CHARACTERIZATION OF NrsZ, A NOVEL SMALL REGULATORY NON-CODING RNA INVOLVED IN THE ADAPTATION OF PSEUDOMONAS AERUGINOSA PAO1

The opportunistic ubiquitous pathogen Pseudomonas aeruginosa strain PAOl is a versatile Gram-negative bacterium that has the extraordinary capacity to colonize a wide diversity of ecological niches and to cause severe and persistent infections in humans. To ensure an optimal coordination of the genes involved in nutrient utilization, this bacterium uses the NtrB/C and/or the CbrA/B two-component systems, to sense nutrients availability and to regulate in consequence the expression of genes involved in their uptake and catabolism. NtrB/C is specialized in nitrogen utilization, while the CbrA/B system is involved in both carbon and nitrogen utilization and both systems activate their target genes expression in concert with the alternative sigma factor RpoN. Moreover, the NtrB/C and CbrA/B two- component systems regulate the secondary metabolism of the bacterium, such as the production of virulence factors. In addition to the fine-tuning transcriptional regulation, P. aeruginosa can rapidly modulate its metabolism using small non-coding regulatory RNAs (sRNAs), which regulate gene expression at the post-transcriptional level by diverse and sophisticated mechanisms and contribute to the fast physiological adaptability of this bacterium. In our search for novel RpoN-dependent sRNAs modulating the nutritional adaptation of P. aeruginosa PAOl, we discovered NrsZ (Nitrogen regulated sRNA), a novel RpoN-dependent sRNA that is induced under nitrogen starvation by the NtrB/C two-component system. NrsZ has a unique architecture, formed of three similar stem-loop structures (SL I, II and II) separated by variant spacer sequences. Moreover, this sRNA is processed in short individual stem-loop molecules, by internal cleavage involving the endoribonuclease RNAse E. Concerning NrsZ functions in P. aeruginosa PAOl, this sRNA was shown to trigger the swarming motility and the rhamnolipid biosurfactants production. This regulation is due to the NrsZ-mediated activation of rhlA expression, a gene encoding for an enzyme essential for swarming motility and rhamnolipids production. Interestingly, the SL I structure of NrsZ ensures its regulatory function on rhlA expression, suggesting that the similar SLs are the functional units of this modular sRNA. However, the regulatory mechanism of action of NrsZ on rhlA expression activation remains unclear and is currently being investigated. Additionally, the NrsZ regulatory network was investigated by a transcriptome analysis, suggesting that numerous genes involved in both primary and secondary metabolism are regulated by this sRNA. To emphasize the importance of NrsZ, we investigated its conservation in other Pseudomonas species and demonstrated that NrsZ is conserved and expressed under nitrogen limitation in Pseudomonas protegens Pf-5, Pseudomonas putida KT2442, Pseudomonas entomophila L48 and Pseudomonas syringae pv. tomato DC3000, strains having different ecological features, suggesting an important role of NrsZ in the adaptation of Pseudomonads to nitrogen starvation. Interestingly the architecture of the different NrsZ homologs is similarly composed by SL structures and variant spacer sequences. However, the number of SL repetitions is not identical, and one to six SLs were predicted on the different NrsZ homologs. Moreover, NrsZ is processed in short molecules in all the strains, similarly to what was previously observed in P. aeruginosa PAOl, and the heterologous expression of the NrsZ homologs restored rhlA expression, swarming motility and rhamnolipids production in the P. aeruginosa NrsZ mutant. In many aspects, NrsZ is an atypical sRNA in the bacterial panorama. To our knowledge, NrsZ is the first described sRNA induced by the NtrB/C. Moreover, its unique modular architecture and its processing in similar short SL molecules suggest that NrsZ belongs to a novel family of bacterial sRNAs. -- L'agent pathogène opportuniste et ubiquitaire Pseudomonas aeruginosa souche PAOl est une bactérie Gram négative versatile ayant l'extraordinaire capacité de coloniser différentes niches écologiques et de causer des infections