Chemical genetic analysis of cdc2p : new insights into the regulation of cytokinesis in "Schizosaccharomyces pombe"

RÉSUMÉ La protéine kinase cyciine-cdc2p (Cdk) joue un rôle fondamental dans la progression du cycle cellulaire dans la levure de fission Schizosaccharomyces pombe. Nous avons étudié le rôle de cdc2p dans la régulation de la cascade de septation ou SIN (septation initiation network) en mitose et en méiose. Le SIN contrôle l'initiation de la cytokinèse à la fin de la mitose, et est supposé être négativement régulé par cdc2p. Nous avons mutagénéisé le site actif de cdc2p afin qu'il puisse lier un analogue de l'ATP (PP1) qui agit comme inhibiteur. Cet analogue ne peut pas lier la kinase de type sauvage. Cette approche dite «chemical genetics» permet une meilleure résolution temporelle comparée à l'approche classique utilisant les mutants sensibles à une température élevée. Nous avons montré que ce mutant cdc2-as (analogue sensitive) est fonctionnel et que, in vitro, l'activité kinase est inhibée en présence de l'analogue. Les cellules portant cette mutation, contrairement aux cellules de type sauvage s'arrêtent de manière irréversible soit en G2 soit en G1 et G2, suivant la concentration de l'inhibiteur. L'inactivation de cdc2p-as dans des cellules arrêtées en métaphase conduit au recrutement asymétrique des protéines du SIN sur le pôle du fuseau mitotique et au recrutement des composants du SIN, ainsi que de la ß-(1,3)glucan synthase à l'anneau contractile. De plus, nos résultats montrent que l'orthologue de la phosphatase cdc14p dans S. pombe, fip1p/clp1p, joue un rôle dans la régulation de la localisation des protéines du SIN suite à l'inactivation de cdc2p. Finalement, l'activité de cdc2p est requise pour maintenir la polo-like kinase plo1p sur les pôles du fuseau mitotique dans les premiers stages de la mitose. C'est pourquoi nous concluons que l'inactivation de cdc2p est suffisante pour activer le SIN et promouvoir la cytokinèse. Dans une étude séparée, nous avons caractérisé des potentiellement nouveaux composants ou régulateurs du SIN qui ont été isolés dans deux criblages génétiques visant à isoler des mutants atténuants la signalisation du SIN. Summary : The cyclin dependent protein kinase (Cdk) cdc2p plays a central role in the cell cycle progression of fission yeast Schizosaccharomyces pombe. We have studied the role of cdc2p in regulating the septation initiation network (SIN) in mitosis and meiosis. The SIN regulates the initiation of cytokinesis at the end of mitosis and is thought to be inhibited by cdc2p. We have mutated the active site of cdc2p to permit binding of an inhibitory ATP analogue (PP1), which is unable to bind unmodified kinases. This "chemical genetic" approach provides a much higher temporal resolution than it can be achieved with classical temperature-sensitive mutants. We demonstrate that cdc2-as (analogue sensitive) is functional and that addition of PP1 inhibits cdc2p kinase activity in vitro. Cells carrying the cdc2-as allele, but not cdc2+, undergo reversible cell cycle arrest following addition of PP1 either in G2, or at both major commitment points in the cell cycle (G1 and G2), depending upon the concentration of PP1. Inactivation of cdc2p-as in cells arrested in early mitosis promotes both the asymmetric recruitment of SIN proteins to the spindle pole bodies (SPBs), and the recruitment of the most downstream SIN components and ß-(1,3)-glucan synthase to the contractile ring. Furthermore, our results indicate that the S. pombe orthologue of Cdc14p, flp1p/clp1p, plays a role in regulating the relocalisation of SIN proteins following inactivation of cdc2p, and that cdc2p activity is required to retain the polo like kinase plot p on the SPBs in early mitosis. Thus, we conclude that inactivation of cdc2p is sufficient to activate the SIN and to promote cytokinesis. In a separate study, we have initially characterised potential novel components or regulators of the SIN pathway identified by two genetic screens for mutants attenuating SIN signaling.

