Combined low initial DNA damage and high radiation-induced apoptosis confers clinical resistance to long-term toxicity in breast cancer patients treated with high-dose radiotherapy

BACKGROUND. Either higher levels of initial DNA damage or lower levels of radiation-induced apoptosis in peripheral blood lymphocytes have been associated to increased risk for develop late radiation-induced toxicity. It has been recently published that these two predictive tests are inversely related. The aim of the present study was to investigate the combined role of both tests in relation to clinical radiation-induced toxicity in a set of breast cancer patients treated with high dose hyperfractionated radical radiotherapy. METHODS. Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma treated with high-dose hyperfractioned radical radiotherapy. Acute and late cutaneous and subcutaneous toxicity was evaluated using the Radiation Therapy Oncology Group morbidity scoring schema. The mean follow-up of survivors (n = 13) was 197.23 months. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radiation-induced apoptosis (RIA) at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. RESULTS. Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). Radiation-induced apoptosis increased with radiation dose (median 12.36, 17.79 and 24.83 for 1, 2, and 8 Gy respectively). We observed that those "expected resistant patients" (DSB values lower than 1.78 DSB/Gy per 200 Mbp and RIA values over 9.58, 14.40 or 24.83 for 1, 2 and 8 Gy respectively) were at low risk of suffer severe subcutaneous late toxicity (HR 0.223, 95%CI 0.073-0.678, P = 0.008; HR 0.206, 95%CI 0.063-0.677, P = 0.009; HR 0.239, 95%CI 0.062-0.929, P = 0.039, for RIA at 1, 2 and 8 Gy respectively) in multivariate analysis. CONCLUSIONS. A radiation-resistant profile is proposed, where those patients who presented lower levels of initial DNA damage and higher levels of radiation induced apoptosis were at low risk of suffer severe subcutaneous late toxicity after clinical treatment at high radiation doses in our series. However, due to the small sample size, other prospective studies with higher number of patients are needed to validate these results.

Interaction between ATM and PARP-1 in response to DNA damage and sensitization of ATM deficient cells through PARP inhibition

ATM and PARP-1 are two of the most important players in the cell's response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after gamma-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced gamma-H2AX foci formation in response to gamma-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced gamma H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway) and activates ATM kinase.

Bacterial-induced protection against allergic inflammation through a multicomponent immunoregulatory mechanism.

Background Airborne microbial products have been reported to promote immune responses that suppress asthma, yet how these beneficial effects take place remains controversial and poorly understood. Methods We exposed mice to the bacterium Escherichia coli and subsequently induced allergic airway inflammation through sensitization and intranasal challenge with ovalbumin. Results Pulmonary exposure to the bacterium Escherichia coli leads to a suppression of allergic airway inflammation. This immune modulation was neither mediated by the induction of a T helper 1 (Th1) response nor regulatory T cells; however, it was dependent on Toll-like receptor 4 (TLR4) but did not involve TLR desensitisation. Dendritic cell migration to the draining lymph nodes and activation of T cells was unaffected by prior exposure to E.coli, while dendritic cells in the lung displayed a less activated phenotype and had impaired antigen presentation capacity. Consequently, in situ Th2 cytokine production was abrogated. The suppression of airway hyper-responsiveness was mediated through the recruitment of gd T cells; however, the suppression of dendritic cells and T cells was mediated through a distinct mechanism that could not be overcome by the local administration of activated dendritic cells, or by the in vivo administration of tumour necrosis factor a. Conclusion Our data reveal a localized immunoregulatory pathway that acts to protect the airways from allergic inflammation.

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.

Context switching accounting mechanism

How many times a given process p preempts, either voluntarily or involuntarily, is an important threat to computer's processes throughput. Whenever running cpu-bound processes on a multi-core system without an actual system grid engine as commonly found on Grid Clusters, their performance and stability are directly related to their accurate implementation and the system reliability which is, to an extend, an important caveat most of the times so difficult to detect. Context Switching is time-consuming. Thus, if we could develop a tool capable of detecting it and gather data from every single performed Context Switch, we would beable to study this data and present some results that should pin-point at whatever their main cause could be.

