p53-induced apoptosis occurs in the absence of p14(ARF) in malignant pleural mesothelioma

Malignant pleural mesotheliomas (MPMs) are usually wild type for the p53 gene but contain homozygous deletions in the INK4A locus that encodes p14(ARF), an inhibitor of p53-MDM2 interaction. Previous findings suggest that lack of p14(ARF) expression and the presence of SV40 large T antigen (L-Tag) result in p53 inactivation in MPM. We did not detect SV40 L-Tag mRNA in either MPM cell lines or primary cultures, and treatment of p14(ARF)-deficient cells with cisplatin (CDDP) increased both total and phosphorylated p53 and enhanced p53 DNA-binding activity. On incubation with CDDP, levels of positively regulated p53 transcriptional targets p21(WAF), PIG3, MDM2, Bax, and PUMA increased in p14(ARF)-deficient cells, whereas negatively regulated survivin decreased. Significantly, p53-induced apoptosis was activated by CDDP in p14(ARF)-deficient cells, and treatment with p53-specific siRNA rendered them more CDDP-resistant. p53 was also activated by: 1) inhibition of MDM2 (using nutlin-3); 2) transient overexpression of p14(ARF); and 3) targeting of survivin using antisense oligonucleotides. However, it is noteworthy that only survivin downregulation sensitized cells to CDDP-induced apoptosis. These results suggest that p53 is functional in the absence of p14(ARF) in MPM and that targeting of the downstream apoptosis inhibitor survivin can sensitize to CDDP-induced apoptosis.

The role of muscle flap in preventing bronchus stump insufficiency after pneumonectomy for malignant pleural mesothelioma in high-risk patients

Bronchus stump insufficiency (BSI) is one of the major complications after pneumonectomy; we analyzed all patients who underwent extra pleural pneumonectomy (EPP) for malignant pleural mesothelioma (MPM) in order to detect the role of muscle flap (MF) on preventing early and late stump insufficiency. From January 2000 until December 2005, there were 42 patients admitted with MPM for further intervention at our institution. Thirty patients were suitable for surgery and thus received a multimodal treatment with neo-adjuvant chemotherapy using Cisplatin and Gemcitabin (Gemzar), EPP followed by 54 Gray (Gy) adjuvant radiotherapy. Data were collected from the surgical and oncological records. There were 37 male patients (88%), the median age was 65 years (range 40-83 years). Seven (17%) patients had concomitant diseases. Forty patients (95%) had asbestos exposition. The operative procedures were EPP with muscle flap through an anterolateral thoracotomy. Univariate and multivariate analyses were done. One patient (3%) died on the 2nd postoperative day due to lung embolism. Mild complications were noticed in the early postoperative phase in 8 (25%) patients. There was no early or late stump insufficiency during the 15-month follow-up. Surgical techniques using muscle flap seems to play a major role in the prevention of bronchus stump insufficiency especially after neo-adjuvant chemotherapy.

A microfluidic platform for chemoresistive testing of multicellular pleural cancer spheroids

This study reports on a microfluidic platform on which single multicellular spheroids from malignant pleural mesothelioma (MPM), an aggressive tumor with poor prognosis, can be loaded, trapped and tested for chemotherapeutic drug response. A new method to detect the spheroid viability cultured on the microfluidic chip as a function of the drug concentration is presented. This approach is based on the evaluation of the caspase activity in the supernatant sampled from the chip and tested using a microplate reader. This simple and time-saving method does only require a minimum amount of manipulations and was established for very low numbers of cells. This feature is particularly important in view of personalised medicine applications for which the number of cells obtained from the patients is low. MPM spheroids were continuously perfused for 48 hours with cisplatin, one of the standard chemotherapeutic drugs used to treat MPM. The 50% growth inhibitory concentration of cisplatin in perfused MPM spheroids was found to be twice as high as in spheroids cultured under static conditions. This chemoresistance increase might be due to the continuous support of nutrients and oxygen to the perfused spheroids.

