Mesure de la biomasse et de l'activité bactérienne dans l'eau de distribution

Afin d'étudier la reviviscence bactérienne dans les réseaux de distribution, des méthodes de mesures de la biomasse et de l'activité bactérienne ont été investiguées sur des eaux provenant d'un réseau de distribution. Trois méthodes d'estimation de la biomasse bactérienne ont été comparées : le comptage sur gélose, selon la norme française d'examen bactériologique des eaux de consommation, le dosage de l'ADN contenu dans les particules retenues sur une membrane de porosité de 0.2 µm et le comptage direct au microscope à épifluorescence après coloration des bactéries à l'acridine orange. Les comptages sur gélose, tout comme en milieu aquatique naturel, sous-estiment très largement le nombre de bactéries; ceci semble principalement lié à la présence de bactéries viables mais non cultivables. Le dosage de l'ADN et les comptages directs corrèlent assez bien avec en moyenne un contenu en ADN par bactérie de 4,1 x 10-15 g d'ADN, mais la première méthode semble moins précise. Le comptage direct semble donc la méthode la plus adaptée à l'estimation du nombre total de bactéries dans ce type de milieu.Afin d'estimer l'activité bactérienne, les protocoles expérimentaux de deux méthodes utilisées en écologie bactérienne ont été adaptés aux conditions particulières de l'eau de distribution : l'incorporation de thymidine tritiée dans l'ADN bactérien et l'incorporation de leucine tritiée dans les protéines. La comparaison des deux méthodes sur une série d'échantillons montre une bonne corrélation, avec un rapport molaire entre incorporation de leucine et de thymidine compatible avec les facteurs de conversion des deux méthodes cités dans la littérature et établis pour les milieux aquatiques naturels. Les deux méthodes sont utilisables pour mesurer l'activité bactérienne dans l'eau potable, néanmoins l'incorporation de thymidine est plus aisée à mettre en oeuvre, car elle ne nécessite de travailler qu'à une seule concentration en traceur radioactif.Bacterial regrowth in distribution systems is an important problem for drinking water producers. It is linked to the more and more frequent utilization of low quality surface waters, containing high concentration of organic matter, as raw water, and also to the increase in size and complexity of the distribution networks with high residence time of the water between its production and utilization. At the present time chlorination of treated water, with sometimes rechlorination in the network, is the usual way to limit growth in distribution systems. This solution however presents disadvantages, the major one is the formation of unpleasant organochlorine compounds which are responsible for tastes and odours of water. An alternative strategy consists of developing treatment lines in which biodegradable dissolved organic carbon is removed. It allows through a reduction of the chlorine demand of the water to increase the stability of the chlorine residual of the water. In this context, it is important to get a good knowledge of the factors controlling bacterial development in distribution networks. Up to now, studies on this subject have met some methodological problems linked to the fact that classical bacteriological methods are inadequate to study this kind of systems.In this paper, various methods have been investigated to estimate bacterial biomass and activity in tap water. For this study, the analyzed water samples have been collected in the distribution system of the Parisian suburbs.Three methods have been tested for the determination of bacterial biomass : plate count, measurement of DNA associated with particles with a size higher than 0.2 µm and direct microscopic enumeration. Heterotrophic plate counts have been performed following the French standard and results are expressed in CFU (Colony Forming Units) per ml; the DNA collected after filtration of 500 ml to 1500 ml of water on a 0.2 µm pore size membrane was estimated using a fluorimetric method, as proposed by Mc COY and OLSON (1985); direct enumerations were performed by epifluorescence microscopy after acridine orange staining (AODC) following the procedure proposed by HOBBIE et al. (1977), the comparison between plate counts and AODC (fig. 1) shows the important underestimation of the bacterial numbers when estimated by the CFU (up to 3 orders of magnitude). Such discrepancy has already been observed in natural aquatic ecosystems and is usually explained by the presence of numerous dead