Structure and function of a "yellow" protein from saliva of the sand fly Lutzomyia longipalpis that confers protective immunity against Leishmania major infection.

LJM11, an abundant salivary protein from the sand fly Lutzomyia longipalpis, belongs to the insect "yellow" family of proteins. In this study, we immunized mice with 17 plasmids encoding L. longiplapis salivary proteins and demonstrated that LJM11 confers protective immunity against Leishmania major infection. This protection correlates with a strong induction of a delayed type hypersensitivity (DTH) response following exposure to L. longipalpis saliva. Additionally, splenocytes of exposed mice produce IFN-γ upon stimulation with LJM11, demonstrating the systemic induction of Th1 immunity by this protein. In contrast to LJM11, LJM111, another yellow protein from L. longipalpis saliva, does not produce a DTH response in these mice, suggesting that structural or functional features specific to LJM11 are important for the induction of a robust DTH response. To examine these features, we used calorimetric analysis to probe a possible ligand binding function for the salivary yellow proteins. LJM11, LJM111, and LJM17 all acted as high affinity binders of prohemostatic and proinflammatory biogenic amines, particularly serotonin, catecholamines, and histamine. We also determined the crystal structure of LJM11, revealing a six-bladed β-propeller fold with a single ligand binding pocket located in the central part of the propeller structure on one face of the molecule. A hypothetical model of LJM11 suggests a positive electrostatic potential on the face containing entry to the ligand binding pocket, whereas LJM111 is negative to neutral over its entire surface. This may be the reason for differences in antigenicity between the two proteins.

IRF6 is a mediator of the Notch pro-differentiation and tumour suppressive function in keratinocytes

