Establishment and characterization of models of chemotherapy resistance in colorectal cancer: Towards a predictive signature of chemoresistance.

Current standard treatments for metastatic colorectal cancer (CRC) are based on combination regimens with one of the two chemotherapeutic drugs, irinotecan or oxaliplatin. However, drug resistance frequently limits the clinical efficacy of these therapies. In order to gain new insights into mechanisms associated with chemoresistance, and departing from three distinct CRC cell models, we generated a panel of human colorectal cancer cell lines with acquired resistance to either oxaliplatin or irinotecan. We characterized the resistant cell line variants with regards to their drug resistance profile and transcriptome, and matched our results with datasets generated from relevant clinical material to derive putative resistance biomarkers. We found that the chemoresistant cell line variants had distinctive irinotecan- or oxaliplatin-specific resistance profiles, with non-reciprocal cross-resistance. Furthermore, we could identify several new, as well as some previously described, drug resistance-associated genes for each resistant cell line variant. Each chemoresistant cell line variant acquired a unique set of changes that may represent distinct functional subtypes of chemotherapy resistance. In addition, and given the potential implications for selection of subsequent treatment, we also performed an exploratory analysis, in relevant patient cohorts, of the predictive value of each of the specific genes identified in our cellular models.

Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models.

The aim of the present study was to identify Candida albicans transcription factors (TFs) involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens (FBs) quantified in kidneys. Mutants of unannotated genes which generated a kidney FB significantly different from that of wild-type were selected and individually examined in Galleria mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25% of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects), a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching FB phenotypes were observed in 50% of the cases, highlighting the bias due to host effects. In contrast, 33.4% concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the "pool effect." After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adapt.

Multidimensional Deprivation in Contemporary Switzerland Across Social Groups and Time

We have investigated the phenomenon of deprivation in contemporary Switzerland through the adoption of a multidimensional, dynamic approach. By applying Self Organizing Maps (SOM) to a set of 33 non-monetary indicators from the 2009 wave of the Swiss Household Panel (SHP), we identified 13 prototypical forms (or clusters) of well-being, financial vulnerability, psycho-physiological fragility and deprivation within a topological dimensional space. Then new data from the previous waves (2003 to 2008) were classified by the SOM model, making it possible to estimate the weight of the different clusters in time and reconstruct the dynamics of stability and mobility of individuals within the map. Looking at the transition probabilities between year t and year t+1, we observed that the paths of mobility which catalyze the largest number of observations are those connecting clusters that are adjacent on the topological space.

Effect of oral nitrate supplementation on pulmonary hemodynamics during exercise and time trial performance in normoxia and hypoxia: a randomized controlled trial.

BACKGROUND: Hypoxia-induced pulmonary vasoconstriction increases pulmonary arterial pressure (PAP) and may impede right heart function and exercise performance. This study examined the effects of oral nitrate supplementation on right heart function and performance during exercise in normoxia and hypoxia. We tested the hypothesis that nitrate supplementation would attenuate the increase in PAP at rest and during exercise in hypoxia, thereby improving exercise performance. METHODS: Twelve trained male cyclists [age: 31 ± 7 year (mean ± SD)] performed 15 km time-trial cycling (TT) and steady-state submaximal cycling (50, 100, and 150 W) in normoxia and hypoxia (11% inspired O2) following 3-day oral supplementation with either placebo or sodium nitrate (0.1 mmol/kg/day). We measured TT time-to-completion, muscle tissue oxygenation during TT and systolic right ventricle to right atrium pressure gradient (RV-RA gradient: index of PAP) during steady state cycling. RESULTS: During steady state exercise, hypoxia elevated RV-RA gradient (p > 0.05), while oral nitrate supplementation did not alter RV-RA gradient (p > 0.05). During 15 km TT, hypoxia lowered muscle tissue oxygenation (p < 0.05). Nitrate supplementation further decreased muscle tissue oxygenation during 15 km TT in hypoxia (p < 0.05). Hypoxia impaired time-to-completion during TT (p < 0.05), while no improvements were observed with nitrate supplementation in normoxia or hypoxia (p > 0.05). CONCLUSION: Our findings indicate that oral nitrate supplementation does not attenuate acute hypoxic pulmonary vasoconstriction nor improve performance during time trial cycling in normoxia and hypoxia.

Influence of transport and time on blood variables commonly measured for the athlete biological passport.