Molecular basis of maturation promoting factor

Maturation promoting factor (MPF), which is functionally defined by its ability to induce Xenopus oocyte maturation, is an M phase (meiosis and mitosis) specific activity that is present in all species tested. It was hypothesized that MPF is a universal trigger of the interphase to M phase transition during the cell cycle. The current model for the molecular basis of MPF is that MPF is a protein kinase having the cdc2 protein as its catalytic subunit and is identical to the M phase-specific histone H1 kinase. In the present study, I have shown that more than just cdc2 kinase contributes to MPF activity, and M phase-specific H1 kinase is composed of at least two entities, instead of just cdc2 kinase. Therefore, the simple model of MPF = cdc2 kinase = M phase-specific H1 kinase should be ruled out.^ My study began with the characterization of the mitosis-specific monoclonal antibody MPM-2. MPM-2 reacts specifically with M phase cells from different species by recognizing a discrete set of proteins once they are phosphorylated at the G$\sb2$/M transition. I found that phosphorylation of MPM-2 antigens coincided with the appearance of MPF activity during oocyte maturation stimulated by progesterone. If MPM-2 was injected into oocytes before the stimulation, MPF activity failed to appear, and the oocytes could not mature. Furthermore, MPM-2 was able to deplete MPF activity from M phase extracts. These results identified MPM-2 as a probe that recognizes either MPF itself or a regulator of MPF.^ Since M phase-specific H1 kinase was believed to be identical to cdc2 kinase and MPF, I proceeded to determine whether MPM-2 recognized the M phase-specific H1 kinase. I found that MPM-2 did recognize an M phase-specific H1 kinase. However, this kinase was not cdc2 kinase. This kinase (MPM-2 kinase) is present in a latent form in immature oocytes and is activated in tandem with the activation of MPF during oocyte maturation. It appears to accelerate progesterone-induced oocyte maturation. Therefore, MPM-2 kinase may be a novel positive regulator of MPF activation.^ MPM-2 depletes MPF activity, but not cdc2 kinase activity. This discrepancy caused me to question the equivalency of MPF with cdc2 kinase. I found that when a high percentage of MPF activity was recovered from gel filtration of mature oocyte extract, the recovered MPF activity was due to two factors, cdc2 kinase and a factor recognized by MPM-2. This factor might activate and stabilize cdc2 kinase. Identification of this factor in the present study may contribute to the understanding of the autoactivation of MPF. ^

Cisplatin-resistant cells in malignant pleural mesothelioma cell lines show ALDH(high)CD44(+) phenotype and sphere-forming capacity

BACKGROUND Conventional chemotherapy in malignant pleural mesothelioma (MPM) has minimal impact on patient survival due to the supposed chemoresistance of cancer stem cells (CSCs). We sought to identify a sub-population of chemoresistant cells by using putative CSC markers, aldehyde dehydrogenase (ALDH) and CD44 in three MPM cell lines; H28, H2052 and Meso4. METHODS The Aldefluor assay was used to measure ALDH activity and sort ALDH(high) and ALDH(low) cells. Drug-resistance was evaluated by cell viability, anchorage-independent sphere formation, flow-cytometry and qRT-PCR analyses. RESULTS The ALDH(high) - and ALDH(low) -sorted fractions were able to demonstrate phenotypic heterogeneity and generate spheres, the latter being less efficient, and both showed an association with CD44. Cis- diamminedichloroplatinum (II) (cisplatin) treatment failed to reduce ALDH activity and conferred only a short-term inhibition of sphere generation in both ALDH(high) and ALDH(low) fractions of the three MPM cell lines. Induction of drug sensitivity by an ALDH inhibitor, diethylaminobenzaldehyde (DEAB) resulted in significant reductions in cell viability but not a complete elimination of the sphere-forming cells, suggestive of the presence of a drug-resistant subpopulation. At the transcript level, the cisplatin + DEAB-resistant cells showed upregulated mRNA expression levels for ALDH1A2, ALDH1A3 isozymes and CD44 indicating the involvement of these markers in conferring chemoresistance in both ALDH(high) and ALDH(low) fractions of the three MPM cell lines. CONCLUSIONS Our study shows that ALDH(high) CD44(+) cells are implicated in conveying tolerance to cisplatin in the three MPM cell lines. The combined use of CD44 and ALDH widens the window for identification and targeting of a drug-resistant population which may improve the current treatment modalities in mesothelioma.

