A downstream mediator in the growth repression limb of the jasmonate pathway.

Wounding plant tissues initiates large-scale changes in transcription coupled to growth arrest, allowing resource diversion for defense. These processes are mediated in large part by the potent lipid regulator jasmonic acid (JA). Genes selected from a list of wound-inducible transcripts regulated by the jasmonate pathway were overexpressed in Arabidopsis thaliana, and the transgenic plants were then assayed for sensitivity to methyl jasmonate (MeJA). When grown in the presence of MeJA, the roots of plants overexpressing a gene of unknown function were longer than those of wild-type plants. When transcript levels for this gene, which we named JASMONATE-ASSOCIATED1 (JAS1), were reduced by RNA interference, the plants showed increased sensitivity to MeJA and growth was inhibited. These gain- and loss-of-function assays suggest that this gene acts as a repressor of JA-inhibited growth. An alternative transcript from the gene encoding a second protein isoform with a longer C terminus failed to repress jasmonate sensitivity. This identified a conserved C-terminal sequence in JAS1 and related genes, all of which also contain Zim motifs and many of which are jasmonate-regulated. Both forms of JAS1 were found to localize to the nucleus in transient expression assays. Physiological tests of growth responses after wounding were consistent with the fact that JAS1 is a repressor of JA-regulated growth retardation.

Tumor-host interactions Part 1: Stromal signatures of breast and prostate cancer Part 2: GLG1 and the control of the secretory pathway in tumor cells

Tumor-host interaction is a key determinant during cancer progression, from primary tumor growth to metastatic dissemination. At each step, tumor cells have to adapt to and subvert different types of microenvironment, leading to major phenotypic and genotypic alterations that affect both tumor and surrounding stromal compartments. Understanding the molecular mechanisms that govern tumor-host interplay may be essential for better comprehension of tumorigenesis in an effort to improve current anti-cancer therapies. The present work is composed of two projects that address tumor-host interactions from two different perspectives, the first focusing on the characterization of tumor-associated stroma and the second on membrane trafficking in tumor cells. Part 1. To selectively address stromal gene expression changes during cancer progression, oligonucleotide-based Affymetrix microarray technology was used to analyze the transcriptomes of laser-microdissected stromal cells derived from invasive human breast and prostate carcinoma. Comparison showed that invasive breast and prostate cancer elicit distinct, tumor-specific stromal responses, with a limited panel of shared induced and/or repressed genes. Both breast and prostate tumor-specific deregulated stromal gene sets displayed statistically significant survival-predictive ability for their respective tumor type. By contrast, a stromal gene signature common to both tumor types did not display prognostic value, although expression of two individual genes within this common signature was found to be associated with patient survival. Part 2. GLG1 is known as an E-selectin ligand and an intracellular FGF receptor, depending on cell type and context. Immunohistochemical and immunofluorescence analyses showed that GLG1 is primarily localized in the Golgi of human tumor cells, a central location in the biosynthetic/secretory pathways. GLG1 has been shown to interact with and to recruit the ARF GEF BIGI to the Golgi membrane. Depletion of GLG1 or BIGI markedly reduced ARF3 membrane localization and activation, and altered the Golgi structure. Interestingly, these perturbations did not impair constitutive secretion in general, but rather seemed to impair secretion of a specific subset of proteins that includes MMP-9. Thus, GLG1 coordinates ARF3 activation by recruiting BIGI to the Golgi membrane, thereby affecting secretion of specific molecules. - Les interactions tumeur-hôte constituent un élément essentiel à la progression tumorale, de la croissance de la tumeur primaire à la dissémination des métastases. A chaque étape, les cellules tumorales doivent s'adapter à différents types de microenvironnement et les détourner à leur propre avantage, donnant lieu à des altérations phénotypiques et génotypiques majeures qui affectent aussi bien la tumeur elle-même que le compartiment stromal environnant. L'étude des mécanismes moléculaires qui régissent les interactions tumeur-hôte constitue une étape essentielle pour une meilleure compréhension du processus de tumorigenèse dans le but d'améliorer les thérapies anti cancer existantes. Le travail présenté ici est composé de deux projets qui abordent la problématique des interactions tumeur-hôte selon différentes perspectives, le premier se concentrant sur la caractérisation du stroma tumoral et le second sur le trafic intracellulaire des cellules tumorales. Partie 1. Pour examiner les changements d'expression des gènes dans le stroma en réponse à la progression du cancer, des puces à ADN Affymetrix ont été utilisées afin d'analyser les transcriptomes des cellules stromales issues de carcinomes invasifs du sein et de la prostate et collectées par microdissection au laser. L'analyse comparative a montré que les cancers invasifs du sein et de la prostate provoquent des réponses stromales spécifiques à chaque type de tumeur, et présentent peu de gènes induits ou réprimés de façon similaire. L'ensemble des gènes dérégulés dans le stroma associé au cancer du sein, ou à celui de la prostate, présente une valeur pronostique pour les patients atteints d'un cancer du sein, respectivement de la prostate. En revanche, la signature stromale commune aux deux types de cancer n'a aucune valeur prédictive, malgré le fait que l'expression de deux gènes présents dans cette liste soit liée à la survie des patients. Partie 2. GLG1 est connu comme un ligand des sélectines E ainsi que comme récepteur intracellulaire pour des facteurs de croissances FGFs selon le type de cellule dans lequel il est exprimé. Des analyses immunohistochimiques et d'immunofluorescence ont montré que dans les cellules tumorales, GLG1 est principalement localisé au niveau de l'appareil de Golgi, une place centrale dans la voie biosynthétique et sécrétoire. Nous avons montré que GLG1 interagit avec la protéine BIGI et participe à son recrutement à la membrane du Golgi. L'absence de GLG1 ou de BIGI réduit drastiquement le pool d'ARF3 associé aux membranes ainsi que la quantité d'ARF3 activés, et modifie la structure de l'appareil de Golgi. Il est particulièrement intéressant de constater que ces perturbations n'ont pas d'effet sur la sécrétion constitutive en général, mais semblent plutôt affecter la sécrétion spécifique d'un sous-groupe défini de protéines comprenant MMP-9. GLG1 coordonne donc l'activation de ARF3 en recrutant BIGI à la membrane du Golgi, agissant par ce moyen sur la sécrétion de molécules spécifiques.