I. Résumé large publicIRF6 est un médiateur de Notch dans la différenciation des kératinocytes et dans sa fonction de suppresseur de tumeursLa peau est l'organe le plus important du corps humain, elle représente chez l'adulte une surface d'environ 1,5 m2 et elle est composée de 2000 milliards de cellules. La peau est composée de plusieurs types cellulaires dont les kératinocvtes. Ces cellules, qui se trouvent dans la couche la plus externe de la peau (Pépiderme), nous protègent de la déshydratation et des agressions externes telles que les infections et rayons ultraviolets. Cette fonction de « barrière » est mise en place grâce à un processus appelé différenciation des kératinocvtes durant lequel les kératinocytes deviennent matures et finalement meurent pour former la couche cornée la plus externe difficilement pénétrable. L'homéostasie tissulaire est un mécanisme qui régule l'équilibre entre prolifération, différentiation et mort cellulaire. Une perturbation de cet équilibre peut mener à la formation d'une tumeur. Il existe différents types de tumeurs de la peau. Nous nous sommes intéressés aux «carcinomes spino-cellulaires» (SCC) qui se développent à partir des keratinocytes en différenciation. Notch est une molécule impliquée positivement dans la différenciation des kératinocytes et joue un rôle prépondérant dans la suppression des tumeurs kératinocytaires comme les SCC dans lesquelles Notch est faiblement exprimé. L'implication de Notch dans la différenciation et dans la carcinogenèse kératinocytaire n'est plus controversée, mais les mécanismes qui sont à la base de ces fonctions restent encore à élucider. IRfF6 est une protéine qui, d'après sa structure, a été classée parmi une famille de régulateurs de la défense de l'organisme (IRFs). Des études ultérieures ont montré qu'IRf 6 n'a pas de rôle dans la réponse immunitaire mais qu'il est plutôt impliqué dans le développement de l'épiderme. Dans ce travail, nous avons établi que, dans les kératinocytes, l'expression d'IPJF6 est contrôlé par Notch et que, comme pour ce dernier, elle est réduite dans les SCCs. De plus, nous avons observé qu'IRF6 régule les mêmes gènes que Notch, et qu'il est en effet un médiateur de la fonction de Notch dans la différenciation des kératinocytes. Parmi les gènes contrôlés par l'axe Notch-IRF6 il y en a trois qui sont sur-exprimés dans les SCCs et qui sont réprimés par cet axe. Il s'agit d'une part d'IRF3 et IRF7, deux autres membres de la famille IRF, et du récepteur EGFR (Epidermal growth factor receptor), un oncogène (un gène impliqué dans l'accélération de la formation de tumeurs). Dans leur ensemble, ces découvertes nous informent sur les mécanismes impliqués dans les fonctions pro-differentiatrice et tumeur suppressive de Notch. Plus encore, elles ouvrent des perspectives intéressantes quant au développement de nouvelles approches thérapeutiques dans le traitement des cancers.II. RésuméLa voie de signalisation de Notch joue un rôle très important dans la différenciation cellulaire et dans la carcinogenèse de nombreux tissus. Dans les kératinocytes, elle agit comme suppresseur de tumeurs, fonction altérée dans les cancers spino cellulaires SCC (tumeurs kératinocytaires) de part la perte d'expression de Notch.Bien que les fonctions pro-différenciatrice et tumeur-suppressive de la voie de signalisation de Notch soient aujourd'hui reconnues, les mécanismes sous-jacents restent à explorer.Dans ce travail, nous montrons qu'IRF6, un membre de la famille des régulateurs de la voie de l'interféron (IRF), ne possédant pas de rôles dans la réponse immunitaire mais essentiel dans le développement de l'épiderme, est d'autant plus exprimé que le kératinocytes sont différenciées alors que son expression est drastiquement diminuée dans les SCC. De façon intéressante, l'expression d'IRF6 durant la différenciation kératinocytaire est directement contrôlée par Notch.Dans les kératinocytes l'expression accrue d'IRP6 a les mêmes effets que 1'activation de la voie de Notch induisant les marqueurs de différentiation des couches supra-basales de l'épiderme et inhibant ceux de la couche basale impliqués dans la prolifération cellulaire. Cependant IRF6 n'est pas impliqué dans la régulation d'autres cibles de Notch, comme p21WAFI/CiP' et Hesl. Comme Notch, IRF6 contrôle négativement l'expression de EGFR et IRF3/7. De ce fait EGFR et IRF3 et IRF7 sont fortement exprimés dans les SCCs humaines où l'expression de Notch et IRF6 est fortement réduite.En conclusion, nous avons démontré qu'IRF6 est une cible directe de Notch/CSL dans les keratinocytes qui medie les effets "non-canonique" de cette voie de signalisation dans la différentiation et dans la suppression tumorale.III. SummaryThe Notch pathway is an important regulator of differentiation and carcinogenesis. In keratinocytes it acts as tumour suppressor and the Notch gene is markedly reduced in keratinocyte-derived squamous cell carcinoma (SCC). While the pro-differentiation and tumour suppressive functions of Notch signalling in keratinocytes are well established, the underlying mechanisms are still poorly understood, We report here that Interferon Regulatory Factor 6 (IRF6), an IRF family member with an essential role in epidermal development, is downmodulated in SCC and is induced in differentiating cells. We observed that the induction of IRF6 in differentiating keratinocytes is suppressed by Notch inhibition. IRF6 expression is also decreased in mice with keratinocyte-specific deletion of the Notch 1/2.Moreover we show that the expression of this gene is induced by Notch activation through a CSL-dependent mechanism even under conditions of protein synthesis inhibition, with endogenous Notch 1 binding to the IRF6 promoter.Increased IRJF6 expression is necessary for the impact of Notch activation on differentiation markers K1 and Involucrin, and proliferation markers integrins and p63, but not on other "canonical" Notch targets like p21WAF1/Cipl, Hes1 and Hey1. Like Notch 1, IRF6 down-modulates expression of epidermal growth factor receptor (EGFR) as well as two other IRF family members, IRF3 and 7, which we previously linked to positive control of p63 expression. Expression of IRF3, IRF7 and EGFR is enhanced in cutaneous squamous cell carcinomas, illustrating a strikingly opposite pattern compared to Notch and IRF6.Thus, IRF6 is a primary Notch target in keratinocytes, which mediates the effects of this pathway on differentiation and contributes to tumor suppression.