An anonymous reputation mechanism for cloud computing networks using volunteer resources

One of the major problems when using non-dedicated volunteer resources in adistributed network is the high volatility of these hosts since they can go offlineor become unavailable at any time without control. Furthermore, the use ofvolunteer resources implies some security issues due to the fact that they aregenerally anonymous entities which we know nothing about. So, how to trustin someone we do not know?.Over the last years an important number of reputation-based trust solutionshave been designed to evaluate the participants' behavior in a system.However, most of these solutions are addressed to P2P and ad-hoc mobilenetworks that may not fit well with other kinds of distributed systems thatcould take advantage of volunteer resources as recent cloud computinginfrastructures.In this paper we propose a first approach to design an anonymous reputationmechanism for CoDeS [1], a middleware for building fogs where deployingservices using volunteer resources. The participants are reputation clients(RC), a reputation authority (RA) and a certification authority (CA). Users needa valid public key certificate from the CA to register to the RA and obtain thedata needed to participate into the system, as now an opaque identifier thatwe call here pseudonym and an initial reputation value that users provide toother users when interacting together. The mechanism prevents not only themanipulation of the provided reputation values but also any disclosure of theusers' identities to any other users or authorities so the anonymity isguaranteed.

Methicillin-resistant Staphylococcus aureus resensitization by protein synthesis inhibitors : proposal for a mechanism

RESUME Staphylococcus aureus est un important pathogène à gram-positif, à la fois responsable d'infections nosocomiales et communautaires. Le S. aureus résistant à la méthicilline est intrinsèquement résistant aux bêta-lactamines, inhibiteurs de la synthèse de la paroi bactérienne, grâce à une enzyme nouvellement acquise, la protéine liant la pénicilline 2A, caractérisée par une faible affinité pour ces agents et pouvant poursuivre la synthèse de la paroi, alors que les autres enzymes sont bloquées. Ce micro-organisme a également développé des résistances contre quasiment tous les antibiotiques couramment utilisés en clinique. Parallèlement au développement de molécules entièrement nouvelles, il peut être utile d'explorer d'éventuelles caractéristiques inattendues de médicaments déjà existants, par exemple en les combinant, dans l'espoir d'un potentiel effet synergique. Comprendre les mécanismes de tels effets synergiques pourrait contribuer à la justification de leur utilisation clinique potentielle. Récemment, un effet synergique contre le S. aureus résistant à la méthicilline a été décrit entre la streptogramine quinupristine-datfopristine et les bêta-lactamines, aussi bien in vitro qu'in vivo. Le présent travail a pour but de proposer un modèle pour le mécanisme de cette interaction positive et de l'étendre à d'autres classes d'antibiotiques. Premièrement, un certain nombre de méthodes microbiologiques ont permis de mieux cerner la nature de cette interaction, en montrant qu'elle agissait spécifiquement sur le S. aureus résistant à la méthicilline et qu'elle était restreinte à l'association entre inhibiteurs de la synthèse des protéines et bêta-lactamines. Deuxièmement, L'observation de l'influence des inhibiteurs de la synthèse des protéines sur la machinerie de la paroi bactérienne, c'est-à-dire sur l'expression des protéines liant la pénicilline, responsables de la synthèse du peptidoglycan, a montré une diminution de la quantité de ta protéine liant la pénicilline 2, connue pour posséder une activité de transglycosylation, indispensable au bon fonctionnement de la protéine liant la pénicilline 2A, responsable de la résistance à la méthicilline. Troisièmement, l'analyse fine de la composition du peptidoglycan extrait de bactéries, avant ou après traitement par des inhibiteurs de la synthèse des protéines, a montré des altérations corrélant avec leur capacité à agir en synergie avec les bêta-lactamines contre S. aureus résistant à ta méthicilline. Ces altérations dans les muropeptides pourraient représenter une signature de la diminution de la quantité de la protéine liant la pénicilline 2. Le modèle mécanistique retenu considère que les inhibiteurs de la synthèse des protéines pourraient diminuer l'expression de la protéine Liant la pénicilline 2, indispensable à la résistance à la méthiciltine, et que ce déséquilibre dans les enzymes synthétisant la paroi bactérienne pourrait générer une signature dans les muropeptides. SUMMARY Staphylococcus aureus is a major gram-positive pathogen causing both hospital-acquired and community-acquired infections. Methicillin- resistant Staphylococcus aureus is intrinsically resistant to the cell wall inhibitors beta-lactams by virtue of a newly acquired cell-wall-building enzyme, tow-affinity penicillin-binding protein 2A, which can build the wall when other penicillin-binding proteins are blocked. Moreover, the microorganism has developed resistance to virtually all non-experimental antibiotics. In addition of producing entirely new molecules, it is useful to explore unexpected features of existing drugs, for example by using them in combination, expecting drug synergisms. Understanding the mechanisms of such synergisms would help justify their putative clinical utilization. Recently, a synergism between the streptogramin quinupristin-dalfopristin and beta-lactams was reported against methicillin-resistant S. aureus, both in vitro and in vivo. The present work intends to propose a model for the mechanism of this positive interaction and to extend it to other drug classes. First, microbiological experimentation helped better defining the nature of this interaction, restricting it to methicillin-resistant S. aureus, and to the association of protein synthesis inhibitors with beta-lactams. Second, the observation of inhibitors of protein synthesis influence on the cell-wall-building machinery, i.e. on the expression of penicillin-binding proteins responsible for peptidoglycan synthesis, showed a decrease in the amount of penicillin-binding protein 2, known to provide a transglycosylase activity for glycan chain elongation, indispensable for the functionality of the low-affinity penicillin-binding protein 2A responsible for methicillin resistance. Third, the fine analysis of the peptidoglycan composition purified from bacteria before or after treatment with inhibitors of protein synthesis showed alterations that correlated with their ability to synergize with beta-lactams against methicillin-resistant S. aureus. These muropeptide alterations could be the signature of decrease in the amount of penicillin-binding protein 2. The retained mechanistic model is that inhibitors of protein synthesis could decrease the expression of penicillin-binding protein 2, wich is indispensable for methicillin-resistance, and that this imbalance in cell-wall-building enzymes could generate a muropeptide signature.