cells enumerated by microscopy. Now, it seems that the difference between, plate counts and direct counts may rather be explained by the presence in water of « viable but non culturable » bacteria.A comparison between DNA estimation and direct counts have also been performed. Figure 2 shows the results of this comparison. In spite of the dispersion, the correlation between both methods is significant and the correlation straight line indicates an average DNA content per bacteria of 4.1 x 10-15g DNA in good accordance with the values quoted in the literature. The dispersion of the data around this average can be explained by various ways : the variability of DNA per cell content for the different bacterial strains present in the water samples, the precision of the DNA method which is not higher than 20 % and possible contamination by other organisms than bacteria, as flagellates or ciliates, which are retained on the 0.2 µm pore size membrane.On the basis of these tests, it seems that the direct count by epifluorescence microscopy is the most adapted method for studying the bacterial regrowth in distribution system.The understanding of bacterial dynamics in a distribution system requires measurements of bacterial activity. Various methods have been developed in order to estimate bacterial activity in natural aquatic ecosystems. They are primarily based on the use of radioactive tracers. At the present time, the tritiated thymidine incorporation method, which measures the replication of bacterial DNA, is the most usually used one, but the incorporation of tritiated leucine into proteins, which measures increase in bacterial biomass, seems to be also an interesting method. These methods have been selected, on one hand, because of their specificity towards bacteria and, on the other hand, because of their high sensibility which is required for measurements of bacterial activity in the conditions of drinking water. Up to now, these methods have never been applied to drinking water. We have modified the experimental procedure of both methods : incubation time, radioactive tracers concentrations and volume of the sample have been tested and adapted in order to allow measurement in the conditions of drinking water samples. For thymidine incorporation, the volume of sample, incubated during 20 hours in the presence of 20 nM concentration of 3H-thymidine, was 100 ml. The incorporation was measured in the DNA, using the biochemical procedure proposed by WICKS and ROBARTS (1977), rather than in the total macromolecules. For leucine incorporation, we measured the incorporation rate at four leucine concentrations (2, 27, 52, 77 nM : 2 nM of 3H-leucine + non radioactive leucine) in 25 ml samples and the incubation lasted 3 to 4 hours. The incorporation rate was calculated as the reciprocal of the angular coefficient of the correlation straight fine obtained when the reciprocal of the fraction of leucine incorporated per hour was plotted against leucine concentration (fig. 3). Comparison of both methods on samples of drinking is presented at figure 4, a good linear correlation was found. The equation of the correlation straigth line is :log [Inc.leu (pmol/l.h)] = 0,97 log [Inc.thy (pmol/l.h)] + 1.35(n = 69, r = 0.84)The molar ratio between leucine and thymidine incorporation found in these samples (20 to 25) seems to be in good agreement with the usual conversion factors found for both methods in natural aquatic ecosystems. Bath methods seem to be available to bacterial activity estimations in drinking water, the triatiated thymidine incorporation method which requires working with only one concentration of radioactive tracer seems easier to use

Studies of BDOC and bacterial dynamics in the drinking water distribution system of the Northern Parisian suburbs

La dégradation de la qualité de l'eau dans les réseaux de distribution, due à la reviviscence bactérienne, est, à l'heure actuelle, un souci majeur pour les producteurs d'eau potable. Dans ce contexte, une bonne connaissance des facteurs de contrôle du développement bactérien dans ce type de milieu s'avère nécessaire. Le but de la présente étude est de comprendre le rôle du carbone organique dissous biodégradable (CODB) dans la dynamique bactérienne en réseau de distribution. Cet article présente les résultats d'une étude en cours, lancée à l'initiative du Syndicat des Eaux de l'Ile de France, sur le réseau de distribution de la banlieue nord de Paris alimenté par l'usine de production de Méry-sur-Oise.Le CODB a été