Análisis de la mortalidad en la unidad de cuidados intensivos del Hospital Central de Mendoza, Argentina

Objetivo: Analizar la mortalidad en la Unidad de Cuidados Intensivos (UCI) del Hospital Central de Mendoza y evaluar el valor predictivo de la escala APACHE II (Evaluación Fisiológica Aguda y de Salud Crónica). Material y Método: Se realizó un estudio retrospectivo y observacional de los pacientes ingresados a la Unidad de Cuidados Intensivos del Hospital Central de Mendoza, desde el 01/11/06 hasta el 31/03/08. Se calculó la distribución de sexos y de edades de la muestra, la estadía promedio, principales motivos de ingreso a la UCI y la puntuación APACHE II en las primeras 24 horas de internación. Se calculó la mortalidad esperada y la mortalidad obtenida global y se analizó el coeficiente entre ambas mortalidades. Resultados: Se incluyeron 904 pacientes, 61,82% masculinos y 38,18% femeninos, con una edad media 46 años (±19,36). Estadía promedio en la UCI 8,5 días promedio. El principal motivo de internación fueron los Traumatismos Encéfalocraneanos (TEC) con un 27,7% del total (86% asociados a politraumatismo grave). La mortalidad global obtenida fue del 41,48% vs. 24,08% esperable, con un coeficiente de mortalidad de 1,72 (p<0,0001). Conclusiones: La UCI estudiada presenta por las características de la población asistida un elevado índice de mortalidad global. La mortalidad obtenida fue 72% mayor a la mortalidad esperable según la puntuación APACHE II, demostrando esta Escala un bajo valor predictivo en nuestra UCI. La diferencia entre mortalidades podría parcialmente explicarse por la alta prevalencia de entidades con mortalidades subvaloradas por este modelo pronóstico, como pacientes politraumatizados y neurocríticos. En nuestro estudio, la Escala APACHE II presentó una franca subestimación de la mortalidad en ambas patologías. Sugerimos la realización de un estudio de regresión logística local para determinar un factor de corrección y/o adicionar puntos al valor APACHE II según el diagnóstico de ingreso del paciente. Asimismo, proponemos evaluar el empleo de medidas alternativas para predecir mortalidad, como sistemas de tercera generación (por ejemplo: APACHE III, MPM II y SAPS II).

Biparental Inheritance of γ-Tubulin during Human Fertilization: Molecular Reconstitution of Functional Zygotic Centrosomes in Inseminated Human Oocytes and in Cell-free Extracts Nucleated by Human Sperm

Human sperm centrosome reconstitution and the parental contributions to the zygotic centrosome are examined in mammalian zygotes and after exposure of spermatozoa to Xenopus laevis cell-free extracts. The presence and inheritance of the conserved centrosomal constituents γ-tubulin, centrin, and MPM-2 (which detects phosphorylated epitopes) are traced, as is the sperm microtubule-nucleating capability on reconstituted centrosomes. γ-Tubulin is biparentally inherited in humans (maternal >> than paternal): Western blots detect the presence of paternal γ-tubulin. Recruitment of maternal γ-tubulin to the sperm centrosome occurs after sperm incorporation in vivo or exposure to cell-free extract, especially after sperm “priming” induced by disulfide bond reduction. Centrin is found in the proximal sperm centrosomal region, demonstrates expected calcium sensitivity, but appears absent from the zygotic centrosome after sperm incorporation or exposure to extracts. Sperm centrosome phosphorylation is detected after exposure of primed sperm to egg extracts as well as during the early stages of sperm incorporation after fertilization. Finally, centrosome reconstitution in cell-free extracts permits sperm aster microtubule assembly in vitro. Collectively, these results support a model of a blended zygotic centrosome composed of maternal constituents attracted to an introduced paternal template after insemination.