Qualité microbiologique de l'air des écuries et conséquences pour la santé des travailleurs du secteur équestre

Les sports équestres sont en plein développement en France où l'on compte plus d'un million de chevaux. Le nombre de cavaliers pratiquant en club ou chez eux est estimé à 600 000 et l'on dénombre plus de 68 000 emplois en France dans tous les domaines : agriculture, environnement, courses, loisirs, sports (www.haras-nationaux.fr). On trouve également dans les métiers du cheval les vétérinaires spécialisés ainsi que les maréchaux-ferrants. De plus, la France est un pays reconnu pour son activité d'élevage avec une production de chevaux de concours de très grande qualité.Récemment, plusieurs études relatant les expositions professionnelles à la poussière des écuries ont paru dans les journaux scientifiques. Les résultats montrent que la poussière issue des écuries contient, entre autres, des concentrations relativement élevées d'endotoxines (1) et de ?(1-3)-glucan (2) pouvant avoir des conséquences sur la santé respiratoire des travailleurs. En effet, des études précédentes ont montré que le fait de travailler ou de côtoyer des chevaux était associé avec une augmentation des risques de problèmes des voies respiratoires supérieures tels que des irritations du nez, de la toux (sèche et productive), de l'asthme, des bronchites chroniques et des épisodes d'" organic dust toxic syndrome (3) " (Gallagher et al., 2007 ; Mazan et al., 2009). [Auteure]

β-Glucan antigenemia assay for the diagnosis of invasive fungal infections in patients with hematological malignancies: a systematic review and meta-analysis of cohort studies from the Third European Conference on Infections in Leukemia (ECIL-3).

BACKGROUND: Invasive fungal infections (IFIs) are life-threatening complications in patients with hemato-oncological malignancies, and early diagnosis is crucial for outcome. The compound 1,3-β-D-glucan (BG), a cell wall component of most fungal species, can be detected in blood during IFI. Four commercial BG antigenemia assays are available (Fungitell, Fungitec-G, Wako, and Maruha). This meta-analysis from the Third European Conference on Infections in Leukemia (ECIL-3) assessed the performance of BG assays for the diagnosis of IFI in hemato-oncological patients. METHODS: Studies reporting the performance of BG antigenemia assays for the diagnosis of IFI (European Organization for Research and Treatment of Cancer and Mycoses Study Group criteria) in hemato-oncological patients were identified. The analysis was focused on high-quality cohort studies with exclusion of case-control studies. Meta-analysis was performed by conventional meta-analytical pooling and bivariate analysis. RESULTS: Six cohort studies were included (1771 adult patients with 414 IFIs of which 215 were proven or probable). Similar performance was observed among the different BG assays. For the cutoff recommended by the manufacturer, the diagnostic performance of the BG assay in proven or probable IFI was better with 2 consecutive positive test results (diagnostic odds ratio for 2 consecutive vs one single positive results, 111.8 [95% confidence interval {CI}, 38.6-324.1] vs 16.3 [95% CI, 6.5-40.8], respectively; heterogeneity index for 2 consecutive vs one single positive results, 0% vs 72.6%, respectively). For 2 consecutive tests, sensitivity and specificity were 49.6% (95% CI, 34.0%-65.3%) and 98.9% (95% CI, 97.4%-99.5%), respectively. Estimated positive and negative predictive values for an IFI prevalence of 10% were 83.5% and 94.6%, respectively. CONCLUSIONS: Different BG assays have similar accuracy for the diagnosis of IFI in hemato-oncological patients. Two consecutive positive antigenemia assays have very high specificity, positive predictive value, and negative predictive value. Because sensitivity is low, the test needs to be combined with clinical, radiological, and microbiological findings.

