The vacuolar transporter chaperone (VTC) complex is required for microautophagy.

Microautophagy involves direct invagination and fission of the vacuolar/lysosomal membrane under nutrient limitation. This occurs by an autophagic tube, a specialized vacuolar membrane invagination that pinches off vesicles into the vacuolar lumen. In this study we have identified the VTC (vacuolar transporter chaperone) complex as required for microautophagy. The VTC complex is present on the ER and vacuoles and at the cell periphery. On induction of autophagy by nutrient limitation the VTC complex is recruited to and concentrated on vacuoles. The VTC complex is inhomogeneously distributed within the vacuolar membranes, showing an enrichment on autophagic tubes. Deletion of the VTC complex blocks microautophagic uptake into vacuoles. The mutants still form autophagic tubes but the production of microautophagic vesicles from their tips is impaired. In line with this, affinity-purified antibodies to the Vtc proteins inhibit microautophagic uptake in a reconstituted system in vitro. Our data suggest that the VTC complex is an important constituent of autophagic tubes and that it is required for scission of microautophagic vesicles from these tubes.

Canakinumab for acute gouty arthritis in patients with limited treatment options: results from two randomised, multicentre, active-controlled, double-blind trials and their initial extensions.

OBJECTIVES: Gouty arthritis patients for whom non-steroidal anti-inflammatory drugs and colchicine are inappropriate have limited treatment options. Canakinumab, an anti-interleukin-1β monoclonal antibody, may be an option for such patients. The authors assessed the efficacy/safety of one dose of canakinumab 150 mg (n=230) or triamcinolone acetonide (TA) 40 mg (n=226) at baseline and upon a new flare in frequently flaring patients contraindicated for, intolerant of, or unresponsive to non-steroidal anti-inflammatory drugs and/or colchicine. Core study co-primary endpoints were pain intensity 72 h postdose (0-100 mm visual analogue scale and time to first new flare. METHODS: Two 12-week randomised, multicentre, active-controlled, double-blind, parallel-group core studies with double-blind 12-week extensions (response in acute flare and in prevention of episodes of re-flare in gout (β-RELIEVED and β-RELIEVED-II)). RESULTS: 82.6% patients had comorbidities. Mean 72-h visual analogue scale pain score was lower with canakinumab (25.0 mm vs 35.7 mm; difference, -10.7 mm; 95% CI -15.4 to -6.0; p<0.0001), with significantly less physician-assessed tenderness and swelling (ORs=2.16 and 2.74; both p≤0.01) versus TA. Canakinumab significantly delayed time to first new flare, reduced the risk of new flares by 62% versus TA (HR: 0.38; 95% CI 0.26 to 0.57) in the core studies and by 56% (HR: 0.44; 95% CI 0.32 to 0.60; both p≤0.0001) over the entire 24-week period, and decreased median C-reactive protein levels (p≤0.0001 at 72 h and 7 days). Over the 24-week period, adverse events were reported in 66.2% (canakinumab) and 52.8% (TA) and serious adverse events were reported in 8.0% (canakinumab) and 3.5% (TA) of patients. Adverse events reported more frequently with canakinumab included infections, low neutrophil count and low platelet count. CONCLUSION: Canakinumab provided significant pain and inflammation relief and reduced the risk of new flares in these patients with acute gouty arthritis.

Anti-TNF therapy in acute sciatica reduces the long-term need for surgery: A three-year follow-up of a randomized double blind placebo controlled trial

Introduction: Two subcutaneous injections of adalimumab in severeacute sciatica have demonstrated a significant benefit on the numberof back surgeries in a short-term randomized controlled clinical trial[1]. This 3-year follow-up study aimed to determine whether theshort-term benefit was sustained over a longer period of time.Methods: Information on surgery was retrieved in 56/61 patients(93%). We used a Cox proportional hazard models to determinefactors predisposing to surgery.Results: Twenty-three (41%) patients had back surgery within 3 years,8/29 (28%) in the adalimumab group and 15/ 27 (56%) in the placebogroup, p = 0.038. Adalimumab injections reduced the need for backsurgery by 61% (Hazard Ratio (HR): 0.39 (95% CI: 0.17-0.92). In amultivariate model, treatment with a TNF-α antagonist remained thestrongest protective factor (HR 0.17, p = 0.002). Other significantpredictors of surgery were a good correlation between symptomsand MRI findings (HR = 11.6, p = 0.04), baseline intensity of leg pain(HR = 1.3, p = 0.06), intensity of back pain (HR = 1.4, p = 0.03)and duration of sickness leave (HR = 1.01 per day, p = 0.03).Conclusion: A short course of adalimumab in patients with severeacute sciatica significantly reduces the need for back surgery.

