Online Survival Analysis Software to Assess the Prognostic Value of Biomarkers Using Transcriptomic Data in Non-Small-Cell Lung Cancer

In the last decade, optimized treatment for non-small cell lung cancer had lead to improved prognosis, but the overall survival is still very short. To further understand the molecular basis of the disease we have to identify biomarkers related to survival. Here we present the development of an online tool suitable for the real-time meta-analysis of published lung cancer microarray datasets to identify biomarkers related to survival. We searched the caBIG, GEO and TCGA repositories to identify samples with published gene expression data and survival information. Univariate and multivariate Cox regression analysis, Kaplan-Meier survival plot with hazard ratio and logrank P value are calculated and plotted in R. The complete analysis tool can be accessed online at: www.kmplot.com/lung. All together 1,715 samples of ten independent datasets were integrated into the system. As a demonstration, we used the tool to validate 21 previously published survival associated biomarkers. Of these, survival was best predicted by CDK1 (p<1E-16), CD24 (p<1E-16) and CADM1 (p = 7E-12) in adenocarcinomas and by CCNE1 (p = 2.3E-09) and VEGF (p = 3.3E-10) in all NSCLC patients. Additional genes significantly correlated to survival include RAD51, CDKN2A, OPN, EZH2, ANXA3, ADAM28 and ERCC1. In summary, we established an integrated database and an online tool capable of uni- and multivariate analysis for in silico validation of new biomarker candidates in non-small cell lung cancer.     

Chronic Increase of Urea Leads to Carbamylated Proteins Accumulation in Tissues in a Mouse Model of CKD

Carbamylation is a general process involved in protein molecular ageing due to the nonenzymatic binding of isocyanic acid, mainly generated by urea dissociation, to free amino groups. In vitro experiments and clinical studies have suggested the potential involvement of carbamylated proteins (CPs) in chronic kidney disease (CKD) complications like atherosclerosis, but their metabolic fate in vivo is still unknown. To address this issue, we evaluated protein carbamylation in the plasma and tissues of control and 75% nephrectomised C57BL/6J mice by LC-MS/MS assay of homocitrulline, the major carbamylation-derived product (CDP). A basal level of carbamylation was evidenced under all conditions, showing that carbamylation is a physiological process of protein modification in vivo. CP plasma concentrations increased in nephrectomized vs. control mice over the 20 weeks of the experiment (e.g. 335±43 vs. 167±19 μmol homocitrulline/mol lysine (p<0.001) 20 weeks after nephrectomy). Simultaneously, CP content increased roughly by two-fold in all tissues throughout the experiment. The progressive accumulation of CPs was specifically noted in long-lived extracellular matrix proteins, especially collagen (e.g. 1264±123 vs. 726±99 μmol homocitrulline/mol lysine (p<0.01) in the skin of nephrectomized vs. control mice after 20 weeks of evolution). These results show that chronic increase of urea, as seen in CKD, increases the carbamylation rate of plasma and tissue proteins. These results may be considered in the perspective of the deleterious effects of CPs demonstrated in vitro and of the correlation evidenced recently between plasma CPs and cardiovascular risk or mortality in CKD patients.     

Lkb1 Loss Promotes Tumor Progression of BRAFV600E-Induced Lung Adenomas

Aberrant activation of MAP kinase signaling pathway and loss of tumor suppressor LKB1 have been implicated in lung cancer development and progression. Although oncogenic KRAS mutations are frequent, BRAF mutations (BRAF^V600E) are found in 3% of human non-small cell lung cancers. Contrary to KRAS mutant tumors, BRAF^V600E-induced tumors are benign adenomas that fail to progess. Interestingly, loss of tumor supressor LKB1 coexists with KRAS oncogenic mutations and synergizes in tumor formation and progression, however, its cooperation with BRAF^V600E oncogene is unknown. Our results describe a lung cell population in neonates mice where expression of BRAF^V600E leads to lung adenoma development. Importantly, expression of BRAF^V600E concomitant with the loss of only a single-copy of Lkb1, overcomes senencence–like features of BRAF^V600E-mutant adenomas leading malignization to carcinomas. These results posit LKB1 haploinsufficiency as a risk factor for tumor progression of BRAF^V600E mutated lung adenomas in human cancer patients.     