ppGpp controlled by the Gac/Rsm regulatory pathway sustains biocontrol activity in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway is instrumental for secondary metabolism and biocontrol of root pathogens via the expression of regulatory small RNAs (sRNAs). Furthermore, in strain CHA0, an imbalance in the Krebs cycle can affect the strain's ability to produce extracellular secondary metabolites, including biocontrol factors. Here, we report the metabolome of wild-type CHA0, a gacA-negative mutant, which has lost Gac/Rsm activities, and a retS-negative mutant, which shows strongly enhanced Gac/Rsm-dependent activities. Capillary electrophoresis-based metabolomic profiling revealed that the gacA and retS mutations had opposite effects on the intracellular levels of a number of central metabolites, suggesting that the Gac/Rsm pathway regulates not only secondary metabolism but also primary metabolism in strain CHA0. Among the regulated metabolites identified, the alarmone guanosine tetraphosphate (ppGpp) was characterized in detail by the construction of relA (for ppGpp synthase) and spoT (for ppGpp synthase/hydrolase) deletion mutants. In a relA spoT double mutant, ppGpp synthesis was completely abolished, the expression of Rsm sRNAs was attenuated, and physiological functions such as antibiotic production, root colonization, and plant protection were markedly diminished. Thus, ppGpp appears to be essential for sustaining epiphytic fitness and biocontrol activity of strain CHA0.

Peroxisome proliferator-activated receptors: a nuclear receptor signaling pathway in lipid physiology.

Peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors that belong to the steroid/thyroid/retinoic acid receptor superfamily. All their characterized target genes encode proteins that participate in lipid homeostasis. The recent finding that antidiabetic thiazolidinediones and adipogenic prostanoids are ligands of one of the PPARs reveals a novel signaling pathway that directly links these compounds to processes involved in glucose homeostasis and lipid metabolism including adipocyte differentiation. A detailed understanding of this pathway could designate PPARs as targets for the development of novel efficient treatments for several metabolic disorders.

Iron-deficiency anemia and hematochezia: when is colonoscopy appropriate?