How to keep the brain awake? The complex molecular pharmacogenetics of wake promotion.

Wake-promoting drugs are widely used to treat excessive daytime sleepiness. The neuronal pathways involved in wake promotion are multiple and often not well characterized. We tested d-amphetamine, modafinil, and YKP10A, a novel wake-promoting compound, in three inbred strains of mice. The wake duration induced by YKP10A and d-amphetamine depended similarly on genotype, whereas opposite strain differences were observed after modafinil. Electroencephalogram (EEG) analysis during drug-induced wakefulness revealed a transient approximately 2 Hz slowing of theta oscillations and an increase in beta-2 (20-35 Hz) activity only after YKP10A. Gamma activity (35-60 Hz) was induced by all drugs in a drug- and genotype-dependent manner. Brain transcriptome and clustering analyses indicated that the three drugs have both common and specific molecular signatures. The correlation between specific EEG and gene-expression signatures suggests that the neuronal pathways activated to stay awake vary among drugs and genetic background.

Identification and characterization of a caspase-2 activating complex

Résumé Les caspases sont des protéases essentielles lors de l'induction de l'apoptose ou pour la maturation de certaines cytokines. Elles peuvent être divisées en deux groupes: les caspases initiatrices, qui sont les premières activées lors d'un signal pro-apoptotique, et les caspases effectrices, qui sont activées par les caspases initiatrices et sont responsables du clivage et de la dégradation des substrats cellulaires. Les caspases initiatrices sont activées dans des complexes de haut poids moléculaire: l'apoptosome pour la caspase-9 et le DISC pour la caspase-8. La caspase-2 est également une caspase initiatrice qui contient un domaine CARD. Cependant son mécanisme d'activation n'est pas encore connu. Lors de cette étude, nous avons découvert et caractérisé le complexe qui permet l'activation de la caspase-2. Ce complexe, appelé le PIDDosome, est composé de PIDD/LRDD, de la protéine adaptatrice RAIDD et de la protéase caspase-2. L'expression forcée de PIDD induit l'activation constitutive de la caspase-2. Cela entraîne la mort ou la sensibilisation à la mort des cellules selon la lignée étudiée. Cet effet est expliqué par une perte du potentiel de membrane de la mitochondrie, certainement dû à un effet direct de la caspase-2. Peu de choses sont connues sur PIDD: c'est une protéine contenant un domaine DD qui peut être induite par p53. Nous avons caractérisé PIDD et montré qu'elle est exprimée de façon ubiquitaire. PIDD est constitutivement auto-clivée environ au milieu de la protéine, ce qui génère deux fragments qui restent liés l'un à l'autre. Le fragment N-terminal a une activité régulatrice et le C-terminal une activité effectrice. De plus, PIDD peut se déplacer entre le cytoplasme et le noyau. Enfin, nous avons découvert que PIDD est également impliquée dans l'induction de NF¬ -κB en réponse à des dommages à l'ADN. PIDD est responsable de la modification par sumo de NEMO, étape nécessaire à l'induction de NF-κB après des dommages à l'ADN. Ainsi PIDD semble être à l'intersection de la décision que prend la cellule entre survivre et réparer les dommages, ou entrer en apoptose. Summary Caspases are a family of proteases that fulfill varied and often critical roles in mammalian apoptosis or proteolytic activation of cytokines. Caspases can be divided into two sub-groups: initiator caspases, which are the first activated after a pro-apoptotic signal, and effector caspases, which are activated by initiator caspases and that are responsible for the cleavage and degradation of cellular components. Initiator caspases are activated in high molecular weight platforms such as the apoptosome for caspase-9 or the DISC for caspase-8. Caspase-2 is a CARD-containing initiator caspase whose mechanism of activation was not yet known. In this study we have identified an activating platform for caspase-2. This high molecular weight complex, called the PIDDosome, is composed of PIDD/LRDD, the adaptor protein RAIDD and caspase-2. Constitutive expression of PIDD led to constitutive activation of caspase-2, which in some cell lines was sufficient to induce cell death while in others it merely sensitizes. Active caspase-2 was found to disturb directly the mitochondria by inducing a partial loss of the transmembrane potential. Very little was known on PIDD. It can be induce by p53 and inhibition of its expression by antisense oligonucleotides diminishes p53-dependent apoptosis. We decided to further characterize PIDD function and expression. PIDD possesses seven LRR, two Zu5 domains and one DD. It is ubiquitously expressed and appears to be constitutively cleaved by auto- processing into two main fragments equal in size. The two fragments remain bound to one another and constitute a regulatory N-terminal fragment and an active C-terminal fragment. In addition, PIDD can shuttle between the cytoplasm and the nucleus. Finally, investigating the possible relevance of new interaction partners, we found that PIDD is implicated in DNA damage-induced NF- κB. PIDD binds to RIP1 and to NEMO. In response to DNA damage, PIDD translocates to the nucleus and mediates sumo- modification of NEMO, a necessary step in DNA damage-induced NF-κB. All together these results raise the possibility that PIDD acts as a molecular switch between proliferation and repair, and apoptosis following DNA damage.

