MRE11-RAD50-NBS1 is a critical regulator of FANCD2 stability and function during DNA double-strand break repair.

Monoubiquitination of the Fanconi anaemia protein FANCD2 is a key event leading to repair of interstrand cross-links. It was reported earlier that FANCD2 co-localizes with NBS1. However, the functional connection between FANCD2 and MRE11 is poorly understood. In this study, we show that inhibition of MRE11, NBS1 or RAD50 leads to a destabilization of FANCD2. FANCD2 accumulated from mid-S to G2 phase within sites containing single-stranded DNA (ssDNA) intermediates, or at sites of DNA damage, such as those created by restriction endonucleases and laser irradiation. Purified FANCD2, a ring-like particle by electron microscopy, preferentially bound ssDNA over various DNA substrates. Inhibition of MRE11 nuclease activity by Mirin decreased the number of FANCD2 foci formed in vivo. We propose that FANCD2 binds to ssDNA arising from MRE11-processed DNA double-strand breaks. Our data establish MRN as a crucial regulator of FANCD2 stability and function in the DNA damage response.

Exercise efficiency relates with mitochondrial content and function in older adults.

Chronic aerobic exercise has been shown to increase exercise efficiency, thus allowing less energy expenditure for a similar amount of work. The extent to which skeletal muscle mitochondria play a role in this is not fully understood, particularly in an elderly population. The purpose of this study was to determine the relationship of exercise efficiency with mitochondrial content and function. We hypothesized that the greater the mitochondrial content and/or function, the greater would be the efficiencies. Thirty-eight sedentary (S, n = 23, 10F/13M) or athletic (A, n = 15, 6F/9M) older adults (66.8 ± 0.8 years) participated in this cross sectional study. V˙O2peak was measured with a cycle ergometer graded exercise protocol (GXT). Gross efficiency (GE, %) and net efficiency (NE, %) were estimated during a 1-h submaximal test (55% V˙O2peak). Delta efficiency (DE, %) was calculated from the GXT. Mitochondrial function was measured as ATPmax (mmol/L/s) during a PCr recovery protocol with (31)P-MR spectroscopy. Muscle biopsies were acquired for determination of mitochondrial volume density (MitoVd, %). Efficiencies were 17% (GE), 14% (NE), and 16% (DE) higher in A than S. MitoVD was 29% higher in A and ATPmax was 24% higher in A than in S. All efficiencies positively correlated with both ATPmax and MitoVd. Chronically trained older individuals had greater mitochondrial content and function, as well as greater exercise efficiencies. GE, NE, and DE were related to both mitochondrial content and function. This suggests a possible role of mitochondria in improving exercise efficiency in elderly athletic populations and allowing conservation of energy at moderate workloads.

Cdk5 controls lymphatic vessel development and function by phosphorylation of Foxc2.

The lymphatic system maintains tissue fluid balance, and dysfunction of lymphatic vessels and valves causes human lymphedema syndromes. Yet, our knowledge of the molecular mechanisms underlying lymphatic vessel development is still limited. Here, we show that cyclin-dependent kinase 5 (Cdk5) is an essential regulator of lymphatic vessel development. Endothelial-specific Cdk5 knockdown causes congenital lymphatic dysfunction and lymphedema due to defective lymphatic vessel patterning and valve formation. We identify the transcription factor Foxc2 as a key substrate of Cdk5 in the lymphatic vasculature, mechanistically linking Cdk5 to lymphatic development and valve morphogenesis. Collectively, our findings show that Cdk5-Foxc2 interaction represents a critical regulator of lymphatic vessel development and the transcriptional network underlying lymphatic vascular remodeling.

Combined loss of the BH3-only proteins Bim and Bmf restores B-cell development and function in TACI-Ig transgenic mice.