sévères et persistantes chez l'être humain. Afin d'assurer une coordination optimale des gènes impliqués dans l'utilisation de différents nutriments, cette bactérie se sert de systèmes à deux composants tel que NtrB/C et CbrA/B afin de détecter la disponibilité des ressources nutritives, puis de réguler en conséquence l'expression des gènes impliqués dans leur importation et leur catabolisme. Le système NtrB/C régule l'utilisation des sources d'azote alors que le système CbrA/B est impliqué à la fois dans l'utilisation des sources de carbone et d'azote. Ces deux systèmes activent l'expression de leurs gènes-cibles de concert avec le facteur sigma alternatif RpoN. En outre, NtrB/C et CbrA/B régulent aussi le métabolisme secondaire, contrôlant notamment la production d'importants facteurs de virulence. En plus de toutes ces régulations génétiques fines ayant lieu au niveau transcriptionnel, P. aeruginosa est aussi capable de moduler son métabolisme en se servant de petits ARNs régulateurs non-codants (ARNncs), qui régulent l'expression génétique à un niveau post- transcriptionnel par divers mécanismes sophistiqués et contribuent à rendre particulièrement rapide l'adaptation physiologique de cette bactérie. Au cours de nos recherches sur de nouveaux ARNncs dépendant du facteur sigma RpoN et impliqués dans l'adaptation nutritionnelle de P. aeruginosa PAOl, nous avons découvert NrsZ (Nitrogen regulated sRNA), un ARNnc induit par la cascade NtrB/C-RpoN en condition de carence en azote. NrsZ a une architecture unique, composée de trois structures en tige- boucle (TB I, II et III) hautement similaires et séparées par des « espaceurs » ayant des séquences variables. De plus, cet ARNnc est clivé en petits fragments correspondant au trois molécules en tige-boucle, par un processus de clivage interne impliquant l'endoribonucléase RNase E. Concernant les fonctions de NrsZ chez P. aeruginosa PAOl, cet ARNnc est capable d'induire la motilité de type « swarming » et la production de biosurfactants, nommés rhamnolipides. Cette régulation est due à l'activation par NrsZ de l'expression de rhlA, un gène essentiel pour la motilité de type swarming et pour la production de rhamnolipides. Étonnamment, la structure TB I est capable d'assurer à elle seule la fonction régulatrice de NrsZ sur l'expression de rhlA, suggérant que ces molécules TBs sont les unités fonctionnelles de cet ARNnc modulaire. Cependant, le mécanisme moléculaire par lequel NrsZ active l'expression de rhlA demeure à ce jour incertain et est actuellement à l'étude. En plus, le réseau de régulations médiées par NrsZ a été étudié par une analyse de transcriptome qui a indiqué que de nombreux gènes impliqués dans le métabolisme primaire ou secondaire seraient régulés par NrsZ. Pour accentuer l'importance de NrsZ, nous avons étudié sa conservation dans d'autres espèces de Pseudomonas. Ainsi, nous avons démontré que NrsZ est conservé et exprimé en situation de carence d'azote par les souches Pseudomonas protegens Pf-5, Pseudomonas putida KT2442, Pseudomonas entomophila L48, Pseudomonas syringae pv. tomato DC3000, quatre espèces ayant des caractéristiques écologiques très différentes, suggérant que NrsZ joue un rôle important dans l'adaptation du genre Pseudomonas envers la carence en azote. Chez toutes les souches étudiées, les différents homologues de NrsZ présentent une architecture similaire faite de TBs conservées et d'espaceurs. Cependant, le nombre de TBs n'est pas identique et peut varier de une à six copies selon la souche. Les différentes versions de NrsZ sont clivées en petites molécules dans ces quatre souches, comme il a été observé chez P. aeruginosa PAOl. De plus, l'expression hétérologue des différentes variantes de NrsZ est capable de restaurer l'expression de rhlA, la motilité swarming et la production de rhamnolipides dans une souche de P. aeruginosa dont nrsZ a été inactivé. Par bien des aspects, NrsZ est un ARNnc atypique dans le monde bactérien. À notre connaissance, NrsZ est le premier ARNnc décrit comme étant régulé par le système NtrB/C. De plus, son unique architecture modulaire et son clivage en petites molécules similaires suggèrent que NrsZ appartient à une nouvelle famille d'ARNncs bactériens.