Two mechanisms of caspase 9 processing in double-stranded RNA- and virus-triggered apoptosis.

Viral double-stranded RNA (dsRNA) is a ubiquitous intracellular "alert signal" used by cells to detect viral infection and to mount anti-viral responses. DsRNA triggers a rapid (complete within 2-4 h) apoptosis in the highly-susceptible HeLa cell line. Here, we demonstrate that the apical event in this apoptotic cascade is the activation of procaspase 8. Downstream of caspase 8, the apoptotic signaling cascade bifurcates into a mitochondria-independent caspase 8/caspase 3 arm and a mitochondria-dependent, caspase 8/Bid/Bax/Bak/cytochrome c arm. Both arms impinge upon, and activate, procaspase 9 via two different cleavage sites within the procaspase 9 molecule (D330 and D315, respectively). This is the first in vivo demonstration that the "effector" caspase 3 plays an "initiator" role in the regulation of caspase 9. The dsRNA-induced apoptosis is potentiated by the inhibition of protein synthesis, whose role is to accelerate the execution of all apoptosis steps downstream of, and including, the activation of caspase 8. Thus, efficient apoptosis in response to viral dsRNA results from the co-operation of the two major apical caspases (8 and 9) and the dsRNA-activated protein kinase R (PKR)/ribonuclease L (RNase L) system that is essential for the inhibition of protein synthesis in response to viral infection.