Failure mechanism under tensile loads in CFRP 0-90º cross-ply laminates containing voids

The failure mechanism of a voided CFRP 0-90° cross-ply laminate under tensile loads applied in one direction was studied in this Final Degree Project. For this purpose, voided coupons were manufactured for being tested and a FEA was done. In both investigations, voids were placed in 90º and 0º direction, in order to understand the void location influence. On the one hand, the behaviour of the voided laminates was investigated through a FEM in order to preview the stress distribution within the material. On the other hand, voided specimens where manufactured by applying blowing agent in between the inner layers. These specimens were tested by a quasi-static step wise tensile test where data showing its real behaviour was collected. Specimens were X-rayed after each step of the test in order to investigate the failure mechanism of the composite. Data from the test was studied so that relations such as strength of the laminates, crack density per stress, void length per first crack at the void and void area per first crack at the specimen could be characterized

Incidence of liver damage of uncertain origin in HIV patients not co-infected with HCV/HBV.

BACKGROUND AND AIMS Several studies have reported that a significant number of HIV patients not co-infected with HCV/HBV develop liver damage of uncertain origin (LDUO). The objective of our study was to evaluate the incidence of and risk factors for the development of LDUO in HIV infected patients not co-infected with HCV/HBV. METHODS Prospective longitudinal study that included HIV-infected patients free of previous liver damage and viral hepatitis B or C co-infections. Patients were followed up at 6-monthly intervals. Liver stiffness was measured at each visit. Abnormal liver stiffness (ALS) was defined as a liver stiffness value greater than 7.2 kPa at two consecutive measurements. For patients who developed ALS, a protocol was followed to diagnose the cause of liver damage. Those patients who could not be diagnosed with any specific cause of liver disease were diagnosed as LDUO and liver biopsy was proposed. RESULTS 210 patients matched the inclusion criteria and were included. 198 patients completed the study. After a median (Q1-Q3) follow-up of 18 (IQR 12-26) months, 21 patients (10.6%) developed ALS. Of these, fifteen patients were diagnosed as LDUO. The incidence of LDUO was 7.64 cases/100 patient-years. Histological studies were performed on ten (66.6%) patients and all showed liver steatosis. A higher HOMA-IR value and body mass index were independently associated with the development of LDUO. CONCLUSION We found a high incidence of LDUO in HIV-infected patients associated with metabolic risk factors. The leading cause of LDUO in our study was non-alcoholic fatty liver disease.

The combination of social and personal contexts affects dominance hierarchy development in shore crabs, Carcinus maenas

Many animals that live in groups maintain competitive relationships, yet avoid continual fighting, by forming dominance hierarchies. We compare predictions of stochastic, individual-based models with empirical experimental evidence using shore crabs to test competing hypotheses regarding hierarchy development. The models test (1) what information individuals use when deciding to fight or retreat, (2) how past experience affects current resource-holding potential, and (3) how individuals deal with changes to the social environment. First, we conclude that crabs assess only their own state and not their opponent's when deciding to fight or retreat. Second, willingness to enter, and performance in, aggressive contests are influenced by previous contest outcomes. Winning increases the likelihood of both fighting and winning future interactions, while losing has the opposite effect. Third, when groups with established dominance hierarchies dissolve and new groups form, individuals reassess their ranks, showing no memory of previous rank or group affiliation. With every change in group composition, individuals fight for their new ranks. This iterative process carries over as groups dissolve and form, which has important implications for the relationship between ability and hierarchy rank. We conclude that dominance hierarchies emerge through an interaction of individual and social factors, and discuss these findings in terms of an underlying mechanism. Overall, our results are consistent with crabs using a cumulative assessment strategy iterated across changes in group composition, in which aggression is constrained by an absolute threshold in energy spent and damage received while fighting.

