Influence possible des protozoaires sur le taux de mortalité des bactéries autotrophes nitrifiantes

Le modèle de l'IAWQ du processus de boues activées représente les mécanismes endogènes de la biomasse nitrifiante par le décès des micro-organismes (équation d'ordre 1 par rapport à la biomasse). La constante de décès, ou taux de mortalité bA, est aujourd'hui encore mal connue, et en particulier les facteurs influants sur sa valeur. De récentes études ont montré que la prédation par la microfaune pourrait être un facteur déterminant sur la valeur de bA. Cette étude se propose donc de quantifier l'effet de la prédation sur la valeur de bA. Deux réacteurs maintenus sans alimentation en substrat ont été caractérisés en parallèle: l'un a reçu une dose d'antibiotique spécifique aux eucaryotes (cycloheximide) afin de diminuer la quantité d'organismes de la microfaune, alors que l'autre n'a reçu aucun antibiotique (témoin). Les résultats obtenus montrent que le cycloheximide inhibe la plupart des organismes de la microfaune sauf les amibes; celles-ci semblent plutôt stimulées par cet antibiotique. En ce qui concerne la nitrification, un ralentissement de la production de nitrate dans le réacteur traité à l'antibiotique est observé à partir du sixième jour. Cette diminution de production de nitrate est probablement causée par une réduction de l'azote nitrifiable (qui est mobilisé par les amibes) couplée à une prédation des organismes nitrifiants par les amibes. D'ailleurs, l'augmentation de la prédation par les amibes à partir du jour 6 a diminué l'activité nitrifiante également mesurée par respirométrie (rO2 Nmax). Cette diminution du taux de respiration indique une augmentation du taux de mortalité (bA) des organismes nitrifiants. En effet, la valeur du taux de mortalité mesurée dans le réacteur témoin est de 0.08 d-1 alors que selon la microfaune présente dans le réacteur inhibé au cycloheximide, la valeur de ce taux de mortalité a varié entre 0.05 d-1 et 0.15 d-1.Designing biological wastewater treatment plants with the aid of the model developed by the IAWQ requires the knowledge of biological kinetic parameters. For nitrifying activated sludge, these parameters are related to nitrifying bacteria: maximum autotrophic growth rate µAmax, yield coefficient YA and the autotrophic decay rate bA. Although variables influencing µAmax and YA values are well known, this is not the case for bA. MARTINAGE and PAUL (2000) have recently shown that the bA value is strongly influenced by the influent quality, leading to the assumption that influent quality has a strong effect on microfauna composition, and consequently on the grazing rate of microfauna on nitrifying bacteria. In fixed-film processes, protozoan grazing reduces the bacterial population considerably (NATUSCKA and WELANDER, 1994). However, although many data are available concerning the grazing rates of different protozoa, the effect of microfaunal grazing on nitrification is still a matter of debate (RATSAK et al., 1994) and its effect on the bA value is still unknown. These two topics are investigated here.Nitrifying activated sludges were grown in two identical batch reactors, but in one, cycloheximide was added to inhibit eucaryotic growth (MAURINES CARBONEILL et al., 1998). Microfauna organism numbers were quantified in both reactors by microscopic observations of flagellated protozoa (>8 µm), amoebae, ciliates, rotifers and higher invertebrates (Fig. 3). Microbial counts were then correlated with the bA value. The latter was determined using the procedure proposed by SALZER (1992) which consists of characterising the time behaviour of the maximum nitrification rate measured by respirometry of activated sludge under substrate starvation. Under these conditions bacteria die and organic nitrogen is released into the bulk phase. This nitrogen is ammonified, and nitrifying bacteria use this substrate to produce nitrate, and then autotrophic bacterial growth occurs. This method takes this growth into account by characterising nitrate production during the experiment (Fig. 2).The effect of cycloheximide on nitrification was first determined to make sure that this compound is not inhibitory toward nitrifiers. Results obtained (Table 1) show that cycloheximide was not inhibitory toward nitrate production or the maximum nitrification oxygen uptake rate (rO2 N) after 4 hours of contact with nitrifying biomass. Cycloheximide addition in the activated sludge had an important impact on rotifers and flagellates but no effect on ciliates; it also seemed to stimulate amoebae growth. In both reactors, flagellates were mainly Peranama, attached ciliates were mainly Opercularia and Epistylis and a few Vorticella. Free ciliates like Aspidisca and Euplotes were found in both reactors.Variation with time of the abundance of microfauna organisms is shown in Figures 4 and 5 for both reactors. In the reference reactor the number of microfauna organisms decreased with time (Fig. 4) probably due to substrate starvation. Microfauna composition remained however diversified. For the inhibited reactor (Fig. 5), three periods were observed. During period I, the microfauna was mainly composed of ciliates and the number of microfaunal organisms decreased rapidly. During period II, an important growth of amoebae was observed. Cycloheximide was then added during this period to reduce their number. This growth of amoebae seems to be caused by the resistance of these micro-organisms toward inhibiting compounds (SRIKANTH et BERK, 1993). During period III, the number of microfaunal organisms was lower than during period II, and microfauna was mainly composed of ciliates.Nitrate concentration behaviour, necessary for bA calculation, is shown on Figure 6. In the reference reactor, nitrate concentrations varied linearly. For the inhibited reactor, the linear pattern was not observed during period II. This result was probably caused by an important nitrogen assimilation need of amoebae (ELDRIGE and JACKSON, 1993). Because organic nitrogen released by bacterial decay is consumed by amoebae assimilation, less nitrogen is available for the ammonification process and therefore for nitrification. Ammonia concentrations remained below 0.2 mg N·l-1 during all the experiment for both reactors. When amoebae disappeared from the inhibited reactor (period III) nitrate concentration varied linearly again.Variations of the maximum nitrification oxygen uptake rate (rO2 Nmax) with time are presented in Figure 7 (A&B) for both reactors. Two curves are plotted on each figure. Empty squares represent the measured rO2 N and black points represent the maximum nitrification rate that would have been measured if no growth on ammonification products had occurred. For the reference reactor (Fig. 7A), a value for bA of 0.08 d-1 can be calculated and can be considered constant for a constant microfauna composition.Three bA values can be estimated for the reactor inhibited with cycloheximide (Fig. 7B), corresponding to the three periods observed for microfauna composition. During period I, the bA value is 0.05 d-1 : a decrease in the microfaunal organism numbers implies a decrease of the bA value. During period II, when a development of amoebae is observed, the bA value increases and reaches 0.15 d-1. During period III with reduced grazing, the bA value is 0.13 d-1. Since during periods I and III the microfauna is mainly composed of ciliates, this difference between bA values is likely due to the observed difference in floc size between periods I and III.The results obtained during this study tend to prove (1) that the use of cycloheximide reduces microfaunal populations but can lead to a development of amoebae, and (2) that microfauna grazing seems to have an influence on the bA value, which can vary from 0.05 to 0.15 d-1 depending on microfaunal composition and abundance

