Molecular phylogenetics and temporal diversification in the genus Aeromonas based on the sequences of five housekeeping genes

Several approaches have been developed to estimate both the relative and absolute rates of speciation and extinction within clades based on molecular phylogenetic reconstructions of evolutionary relationships, according to an underlying model of diversification. However, the macroevolutionary models established for eukaryotes have scarcely been used with prokaryotes. We have investigated the rate and pattern of cladogenesis in the genus Aeromonas (γ-Proteobacteria, Proteobacteria, Bacteria) using the sequences of five housekeeping genes and an uncorrelated relaxed-clock approach. To our knowledge, until now this analysis has never been applied to all the species described in a bacterial genus and thus opens up the possibility of establishing models of speciation from sequence data commonly used in phylogenetic studies of prokaryotes. Our results suggest that the genus Aeromonas began to diverge between 248 and 266 million years ago, exhibiting a constant divergence rate through the Phanerozoic, which could be described as a pure birth process.

Single cell tuning of Myc expression by antigen receptor signal strength and interleukin-2 in T lymphocytes.

Myc controls the metabolic reprogramming that supports effector T cell differentiation. The expression of Myc is regulated by the T cell antigen receptor (TCR) and pro-inflammatory cytokines such as interleukin-2 (IL-2). We now show that the TCR is a digital switch for Myc mRNA and protein expression that allows the strength of the antigen stimulus to determine the frequency of T cells that express Myc. IL-2 signalling strength also directs Myc expression but in an analogue process that fine-tunes Myc quantity in individual cells via post-transcriptional control of Myc protein. Fine-tuning Myc matters and is possible as Myc protein has a very short half-life in T cells due to its constant phosphorylation by glycogen synthase kinase 3 (GSK3) and subsequent proteasomal degradation. We show that Myc only accumulates in T cells exhibiting high levels of amino acid uptake allowing T cells to match Myc expression to biosynthetic demands. The combination of digital and analogue processes allows tight control of Myc expression at the population and single cell level during immune responses.

Population Variation and Genetic Control of Modular Chromatin Architecture in Humans.

Chromatin state variation at gene regulatory elements is abundant across individuals, yet we understand little about the genetic basis of this variability. Here, we profiled several histone modifications, the transcription factor (TF) PU.1, RNA polymerase II, and gene expression in lymphoblastoid cell lines from 47 whole-genome sequenced individuals. We observed that distinct cis-regulatory elements exhibit coordinated chromatin variation across individuals in the form of variable chromatin modules (VCMs) at sub-Mb scale. VCMs were associated with thousands of genes and preferentially cluster within chromosomal contact domains. We mapped strong proximal and weak, yet more ubiquitous, distal-acting chromatin quantitative trait loci (cQTL) that frequently explain this variation. cQTLs were associated with molecular activity at clusters of cis-regulatory elements and mapped preferentially within TF-bound regions. We propose that local, sequence-independent chromatin variation emerges as a result of genetic perturbations in cooperative interactions between cis-regulatory elements that are located within the same genomic domain.

Germination, senescence and pathogenic attack in soybean (Glycine max. L.): Identification of the cytosolic aconitase participating in the glyoxylate cycle

During our study of the glyoxylate cycle in soybean (Glycine max. L. var. Maple arrow), two mitochondrial and three cytosolic aconitase molecular species (EC 4.2.1.3) were detected, designated as M1, M2, C1, C2 and C3 isoforms, respectively, according to their intracellular locations and electrophoretic mobilities. Using the glyoxylate cycle marker enzymes isocitrate lyase (ICL, EC 4.1.3.1) and malate synthase (MS, EC 4.1.3.2), the activity of this pathway providing the essential link between P-oxidation and gluconeogenesis was confirmed during germination (cotyledons) and senescence (leaves). It was then established that, in both cases, the activity of the CI aconitase isoform developed concomitantly with the transcription and translation levels of the icl and ms genes. This strongly suggests that C1 aconitase is constitutive of the glyoxylate cycle. In addition, the same isoform was found to be active during pathogenic attack as well (hypocotyls). It might be assumed that in such a case the glyoxylate cycle is reinitiated as a part of a carbon reallocation system feeding on the diseased tissue cellular components.