What Counts Versus What Can Be Counted : The Complex Interplay of Market Orientation and Marketing Performance Measurement

Market orientation (MO) and marketing performance measurement (MPM) are two of the most widespread strategic marketing concepts among practitioners. However, some have questioned the benefits of extensive investments in MO and MPM. More importantly, little is known about which combinations of MO and MPM are optimal in ensuring high business performance. To address this research gap, the authors analyze a unique data set of 628 firms with a novel method of configurational analysis: fuzzy-set qualitative comparative analysis. In line with prior research, the authors find that MO is an important determinant of business performance. However, to reap its benefits, managers need to complement it with appropriate MPM, the level and focus of which vary across firms. For example, whereas large firms and market leaders generally benefit from comprehensive MPM, small firms may benefit from measuring marketing performance only selectively or by focusing on particular dimensions of marketing performance. The study also finds that many of the highest-performing firms do not follow any of the particular best practices identified.

Weight vs magnetization enumerator for Gallager codes

We propose a method to determine the critical noise level for decoding Gallager type low density parity check error correcting codes. The method is based on the magnetization enumerator (¸M), rather than on the weight enumerator (¸W) presented recently in the information theory literature. The interpretation of our method is appealingly simple, and the relation between the different decoding schemes such as typical pairs decoding, MAP, and finite temperature decoding (MPM) becomes clear. Our results are more optimistic than those derived via the methods of information theory and are in excellent agreement with recent results from another statistical physics approach.

Critical noise levels for LDPC decoding

We determine the critical noise level for decoding low density parity check error correcting codes based on the magnetization enumerator , rather than on the weight enumerator employed in the information theory literature. The interpretation of our method is appealingly simple, and the relation between the different decoding schemes such as typical pairs decoding, MAP, and finite temperature decoding (MPM) becomes clear. In addition, our analysis provides an explanation for the difference in performance between MN and Gallager codes. Our results are more optimistic than those derived via the methods of information theory and are in excellent agreement with recent results from another statistical physics approach.

Critical noise levels for low-density parity check decoding

We determine the critical noise level for decoding low-density parity check error-correcting codes based on the magnetization enumerator (M), rather than on the weight enumerator (W) employed in the information theory literature. The interpretation of our method is appealingly simple, and the relation between the different decoding schemes such as typical pairs decoding, MAP, and finite temperature decoding (MPM) becomes clear. In addition, our analysis provides an explanation for the difference in performance between MN and Gallager codes. Our results are more optimistic than those derived using the methods of information theory and are in excellent agreement with recent results from another statistical physics approach.

What counts versus what can be counted:the complex interplay of market orientation and marketing performance measurement

Market orientation (MO) and marketing performance measurement (MPM) are two of the most widespread strategic marketing concepts among practitioners. However, some have questioned the benefits of extensive investments in MO and MPM. More importantly, little is known about which combinations of MO and MPM are optimal in ensuring high business performance. To address this research gap, the authors analyze a unique data set of 628 firms with a novel method of configurational analysis: fuzzy-set qualitative comparative analysis. In line with prior research, the authors find that MO is an important determinant of business performance. However, to reap its benefits, managers need to complement it with appropriate MPM, the level and focus of which vary across firms. For example, whereas large firms and market leaders generally benefit from comprehensive MPM, small firms may benefit from measuring marketing performance only selectively or by focusing on particular dimensions of marketing performance. The study also finds that many of the highest-performing firms do not follow any of the particular best practices identified.