Simple and Reliable Determination of Intravoxel Incoherent Motion Parameters for the Differential Diagnosis of Head and Neck Tumors

Intravoxel incoherent motion (IVIM) imaging can characterize diffusion and perfusion of normal and diseased tissues, and IVIM parameters are authentically determined by using cumbersome least-squares method. We evaluated a simple technique for the determination of IVIM parameters using geometric analysis of the multiexponential signal decay curve as an alternative to the least-squares method for the diagnosis of head and neck tumors. Pure diffusion coefficients (D), microvascular volume fraction (f), perfusion-related incoherent microcirculation (D), and perfusion parameter that is heavily weighted towards extravascular space (P) were determined geometrically (Geo D, Geo f, and Geo P) or by least-squares method (Fit D, Fit f, and Fit D) in normal structures and 105 head and neck tumors. The IVIM parameters were compared for their levels and diagnostic abilities between the 2 techniques. The IVIM parameters were not able to determine in 14 tumors with the least-squares method alone and in 4 tumors with the geometric and least-squares methods. The geometric IVIM values were significantly different (p<0.001) from Fit values (+2±64% and 7±24% for D and f values, respectively). Geo D and Fit D differentiated between lymphomas and SCCs with similar efficacy (78% and 80% accuracy, respectively). Stepwise approaches using combinations of Geo D and Geo P, Geo D and Geo f, or Fit D and Fit Ddifferentiated between pleomorphic adenomas, Warthin tumors, and malignant salivary gland tumors with the same efficacy (91% accuracy = 21/ 23). However, a stepwise differentiation using Fit D and Fit f was less effective (83% accuracy = 19/23). Considering cumbersome procedures with the least squares method compared with the geometric method, we concluded that the geometric determination of IVIM parameters can be an alternative to least-squares method in the diagnosis of head and neck tumors

Repeatability of IVIM biomarkers from diffusion-weighted MRI in head and neck:Bayesian probability versus neural network

Purpose: The intravoxel incoherent motion (IVIM) model for DWI might provide useful biomarkers for disease management in head and neck cancer. This study compared the repeatability of three IVIM fitting methods to the conventional nonlinear least-squares regression: Bayesian probability estimation, a recently introduced neural network approach, IVIM-NET, and a version of the neural network modified to increase consistency, IVIM-NETmod. Methods: Ten healthy volunteers underwent two imaging sessions of the neck, two weeks apart, with two DWI acquisitions per session. Model parameters (ADC, diffusion coefficient (Formula presented.), perfusion fraction (Formula presented.), and pseudo-diffusion coefficient (Formula presented.)) from each fit method were determined in the tonsils and in the pterygoid muscles. Within-subject coefficients of variation (wCV) were calculated to assess repeatability. Training of the neural network was repeated 100 times with random initialization to investigate consistency, quantified by the coefficient of variance. Results: The Bayesian and neural network approaches outperformed nonlinear regression in terms of wCV. Intersession wCV of (Formula presented.) in the tonsils was 23.4% for nonlinear regression, 9.7% for Bayesian estimation, 9.4% for IVIM-NET, and 11.2% for IVIM-NETmod. However, results from repeated training of the neural network on the same data set showed differences in parameter estimates: The coefficient of variances over the 100 repetitions for IVIM-NET were 15% for both (Formula presented.) and (Formula presented.), and 94% for (Formula presented.); for IVIM-NETmod, these values improved to 5%, 9%, and 62%, respectively. Conclusion: Repeatabilities from the Bayesian and neural network approaches are superior to that of nonlinear regression for estimating IVIM parameters in the head and neck

Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70.