Pathogenic attack and carbon reallocation in soybean leaves (Glycine max. L.): Reinitiation of the glyoxylate cycle as a defence reaction

Pathogenic attack by the fungus Botrytis cinerea (primary pathogen) on soybean leaves (Glycine max. L.; cv. Maple arrow) results in a hypersensitive response (necrotising infected leaves), in the establishment of local acquired resistance, as well as in the systemic induction of genes coding for pathogenesis-related proteins. It now appears that, concomitantly with these already well documented defence reactions, the pathogenic attack also induces the carbon reallocation mechanism based on the reinitiation of the glyoxylate cycle (pseudo-senescence of the infected leaves).

High c-Met expression in stage I-II pancreatic adenocarcinoma: proposal for an immunostaining scoring method and correlation with poor prognosis.

AIMS: c-Met is an emerging biomarker in pancreatic ductal adenocarcinoma (PDAC); there is no consensus regarding the immunostaining scoring method for this marker. We aimed to assess the prognostic value of c-Met overexpression in resected PDAC, and to elaborate a robust and reproducible scoring method for c-Met immunostaining in this setting. METHODS AND RESULTS: c-Met immunostaining was graded according to the validated MetMab score, a classic visual scale combining surface and intensity (SI score), or a simplified score (high c-Met: ≥20% of tumour cells with strong membranous staining), in stage I-II PDAC. A computer-assisted classification method (Aperio software) was developed. Clinicopathological parameters were correlated with disease-free survival (DFS) and overall survival(OS). One hundred and forty-nine patients were analysed retrospectively in a two-step process. Thirty-seven samples (whole slides) were analysed as a pre-run test. Reproducibility values were optimal with the simplified score (kappa = 0.773); high c-Met expression (7/37) was associated with shorter DFS [hazard ratio (HR) 3.456, P = 0.0036] and OS (HR 4.257, P = 0.0004). c-Met expression was concordant on whole slides and tissue microarrays in 87.9% of samples, and quantifiable with a specific computer-assisted algorithm. In the whole cohort (n = 131), patients with c-Met(high) tumours (36/131) had significantly shorter DFS (9.3 versus 20.0 months, HR 2.165, P = 0.0005) and OS (18.2 versus 35.0 months, HR 1.832, P = 0.0098) in univariate and multivariate analysis. CONCLUSIONS: Simplified c-Met expression is an independent prognostic marker in stage I-II PDAC that may help to identify patients with a high risk of tumour relapse and poor survival.

Frequent nitric oxide synthase-2 expression in human colon adenomas: implication for tumor angiogenesis and colon cancer progression.

An increased expression of nitric oxide synthase (NOS) has been observed in human colon carcinoma cell lines as well as in human gynecological, breast, and central nervous system tumors. This observation suggests a pathobiological role of tumor-associated NO production. Hence, we investigated NOS expression in human colon cancer in respect to tumor staging, NOS-expressing cell type(s), nitrotyrosine formation, inflammation, and vascular endothelial growth factor expression. Ca2+-dependent NOS activity was found in normal colon and in tumors but was significantly decreased in adenomas (P < 0.001) and carcinomas (Dukes' stages A-D: P < 0.002). Ca2+-independent NOS activity, indicating inducible NOS (NOS2), is markedly expressed in approximately 60% of human colon adenomas (P < 0.001 versus normal tissues) and in 20-25% of colon carcinomas (P < 0.01 versus normal tissues). Only low levels were found in the surrounding normal tissue. NOS2 activity decreased with increasing tumor stage (Dukes' A-D) and was lowest in colon metastases to liver and lung. NOS2 was detected in tissue mononuclear cells (TMCs), endothelium, and tumor epithelium. There was a statistically significant correlation between NOS2 enzymatic activity and the level of NOS2 protein detected by immunohistochemistry (P < 0.01). Western blot analysis of tumor extracts with Ca2+-independent NOS activity showed up to three distinct NOS2 protein bands at Mr 125,000-Mr 138,000. The same protein bands were heavily tyrosine-phosphorylated in some tumor tissues. TMCs, but not the tumor epithelium, were immunopositive using a polyclonal anti-nitrotyrosine antibody. However, only a subset of the NOS2-expressing TMCs stained positively for 3-nitrotyrosine, which is a marker for peroxynitrite formation. Furthermore, vascular endothelial growth factor expression was detected in adenomas expressing NOS2. These data are consistent with the hypothesis that excessive NO production by NOS2 may contribute to the pathogenesis of colon cancer progression at the transition of colon adenoma to carcinoma in situ.