Is (1→3)-β-D-glucan the missing link from bedside assessment to pre-emptive therapy of invasive candidiasis?

ABSTRACT: Invasive candidiasis is a frequent life-threatening complication in critically ill patients. Early diagnosis followed by prompt treatment aimed at improving outcome by minimizing unnecessary antifungal use remains a major challenge in the ICU setting. Timely patient selection thus plays a key role for clinically efficient and cost-effective management. Approaches combining clinical risk factors and Candida colonization data have improved our ability to identify such patients early. While the negative predictive value of scores and predicting rules is up to 95 to 99%, the positive predictive value is much lower, ranging between 10 and 60%. Accordingly, if a positive score or rule is used to guide the start of antifungal therapy, many patients may be treated unnecessarily. Candida biomarkers display higher positive predictive values; however, they lack sensitivity and are thus not able to identify all cases of invasive candidiasis. The (1→3)-β-D-glucan (BG) assay, a panfungal antigen test, is recommended as a complementary tool for the diagnosis of invasive mycoses in high-risk hemato-oncological patients. Its role in the more heterogeneous ICU population remains to be defined. More efficient clinical selection strategies combined with performant laboratory tools are needed in order to treat the right patients at the right time by keeping costs of screening and therapy as low as possible. The new approach proposed by Posteraro and colleagues in the previous issue of Critical Care meets these requirements. A single positive BG value in medical patients admitted to the ICU with sepsis and expected to stay for more than 5 days preceded the documentation of candidemia by 1 to 3 days with an unprecedented diagnostic accuracy. Applying this one-point fungal screening on a selected subset of ICU patients with an estimated 15 to 20% risk of developing candidemia is an appealing and potentially cost-effective approach. If confirmed by multicenter investigations, and extended to surgical patients at high risk of invasive candidiasis after abdominal surgery, this Bayesian-based risk stratification approach aimed at maximizing clinical efficiency by minimizing health care resource utilization may substantially simplify the management of critically ill patients at risk of invasive candidiasis.

B1-insensitive T2 preparation for improved coronary magnetic resonance angiography at 3 T.

At 3 T, the effective wavelength of the RF field is comparable to the dimension of the human body, resulting in B1 standing wave effects and extra variations in phase. This effect is accompanied by an increase in B0 field inhomogeneity compared to 1.5 T. This combination results in nonuniform magnetization preparation by the composite MLEV weighted T2 preparation (T2 Prep) sequence used for coronary magnetic resonance angiography (MRA). A new adiabatic refocusing T2 Prep sequence is presented in which the magnetization is tipped into the transverse plane with a hard RF pulse and refocused using a pair of adiabatic fast-passage RF pulses. The isochromats are subsequently returned to the longitudinal axis using a hard RF pulse. Numerical simulations predict an excellent suppression of artifacts originating from B1 inhomogeneity while achieving good contrast enhancement between coronary arteries and surrounding tissue. This was confirmed by an in vivo study, in which coronary MR angiograms were obtained without a T2 Prep, with an MLEV weighted T2 Prep and the proposed adiabatic T2 Prep. Improved quantitative and qualitative coronary MRA image measurement was achieved using the adiabatic T2 Prep at 3 T.

β-Glucan Antigenemia Anticipates Diagnosis of Blood Culture-Negative Intraabdominal Candidiasis.