Resting-brain functional connectivity predicted by analytic measures of network communication.

The complex relationship between structural and functional connectivity, as measured by noninvasive imaging of the human brain, poses many unresolved challenges and open questions. Here, we apply analytic measures of network communication to the structural connectivity of the human brain and explore the capacity of these measures to predict resting-state functional connectivity across three independently acquired datasets. We focus on the layout of shortest paths across the network and on two communication measures-search information and path transitivity-which account for how these paths are embedded in the rest of the network. Search information is an existing measure of information needed to access or trace shortest paths; we introduce path transitivity to measure the density of local detours along the shortest path. We find that both search information and path transitivity predict the strength of functional connectivity among both connected and unconnected node pairs. They do so at levels that match or significantly exceed path length measures, Euclidean distance, as well as computational models of neural dynamics. This capacity suggests that dynamic couplings due to interactions among neural elements in brain networks are substantially influenced by the broader network context adjacent to the shortest communication pathways.

Citrus fruit and cancer risk in a network of case-control studies.

BACKGROUND: Citrus fruit has shown a favorable effect against various cancers. To better understand their role in cancer risk, we analyzed data from a series of case-control studies conducted in Italy and Switzerland. PATIENTS AND METHODS: The studies included 955 patients with oral and pharyngeal cancer, 395 with esophageal, 999 with stomach, 3,634 with large bowel, 527 with laryngeal, 2,900 with breast, 454 with endometrial, 1,031 with ovarian, 1,294 with prostate, and 767 with renal cell cancer. All cancers were incident and histologically confirmed. Controls were admitted to the same network of hospitals for acute, nonneoplastic conditions. Odds ratios (OR) were estimated by multiple logistic regression models, including terms for major identified confounding factors for each cancer site, and energy intake. RESULTS: The ORs for the highest versus lowest category of citrus fruit consumption were 0.47 (95% confidence interval, CI, 0.36-0.61) for oral and pharyngeal, 0.42 (95% CI, 0.25-0.70) for esophageal, 0.69 (95% CI, 0.52-0.92) for stomach, 0.82 (95% CI, 0.72-0.93) for colorectal, and 0.55 (95% CI, 0.37-0.83) for laryngeal cancer. No consistent association was found with breast, endometrial, ovarian, prostate, and renal cell cancer. CONCLUSIONS: Our findings indicate that citrus fruit has a protective role against cancers of the digestive and upper respiratory tract.