Birth Weight, Working Memory and Epigenetic Signatures in IGF2 and Related Genes: A MZ Twin Study.

Neurodevelopmental disruptions caused by obstetric complications play a role in the etiology of several phenotypes associated with neuropsychiatric diseases and cognitive dysfunctions. Importantly, it has been noticed that epigenetic processes occurring early in life may mediate these associations. Here, DNA methylation signatures at IGF2 (insulin-like growth factor 2) and IGF2BP1-3 (IGF2-binding proteins 1-3) were examined in a sample consisting of 34 adult monozygotic (MZ) twins informative for obstetric complications and cognitive performance. Multivariate linear regression analysis of twin data was implemented to test for associations between methylation levels and both birth weight (BW) and adult working memory (WM) performance. Familial and unique environmental factors underlying these potential relationships were evaluated. A link was detected between DNA methylation levels of two CpG sites in the IGF2BP1 gene and both BW and adult WM performance. The BW-IGF2BP1 methylation association seemed due to non-shared environmental factors influencing BW, whereas the WM-IGF2BP1 methylation relationship seemed mediated by both genes and environment. Our data is in agreement with previous evidence indicating that DNA methylation status may be related to prenatal stress and later neurocognitive phenotypes. While former reports independently detected associations between DNA methylation and either BW or WM, current results suggest that these relationships are not confounded by each other.

Prevalence and clinical outcomes for patients with MET protein expression in patients with non-small cell lung cancer in Europe: Results from the European Thoracic Oncology Platform Lungscape Project.

Aim The reported prevalence of MET overexpression varies from 25-55% in non-small cell lung cancer (NSCLC) and clinical correlations are emerging slowly. In a well-defined NSCLC cohort of the Lungscape program, we explore the epidemiology, the natural history of IHC MET positivity and its association to OS, RFS and TTR. Methods Resected stage I-III NSCLC identified based on the quality of clinical data and FFPE tissue availability were assessed for MET expression using immunohistochemistry (IHC) on TMAs (CONFIRM anti total c-MET assay, clone SP44, Ventana BenchMark platform). All cases were analysed at participating pathology laboratories using the same protocol, after passing an external quality assurance program. MET positive status is defined as ≥ 50% of tumor cells staining with 2+ or 3+ intensity. Results A total of 2709 cases are included in the iBiobank and will be analysed. IHC MET expression is currently available for 1552 patients, with positive MET IHC staining in 380 cases [24.5%; IHC 3+ in 157 cases (41.3%) and 2+ in 223 cases (58.7%)]. The cohort of 1552 patients includes 48.2%, 44.7% and 4.4% cases of adenocarcinoma, squamous and large cell histologies, respectively. IHC MET status was independent of stage, age and smoking history. Significant differences in MET positivity were associated with gender (32% vs. 21% for female vs. male, p < 0.001), with performance status (25% vs. 18% for 0 vs. 1-3, p = 0.006), and histology (34%, 14% and 24% for adenocarcinoma, squamous and large cell carcinoma, p < 0.001). IHC MET positivity was independent of the IHC ALK status (p = 0.08). At last FU, 52% of patients were still alive, with a median FU of 4.8 yrs. No association of IHC MET was found with OS, RFS or TTR. Conclusions The preliminary results for this large multicentre European cohort describe a prevalence of MET overexpression that seems lower than previous observations in NSCLC, such as reported for the OAM4971g trial, suggesting potential biological differences between surgically resected and metastatic disease. Analysis for the full cohort is ongoing and results will be presented. Disclosure L. Bubendorf: Disclosures: Stock ownership: Roche Advisory boards: Roche, Pfizer Research support: Roche; K. Schulze: Full time employee of Roche; A. Das-Gupta: I am a full time employee of Roche. All other authors have declared no conflicts of interest.

Gibberellic acid signaling is required for ambient temperature-mediated induction of flowering in Arabidopsis thaliana.