Terminal differentiation of B cells depends on two interconnected survival pathways, elicited by the B-cell receptor (BCR) and the BAFF receptor (BAFF-R), respectively. Loss of either signaling pathway arrests B-cell development. Although BCR-dependent survival depends mainly on the activation of the v-AKT murine thymoma viral oncogene homolog 1 (AKT)/PI3-kinase network, BAFF/BAFF-R-mediated survival engages non-canonical NF-κB signaling as well as MAPK/extracellular-signal regulated kinase and AKT/PI3-kinase modules to allow proper B-cell development. Plasma cell survival, however, is independent of BAFF-R and regulated by APRIL that signals NF-κB activation via alternative receptors, that is, transmembrane activator and CAML interactor (TACI) or B-cell maturation (BCMA). All these complex signaling events are believed to secure survival by increased expression of anti-apoptotic B-cell lymphoma 2 (Bcl2) family proteins in developing and mature B cells. Curiously, how lack of BAFF- or APRIL-mediated signaling triggers B-cell apoptosis remains largely unexplored. Here, we show that two pro-apoptotic members of the 'Bcl2 homology domain 3-only' subgroup of the Bcl2 family, Bcl2 interacting mediator of cell death (Bim) and Bcl2 modifying factor (Bmf), mediate apoptosis in the context of TACI-Ig overexpression that effectively neutralizes BAFF as well as APRIL. Surprisingly, although Bcl2 overexpression triggers B-cell hyperplasia exceeding the one observed in Bim(-/-)Bmf(-/-) mice, Bcl2 transgenic B cells remain susceptible to the effects of TACI-Ig expression in vivo, leading to ameliorated pathology in Vav-Bcl2 transgenic mice. Together, our findings shed new light on the molecular machinery restricting B-cell survival during development, normal homeostasis and under pathological conditions. Our data further suggest that Bcl2 antagonists might improve the potency of BAFF/APRIL-depletion strategies in B-cell-driven pathologies.

Effect of aerobic exercise on skeletal muscle mitochondrial content and function in older adults

Regular aerobic exercise training, which is touted as a way to ameliorate metabolic diseases, increases aerobic capacity. Aerobic capacity usually declines with advanced age. The decline in aerobic capacity is typically associated by a decrease in the quality of skeletal muscle. At the molecular level, this decreased quality comes in part from perturbations in skeletal muscle mitochondria. Of particular is a decrease in the total amount of mitochondria that occupy the skeletal muscle volume. What is not well established is if this decrease in mitochondrial content is due to inactive lifestyle or the process of aging. Herein, the work of the thesis shows a clear connection between mitochondrial content and aerobic capacity. This indicates that active individuals with higher VChmax levels also contain higher volumes of mitochondria inside their muscle as opposed to sedentary counterparts who have lower levels of mitochondrial content. Upon taking these previously sedentary individuals and entering them into an aerobic exercise intervention, they are able to recover their mitochondrial content as well as function to similar levels of lifelong athletes of the same age. Furthermore, the results of this thesis show that mitochondrial content and function also correlate with exercise efficiency. If one is more efficient, he/she is able to expend less energy for a similar power output. Furthermore, individuals who increase in efficiency also increase in the ability to oxidize and utilize fat during pro-longed exercise. This increased reliance on fat after the intervention is associated with an increased amount of mitochondria, particularly in the intermyofibrillar region of skeletal muscle. Therefore, elderly adults who were once sedentary were able to recover mitochondrial content and function and are able to reap other health benefits from regular aerobic exercise training. Aging per se does not seem to be the culprit that will lead to metabolic diseases but rather it seems to be a lack of physical activity. -- Un entraînement sportif d'endurance, connu pour réduire le risque de développer des maladies métaboliques, augmente notre capacité aérobie. La capacité aérobie diminue généralement avec l'âge. Ce déclin est typiquement associé d'une diminution de la qualité du muscle squelettique. Au niveau moléculaire, cette diminution est due à des perturbations dans les mitochondries du muscle squelettique,, ce qui conduit à une diminution de la quantité totale des mitochondries présentes dans le muscle squelettique. Il n'a pas encore été établi si cette diminution de la teneur mitochondriale est due à un mode de vie sédentaire ou au processus du vieillissement. Ce travail de thèse montre un lien clair entre le contenu mitochondrial et la capacité aérobie. Il indique que des personnes âgées actives, avec des niveaux de V02max plus élevés, possèdent également un volume plus élevé de mitochondries dans leurs muscles en comparaison à leurs homologues sédentaires. En prenant des individus sédentaires et leur faisant pratiquer une activité physique aérobie, il est possible d'accroître leur contenu de même que leur fonction mitochondriale à des niveaux similaires à ceux d'athlètes du même âge ayant pratiqué une activité physique tout au long de leur vie. De plus, les résultats de ce travail démontrent que le contenu et la fonction mitochondriale sont en corrélation avec l'efficiscience lors d'exercice physique. En agumentant l'effiscience, les personnes sont alors capables de dépenser moins d'énergie pour une puissance d'exercice similaire. Donc, un volume mitochondrial accru dans le muscle squelettique, associé à une fonction mitochondriale améliorée, est associté à une augmentation de l'effiscience. En outre, les personnes qui augmentent leur effiscience, augmentent aussi leur capacité à oxyder les graisses durant l'exercice prolongé. Une augmentation du recours au graisses après l'intervention est associée à une quantité accrue de mitochondries, en particulier dans la région inter-myofibrillaire du muscle squelettique. Par conséquent, les personnes âgées autrefois sédentaires sont en mesure de récupérer leur contenu et leur fonction mitochondriale ainsi que d'autres avantages pour la santé grâce à un entraînement aérobie régulier. Le vieillissement en soi ne semble donc pas être le coupable conduisant aux maladies métaboliques qui semblent plutôt être lié à un manque d'activité physique.