Molecular players shaping the arbuscular mycorrhizal symbiosis of rice

SUMMARY : The arbuscular mycorrhizal (AM) symbiosis is an evolutionarily ancient association between most land plants and Glomeromycotan fungi that is based on the mutual exchange of nutrients between the two partners. Its structural and physiological establishment is a multi-step process involving a tightly regulated signal exchange leading to intracellular colonization of roots by the fungi. Most research on the molecular biology and genetics of symbiosis development has been performed in dicotyledonous model legumes. In these, a plant signaling pathway, the common SYM pathway, has been found to be required for accommodation of both root symbionts rhizobia and AM fungi. Rice, a monocotyledon model and the world's most important staple crop also forms AM symbioses, has been largely ignored for studies of the AM symbiosis. Therefore in this PhD work functional conservation of the common SYM pathway in rice was addressed and demonstrated. Mycorrhiza-specific marker genes were established that are expressed at different stages of AM development and therefore represent readouts for various AM-specific signaling events. These tools were successfully used to obtain evidence for a yet unknown signaling network comprising common SYM-dependent and -independent events. In legumes AM colonization induces common SYM signaling dependent changes in root system architecture. It was demonstrated that also in rice, root system architecture changes in response to AM colonization but these alterations occur independently of common SYM signaling. The rice root system is complex and contains three different root types. It was shown that root type identity influences the quantity of AM colonization, indicating root type specific symbiotic properties. Interestingly, the root types differed in their transcriptional responses to AM colonization and the less colonized root type responded more dramatically than the more strongly colonized root type. Finally, in an independent project a novel mutant, inhospitable (iho), was discovered. It is perturbed at the most early step of AM colonization, namely differentiation of the AM fungal hyphae into a hyphopodium at the root surface. As plant factors required for this early step are not known, identification of the IHO gene will greatly contribute to the advance of mycorrhiza RÉSUMÉ : La symbiose mycorhizienne arbusculaire (AM) est une association évolutionnairement ancienne entre la majorité des plantes terrestres et les champignons du type Glomeromycota, basée sur l'échange mutuel d'éléments nutritifs entre les deux partenaires. Son établissement structural et physiologique est un processus en plusieurs étapes, impliquant des échanges de signaux étroitement contrôlés, aboutissant à la colonisation intracellulaire des racines par le champignon. La plupart des recherches sur la biologie moléculaire et la génétique du développement de la symbiose ont été effectuées sur des légumineuses dicotylédones modèles. Dans ces dernières, une voie de signalisation, la voie SYM, s'est avérée nécessaire pour permettre la mise en place de la symbiose mycorhizienne. Chez les plantes monocotylédones, comme le riz, une des céréales les plus importantes, nourrissant la moitié de la population mondiale, peu de recherches ont été effectuées sur les bases de la cette symbiose. Dans ce travail de thèse, la conservation fonctionnelle de la voie commune SYM chez le riz a été étudiée et démontrée. De plus, des gènes marqueurs spécifiques des différentes étapes du développement de l'AM ont été identifiés, permettant ainsi d'avoir des traceurs de la colonisation. Ces outils ont été utilisés avec succès pour démontrer l'existence d'un nouveau réseau de signalisation, comprenant des éléments SYM dépendant et indépendant. Chez les légumineuses, la colonisation par les AM induit des changements dans l'architecture du système racinaire, via la signalisation SYM dépendantes. Cependant chez le riz, il a été démontré que l'architecture de système racinaire changeait suite à la colonisation de l'AM, mais ceux, de façon SYM indépendante. Le système racinaire du riz est complexe et contient trois types différents de racines. Il a été démontré que le type de racine pouvait influencer l'efficacité de la colonisation par l'AM, indiquant que les racines ont des propriétés symbiotiques spécifiques différentes. De façon surprenante, les divers types de racines répondent de différemment suite à colonisation par l'AM avec des changements de la expression des gènes. Le type de racine le moins colonisé, répondant le plus fortement a la colonisation, et inversement. En parallèle, dans un projet indépendant, un nouveau mutant, inhospitable (iho), a été identifié. Ce mutant est perturbé lors de l'étape la plus précoce de la colonisation par l'AM, à savoir la différentiation des hyphes fongiques de l'AM en hyphopodium, à la surface des racines. Les facteurs d'origine végétale requis pour cette étape étant encore inconnus, l'identification du gène IHO contribuera considérablement a accroître nos connaissance sur les bases de la mise en place de cette symbiose.

Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle.

Plants constantly adjust their repertoire of plasma membrane proteins that mediates transduction of environmental and developmental signals as well as transport of ions, nutrients, and hormones. The importance of regulated secretory and endocytic trafficking is becoming increasingly clear; however, our knowledge of the compartments and molecular machinery involved is still fragmentary. We used immunogold electron microscopy and confocal laser scanning microscopy to trace the route of cargo molecules, including the BRASSINOSTEROID INSENSITIVE1 receptor and the REQUIRES HIGH BORON1 boron exporter, throughout the plant endomembrane system. Our results provide evidence that both endocytic and secretory cargo pass through the trans-Golgi network/early endosome (TGN/EE) and demonstrate that cargo in late endosomes/multivesicular bodies is destined for vacuolar degradation. Moreover, using spinning disc microscopy, we show that TGN/EEs move independently and are only transiently associated with an individual Golgi stack.

Outcome and prognostic factors in olfactory neuroblastoma: a rare cancer network study.