Pyochelin-mediated signalling in Pseudomonas aeruginosa

SUMMARY: Iron is an essential element for nearly all organisms but it is poorly available in most environments and not sufficient to support microbial growth. Bacteria have evolved a range of strategies to acquire this important metal, the most common of these being siderophore-mediated iron uptake. Siderophores are high-affinity iron chelators which are released to the extracellular environment where they complex iron and deliver it to the bacterial cell, via specific uptake systems. The Gram-negative bacterium Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, which both contribute to the virulence of this opportunistic human pathogen. The genes responsible for pyochelin-mediated iron uptake are grouped in the P. aeruginosa chromosome. The pyochelin biosynthetic genes are organized in two divergent operons, pchDCBA and pchEFGHI, which flank the regulatory gene pchR. The fptA gene, encoding the ferric pyochelin outer membrane receptor, occurs immediately downstream of the pchEFGHI genes. The biosynthesis of the siderophore and its receptor is subjected to dual regulation enabling P. aeruginosa to respond not only to the intracellular iron level but also to the presence of the siderophore in the extracellular environment. Negative regulation is mediated by the widespread Fur protein which employs ferrous iron as a corepressor and binds to a consensus sequence in the promoter region of iron-regulated genes. Positive regulation occurs during iron starvation and requires the AraC-type transcriptional regulator PchR. This regulator, together with pyochelin, induces the expression of pyochelin biosynthesis and uptake genes via a mechanism which was partly unraveled during this thesis. A 32-bp conserved sequence element (PchR-box) was identified in promoter regions of pyochelin-controlled genes. The PchR-box in the pchR-pchDCBA intergenic region was found to be essential for the induction of the pchDCBA operon and for the repression of the divergently transcribed pchR gene. PchR was purified as a fusion with maltose-binding protein (MBP). Mobility shift assays demonstrated specific binding of MBP-PchR to the PchR-box in the presence, but not in the absence of pyochelin. PchR-box mutations which interfered with pyochelin-dependent regulation in vivo, also affected pyochelin-dependent PchR-box recognition in vitro. These results show that pyochelin is the intracellular effector required for PchR-mediated regulation. The fact that extracellular pyochelin triggers this regulation implies that the siderophore can enter the cytoplasm. This conclusion was corroborated by analysing the importance of known and putative pyochelin uptake genes for pyochelin-dependent gene regulation. The pyochelin receptor gene fptA is followed by three genes, fptB, fptC, and fptX, which were shown here to be co-transcribed with fPtA. While fPtX encodes an inner membrane pen-I-lease, the functions of FptB and FptC are currently unknown. FptA and FptX, which are both required for pyochelin-mediated iron uptake, were found to be also needed for pyochelin-dependent gene regulation. FptB and FptC however, were not required and their role, if any, in the uptake of the PchR effector pyochelin remains elusive. RESUME Le fer est un élément essentiel pour la quasi-totalité des organismes, mais dans la plupart des environnements, il est difficilement accessible et insuffisant à la croissance microbienne. Les bactéries ont développé de multiples stratégies pour acquérir ce précieux métal, la plus commune étant l'acquisition au moyen de sidérophores. Les sidérophores sont des petites molécules dotées d'une forte affinité pour le fer qui, une fois relâchées dans l'environnement extracellulaire, vont complexer le fer et le délivrer à la cellule bactérienne par l'intermédiaire de systèmes d'acquisition spécifiques. La bactérie Gram-négative Pseudomonas aeruginosa produit deux sidérophores, la pyoverdine et la pyochéline, qui contribuent également à la virulence de ce pathogène opportuniste. Les gènes impliqués dans l'acquisition du fer à l'aide de la pyochéline sont regroupés sur t. le chromosome de P. aeruginosa. Les gènes de biosynthèse de la pyochéline sont organisés en deux opérons divergents, pchDCBA et pchEFGHI, qui flanquent le gène régulateur pchR. Le gène fptA, codant pour le récepteur de la pyochéline dans la membrane externe, est situé immédiatement en aval des gènes pchEFGHL La biosynthèse du sidérophore et de son récepteur est soumise à une double régulation permettant à P. aeruginosa de réagir non seulement à la quantité de fer intracellulaire, mais également à la présence du sidérophore dans le milieu extracellulaire. La répression se fait par l'intermédiaire de la protéine Fur, qui nécessite le fer ferreux comme co-répresseur et se lie à une séquence consensus dans la région promotrice des gènes régulés par le fer. L'induction se produit lorsque le fer est limitant, et requiert PchR, un régulateur transcriptionnel de la famille AraC. En présence de pyochéline, ce régulateur induit l'expression des gènes de biosynthèse et du récepteur de la pyochéline par l'intermédiaire d'un mécanisme partiellement résolu dans ce travail. Une séquence conservée (PchR-box) a été identifiée dans la région promotrice des gènes régulés par la pyochéline. La PchR-box située dans la région intergénique pchR-pchDCBA s'est révélée être importante pour l'induction de l'opéron pchDCBA et la répression du gène divergent pchR. PchR a été purifiée en tant que protéine de fusion avec une protéine liant le maltose (MBP). Des expériences de gel retard ont démontré la liaison spécifique de la protéine MBP-PchR sur la PchR-box en présence, mais non en absence de pyochéline. Les mutations de la PchR-box qui ont affecté la régulation pyochéline-dépendante in vivo, ont également eu un effet sur la liaison de la protéine in vitro. Ces résultats démontrent que la pyochéline est l'effecteur intracellulaire nécessaire à la régulation par PchR. Le fait que la pyochéline extracellulaire soit capable d'activer cette régulation implique que le sidérophore entre dans le cytoplasme. Cette conclusion a été corroborée par l'évaluation du rôle des gènes connus ou putatifs de l'incorporation du fer via la pyochéline sur la régulation pyochéline-dépendente. Le gène fPtA, codant pour le récepteur de la pyochéline, est suivi de trois gènes, fptB,fptC, et fptX, co-transcrits avec,ffitA. Si sffitX code pour une perméase de la membrane interne, la fonction de FptB et FptC reste obscure. FptA et FptX, nécessaires à l'acquisition du fer par l'intermédiaire de la pyochéline, se sont également révélés être requis pour la régulation pyochéline-dépendante des gènes pchDCBA, pchEFGHI et fptABCX. FptB et FptC n'ont quant à eux vraisemblablement pas de rôle majeur à jouer, si ce n'est aucun, dans l'incorporation de la pyochéline.