Role of lipase in the regulation of upper gastrointestinal function in humans.

The role of lipase in the regulation of upper gastrointestinal function is poorly understood. We studied the effect of orlistat, a new, potent, and highly specific lipase inhibitor, on gastric emptying, cholecystokinin (CCK) release, and pancreaticobiliary secretion. Three groups of studies were performed in nine healthy volunteers, using the double-indicator technique with a triple-lumen duodenal tube, polyethylene glycol 4000 as a duodenal perfusion marker, and 99mTc-diethylenetriamine pentaacetic acid as a meal marker. Gastric emptying, pancreaticobiliary output, and postprandial plasma CCK levels were measured after ingestion of the following isocaloric 500-ml liquid meals with or without 200 mg orlistat: 1) a pure fat meal (10% Intralipid), 2) a meal containing free fatty acids, or 3) an albumin-glucose meal. All experiments were performed in a randomized, placebo-controlled, crossover design. Orlistat markedly inhibited lipase activity in all three experiments. Orlistat given with the fat meal reduced CCK release and output of lipase, trypsin, and bilirubin and accelerated the rate of gastric emptying (P < 0.05). After ingestion of the free fatty acid or albumin-glucose meal, orlistat had no significant effect on any of these parameters. We conclude that lipase plays an important, nutrient-specific role in the regulation of gastric emptying and pancreaticobiliary secretion after ingestion of fatty meals in humans.

Chirurgie viscérale. Chirurgie thyroïdienne et parathyroïdienne: place de la chirurgie minimale invasive [Update in thyroid and parathyroid surgery: the role for minimally invasive surgery].

Thyroid and parathyroid surgery takes an important place in general surgery. Preservation of essential cervical structures and cosmetic of outcomes of cervical incisions are major concern. Several minimally invasive strategies have been developed recently but their acceptance in the surgical community is low. Their main advantages are focused tissue dissection and improved cosmetics. In this review, management and surgical indications of thyroid nodules and parathyroid adenomas are discussed. The various minimally invasive techniques are described and their current indications are reviewed.

BAFF, APRIL and their receptors: structure, function and signaling.

BAFF, APRIL and their receptors play important immunological roles, especially in the B cell arm of the immune system. A number of splice isoforms have been described for both ligands and receptors in this subfamily, some of which are conserved between mouse and human, while others are species-specific. Structural and mutational analyses have revealed key determinants of receptor-ligand specificity. BAFF-R has a strong selectivity for BAFF; BCMA has a higher affinity for APRIL than for BAFF, while TACI binds both ligands equally well. The molecular signaling events downstream of BAFF-R, BCMA and TACI are still incompletely characterized. Survival appears to be mediated by upregulation of Bcl-2 family members through NF-kappaB activation, degradation of the pro-apototic Bim protein, and control of subcellular localization of PCKdelta. Very little is known about other signaling events associated with receptor engagement by BAFF and APRIL that lead for example to B cell activation or to CD40L-independent Ig switch.

Resolving the ortholog conjecture: orthologs tend to be weakly, but significantly, more similar in function than paralogs.

The function of most proteins is not determined experimentally, but is extrapolated from homologs. According to the "ortholog conjecture", or standard model of phylogenomics, protein function changes rapidly after duplication, leading to paralogs with different functions, while orthologs retain the ancestral function. We report here that a comparison of experimentally supported functional annotations among homologs from 13 genomes mostly supports this model. We show that to analyze GO annotation effectively, several confounding factors need to be controlled: authorship bias, variation of GO term frequency among species, variation of background similarity among species pairs, and propagated annotation bias. After controlling for these biases, we observe that orthologs have generally more similar functional annotations than paralogs. This is especially strong for sub-cellular localization. We observe only a weak decrease in functional similarity with increasing sequence divergence. These findings hold over a large diversity of species; notably orthologs from model organisms such as E. coli, yeast or mouse have conserved function with human proteins.