Purpose: To assess the outcome in patients with olfactory neuroblastoma (ONB). Methods and Materials: Seventy-seven patients treated for nonmetastatic ONB between 1971 and 2004 were included. According to Kadish classification, there were 11 patients with Stage A, 29 with Stage B, and 37 with Stage C. T-classification included 9 patients with T1, 26 with T2, 16 with T3, 15 with T4a, and 11 with T4b tumors. Sixty-eight patients presented with N0 (88%) disease. Results: Most of the patients (n = 56, 73 %) benefited from surgery (S), and total excision was possible in 44 patients (R0 in 32, R1 in 13, R2 in 11). All but five patients benefited from RT, and chemotherapy was given in 21(27%). Median follow-up period was 72 months (range, 6-315). The 5-year overall survival (OS), disease-free survival (DES), locoregional control, and local control were 64%, 57%, 62%, and 70%, respectively. In univariate analyses, favorable factors were Kadish A or B disease, T1 T3 tumors, no nodal involvement, curative surgery, R0/R1 resection, and RT-dose 54 Gy or higher. Multivariate analysis revealed that the best independent factors predicting the outcome were T1 T3, N0, R0/R1 resection, and total RT dose (54 Gy or higher). Conclusion: In this multicenter retrospective study, patients with ONB treated with R0 or R1 surgical resection followed by at least 54-Gy postoperative RT had the best outcome. Novel strategies including concomitant chemotherapy and/or higher dose RT should be prospectively investigated in this rare disease for which local failure remains a problem.

Early stage primary bone lymphoma - a retrospective, multicenter rare cancer network (rcn) study

Purpose: Primary bone lymphoma (PBL) accounts for less than 1% of all malignant lymphomas, and 4-5% of all extra-nodal lymphomas. In this study, the disease profile, outcome, and prognostic factors were assessed in patients with stage I and II PBL.Patients and Methods: Thirteen Rare Cancer Network (RCN) institutions enrolled 116 consecutive patients with PBL treated between 1987 and 2008 in this study. Inclusion criteria were age > 16 years, stage I and II, minimum 6 months follow-up and a biopsy-proven confirmation of non-Hodgkin's lymphoma (NHL). Eighty-seven patients underwent chemoradiotherapy (CXRT), 15 radiotherapy (RT) without (13) or with (2) surgery, 14 chemotherapy (CXT) without (9) or with (5) surgery. Median RT dose was 40 Gy (range: 4-60). The median number of CXT cycles was 6 (range: 2-8). Median follow-up was 41 months (range: 6-242).Results: The overall response rate at the end of treatment was 91% (CR 74%, PR 17%). Local recurrence or progression was observed in 12 (10%) patients, and systemic recurrence in 17 (15%). Causes of death included disease progression in 21, unrelated in 5, CXT-related toxicity in 1, and second primary cancer in 2 patients. The 5-yr overall survival (OS), lymphoma-specific survival (LSS), and local control (LC) were 76%, 78% and 92%, respectively. In univariate analyses (log-rank test), favorable prognostic factors for OS were age <50 years (P=0.008), international prognostic index (IPI) score ≤1 (P=0.009), high grade histology (P=0.04), CXRT (P=0.05), CXT (P=0,0004), complete response (CR) (P<0.0001), number of CXT cycles ( ≥6 ) (P=0.01), and RT dose > 40 Gy (P=0.005). All above-mentioned parameters were also significant for LSS except for age and number of chemotherapy cycles. For LC, only CR and stage I were favorable factors. In multivariate analysis, IPI score, RT dose, complete response, and chemotherapy were independently influencing the outcome (OS and LSS). Complete response at the end of treatment was the only predicting factor for LC. Six patients developed grade 3 or more toxicities, according to Common Terminology Criteria for Adverse Events (CTCAE) V3.0.Conclusion: This large multicenter study confirms the relatively good prognosis of early stage PBL treated with combined CXRT. Local control was excellent, while systemic failures were rare. An adequate dose of RT (40 Gy or more) and complete CXT regime (≥ 6 cycles) were associated with better outcome.