Some recent studies have characterized the stability of blood variables commonly measured for the Athlete Biological Passport. The aim of this study was to characterize the impact of different shipments conditions and the quality of the results returned by the haematological analyzer. Twenty-two healthy male subjects provided five EDTA tubes each. Four shipment conditions (24, 36, 48, 72 h) under refrigerated conditions were tested and compared to a set of samples left in the laboratory also under refrigerated conditions (group control). All measurements were conducted using two Sysmex XT-2000i analyzers. Haemoglobin concentration, reticulocytes percentage, and OFF-score numerical data were the same for samples analyzed just after collection and after a shipment under refrigerated conditions up to 72 h. Detailed information reported especially by the differential (DIFF) channel scatterplot of the Sysmex XT-2000i indicated that there were signs of blood deterioration, but were not of relevance for the variables used in the Athlete Biological Passport. As long as the cold chain is guaranteed, the time delay between the collection and the analyses of blood variables can be extended. Copyright© 2015 John Wiley & Sons, Ltd.

A gamma camera count rate saturation correction method for whole-body planar imaging.

Whole-body (WB) planar imaging has long been one of the staple methods of dosimetry, and its quantification has been formalized by the MIRD Committee in pamphlet no 16. One of the issues not specifically addressed in the formalism occurs when the count rates reaching the detector are sufficiently high to result in camera count saturation. Camera dead-time effects have been extensively studied, but all of the developed correction methods assume static acquisitions. However, during WB planar (sweep) imaging, a variable amount of imaged activity exists in the detector's field of view as a function of time and therefore the camera saturation is time dependent. A new time-dependent algorithm was developed to correct for dead-time effects during WB planar acquisitions that accounts for relative motion between detector heads and imaged object. Static camera dead-time parameters were acquired by imaging decaying activity in a phantom and obtaining a saturation curve. Using these parameters, an iterative algorithm akin to Newton's method was developed, which takes into account the variable count rate seen by the detector as a function of time. The algorithm was tested on simulated data as well as on a whole-body scan of high activity Samarium-153 in an ellipsoid phantom. A complete set of parameters from unsaturated phantom data necessary for count rate to activity conversion was also obtained, including build-up and attenuation coefficients, in order to convert corrected count rate values to activity. The algorithm proved successful in accounting for motion- and time-dependent saturation effects in both the simulated and measured data and converged to any desired degree of precision. The clearance half-life calculated from the ellipsoid phantom data was calculated to be 45.1 h after dead-time correction and 51.4 h with no correction; the physical decay half-life of Samarium-153 is 46.3 h. Accurate WB planar dosimetry of high activities relies on successfully compensating for camera saturation which takes into account the variable activity in the field of view, i.e. time-dependent dead-time effects. The algorithm presented here accomplishes this task.

Glucose represses connexin36 in insulin-secreting cells.

The gap-junction protein connexin36 (Cx36) contributes to control the functions of insulin-producing cells. In this study, we investigated whether the expression of Cx36 is regulated by glucose in insulin-producing cells. Glucose caused a significant reduction of Cx36 in insulin-secreting cell lines and freshly isolated pancreatic rat islets. This decrease appeared at the mRNA and the protein levels in a dose- and time-dependent manner. 2-Deoxyglucose partially reproduced the effect of glucose, whereas glucosamine, 3-O-methyl-D-glucose and leucine were ineffective. Moreover, KCl-induced depolarization of beta-cells had no effect on Cx36 expression, indicating that glucose metabolism and ATP production are not mandatory for glucose-induced Cx36 downregulation. Forskolin mimicked the repression of Cx36 by glucose. Glucose or forskolin effects on Cx36 expression were not suppressed by the L-type Ca(2+)-channel blocker nifedipine but were fully blunted by the cAMP-dependent protein kinase (PKA) inhibitor H89. A 4 kb fragment of the human Cx36 promoter was identified and sequenced. Reporter-gene activity driven by various Cx36 promoter fragments indicated that Cx36 repression requires the presence of a highly conserved cAMP responsive element (CRE). Electrophoretic-mobility-shift assays revealed that, in the presence of a high glucose concentration, the binding activity of the repressor CRE-modulator 1 (CREM-1) is enhanced. Taken together, these data provide evidence that glucose represses the expression of Cx36 through the cAMP-PKA pathway, which activates a member of the CRE binding protein family.

Molecular basis of selective PPARgamma modulation for the treatment of Type 2 diabetes.