Network Bursting Dynamics in Excitatory Cortical Neuron Cultures Results from the Combination of Different Adaptive Mechanism

In the brain, synchronization among cells of an assembly is a common phenomenon, and thought to be functionally relevant. Here we used an in vitro experimental model of cell assemblies, cortical cultures, combined with numerical simulations of a spiking neural network (SNN) to investigate how and why spontaneous synchronization occurs. In order to deal with excitation only, we pharmacologically blocked GABA_Aergic transmission using bicuculline. Synchronous events in cortical cultures tend to involve almost every cell and to display relatively constant durations. We have thus named these “network spikes” (NS). The inter-NS-intervals (INSIs) proved to be a more interesting phenomenon. In most cortical cultures NSs typically come in series or bursts (“bursts of NSs”, BNS), with short (∼1 s) INSIs and separated by long silent intervals (tens of s), which leads to bimodal INSI distributions. This suggests that a facilitating mechanism is at work, presumably short-term synaptic facilitation, as well as two fatigue mechanisms: one with a short timescale, presumably short-term synaptic depression, and another one with a longer timescale, presumably cellular adaptation. We thus incorporated these three mechanisms into the SNN, which, indeed, produced realistic BNSs. Next, we systematically varied the recurrent excitation for various adaptation timescales. Strong excitability led to frequent, quasi-periodic BNSs (CV∼0), and weak excitability led to rare BNSs, approaching a Poisson process (CV∼1). Experimental cultures appear to operate within an intermediate weakly-synchronized regime (CV∼0.5), with an adaptation timescale in the 2–8 s range, and well described by a Poisson-with-refractory-period model. Taken together, our results demonstrate that the INSI statistics are indeed informative: they allowed us to infer the mechanisms at work, and many parameters that we cannot access experimentally.     

The Co-Occurrence of Species and the Co-Diversity of Sites in Neutral Models of Biodiversity

Patterns of co-occurrence of species are widely used to assess the fit of ecological neutral models to empirical patterns. The mathematically equivalent patterns of co-diversity of sites, in contrast, have been considered only indirectly and analyses normally are focused on the spatial distribution of species richness, rather than on the patterns of species sharing. Here we use two analytical tools (range-diversity plots and rank plots) to assess the predictions of simple neutral models in relation to patterns of co-occurrence and co-diversity. Whereas a fully stochastic null model predicts zero average among species and among sites, neutral models generate systems with low levels of covariance among species and high levels of positive covariance among sites. These patterns vary with different combinations of dispersal and speciation rates, but are always linked to the shape, symmetry, and spread of the range-size and species-richness frequency distributions. Non-homogeneous patterns of diversity and distribution arise in neutral models because of the spatial arrangement of sites and their concomitant similarity, which is reflected also in the spread of the range-size frequency distribution. The nearly null covariance among species, in contrast, implies low variance in species richness of sites and very slim frequency distributions. In real world assemblages of Mexican volant and non-volant mammals, patterns of range-size and species-richness frequency distribution are similar to those generated by neutral models. However, when the comparison includes the covariance both for species (co-occurrence) and for sites (co-diversity), empirical patterns differ significantly from the predictions of neutral models. Because of the mathematical links between the covariance in the distribution of species and the variance of species-richness values and between the covariance in species sharing among sites and the variance of range-size values, a full understanding of patterns of diversity calls for the simultaneous analysis of co-occurrence and co-diversity.     

Heterotrophic Compensation: A Possible Mechanism for Resilience of Coral Reefs to Global Warming or a Sign of Prolonged Stress?