Rationale: Life-threatening intraabdominal candidiasis (IAC) occurs in 30 to 40% of high-risk surgical intensive care unit (ICU) patients. Although early IAC diagnosis is crucial, blood cultures are negative, and the role of Candida score/colonization indexes is not established. Objectives: The aim of this prospective Fungal Infection Network of Switzerland (FUNGINOS) cohort study was to assess accuracy of 1,3-β-d-glucan (BG) antigenemia for diagnosis of IAC. Methods: Four hundred thirty-four consecutive adults with abdominal surgery or acute pancreatitis and ICU stay 72 hours or longer were screened: 89 (20.5%) at high risk for IAC were studied (68 recurrent gastrointestinal tract perforation, 21 acute necrotizing pancreatitis). Diagnostic accuracy of serum BG (Fungitell), Candida score, and colonization indexes was compared. Measurements and Main Results: Fifty-eight of 89 (65%) patients were colonized by Candida; 29 of 89 (33%) presented IAC (27 of 29 with negative blood cultures). Nine hundred twenty-one sera were analyzed (9/patient): median BG was 253 pg/ml (46-9,557) in IAC versus 99 pg/ml (8-440) in colonization (P < 0.01). Sensitivity and specificity of two consecutive BG measurements greater than or equal to 80 pg/ml were 65 and 78%, respectively. In recurrent gastrointestinal tract perforation it was 75 and 77% versus 90 and 38% (Candida score ≥ 3), 79 and 34% (colonization index ≥ 0.5), and 54 and 63% (corrected colonization index ≥ 0.4), respectively. BG positivity anticipated IAC diagnosis (5 d) and antifungal therapy (6 d). Severe sepsis/septic shock and death occurred in 10 of 11 (91%) and 4 of 11 (36%) patients with BG 400 pg/ml or more versus 5 of 18 (28%, P = 0.002) and 1 of 18 (6%, P = 0.05) with BG measurement less than 400 pg/ml. β-Glucan decreased in IAC responding to therapy and increased in nonresponse. Conclusions: BG antigenemia is superior to Candida score and colonization indexes and anticipates diagnosis of blood culture-negative IAC. This proof-of-concept observation in strictly selected high-risk surgical ICU patients deserves investigation of BG-driven preemptive therapy.

Robust volume-targeted balanced steady-state free-precession coronary magnetic resonance angiography in a breathhold at 3.0 Tesla: a reproducibility study.

BACKGROUND: Transient balanced steady-state free-precession (bSSFP) has shown substantial promise for noninvasive assessment of coronary arteries but its utilization at 3.0 T and above has been hampered by susceptibility to field inhomogeneities that degrade image quality. The purpose of this work was to refine, implement, and test a robust, practical single-breathhold bSSFP coronary MRA sequence at 3.0 T and to test the reproducibility of the technique. METHODS: A 3D, volume-targeted, high-resolution bSSFP sequence was implemented. Localized image-based shimming was performed to minimize inhomogeneities of both the static magnetic field and the radio frequency excitation field. Fifteen healthy volunteers and three patients with coronary artery disease underwent examination with the bSSFP sequence (scan time = 20.5 ± 2.0 seconds), and acquisitions were repeated in nine subjects. The images were quantitatively analyzed using a semi-automated software tool, and the repeatability and reproducibility of measurements were determined using regression analysis and intra-class correlation coefficient (ICC), in a blinded manner. RESULTS: The 3D bSSFP sequence provided uniform, high-quality depiction of coronary arteries (n = 20). The average visible vessel length of 100.5 ± 6.3 mm and sharpness of 55 ± 2% compared favorably with earlier reported navigator-gated bSSFP and gradient echo sequences at 3.0 T. Length measurements demonstrated a highly statistically significant degree of inter-observer (r = 0.994, ICC = 0.993), intra-observer (r = 0.894, ICC = 0.896), and inter-scan concordance (r = 0.980, ICC = 0.974). Furthermore, ICC values demonstrated excellent intra-observer, inter-observer, and inter-scan agreement for vessel diameter measurements (ICC = 0.987, 0.976, and 0.961, respectively), and vessel sharpness values (ICC = 0.989, 0.938, and 0.904, respectively). CONCLUSIONS: The 3D bSSFP acquisition, using a state-of-the-art MR scanner equipped with recently available technologies such as multi-transmit, 32-channel cardiac coil, and localized B0 and B1+ shimming, allows accelerated and reproducible multi-segment assessment of the major coronary arteries at 3.0 T in a single breathhold. This rapid sequence may be especially useful for functional imaging of the coronaries where the acquisition time is limited by the stress duration and in cases where low navigator-gating efficiency prohibits acquisition of a free breathing scan in a reasonable time period.