Occurrence and new mutations involved in rifampicin-resistant Propionibacterium acnes strains isolated from biofilm or device-related infections.

We described for the first time the amino acid substitutions conferring rifampicin resistance in eight Propionibacterium acnes strains isolated from patients with biofilm or device-related infections. We identified different mutations in cluster I and one mutation, never reported, in cluster II of the rpoB gene (I480V) associated with the most frequent one in cluster I (S442L). Half of the patients previously received treatment with rifampicin.

Autocrine Action of IGF2 Regulates Adult β-Cell Mass and Function.

Insulin-like growth factor 2 (IGF2), produced and secreted by adult β-cells, functions as an autocrine activator of the β-cell insulin-like growth factor 1 receptor signaling pathway. Whether this autocrine activity of IGF2 plays a physiological role in β-cell and whole-body physiology is not known. Here, we studied mice with β-cell-specific inactivation of Igf2 (βIGF2KO mice) and assessed β-cell mass and function in aging, pregnancy, and acute induction of insulin resistance. We showed that glucose-stimulated insulin secretion (GSIS) was markedly reduced in old female βIGF2KO mice; glucose tolerance was, however, normal because of increased insulin sensitivity. While on a high-fat diet, both male and female βIGF2KO mice displayed lower GSIS compared with control mice, but reduced β-cell mass was observed only in female βIGF2KO mice. During pregnancy, there was no increase in β-cell proliferation and mass in βIGF2KO mice. Finally, β-cell mass expansion in response to acute induction of insulin resistance was lower in βIGF2KO mice than in control mice. Thus, the autocrine action of IGF2 regulates adult β-cell mass and function to preserve in vivo GSIS in aging and to adapt β-cell mass in response to metabolic stress, pregnancy hormones, and acute induction of insulin resistance.

Aldosterone effects on glomerular structure and function.

OBJECTIVE: Experimental evidence suggests that aldosterone directly contributes to organ damage by promoting cell growth, fibrosis, and inflammation. Based on these premises, this work aimed to assess the glomerular effects of aldosterone, alone and in combination with salt. METHODS: After undergoing uninephrectomy, 75 rats were allocated to five groups: control, salt diet, aldosterone, aldosterone + salt diet, aldosterone + salt diet and eplerenone, and they were all studied for four weeks. We focused on glomerular structural, functional, and molecular changes, including slit diaphragm components, local renin-angiotensin system activation, as well as pro-oxidative and profibrotic changes. RESULTS: Aldosterone significantly increased systolic blood pressure, led to glomerular hypertrophy, mesangial expansion, and it significantly increased the glomerular permeability to albumin and the albumin excretion rate, indicating the presence of glomerular damage. These effects were worsened by adding salt to aldosterone, while they were reduced by eplerenone. Aldosterone-induced glomerular damage was associated with glomerular angiotensin-converting enzyme (ACE) 2 downregulation, with ACE/ACE2 ratio increase, ANP decrease, as well as with glomerular pro-oxidative and profibrotic changes. CONCLUSIONS: Aldosterone damages not only the structure but also the function of the glomerulus. ACE/ACE2 upregulation, ACE2 and ANP downregulation, and pro-oxidative and profibrotic changes are possible mechanisms accounting for aldosterone-induced glomerular injury.