Herpetofauna associada à bromélia rupícola, encholirium spectabile, no semiárido brasileiro: revisão da literatura, ecologia das espécies e comportamento de psychosaura agmosticha (squamata: mabuyidae)

Bromeliads are an important microhabitat for the herpetofauna, for being widely used as refuge from predators and their leaf architecture allows humidity maintenance and relatively constant temperature inside, setting a favorable environment for amphibians and reptiles, especially in areas under hydric stress. However, studies addressing this relationship are still incipient and more concentrated in fitotelmatas bromeliad. For non-fitotelmatas rupicolous bromeliads of the gender Encholirium, which develops into rocky outcrops and contains species of semi-arid regions such as the Caatinga, animal-plant relationships are almost unknown. In this context, this study aimed to know the herpetological fauna inhabitant of macambiras bromeliads, Encholirium spectabile, analyzing occupation and use of these bromeliads by different taxa, and the behavioral ecology of the lizard Psychosaura agmosticha, seeking to identify factors associated with this strict relationship in Caatinga. An extensive review of the world literature on the subject “lizards in bromeliads” subsidized this study from the ecological perspective of this association. The field work was carried out at Fazenda Tanques, municipality of Santa Maria / RN, mesoregion of Agreste Potiguar. The observations and/or data collection in daytime and in the evening was conducted monthly during three consecutive days, from January 2011 to August 2012, totaling 450 hour.man of sampling effort. Sixteen species were registered: six lizards (Mabuyidae, Tropiduridae, Gekkonidae and Phyllodactylidae Families), six snakes (Boidae and Dipsadidae Families) and four of amphibians of Hylidae Family. The effect of the forest edge on the distribution of species along the outcrop was significant, with most species found in outcrop edges. Significant difference was found between some pairs of species concerning use of bromeliads, and almost total niche overlap in the use of microhabitat. 62.5% of the species are nocturnal and use these plants for sheltering, breeding and feeding. Regarding the relations between 4 the lizard Psychosaura agmosticha and macambiras bromeliads, behaviors of thermoregulation and foraging in the dry and wet seasons were recorded. Activity periods were concentrated between 7 and 10 am and between 3 and 5 pm in both seasons, showing a clear bimodal pattern. The species basically used the green leaves and there were no significant differences between males and females in the use of bromeliads. Positive associations were found between body temperature and temperatures of bromeliads and air. This species spent 1.95% ± 3.8 of the time moving (PTM) and moved on average 0:36 ± 2.1 seconds per minute (MPM), with significant differences between the wet and dry to PTM, and between the average time of stop and average duration of movements, being considered a sedentary forager. Psychosaura agmosticha, in the study area, is bromelicolous and uses macambiras primarily for thermoregulation and foraging. The results of this study elevate the rupicolous bromeliads Encholirium spectabile as key elements for the maintenance of amphibians and reptiles associated with it, and a clear advantageous association for the conservation of the groups involved.

Conception et synthèse d'inhibiteurs de métalloprotéases

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Large volume metrology process model:measurability analysis with integration of metrology classification model and feature-based selection model

Aircraft manufacturing industries are looking for solutions in order to increase their productivity. One of the solutions is to apply the metrology systems during the production and assembly processes. Metrology Process Model (MPM) (Maropoulos et al, 2007) has been introduced which emphasises metrology applications with assembly planning, manufacturing processes and product designing. Measurability analysis is part of the MPM and the aim of this analysis is to check the feasibility for measuring the designed large scale components. Measurability Analysis has been integrated in order to provide an efficient matching system. Metrology database is structured by developing the Metrology Classification Model. Furthermore, the feature-based selection model is also explained. By combining two classification models, a novel approach and selection processes for integrated measurability analysis system (MAS) are introduced and such integrated MAS could provide much more meaningful matching results for the operators. © Springer-Verlag Berlin Heidelberg 2010.