Background: Colonoscopy is usually proposed for the evaluation of lower gastrointestinal blood loss (hematochezia) or iron deficiency anemia (IDA). Clinical practice guidelines support this approach but formal evidence is lacking. Real clinical scenarios made available on the web would be of great help in decision-making in clinical practice as to whether colonoscopy is appropriate for a given patient. Method: A multidisciplinary multinational expert panel (EPAGE II) developed appropriateness criteria based on best published evidence (systematic reviews, clinical trials, guidelines) and experts' judgement. Using the explicit RAND Appropriateness Method (3 round of experts' votes and a panel meeting) 102 clinical scenarios were judged inappropriate, uncertain, appropriate, or necessary. Results: In IDA, colonoscopy was appropriate in patients >50 years and necessary in the presence of lower abdominal symptoms. In both men and women aged <50 years, colonoscopy was appropriate if prior sigmoidoscopy and/or gastroscopy did not explain the IDA, and necessary if lower gastrointestinal symptoms were present. In women <50 years with a potential gynecological cause, additional lower gastrointestinal symptoms rendered colonoscopy appropriate. In patients >50 years with hematochezia, colonoscopy was always appropriate and mostly necessary, except if a prior colonoscopy was normal within the previous 5 years. Under age 50 years, the presence of any risk factor for colorectal cancer (CRC) and no previous normal colonoscopy (within the last 5 years) made this procedure appropriate and necessary. Conclusion: Colonoscopy is appropriate and even necessary for many indications related to iron deficiency anemia or hematochezia, in particular in patients aged >50 years. The main factors influencing appropriateness are age, results of prior investigations (sigmoidoscopy, gastroscopy, previous colonoscopy), CRC risk and sex. EPAGE II appropriateness criteria are available on www.epage.ch

Pathway databases and tools for their exploitation: benefits, current limitations and challenges

In past years, comprehensive representations of cell signalling pathways have been developed by manual curation from literature, which requires huge effort and would benefit from information stored in databases and from automatic retrieval and integration methods. Once a reconstruction of the network of interactions is achieved, analysis of its structural features and its dynamic behaviour can take place. Mathematical modelling techniques are used to simulate the complex behaviour of cell signalling networks, which ultimately sheds light on the mechanisms leading to complex diseases or helps in the identification of drug targets. A variety of databases containing information on cell signalling pathways have been developed in conjunction with methodologies to access and analyse the data. In principle, the scenario is prepared to make the most of this information for the analysis of the dynamics of signalling pathways. However, are the knowledge repositories of signalling pathways ready to realize the systems biology promise? In this article we aim to initiate this discussion and to provide some insights on this issue.

Role of homer 1 proteins in the calcium signaling pathway(s) underlying exo-endocytosis processes of glutamatergic slmvs in astrocytes

The discovery that astrocytes possess a nonelectrical form of excitability (calcium excitability) that leads to the release of chemical transmitters, an activity called gliotransmission, indicates that these cells may have additional important roles in brain function. Elucidating the stimulussecretion coupling leading to the exocytic release of chemical transmitters (such as glutamate, Bezzi et al., Nature Neurosci, 2004) may therefore clarify i) whether astrocytes represent in full a new class of secretory cells in the brain and ii) whether they can participate to the fast brain signaling in the brain. We have recently discovered the existence in astrocytes of functional sub-membrane microdomains of calcium release from the internal stores in response to mGluR5 activation (Marchaland et al., J of Neurosci., 2008). Such sub-plasma membrane calcium microdomains control exocytosis of astrocytic glutamate signaling to neurons. Homer proteins are scaffold proteins controlling calcium signaling in different cellular microdomains, including dendritic spines in neurons (Sala et al., J of Neurosci., 2005). Thus, similarly to dendritic pines, Homer1 could be implicated in the coupling between astrocytic mGluR5 and IP3Rs on the ER. Here, by using a recently developed approach for studying vesicle recycling dynamics at synapses (Voglmaier et al., Neuron, 2006; Balaji and Ryan, PNAS, 2007) combined with epifluorescence and total internal reflection fluorescence (TIRF) imaging, we investigated the involvement of Homer1 proteins in the calcium dependent stimulus-secretion coupling leading glutamate exocytosis of synaptic-like microvesicles (SLMVs) in astrocytes.