Sodium-dependent phosphate transporters in osteoclast differentiation and function.

Osteoclasts are multinucleated bone degrading cells. Phosphate is an important constituent of mineralized bone and released in significant quantities during bone resorption. Molecular contributors to phosphate transport during the resorptive activity of osteoclasts have been controversially discussed. This study aimed at deciphering the role of sodium-dependent phosphate transporters during osteoclast differentiation and bone resorption. Our studies reveal RANKL-induced differential expression of sodium-dependent phosphate transport protein IIa (NaPi-IIa) transcript and protein during osteoclast development, but no expression of the closely related NaPi-IIb and NaPi-IIc SLC34 family isoforms. In vitro studies employing NaPi-IIa-deficient osteoclast precursors and mature osteoclasts reveal that NaPi-IIa is dispensable for bone resorption and osteoclast differentiation. These results are supported by the analysis of structural bone parameters by high-resolution microcomputed tomography that yielded no differences between adult NaPi-IIa WT and KO mice. By contrast, both type III sodium-dependent phosphate transporters Pit-1 and Pit-2 were abundantly expressed throughout osteoclast differentiation, indicating that they are the relevant sodium-dependent phosphate transporters in osteoclasts and osteoclast precursors. We conclude that phosphate transporters of the SLC34 family have no role in osteoclast differentiation and function and propose that Pit-dependent phosphate transport could be pivotal for bone resorption and should be addressed in further studies.

Impact of T cell receptor affinity on the molecular mechanisms modulating CD8 T cell signalling and function against NY-ESO-1 tumour antigen