Peroxisome proliferator-activated receptors (PPARs) (alpha, beta/delta and gamma) are lipid sensors capable of adapting gene expression to integrate various lipid signals. As such, PPARs are also very important pharmaceutical targets, and specific synthetic ligands exist for the different isotypes and are either currently used or hold promises in the treatment of major metabolic disorders. In particular, compounds of the class of the thiazolinediones (TZDs) are PPARgamma agonists and potent insulin-sensitizers. The specific but still broad expression patterns of PPARgamma, as well as its implication in numerous pathways, constitutes also a disadvantage regarding drug administration, since this potentially increases the chance to generate side-effects through the activation of the receptor in tissues or cells not affected by the disease. Actually, numerous side effects associated with the administration of TZDs have been reported. Today, a new generation of PPARgamma modulators is being actively developed to activate the receptor more specifically, in a cell and time-dependent manner, in order to induce a specific subset of target genes only and modulate a restricted number of metabolic pathways. We will discuss here why and how the development of such selective PPARgamma modulators is possible, and summarize the results obtained with the published molecules.

Thrombosis in paroxysmal nocturnal hemoglobinuria at a glance: a clinical review.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired stem cell disorder, with its primary clinical manifestations being hemolytic anemia, marrow failure and thrombophilia. Chronic hemolysis, failures of the fibrinolytic system, increased leukocyte-derived tissue factor levels in plasma, procoagulant microparticles generated through complement-mediated damage of platelets and venous endothelium are related to the acquired hypercoagulable state. Visceral thrombosis (including hepatic veins and mesenteric veins), cerebrovascular events and pulmonary embolism predict a poor outcome. Thrombosis is also associated with significant morbidity during pregnancy. Depending on the sites of thrombosis, a score-based probability to predict outcome can be assigned. Abdominal vein thromboses account for the majority of morbidity and mortality related to thrombosis, and time-dependent trends suggest that mortality rates tend to decline, with the advent of evolution of therapeutic and diagnostic strategies. In contrast, mortality rates from cerebrovascular events display no significant decline. Prompt diagnosis requires both clinical suspicion and sophisticated imaging techniques, along with multidisciplinary therapeutic intervention. In the eculizumab era, a significant reduction of thrombotic events was observed during therapy, and long-term follow up is needed to establish any benefit in rates and pattern of this complication. However, up to now, only bone marrow transplantation permanently abolishes the coagulation defect.

The 2013 Arvid Wretlind lecture: evolving concepts in parenteral nutrition.

Fifty years after the clinical introduction of total parenteral nutrition (TPN) the Arvid Wretlind lecture is an opportunity to critically analyse the evolution and changes that have marked its development and clinical use. The standard crystalline amino acid solutions, while devoid of side effects, remain incomplete regarding their composition (e.g. glutamine). Lipid emulsions have evolved tremendously and are now included in bi- and tri-compartmental feeding bags enabling a true "total" PN provided daily micronutrients are prescribed. The question of exact individual energy, macro- and micro-nutrient requirements is still unsolved. Many complications attributed to TPN are in fact the consequence of under- or over-feeding: the historical hyperalimentation concept is the main cause, along with the use of fixed weight based predictive equations (incorrect in 70% of the critically ill patients). In the late 80's many complications (hyperglycemia, sepsis, fatty liver, exacerbation of inflammation, mortality) were attributed to TPN leading to its near abandon in favour of enteral nutrition (EN). Enteral feeding, although desirable for many reasons, is difficult causing a worldwide recurrence of malnutrition by insufficient feed delivery. TPN indications have evolved towards its use either alone or in combination with EN: several controversial trials published 2011-13 have investigated TPN timing, an issue which is not yet resolved. The initiation time varies according to the country between admission (Australia and Israel), day 4 (Swiss) and day 7 (Belgium, USA). The most important issue may prove to be and individualized and time dependent prescription of feeding route, energy and substrates.

Na(+)-K(+)-ATPase gene expression during in vitro development of rat fetal forebrain.