Evaluation of the role of ATP-binding cassette transporters as a defence mechanism against temephos in populations of Aedes aegypti

The role of ATP-binding cassette (ABC) transporters in the efflux of the insecticide, temephos, was assessed in the larvae of Aedes aegypti. Bioassays were conducted using mosquito populations that were either susceptible or resistant to temephos by exposure to insecticide alone or in combination with sublethal doses of the ABC transporter inhibitor, verapamil (30, 35 and 40 μM). The best result in the series was obtained with the addition of verapamil (40 μM), which led to a 2x increase in the toxicity of temephos, suggesting that ABC transporters may be partially involved in conferring resistance to the populations evaluated.

Poly(ADP-ribose) polymerases as modulators of mitochondrial activity.

Mitochondria are essential in cellular stress responses. Mitochondrial output to environmental stress is a major factor in metabolic adaptation and is regulated by a complex network of energy and nutrient sensing proteins. Activation of poly(ADP-ribose) polymerases (PARPs) has been known to impair mitochondrial function; however, our view of PARP-mediated mitochondrial dysfunction and injury has only recently fundamentally evolved. In this review, we examine our current understanding of PARP-elicited mitochondrial damage, PARP-mediated signal transduction pathways, transcription factors that interact with PARPs and govern mitochondrial biogenesis, as well as mitochondrial diseases that are mediated by PARPs. With PARP activation emerging as a common underlying mechanism in numerous pathologies, a better understanding the role of various PARPs in mitochondrial regulation may help open new therapeutic avenues.

Comparison of skin microvascular reactivity with hemostatic markers of endothelial dysfunction and damage in type 2 diabetes.

AIM: Patients with non-insulin-dependent diabetes mellitus (NIDDM) are at increased cardiovascular risk due to an accelerated atherosclerotic process. The present study aimed to compare skin microvascular function, pulse wave velocity (PWV), and a variety of hemostatic markers of endothelium injury [von Willebrand factor (vWF), plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (t-PA), tissue factor pathway inhibitor (TFPI), and the soluble form of thrombomodulin (s-TM)] in patients with NIDDM. METHODS: 54 patients with NIDDM and 38 sex- and age-matched controls were studied. 27 diabetics had no overt micro- and/or macrovascular complications, while the remainder had either or both. The forearm skin blood flow was assessed by laser-Doppler imaging, which allowed the measurement of the response to iontophoretically applied acetylcholine (endothelium-dependent vasodilation) and sodium nitroprusside (endothelium-independent vasodilation), as well as the reactive hyperemia triggered by the transient occlusion of the circulation. RESULTS: Both endothelial and non-endothelial reactivity were significantly blunted in diabetics, regardless of the presence or the absence of vascular complications. Plasma vWF, TFPI and s-TM levels were significantly increased compared with controls only in patients exhibiting vascular complications. Concentrations of t-PA and PAI-1 were significantly increased in the two groups of diabetics versus controls. CONCLUSION: In NIDDM, both endothelium-dependent and -independent microvascular skin reactivity are impaired, whether or not underlying vascular complications exist. It also appears that microvascular endothelial dysfunction is not necessarily associated in NIDDM with increased circulating levels of hemostatic markers of endothelial damage known to reflect a hypercoagulable state.

T cells dampen innate immune responses through inhibition of NLRP1 and NLRP3 inflammasomes.

Inflammation is a protective attempt by the host to remove injurious stimuli and initiate the tissue healing process. The inflammatory response must be actively terminated, however, because failure to do so can result in 'bystander' damage to tissues and diseases such as arthritis or type-2 diabetes. Yet the mechanisms controlling excessive inflammatory responses are still poorly understood. Here we show that mouse effector and memory CD4(+) T cells abolish macrophage inflammasome-mediated caspase-1 activation and subsequent interleukin 1beta release in a cognate manner. Inflammasome inhibition is observed for all tested NLRP1 (commonly called NALP1) and NLRP3 (NALP3 or cryopyrin) activators, whereas NLRC4 (IPAF) inflammasome function and release of other inflammatory mediators such as CXCL2, interleukin 6 and tumour necrosis factor are not affected. Suppression of the NLRP3 inflammasome requires cell-to-cell contact and can be mimicked by macrophage stimulation with selected ligands of the tumour necrosis factor family, such as CD40L (also known as CD40LG). In a NLRP3-dependent peritonitis model, effector CD4(+) T cells are responsible for decreasing neutrophil recruitment in an antigen-dependent manner. Our findings reveal an unexpected mechanism of inflammasome inhibition, whereby effector and memory T cells suppress potentially damaging inflammation, yet leave the primary inflammatory response, crucial for the onset of immunity, intact.