Enhanced recovery pathway for urgent colectomy.

BACKGROUND: Enhanced recovery protocols have been proven to decrease complications and hospital stay following elective colorectal surgery. However, these principles have not yet been reported for urgent surgery procedures. We aimed to assess our initial experience with urgent colectomies performed within an established enhanced recovery pathway. METHODS: In a prospective cohort study, all patients undergoing colonic resection between April 2012 and March 2013 were treated according to a standardized enhanced recovery protocol. Urgent surgeries were compared with the elective procedures with regards to baseline characteristics, compliance with enhanced recovery items, and clinical outcome. RESULTS: Patients (N = 28) requiring urgent colonic resection were included and compared with patients undergoing elective colectomy (N = 63). Overall compliance with the protocol was 57% for the urgent compared with 77% for the elective procedures (p = 0.006). The pre-operative compliance was 64 versus 96% (p < 0.001), the intra-operative compliance was 77 versus 86% (p = 0.145), and the post-operative compliance was 49 versus 67% (p = 0.015), for the urgent and elective resections, respectively. Overall, 18 urgent patients (64%) and 32 elective patients (51%) developed postoperative complications (p = 0.261). Median postoperative length of stay was 8 days in the urgent setting compared with 5 days in the elective setting (p = 0.006). CONCLUSIONS: Many of the intra-operative and post-operative enhanced recovery items can also be applied to urgent colectomy, entailing outcomes that approach the results achieved in the elective setting.

Neutralizing antibodies in Multiple Sclerosis a model-based analysis of Interferon beta signaling pathway in macrophages

Multiple Sclerosis is the most common non-traumatic cause of neurologicaldisability in young people. There is no cure yet, and until recently, few long-termtherapies existed. Interferon beta (IFNβ) was the first treatment, and remains the mostcommonly prescribed. One of the most significant problems of IFNβ therapy is theproduction of drug specific antibodies. Up to 45% of patients develop neutralizingantibodies (NAbs) to IFNβ products. The neutralizing antibody binds to the biologicalagent preventing its interaction with its receptor, inhibiting the biological action of theprotein, which abrogates the clinical efficacy of IFNβ treatment. Interferon-betamediates its response by binding to its high affinity cell surface receptor and initiatingthe JAK/STAT signalling cascade. In this project we have analyzed the IFNβ signalingpathway in macrophages when neutralizing antibodies are present. The response tothis pathway after IFNβ stimulation shows a transient oscillatory rhythm of STAT1phosphorylation, which varies as NAbs concentration increases. To improve ourunderstanding of that behavior, we extended an existing mathematical model based onnonlinear ordinary differential equations of JAK/STAT pathway by including IFN-NAbassociation and IFN-activation receptor. Combining our theoretical model withexperimental data we could study the role of neutralizing antibodies on the molecularresponse and determine its lifetime after cytokine stimulation.

Appropriateness of colonoscopy in Europe (EPAGE II). Iron-deficiency anemia and hematochezia.

BACKGROUND AND STUDY AIMS: To summarize the published literature on assessment of appropriateness of colonoscopy for the investigation of iron-deficiency anemia (IDA) and hematochezia, and report appropriateness criteria developed by an expert panel, the 2008 European Panel on the Appropriateness of Gastrointestinal Endoscopy, EPAGE II. METHODS: A systematic search of guidelines, systematic reviews and primary studies regarding the evaluation and management of IDA and hematochezia was performed. The RAND/UCLA Appropriateness Method was applied to develop appropriateness criteria for colonoscopy for these conditions. RESULTS: IDA occurs in 2 %-5 % of adult men and postmenopausal women. Examination of both the upper and lower gastrointestinal tract is recommended in patients with iron deficiency. Colonoscopy for IDA yields one colorectal cancer (CRC) in every 9-13 colonoscopies. Hematochezia is a well-recognized alarm symptom and such patients are likely to be referred for colonoscopy. Colonoscopy is unanimously recommended in patients aged > or = 50. Diverticulosis, vascular ectasias, and ischemic colitis are common causes of acute lower gastrointestinal bleeding (LGIB); CRC is found in 0.2 %-11 % of the colonoscopies performed for LGIB. Most patients with scant hematochezia have an anorectal or a distal source of bleeding. The expert panel considered most clinical indications for colonoscopy as appropriate in the presence of IDA (58 %) or hematochezia (83 %). CONCLUSION: Despite the limitations of the published studies, guidelines unanimously recommend colonoscopy for the investigation of IDA and hematochezia in patients aged > or = 50 years. These indications were also considered appropriate by EPAGE II, as were indications in patients at low risk for CRC with no obvious cause of bleeding found during adequate previous investigations.