Thermally induced bleaching has caused a global decline in corals and the frequency of such bleaching events will increase. Thermal bleaching severely disrupts the trophic behaviour of the coral holobiont, reducing the photosynthetically derived energy available to the coral host. In the short term this reduction in energy transfer from endosymbiotic algae results in an energy deficit for the coral host. If the bleaching event is short-lived then the coral may survive this energy deficit by depleting its lipid reserves, or by increasing heterotrophic energy acquisition. We show for the first time that the coral animal is capable of increasing the amount of heterotrophic carbon incorporated into its tissues for almost a year following bleaching. This prolonged heterotrophic compensation could be a sign of resilience or prolonged stress. If the heterotrophic compensation is in fact an acclimatization response, then this physiological response could act as a buffer from future bleaching by providing sufficient heterotrophic energy to compensate for photoautotrophic energy losses during bleaching, and potentially minimizing the effect of subsequent elevated temperature stresses. However, if the elevated incorporation of zooplankton is a sign that the effects of bleaching continue to be stressful on the holobiont, even after 11 months of recovery, then this physiological response would indicate that complete coral recovery requires more than 11 months to achieve. If coral bleaching becomes an annual global phenomenon by mid-century, then present temporal refugia will not be sufficient to allow coral colonies to recover between bleaching events and coral reefs will become increasingly less resilient to future climate change. If, however, increasing their sequestration of zooplankton-derived nutrition into their tissues over prolonged periods of time is a compensating mechanism, the impacts of annual bleaching may be reduced. Thus, some coral species may be better equipped to face repeated bleaching stress than previously thought.     

Characterization of a surrogate murine antibody to model anti-human CD3 therapies

Fc-modified anti-human CD3ε monoclonal antibodies (mAbs) are in clinical development for the treatment of autoimmune diseases. These next generation mAbs have completed clinical trials in patients with type-1 diabetes and inflammatory bowel disease demonstrating a narrow therapeutic window. Lowered doses are ineffective, yet higher pharmacologically-active doses cause an undesirable level of adverse events. Thus, there is a critical need for a return to bench research to explore ways of improving clinical outcomes. Indeed, we recently reported that a short course of treatment affords synergy, providing long-term disease amelioration when combining anti-mouse CD3 and anti-mouse tumor necrosis factor mAbs in experimental arthritis. Such strategies may widen the window between risk and benefit; however, to more accurately assess experimentally the biology and pharmacology, reagents that mimic the current development candidates were required. Consequently, we engineered an Fc-modified anti-mouse CD3ε mAb, 2C11-Novi. Here, we report the functional characterization of 2C11-Novi demonstrating that it does not bind FcγR in vitro and elicits little cytokine release in vivo, while maintaining classical pharmacodynamic effects (CD3-TCR downregulation and T cell killing). Furthermore, we observed that oral administration of 2C11-Novi ameliorated progression of remitting-relapsing experimental autoimmune encephalitis in mice, significantly reducing the primary acute and subsequent relapse phase of the disease. With innovative approaches validated in two experimental models of human disease, 2C11-Novi represents a meaningful tool to conduct further mechanistic studies aiming at exploiting the immunoregulatory properties of Fc-modified anti-CD3 therapies via combination therapy using parenteral or oral routes of administration.     

Altered Dihydropyrimidine Dehydrogenase Activity Associated with Mild Toxicity in Patients Treated with 5-Fluorouracil Containing Chemotherapy

Dihydropyrimidine dehydrogenase (DPD) plays a pivotal role in the metabolism of 5-fluorouracil (5FU). In patients treated with capecitabine or 5FU combined with other chemotherapeutic drugs, DPD activity in peripheral blood mononuclear cells was increased in patients experiencing grade I/II neutropenia. In contrast, decreased DPD activity proved to be associated with grade I/II dermatological toxicity, including hand-foot syndrome. Thus, patients with a low-normal or high-normal DPD activity proved to be at risk of developing mild toxicity upon treatment with 5FU-based chemotherapy, demonstrating the important role of DPD in the etiology of toxicity associated with 5FU and the catabolites of 5FU.