An HMG-box-containing T-cell factor required for thymocyte differentiation.

Two candidate genes for controlling thymocyte differentiation, T-cell factor-1 (Tcf-1) and lymphoid enhancer-binding factor (Lef-1), encode closely related DNA-binding HMG-box proteins. Their expression pattern is complex and largely overlapping during embryogenesis, yet restricted to lymphocytes postnatally. Here we generate two independent germline mutations in Tcf-1 and find that thymocyte development in (otherwise normal) mutant mice is blocked at the transition from the CD8+, immature single-positive to the CD4+/CD8+ double-positive stage. In contrast to wild-type mice, most of the immature single-positive cells in the mutants are not in the cell cycle and the number of immunocompetent T cells in peripheral lymphoid organs is reduced. We conclude that Tcf-1 controls an essential step in thymocyte differentiation.

Function of TRADD in tumor necrosis factor receptor 1 signaling and in TRIF-dependent inflammatory responses.

Tumor necrosis factor receptor 1 (TNFR1) and Toll-like receptors (TLRs) regulate immune and inflammatory responses. Here we show that the TNFR1-associated death domain protein (TRADD) is critical in TNFR1, TLR3 and TLR4 signaling. TRADD deficiency abrogated TNF-induced apoptosis, prevented recruitment of the ubiquitin ligase TRAF2 and ubiquitination of the adaptor RIP1 in the TNFR1 signaling complex, and considerably inhibited but did not completely abolish activation of the transcription factor NF-kappaB and mitogen-activated protein kinases 'downstream' of TNFR1. TRIF-dependent cytokine production induced by the synthetic double-stranded RNA poly(I:C) and lipopolysaccharide was lower in TRADD-deficient mice than in wild-type mice. Moreover, TRADD deficiency inhibited poly(I:C)-mediated RIP1 ubiquitination and activation of NF-kappaB and mitogen-activated protein kinase signaling in fibroblasts but not in bone marrow macrophages. Thus, TRADD is an essential component of TNFR1 signaling and has a critical but apparently cell type-specific function in TRIF-dependent TLR responses.

Mapping complex tissue architecture with diffusion spectrum magnetic resonance imaging.

Methods are presented to map complex fiber architectures in tissues by imaging the 3D spectra of tissue water diffusion with MR. First, theoretical considerations show why and under what conditions diffusion contrast is positive. Using this result, spin displacement spectra that are conventionally phase-encoded can be accurately reconstructed by a Fourier transform of the measured signal's modulus. Second, studies of in vitro and in vivo samples demonstrate correspondence between the orientational maxima of the diffusion spectrum and those of the fiber orientation density at each location. In specimens with complex muscular tissue, such as the tongue, diffusion spectrum images show characteristic local heterogeneities of fiber architectures, including angular dispersion and intersection. Cerebral diffusion spectra acquired in normal human subjects resolve known white matter tracts and tract intersections. Finally, the relation between the presented model-free imaging technique and other available diffusion MRI schemes is discussed.