Utility of beta-glucan for the diagnosis of invasive fungal infections

(1,3)-b-D-glucan is a component of the fungal cell wall. New assays have made it possible to detect this molecule in a variety of clinical samples such as blood, cerebrospinal fluid, and bronchioalveolar lavage fluid. Detection of this molecule through several assays has been validated as an adjunct method to diagnose invasive fungal infections. With several decades of data and recent positive meta-analyses, these assays have now been sufficiently studied and are ready to enter the mainstream of diagnosis in medical mycology.

Scaffold attachment factor B1 directly interacts with nuclear receptors in living cells and represses transcriptional activity.

Transcriptional activity relies on coregulators that modify the chromatin structure and serve as bridging factors between transcription factors and the basal transcription machinery. Using the DE domain of human peroxisome proliferator-activated receptor gamma (PPARgamma) as bait in a yeast two-hybrid screen of a human adipose tissue library, we isolated the scaffold attachment factor B1 (SAFB1/HET/HAP), which was previously shown to be a corepressor of estrogen receptor alpha. We show here that SAFB1 has a very broad tissue expression profile in human and is also expressed all along mouse embryogenesis. SAFB1 interacts in pull-down assays not only with PPARgamma but also with all nuclear receptors tested so far, albeit with different affinities. The association of SAFB1 and PPARgamma in vivo is further demonstrated by fluorescence resonance energy transfer (FRET) experiments in living cells. We finally show that SAFB1 is a rather general corepressor for nuclear receptors. Its change in expression during the early phases of adipocyte and enterocyte differentiation suggests that SAFB1 potentially influences cell proliferation and differentiation decisions.

Determinants of vitellogenin B1 promoter architecture. HNF3 and estrogen responsive transcription within chromatin.

The liver-specific vitellogenin B1 promoter is efficiently activated by estrogen within a nucleosomal environment after microinjection into Xenopus laevis oocytes, consistent with the hypothesis that significant nucleosome remodeling over this promoter is not a prerequisite for the activation by the estrogen receptor (ERalpha). This observation lead us to investigate determinants other than ERalpha of chromatin structure and transcriptional activation of the vitellogenin B1 promoter in this system and in vitro. We find that the liver-enriched transcription factor HNF3 has an important organizational role for chromatin structure as demonstrated by DNase I-hypersensitive site mapping. Both HNF3 and the estrogen receptor activate transcription synergistically and are able to interact with chromatin reconstituted in vitro with three positioned nucleosomes. We propose that HNF3 is the cellular determinant which establishes a promoter environment favorable to a rapid transcriptional activation by the estrogen receptor.

Vitellogenin genes A1 and B1 are linked in the Xenopus laevis genome.

Genomic clones containing the Xenopus laevis vitellogenin gene B1 have been isolated from DNA libraries and characterized by heteroduplex mapping in the electron microscope, restriction endonuclease analysis, and in vitro transcription in a HeLa whole-cell extract. Sequences from the 3'-flanking region of the previously isolated A1 vitellogenin gene were found in the 5'-flanking region of this B1 gene. Thus, the two genes are linked, with 15.5 kilobase pairs of DNA between them. Their length is about 22 kilobase pairs (A1 gene) and 16.5 kilobase pairs (B1 gene) and they have the following arrangement: 5'-A1 gene-spacer-B1 gene-3'. The analysis of heteroduplexes formed between the two genes revealed several regions of homology. Both genes are in the same orientation and, therefore, are transcribed from the same DNA strand. The possible events by which the vitellogenin gene family arose in Xenopus laevis are discussed.