It is well established that cytotoxic T lymphocytes play a pivotal role in the protection against intracellular pathogens and tumour cells. Such protective immune responses rely on the specific T cell receptor (TCR)-mediated recognition by CD8 T cells of small antigenic peptides presented in the context of class-I Major Histocompatibility Complex molecules (pMHCs) on the surface of infected or malignant cells. The strength (affinity/avidity) of this interaction is a major correlate of protection. Although tumour-reactive CD8 T cells can be observed in cancer patients, anti-tumour immune responses are often ineffective in controlling or eradicating the disease due to the relative low TCR affinity of these cells. To overcome this limitation, tumour-specific CD8 T cells can be genetically modified to express TCRs of improved binding strength against a defined tumour antigen before adoptive cell transfer into cancer patients. We previously generated a panel of TCRs specific for the cancer-testis antigen NY-ESO-l,57.165 with progressively increased affinities for the pMHC complex, thus providing us with a unique tool to investigate the causal link between the surface expression of such TCRs and T cell activation and function. We recently demonstrated that anti-tumour CD8 T cell reactivity could only be improved within physiological affinity limits, beyond which drastic functional declines were observed, suggesting the presence of multiple regulatory mechanisms limiting T cell activation and function in a TCR affinity-dependent manner. The overarching goal of this thesis was (i) to assess the precise impact of TCR affinity on T cell activation and signalling at the molecular level and (ii) to gain further insights on the mechanisms that regulate and delimitate maximal/optimized CD8 T cell activation and signalling. Specifically, by combining several technical approaches we characterized the activation status of proximal (i.e. CD3Ç, Lek, and ZAP-70) and distal (i.e. ERK1/2) signalling molecules along the TCR affinity gradient. Moreover, we assessed the extent of TCR downmodulation, a critical step for initial T cell activation. CD8 T cells engineered with the optimal TCR affinity variants showed increased activation levels of both proximal and distal signalling molecules when compared to the wild-type T cells. Our analyses also highlighted the "paradoxical" status of tumour-reactive CD8 T cells bearing very high TCR affinities, which retained strong proximal signalling capacity and TCR downmodulation, but were unable to propagate signalling distally (i.e. pERKl/2), resulting in impaired cell-mediated functions. Importantly, these very high affinity T cells displayed maximal levels of SHP-1 and SHP-2 phosphatases, two negative regulatory molecules, and this correlated with a partial pERKl/2 signalling recovery upon pharmacological SHP-l/SHP-2 inhibition. These findings revealed the putative presence of inhibitory regulators of the TCR signalling cascade acting very rapidly following tumour-specific stimulation. Moreover, the very high affinity T cells were only able to transiently express enhanced proximal signalling molecules, suggesting the presence of an additional level of regulation that operates through the activation of negative feedback loops over time, limiting the duration of the TCR-mediated signalling. Overall, the determination of TCR-pMHC binding parameters eliciting optimal CD8 T cell activation, signalling, and effector function while guaranteeing high antigen specificity, together with the identification of critical regulatory mechanisms acting proximally in the TCR signalling cascade, will directly contribute to optimize and support the development of future TCR-based adoptive T cell strategies for the treatment of malignant diseases. -- Les lymphocytes T CD8 cytotoxiques jouent un rôle prédominant dans la protection contre les pathogènes intracellulaires et les cellules tumorales. Ces réponses immunitaires dépendent de la spécificité avec laquelle les récepteurs T (TCR) des lymphocytes CD8 reconnaissent les peptides antigéniques présentés par les molécules du complexe Majeur de Histocompatibilité de classe I (pCMH) à la surface des cellules infectées ou malignes. La force (ou affinité/avidité) de l'interaction du TCR-pCMH est un corrélat majeur de protection. Les réponses immunitaires sont cependant souvent inefficaces et ne permettent pas de contrôler ou d'éliminer les cellules tumorales chez les patients atteint du cancer, et ce à cause de la relative faible reconnaissance des TCRs exprimés par les lymphocytes T CD8 envers les antigènes tumoraux. Afin de surmonter cette limitation, les cellules T anti-tumorales peuvent être génétiquement modifiées en les dotant de TCRs préalablement optimisés afin d'augmenter leur reconnaissance ou affinité contre les antigènes tumoraux, avant leur ré¬infusion dans le patient. Nous avons récemment généré des cellules T CD8 exprimant un panel de TCRs spécifiques pour l'antigène tumoral NY-ESO-l157.16J avec des affinités croissantes, permettant ainsi d'investiguer la causalité directe entre l'affinité du TCR-pCMH et la fonction des cellules T CD8. Nous avons démontré que la réactivité anti-tumorale pouvait être améliorée en augmentant l'affinité du TCR dans une intervalle physiologique, mais au delà duquel nous observons un important déclin fonctionnel. Ces résultats suggèrent la présence de mécanismes de régulation limitant l'activation des cellules T de manière dépendante de l'affinité du TCR. Le but de cette thèse a été (i) de définir l'impact précis de l'affinité du TCR sur l'activation et la signalisation des cellules T CD8 au niveau moléculaire et (ii) d'acquérir de nouvelles connaissances sur les mécanismes qui régulent et délimitent l'activation et la signalisation maximale des cellules T CD8 optimisées. Spécifiquement, en combinant plusieurs approches technologiques, nous avons caractérisé l'état d'activation de différentes protéines de la voie de signalisation proximale (CD3Ç, Lek et ZAP-70) et distale (ERK1/2) le long du gradient d'affinité du TCR, ainsi que l'internalisation du TCR, une étape clef dans l'activation initiale des cellules T. Les lymphocytes T CD8 exprimant des TCRs d'affinité optimale ont montré des niveaux d'activation augmentés des molécules proximales et distales par rapport aux cellules de type sauvage (wild-type). Nos analyses ont également mis en évidence un paradoxe chez les cellules T CD8 équipées avec des TCRs de très haute affinité. En effet, ces cellules anti-tumorales sont capables d'activer leurs circuits biochimiques au niveau proximal et d'internaliser efficacement leur TCR, mais ne parviennent pas à propager les signaux biochimiques dépendants du TCR jusqu'au niveau distal (via phospho-ERKl/2), avec pour conséquence une limitation de leur capacité fonctionnelle. Finalement, nous avons démontré que SHP-1 et SHP-2, deux phosphatases avec des propriétés régulatrices négatives, étaient majoritairement exprimées dans les cellules T CD8 de très hautes affinités. Une récupération partielle des niveaux d'activation de ERK1/2 a pu être observée après l'inhibition pharmacologique de ces phosphatases. Ces découvertes révèlent la présence de régulateurs moléculaires qui inhibent le complexe de signalisation du TCR très rapidement après la stimulation anti-tumorale. De plus, les cellules T de très hautes affinités ne sont capables d'activer les molécules de la cascade de signalisation proximale que de manière transitoire, suggérant ainsi un second niveau de régulation via l'activation de mécanismes de rétroaction prenant place progressivement au cours du temps et limitant la durée de la signalisation dépendante du TCR. En résumé, la détermination des paramètres impliqués dans l'interaction du TCR-pCMH permettant l'activation de voies de signalisation et des fonctions effectrices optimales ainsi que l'identification des mécanismes de régulation au niveau proximal de la cascade de signalisation du TCR contribuent directement à l'optimisation et au développement de stratégies anti-tumorales basées sur l'ingénierie des TCRs pour le traitement des maladies malignes.