Heat Shock Protein 70s (HSP70s) are key molecular chaperones that are overexpressed in many cancers and often associated with metastasis and poor prognosis. It has proven difficult to develop ATP-competitive, drug-like small molecule inhibitors of HSP70s due to the flexible and hydrophilic nature of the HSP70 ATP-binding site and its high affinity for endogenous nucleotides. The aim of this study was to explore the potential for the inhibition of HSP70 through alternative binding sites using fragment-based approaches. A surface plasmon resonance (SPR) fragment screen designed to detect secondary binding sites in HSP70 led to the identification by X-ray crystallography of a cryptic binding site in the nucleotide-binding domain (NBD) of HSP70 adjacent to the ATP-binding site. Fragment binding was confirmed and characterized as ATP-competitive using SPR and ligand-observed NMR methods. Molecular dynamics simulations were applied to understand the interactions with the protein upon ligand binding, and local secondary structure changes consistent with interconversion between the observed crystal structures with and without the cryptic pocket were detected. A virtual high-throughput screen (vHTS) against the cryptic pocket was conducted, and five compounds with diverse chemical scaffolds were confirmed to bind to HSP70 with micromolar affinity by SPR. These results identified and characterized a new targetable site on HSP70. While targeting HSP70 remains challenging, the new site may provide opportunities to develop allosteric ATP-competitive inhibitors with differentiated physicochemical properties from current series

Diffusion-weighted magnetic resonance imaging to detect synchronous uterine endometrial and endocervical adenocarcinoma

Synchronous endometrial and cervical cancer is a very rare condition. This report describes a case of a 46-year-old woman who presented with a cervical mass that measured 5.6 cm along its longest diameter, whose biopsy analysis revealed an endocervical mucinous adenocarcinoma. She was classified as having an IB2 cervical carcinoma and treated with concurrent chemoradiation plus hysterectomy. Pathological and immunohistochemical analysis of the surgical specimens revealed a synchronous endometrioid grade 2 adenocarcinoma in the endometrium, and a well-differentiated mucinous adenocarcinoma in the cervix. Magnetic resonance imaging (MRI) studies performed prior to treatment were reviewed and apparent diffusion coefficient (ADC) maps were generated. The ADC values demonstrated distinct signal intensity differences between the endometrial and endocervical tumors. In conclusion, diffusion-weighted MRI and ADC maps can help to distinguish the site of origin of synchronous tumors

Achieving In Vivo Target Depletion through the Discovery and Optimization of Benzimidazolone BCL6 Degraders.

Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein-protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing

G-CSF Prevents the Progression of Structural Disintegration of White Matter Tracts in Amyotrophic Lateral Sclerosis: A Pilot Trial

Background: The hematopoietic protein Granulocyte-colony stimulating factor (G-CSF) has neuroprotective and regenerative properties. The G-CSF receptor is expressed by motoneurons, and G-CSF protects cultured motoneuronal cells from apoptosis. It therefore appears as an attractive and feasible drug candidate for the treatment of amyotrophic lateral sclerosis (ALS). The current pilot study was performed to determine whether treatment with G-CSF in ALS patients is feasible.Methods: Ten patients with definite ALS were entered into a double-blind, placebo-controlled, randomized trial. Patients received either 10 mu g/kg BW G-CSF or placebo subcutaneously for the first 10 days and from day 20 to 25 of the study. Clinical outcome was assessed by changes in the ALS functional rating scale (ALSFRS), a comprehensive neuropsychological test battery, and by examining hand activities of daily living over the course of the study (100 days). The total number of adverse events (AE) and treatment-related AEs, discontinuation due to treatment-related AEs, laboratory parameters including leukocyte, erythrocyte, and platelet count, as well as vital signs were examined as safety endpoints. Furthermore, we explored potential effects of G-CSF on structural cerebral abnormalities on the basis of voxel-wise statistics of Diffusion Tensor Imaging (DTI), brain volumetry, and voxel-based morphometry.Results: Treatment was well-tolerated. No significant differences were found between groups in clinical tests and brain volumetry from baseline to day 100. However, DTI analysis revealed significant reductions of fractional anisotropy (FA) encompassing diffuse areas of the brain when patients were compared to controls. On longitudinal analysis, the placebo group showed significant greater and more widespread decline in FA than the ALS patients treated with G-CSF.Conclusions: Subcutaneous G-CSF treatment in ALS patients appears as feasible approach. Although exploratory analysis of clinical data showed no significant effect, DTI measurements suggest that the widespread and progressive microstructural neural damage in ALS can be modulated by G-CSF treatment. These findings may carry significant implications for further clinical trials on ALS using growth factors