The existence of at least three isoforms of Na(+)-K(+)-ATPase in adult brain tissues [alpha 1, kidney type; alpha 2 [or alpha(+)]; alpha 3] suggests that these genes might be regulated in a cell-specific and time-dependent manner during development. We have studied this question in serum-free aggregating cell cultures of mechanically dissociated rat fetal telencephalon. At the protein level, the relative rate of synthesis of the pool of alpha 1-, alpha 2-, and alpha 3-subunits increased approximately twofold over 15 days of culture, leading to a marked increase in the immunochemical pool of alpha-subunits as measured by a panspecific polyclonal antibody. Concomitantly, Na(+)-K(+)-ATPase enzyme-specific activity increased three- (lower forebrain) to sixfold (upper forebrain). The transcripts of all three alpha-isoforms and beta-subunit were detected in vitro in similar proportion to the level observed in vivo. alpha 3-mRNA (3.7 kb) was more abundant than alpha 1 (3.7 kb) or alpha 2 (5.3 and 3.4 kb). Cytosine arabinoside (0.4 microM) and cholera toxin (0.1 microM) were used to selectively eliminate glial cells or neurons, respectively. It was found that alpha 2-mRNA is predominantly transcribed in glial cell cultures, whereas alpha 3- and beta 1-mRNA (2.7, 2.3, and 1.8 kb) are predominant in neuronal cultures.

Atorvastatin-loaded hydrogel affects the smooth muscle cells of human veins.

Intimal hyperplasia (IH) is the major cause of stenosis of vein grafts. Drugs such as statins prevent stenosis, but their systemic administration has limited effects. We developed a hyaluronic acid hydrogel matrix, which ensures a controlled release of atorvastatin (ATV) at the site of injury. The release kinetics demonstrated that 100% of ATV was released over 10 hours, independent of the loading concentration of the hydrogel. We investigated the effects of such a delivery on primary vascular smooth muscle cells isolated from human veins. ATV decreased the proliferation, migration, and passage of human smooth muscle cells (HSMCs) across a matrix barrier in a similar dose-dependent (5-10 µM) and time-dependent manner (24-72 hours), whether the drug was directly added to the culture medium or released from the hydrogel. Expression analysis of genes known to be involved in the development of IH demonstrated that the transcripts of both the gap junction protein connexin43 (Cx43) and plasminogen activator inhibitor-1 (PAI-1) were decreased after a 24-48-hour exposure to the hydrogel loaded with ATV, whereas the transcripts of the heme oxygenase (HO-1) and the inhibitor of tissue plasminogen activator were increased. At the protein level, Cx43, PAI-1, and metalloproteinase-9 expression were decreased, whereas HO-1 was upregulated in the presence of ATV. The data demonstrate that ATV released from a hydrogel has effects on HSMCs similar to the drug being freely dissolved in the environment.

Modeling morphogen gradient formation from arbitrary realistically shaped sources.

Much of the analytical modeling of morphogen profiles is based on simplistic scenarios, where the source is abstracted to be point-like and fixed in time, and where only the steady state solution of the morphogen gradient in one dimension is considered. Here we develop a general formalism allowing to model diffusive gradient formation from an arbitrary source. This mathematical framework, based on the Green's function method, applies to various diffusion problems. In this paper, we illustrate our theory with the explicit example of the Bicoid gradient establishment in Drosophila embryos. The gradient formation arises by protein translation from a mRNA distribution followed by morphogen diffusion with linear degradation. We investigate quantitatively the influence of spatial extension and time evolution of the source on the morphogen profile. For different biologically meaningful cases, we obtain explicit analytical expressions for both the steady state and time-dependent 1D problems. We show that extended sources, whether of finite size or normally distributed, give rise to more realistic gradients compared to a single point-source at the origin. Furthermore, the steady state solutions are fully compatible with a decreasing exponential behavior of the profile. We also consider the case of a dynamic source (e.g. bicoid mRNA diffusion) for which a protein profile similar to the ones obtained from static sources can be achieved.

Nitric oxide sustains IL-1β expression in human dendritic cells enhancing their capacity to induce IL-17-producing T-cells.

The role played by lung dendritic cells (DCs) which are influenced by external antigens and by their redox state in controlling inflammation is unclear. We studied the role played by nitric oxide (NO) in DC maturation and function. Human DCs were stimulated with a long-acting NO donor, DPTA NONOate, prior to exposure to lipopolysaccharide (LPS). Dose-and time-dependent experiments were performed with DCs with the aim of measuring the release and gene expression of inflammatory cytokines capable of modifying T-cell differentiation, towardsTh1, Th2 and Th17 cells. NO changed the pattern of cytokine release by LPS-matured DCs, dependent on the concentration of NO, as well as on the timing of its addition to the cells during maturation. Addition of NO before LPS-induced maturation strongly inhibited the release of IL-12, while increasing the expression and release of IL-23, IL-1β and IL-6, which are all involved in Th17 polarization. Indeed, DCs treated with NO efficiently induced the release of IL-17 by T-cells through IL-1β. Our work highlights the important role that NO may play in sustaining inflammation during an infection through the preferential differentiation of the Th17 lineage.