Anemia and chronic kidney disease are potential risk factors for mortality in stroke patients: a historic cohort study.

BACKGROUND: Chronic kidney disease (CKD) is associated to a higher stroke risk. Anemia is a common consequence of CKD, and is also a possible risk factor for cerebrovascular diseases. The purpose of this study was to examine if anemia and CKD are independent risk factors for mortality after stroke. METHODS: This historic cohort study was based on a stroke registry and included patients treated for a first clinical stroke in the stroke unit of one academic hospital over a three-year period. Mortality predictors comprised demographic characteristics, CKD, glomerular filtration rate (GFR), anemia and other stroke risk factors. GFR was estimated by means of the simplified Modification of Diet in Renal Disease formula. Renal function was assessed according to the Kidney Disease Outcomes Quality Initiative (K/DOQI)-CKD classification in five groups. A value of hemoglobin < 120 g/L in women and < 130 g/L in men on admission defined anemia. Kaplan-Meier survival curves and Cox models were used to describe and analyze one-year survival. RESULTS: Among 890 adult stroke patients, the mean (Standard Deviation) calculated GFR was 64.3 (17.8) ml/min/1.73 m2 and 17% had anemia. Eighty-two (10%) patients died during the first year after discharge. Among those, 50 (61%) had K/DOQI CKD stages 3 to 5 and 32 (39%) stages 1 or 2 (p < 0.001). Anemia was associated with an increased risk of death one year after discharge (p < 0.001). After adjustment for other factors, a higher hemoglobin level was independently associated with decreased mortality one year after discharge [hazard ratio (95% CI) 0.98 (0.97-1.00)]. CONCLUSIONS: Both CKD and anemia are frequent among stroke patients and are potential risk factors for decreased one-year survival. The inclusion of patients with a first-ever clinical stroke only and the determination of anemia based on one single measure, on admission, constitute limitations to the external validity. We should investigate if an early detection and management of both CKD and anemia could improve survival in stroke patients.

Ubiquitin Ligases of the N-End Rule Pathway: Assessment of Mutations in UBR1 That Cause the Johanson-Blizzard Syndrome.

Background: Johanson-Blizzard syndrome (JBS; OMIM 243800) is an autosomal recessive disorder that includes congenital exocrine pancreatic insufficiency, facial dysmorphism with the characteristic nasal wing hypoplasia, multiple malformations, and frequent mental retardation. Our previous work has shown that JBS is caused by mutations in human UBR1, which encodes one of the E3 ubiquitin ligases of the N-end rule pathway. The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. One class of degradation signals (degrons) recognized by UBR1 are destabilizing N-terminal residues of protein substrates.Methodology/Principal Findings: Most JBS-causing alterations of UBR1 are nonsense, frameshift or splice-site mutations that abolish UBR1 activity. We report here missense mutations of human UBR1 in patients with milder variants of JBS. These single-residue changes, including a previously reported missense mutation, involve positions in the RING-H2 and UBR domains of UBR1 that are conserved among eukaryotes. Taking advantage of this conservation, we constructed alleles of the yeast Saccharomyces cerevisiae UBR1 that were counterparts of missense JBS-UBR1 alleles. Among these yeast Ubr1 mutants, one of them (H160R) was inactive in yeast-based activity assays, the other one (Q1224E) had a detectable but weak activity, and the third one (V146L) exhibited a decreased but significant activity, in agreement with manifestations of JBS in the corresponding JBS patients.Conclusions/Significance: These results, made possible by modeling defects of a human ubiquitin ligase in its yeast counterpart, verified and confirmed the relevance of specific missense UBR1 alleles to JBS, and suggested that a residual activity of a missense allele is causally associated with milder variants of JBS.