Barn owls do not interrupt their siblings

Animals communicate with conspecifics to resolve conflicts over how resources are shared. Since signals reflect individuals' resource-holding potential and motivation to compete, it is crucial that opponents efficiently transmit and receive information to adjust investment optimally in competitive interactions. Acoustic communication is particularly flexible as it can be quickly modulated according to background noise and audience. Diverse mechanisms have evolved to minimize acoustic signal interference, one being the avoidance of signal overlap by adjusting the timing of call production to alternate calls with those of competitors. However, the occurrence and function of overlap avoidance in the resolution of competition among relatives have barely been studied. Using young barn owl siblings, Tyto alba, which vocally negotiate over who will have priority access to food provided by parents, we investigated the extent to which nestlings avoid calling simultaneously and the function of this behaviour. We found that nestlings overlapped both their live siblings' calls and experimentally broadcast calls at least five times less often than expected at random. Furthermore, a focal nestling engaged more intensely in vocal negotiation when competing with nestmates that called simultaneously compared to those that did not overlap their respective calls. This suggests that barn owl nestlings avoid calling simultaneously, as overlapped calls are less efficient at deterring siblings from competing. Overlap avoidance reduces signal interference and, as a consequence, would improve the efficiency of communication among kin.

The complement inhibitor low molecular weight dextran sulfate prevents TLR4-induced phenotypic and functional maturation of human dendritic cells.

Low molecular weight dextran sulfate (DXS) has been reported to inhibit the classical, alternative pathway as well as the mannan-binding lectin pathway of the complement system. Furthermore, it acts as an endothelial cell protectant inhibiting complement-mediated endothelial cell damage. Endothelial cells are covered with a layer of heparan sulfate (HS), which is rapidly released under conditions of inflammation and tissue injury. Soluble HS induces maturation of dendritic cells (DC) via TLR4. In this study, we show the inhibitory effect of DXS on human DC maturation. DXS significantly prevents phenotypic maturation of monocyte-derived DC and peripheral myeloid DC by inhibiting the up-regulation of CD40, CD80, CD83, CD86, ICAM-1, and HLA-DR and down-regulates DC-SIGN in response to HS or exogenous TLR ligands. DXS also inhibits the functional maturation of DC as demonstrated by reduced T cell proliferation, and strongly impairs secretion of the proinflammatory mediators IL-1beta, IL-6, IL-12p70, and TNF-alpha. Exposure to DXS leads to a reduced production of the complement component C1q and a decreased phagocytic activity, whereas C3 secretion is increased. Moreover, DXS was found to inhibit phosphorylation of IkappaB-alpha and activation of NF-kappaB. These findings suggest that DXS prevents TLR-induced maturation of human DC and may therefore be a useful reagent to impede the link between innate and adaptive immunity.