive value of the reagent strip at fifth day was 97% and the LR- 0.13. CONCLUSIONS: Almost 20% of episodes of spontaneous bacterial peritonitis do not resolve with a short-course of antibiotic treatment. In view of the high negative predictive value and low likelihood ratio for a negative test, reagent strips analysis may be an alternative to conventional cytology if a 5-day antibiotic therapy is planned.

Hazardous gas dispersion: a CFD model accounting for atmospheric stability classes.

Nowadays, thanks to the increasing CPU power the use of Computational Fluid Dynamics (CFD) is rapidly imposing also in the industrial risk assessment area, replacing integral models when particular situations, such as those involving complex terrains or large obstacles, are involved. Nevertheless, commercial CFD codes usually do not provide specific turbulence model for simulating atmospheric stratification effects, which are accounted of by the integral models through the well-known stability-class approach. In this work, a new approach able to take account of atmospheric features in CFD simulations has been developed and validated by comparison with available experimental data.

Targeting Id protein interactions by an engineered HLH domain induces human neuroblastoma cell differentiation.

Inhibitor of DNA-binding (Id) proteins prevent cell differentiation, promote growth and sustain tumour development. They do so by binding to E proteins and other transcription factors through the helix-loop-helix (HLH) domain, and inhibiting transcription. This makes HLH-mediated Id protein interactions an appealing therapeutic target. We have used the dominant interfering HLH dimerization mutant 13I to model the impact of Id inhibition in two human neuroblastoma cell lines: LA-N-5, similar to immature neuroblasts, and SH-EP, resembling more immature precursor cells. We have validated 13I as an Id inhibitor by showing that it selectively binds to Ids, impairs complex formation with RB, and relieves repression of E protein-activated transcription. Id inactivation by 13I enhances LA-N-5 neural features and causes SH-EP cells to acquire neuronal morphology, express neuronal proteins such as N-CAM and NF-160, proliferate more slowly, and become responsive to retinoic acid. Concomitantly, 13I augments the cell-cycle inhibitor p27(Kip1) and reduces the angiogenic factor vascular endothelial growth factor. These effects are Id specific, being counteracted by Id overexpression. Furthermore, 13I strongly impairs tumorigenic properties in agar colony formation and cell invasion assays. Targeting Id dimerization may therefore be effective for triggering differentiation and restraining neuroblastoma cell tumorigenicity.

Treatment of chronic he1patitis C genotype 1 with peginterferon-alpha2a (40 kDa) plus ribavirin under routine clinical practice in Spain: early prediction of sustained virological response rate.

BACKGROUND: Sustained virological response rates of up to 52% have been obtained with peginterferon alpha2a (40 kDa) plus ribavirin in patients suffering from chronic hepatitis C genotype 1 in randomized-controlled trials. AIM: To assess early virological response and its clinical utility in predicting an sustained virological response in patients suffering from chronic hepatitis C genotype 1 in routine clinical practice in Spain. METHODS: Treatment-naïve patients received pegylated interferon alpha2a (40 kDa) 180 microg/week plus ribavirin 1000/1200 mg/day for 48 weeks, and were followed for a further 24 weeks. Overall, 475 patients received at least one dose of medication and were included in the efficacy population. RESULTS: The overall sustained virological response rate was 48%. Of those with week 12 virological data, 83% had an early virological response. The negative predictive value of an early virological response was 93%. CONCLUSION: If sustained virological response is the goal, a treatment-decision based on a 12-week evaluation during routine clinical practice is feasible.

Molecular epidemiology of group A rotavirus diarrhea among children in Buenos Aires, Argentina, from 1999 to 2003 and emergence of the infrequent genotype G12.

To examine the epidemiology of rotaviruses in Buenos Aires, Argentina, we screened 1,212 stool samples from children with diarrhea in the southern district of Buenos Aires from 1999 to 2003. We identified 187 samples (15.4%) that were positive for group A rotavirus by use of antigen enzyme-linked immunosorbent assay. Among these specimens, 112 were available for typing: 93 (83.0%) were single-type infections, 9 (8.0%) were mixed-type infections with more than one G or P type, and 10 (8.9%) were G and/or P nontypeable. In contrast to the findings in our last study, from 1996 to 1998, genotype P[4], G2 strains were almost completely absent and P[8], G1 and P[8], G4 strains were dominant, representing more than 80% of the G and P types found. Genotypes G2 and G9 were detected in few samples, and type G3 was completely absent. We identified several uncommon genotype G12 strains, representing the first detections outside of Asia and the United States, by sequencing. Using a genotype G12-specific reverse transcription-PCR, we identified eight (6.7%) positive samples for the 1999 to 2003 period. The high degree of sequence identity between recent G12 isolates from Argentina, the United States, and Asian countries suggests a relatively recent introduction(s) of these strains into humans from a common progenitor. The Argentinean G12 strains belonged to genotype P[9], similar to most of the recently described Asian G12 strains. The finding of G12 strains in several other regions of the world raises the possibility that G12 may be emerging globally and suggests that surveillance for this strain should be conducted routinely.

Detection of group a human rotavirus G9 genotype circulating in Córdoba, Argentina, as early as 1980.

The incidence of human rotavirus G types was determined over a 25-year period (1979-2003) by using reverse transcription-PCR (RT-PCR) to examine 519 stool specimens found to be positive for rotavirus by enzyme linked immunosorbent assay (ELISA) or polyacrylamide gel electrophoresis (PAGE). These stool samples were obtained from children under 3 years old who had been treated for acute diarrhea at public hospitals in Córdoba, Argentina. The present study describes the continued circulation of the common human G types G1 (53.8%), G2 (10.2%), G3 (4.4%), and G4 (27%), and also the detection of the unusual types G8 (0.5%) and G9 (4.2%). Genotype G9 was detected during the 1980-1988 and 1997-2003 periods at relatively low rates. Rotavirus G types distribution was independent of age (1-18 months), gender or out-patient or in-patient status. Unexpectedly, 44.6% of mixed infections were detected, involving common and unusual genotypes. Overall, 95.4% of the typed strains belonged to the most prevalent human serotypes (G1-G4) but the detection of G9 infection throughout this study period highlights the importance of this serotype as a human pathogen.

Hazard assessment of substances produced from the accidental heating of chemical compounds.

Accidental events concerning process industries can affect not only the staff working in, but also the environment and people living next to the factory. For this reason a regulation is imposed by the European Community to prevent accidents that could represent a risk for the population and the environment. In particular, Directive 96/82/CE, the so-called 'Seveso II directive', requests a risk analysis involving also the hazardous materials generated in accidental events. Therefore, it is necessary to develop simple and economic procedure to foresee the hazardous materials that can be produced in the case of major accidents, among which the accidental heating of a chemical due to a fire or a runaway reaction is one of the most frequent. The procedure proposed in this work is based on evolved gas analysis methodology that consists in coupling two instruments: a thermogravimetric analyzer or a flash pyrolyzer, that are employed to simulate accident conditions, and a FTIR spectrometer that can be used to detect the evolved gas composition. More than 40 materials have been examined in various accident scenarios and the obtained data have been statistically analyzed in order to identify meaningful correlations between the presence of a chemical group in the molecule of a chemical and the presence of a given hazardous species in the fume produced.