Distinct molecular mechanisms integrate changes in ambient temperature into the genetic pathways that govern flowering time in Arabidopsis thaliana. Temperature-dependent eviction of the histone variant H2A.Z from nucleosomes has been suggested to facilitate the expression of FT by PIF4 at elevated ambient temperatures. Here we show that, in addition to PIF4, PIF3 and PIF5, but not PIF1 and PIF6, can promote flowering when expressed specifically in phloem companion cells (PCC), where they can induce FT and its close paralog, TSF. However, despite their strong potential to promote flowering, genetic analyses suggest that the PIF genes seem to have only a minor role in adjusting flowering in response to photoperiod or high ambient temperature. In addition, loss of PIF function only partially suppressed the early flowering phenotype and FT expression of the arp6 mutant, which is defective in H2A.Z deposition. In contrast, the chemical inhibition of gibberellic acid (GA) biosynthesis resulted in a strong attenuation of early flowering and FT expression in arp6. Furthermore, GA was able to induce flowering at low temperature (15°C) independently of FT, TSF, and the PIF genes, probably directly at the shoot apical meristem. Together, our results suggest that the timing of the floral transition in response to ambient temperature is more complex than previously thought and that GA signaling might play a crucial role in this process.

DNA Topoisomerase I Gene Copy Number and mRNA Expression Assessed as Predictive Biomarkers for Adjuvant Irinotecan in Stage II/III Colon Cancer.

PURPOSE: Prospective-retrospective assessment of theTOP1gene copy number andTOP1mRNA expression as predictive biomarkers for adjuvant irinotecan in stage II/III colon cancer. EXPERIMENTAL DESIGN: Formalin-fixed, paraffin-embedded tissue microarrays were obtained from an adjuvant colon cancer trial (PETACC3) where patients were randomized to 5-fluorouracil/folinic acid with or without additional irinotecan.TOP1copy number status was analyzed by fluorescencein situhybridization (FISH) using aTOP1/CEN20 dual-probe combination.TOP1mRNA data were available from previous analyses. RESULTS: TOP1FISH and follow-up data were obtained from 534 patients.TOP1gain was identified in 27% using a single-probe enumeration strategy (≥4TOP1signals per cell) and in 31% when defined by aTOP1/CEN20 ratio ≥ 1.5. The effect of additional irinotecan was not dependent onTOP1FISH status.TOP1mRNA data were available from 580 patients with stage III disease. Benefit of irinotecan was restricted to patients characterized byTOP1mRNA expression ≥ third quartile (RFS: HRadjusted, 0.59;P= 0.09; OS: HRadjusted, 0.44;P= 0.03). The treatment byTOP1mRNA interaction was not statistically significant, but in exploratory multivariable fractional polynomial interaction analysis, increasingTOP1mRNA values appeared to be associated with increasing benefit of irinotecan. CONCLUSIONS: In contrast to theTOP1copy number, a trend was demonstrated for a predictive property ofTOP1mRNA expression. On the basis ofTOP1mRNA, it might be possible to identify a subgroup of patients where an irinotecan doublet is a clinically relevant option in the adjuvant setting of colon cancer.Clin Cancer Res; 22(7); 1621-31. ©2015 AACR.

Identification of molecular subtypes and gene expression patterns of breast cancer analysing RNA-seq data

Breast cancer is the most common diagnosed cancer and the leading cause of cancer death among females worldwide. It is considered a highly heterogeneous disease and it must be classified into more homogeneous groups. Hence, the purpose of this study was to classify breast tumors based on variations in gene expression patterns derived from RNA sequencing by using different class discovery methods. 42 breast tumors paired-samples were sequenced by Illumine Genome Analyzer and the data was analyzed and prepared by TopHat2 and htseq-count. As reported previously, breast cancer could be grouped into five main groups known as basal epithelial-like group, HER2 group, normal breast-like group and two Luminal groups with a distinctive expression profile. Classifying breast tumor samples by using PAM50 method, the most common subtype was Luminal B and was significantly associated with ESR1 and ERBB2 high expression. Luminal A subtype had ESR1 and SLC39A6 significant high expression, whereas HER2 subtype had a high expression of ERBB2 and CNNE1 genes and low luminal epithelial gene expression. Basal-like and normal-like subtypes were associated with low expression of ESR1, PgR and HER2, and had significant high expression of cytokeratins 5 and 17. Our results were similar compared with TGCA breast cancer data results and with known studies related with breast cancer classification. Classifying breast tumors could add significant prognostic and predictive information to standard parameters, and moreover, identify marker genes for each subtype to find a better therapy for patients with breast cancer.