Labor market heterogeneity and the aggregate matching function

The matching function -a key building block in models of labor market frictions- impliesthat the job finding rate depends only on labor market tightness. We estimate such amatching function and find that the relation, although remarkably stable over 1967-2007,broke down spectacularly after 2007. We argue that labor market heterogeneities are notfully captured by the standard matching function, but that a generalized matching functionthat explicitly takes into account worker heterogeneity and market segmentation is fullyconsistent with the behavior of the job finding rate. The standard matching function canbreak down when, as in the Great Recession, the average characteristics of the unemployedchange too much, or when dispersion in labor market conditions -the extent to which somelabor markets fare worse than others- increases too much.

Effects of epidural analgesia on pelvic floor function after spontaneous delivery : a longitudinal retrospective study

Abstract: The aim of the study was to assess the effects of epidural analgesia on pelvic floor function. Eighty- two primiparous women (group 1, consisting of 41 given an epidural, and group 2 of 41 not given an epidural) were investigated during pregnancy and at 2 and 10 months after delivery by a questionnaire, clinical examination, and assessment of bladder neck behavior, urethral sphincter function and intravaginal/intra-anal pressures. The prevalence of stress urinary incontinence was similar in both groups at 2 months (24% vs. 17%, P = 0.6) and 10 months (22% vs. 7%, P = 0.1), as was the prevalence of decreased sexual vaginal response at 10 months (27% vs. 10%, P= 0.08). Bladder neck behavior, urethral sphincter function and intravaginal and intra-anal pressures showed no significant differences between the two groups. Ten months after spontaneous delivery, there were no significant differences in the prevalence of stress urinary incontinence and decreased sexual vaginal response, or in bladder neck behavior, urethral sphincter function and pelvic floor muscle strength between women who had or had not had epidural analgesia.

Health-related quality of life in survivors of childhood cancer: the role of chronic health problems.

INTRODUCTION: The influence of specific health problems on health-related quality of life (HRQoL) in childhood cancer survivors is unknown. We compared HRQoL between survivors of childhood cancer and their siblings, determined factors associated with HRQoL, and investigated the influence of chronic health problems on HRQoL. METHODS: Within the Swiss Childhood Cancer Survivor Study, we sent a questionnaire to all survivors (≥16 years) registered in the Swiss Childhood Cancer Registry, who survived >5 years and were diagnosed 1976-2005 aged <16 years. Siblings received similar questionnaires. We assessed HRQoL using Short Form-36 (SF-36). Health problems from a standard questionnaire were classified into overweight, vision impairment, hearing, memory, digestive, musculoskeletal or neurological, and thyroid problems. RESULTS: The sample included 1,593 survivors and 695 siblings. Survivors scored significantly lower than siblings in physical function, role limitation, general health, and the Physical Component Summary (PCS). Lower score in PCS was associated with a diagnosis of central nervous system tumor, retinoblastoma or bone tumor, having had surgery, cranio-spinal irradiation, or bone marrow transplantation. Lower score in Mental Component Summary was associated with older age. All health problems decreased HRQoL in all scales. Most affected were survivors reporting memory problems and musculoskeletal or neurological problems. Health problems had the biggest impact on physical functioning, general health, and energy and vitality. CONCLUSIONS: In this study, we showed the negative impact of specific chronic health problems on survivors' HRQoL. IMPLICATIONS FOR CANCER SURVIVORS: Therapeutic preventive measures, risk-targeted follow-up, and interventions might help decrease health problems and, consequently, improve survivors' quality of life.

Gain of function mutations in CgPDR1 of Candida glabrata not only mediate antifungal resistance but also enhance virulence.

CgPdr1p is a Candida glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2, and CgSNQ2, which are mediators of azole resistance. Single-point mutations in CgPDR1 are known to increase the expression of at least CgCDR1 and CgCDR2 and thus to contribute to azole resistance of clinical isolates. In this study, we investigated the incidence of CgPDR1 mutations in a large collection of clinical isolates and tested their relevance, not only to azole resistance in vitro and in vivo, but also to virulence. The comparison of CgPDR1 alleles from azole-susceptible and azole-resistant matched isolates enabled the identification of 57 amino acid substitutions, each positioned in distinct CgPDR1 alleles. These substitutions, which could be grouped into three different "hot spots," were gain of function (GOF) mutations since they conferred hyperactivity to CgPdr1p revealed by constitutive high expression of ABC-transporter genes. Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2, and CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF mutations, thus suggesting that these are rather trans-acting elements (GOF in CgPDR1) than cis-acting elements (promoters) that lead to azole resistance by upregulating specific combinations of ABC-transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model. The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model. In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions.

Role of CD8 in aberrant function of cytotoxic T lymphocytes.

Using H-2Kd-restricted photoprobe-specific cytotoxic T lymphocyte (CTL) clones, which permit assessment of T cell receptor (TCR)-ligand interactions by TCR photoaffinity labeling, we observed that the efficiency of antigen recognition by CTL was critically dependent on the half-life of TCR-ligand complexes. We show here that antigen recognition by CTL is essentially determined by the frequency of serial TCR engagement, except for very rapid dissociations, which resulted in aberrant TCR signaling and antagonism. Thus agonists that were efficiently recognized exhibited rapid TCR-ligand complex dissociation, and hence a high frequency of serial TCR engagement, whereas the opposite was true for weak agonists. Surprisingly, these differences were largely accounted for by the coreceptor CD8. While it was known that CD8 substantially decreases TCR-ligand complex dissociation, we observed in this study that this effect varied considerably among ligand variants, indicating that epitope modifications can alter the CD8 contribution to TCR-ligand binding, and hence the efficiency of antigen recognition by CTL.

Three-dimensional ordered-subset expectation maximization iterative protocol for evaluation of left ventricular volumes and function by quantitative gated SPECT: a dynamic phantom study.

The purposes of this study were to characterize the performance of a 3-dimensional (3D) ordered-subset expectation maximization (OSEM) algorithm in the quantification of left ventricular (LV) function with (99m)Tc-labeled agent gated SPECT (G-SPECT), the QGS program, and a beating-heart phantom and to optimize the reconstruction parameters for clinical applications. METHODS: A G-SPECT image of a dynamic heart phantom simulating the beating left ventricle was acquired. The exact volumes of the phantom were known and were as follows: end-diastolic volume (EDV) of 112 mL, end-systolic volume (ESV) of 37 mL, and stroke volume (SV) of 75 mL; these volumes produced an LV ejection fraction (LVEF) of 67%. Tomographic reconstructions were obtained after 10-20 iterations (I) with 4, 8, and 16 subsets (S) at full width at half maximum (FWHM) gaussian postprocessing filter cutoff values of 8-15 mm. The QGS program was used for quantitative measurements. RESULTS: Measured values ranged from 72 to 92 mL for EDV, from 18 to 32 mL for ESV, and from 54 to 63 mL for SV, and the calculated LVEF ranged from 65% to 76%. Overall, the combination of 10 I, 8 S, and a cutoff filter value of 10 mm produced the most accurate results. The plot of the measures with respect to the expectation maximization-equivalent iterations (I x S product) revealed a bell-shaped curve for the LV volumes and a reverse distribution for the LVEF, with the best results in the intermediate range. In particular, FWHM cutoff values exceeding 10 mm affected the estimation of the LV volumes. CONCLUSION: The QGS program is able to correctly calculate the LVEF when used in association with an optimized 3D OSEM algorithm (8 S, 10 I, and FWHM of 10 mm) but underestimates the LV volumes. However, various combinations of technical parameters, including a limited range of I and S (80-160 expectation maximization-equivalent iterations) and low cutoff values (< or =10 mm) for the gaussian postprocessing filter, produced results with similar accuracies and without clinically relevant differences in the LV volumes and the estimated LVEF.