déterminé par la méthode de bioessai proposée par SERVAIS et al. (1987, 1989). La biomasse bactérienne libre a été estimée par microscopie à épifluorescence après coloration des bactéries à l'acridine orange, et la méthode d'incorporation de thymidine tritiée utilisée en écologie bactérienne a été adaptée, afin d'estimer la production bactérienne des bactéries présentes dans l'eau du réseau. De plus, la biomasse et l'activité des bactéries fixées ont été étudiées. Une méthode d'estimation de la biomasse, basée sur la mesure de l'activité exoprotéolytique potentielle des bactéries, a été développée. Pour l'estimation de la production bactérienne, la méthode d'incorporation de thymidine tritiée a été adaptée pour être utilisée pour les bactéries fixées.Les résultats obtenus mettent clairement en évidence une décroissance significative de la teneur en CODB dans les canalisations de faible diamètre dans la plupart des situations. Lorsque l'on porte la décroissance du CODB entre l'eau refoulée et l'eau présente dans les canalisations de faible diamètre en fonction du CODB dans l'eau refoulée, une corrélation significative est observée (fig. 1); l'intersection de la droite de corrélation avec l'abscisse indique la présence d'un seuil (environ 0,16 mgC.L-1) en-dessous duquel aucune décroissance de CODB n'est observée. Ce résultat, qui doit encore être confirmé, est important en vue de définir un objectif à atteindre en fin de filière, en terme de teneur en CODB.Dans l'eau refoulée, l'abondance bactérienne est proche de 1 x 104 cellules par mL. Dans le réseau de distribution, elle est toujours supérieure avec des valeurs observées allant jusqu'à 7 x 105 bact.mL-1; elle semble surtout liée à l'absence d'un résiduel de chlore libre (fig. 2). La température et la concentration en CODB dans l'eau refoulée sont aussi déterminantes comme le montre la figure 3 où l'abondance bactérienne dans les canalisations de faibles dia-mètres a été portée en fonction de la température pour deux gammes de concentration en CODB dans l'eau refoulée. Les taux de croissance des bactéries (calculés à .partir des estimations de production bactérienne et de biomasse) sont dans la gamme 0,005 à 0,1 h-1 (fig. 4), en l'absence de chlore libre ce qui correspond à des temps de génération compris entre 7 et 140 heures. La température semble fixer la valeur maximale du taux de croissance, sous ce maximum une large gamme de valeurs est observée traduisant la variabilité des conditions nutritionnelles. Les plus hauts taux de croissance observés dans le réseau sont proches des taux de croissance de bactéries mesurés dans les milieux aquatiques naturels.Les résultats obtenus sur les bactéries fixées montrent une biomasse bactérienne fixée dans la gamme 0,25 à 0,65 µgC.cm-2, ce qui correspond à une abondance de 1 x 107 à 2,6 x 107 bact.cm-2 (tableau 1). Ainsi donc, dans une canalisation de 100mm de diamètre, on peut dire que la biomasse fixée est, en moyenne, approximativement de 50 à 75 fois plus élevée que la biomasse moyenne des bactéiesJibres (2 x 105 bact.mL-1) (tableau 2). Le taux de croissance des bactéries fixées apparaît du même ordre de grandeur que celui des bactéries en suspension. Ceci signifie que dans un réseau de distribution, l'essentiel de la production de biomasse bactérienne s'effectue sur les parois des canalisations, les bactéries en suspension résultant principalement d'un décrochage de bactéries.Un modèle mathématique de la dynamique du CODB et des bactéries dans les réseaux de distribution, incluant les connaissances acquises concernant le contrôle de l'activité bactérienne par la matière organique dissoute, est actuellement développé (fig. 5). Il inclut une représentation mathématique des cinétiques des processus d'adsorption-désorption des bactéries (tableau 4), de fixation des bactéries, d'utilisation de la matière organique biodégradable et de la croissance bactérienne (tableau 3), ainsi que de l'impact du chlore libre sur les bactéries libres et fixées (fig. 6). Bien que préliminaire, il permet de simuler l'évolution longitudinale de la biomasse bactérienne libre et fixée, du CODB et du taux de chlore libre dans le cas simplifié d'une canalisation de diamètre fixé parcourue par un flux d'eau à vitesse constante (fig. 7). Une de ses applications permet de simuler l'impact de la teneur en CODB de l'eau injectée dans le réseau sur les biomasses libres et fixées (fig. 8).The deterioration of water quality in distribution systems due to bacterial regrowth is, at the present time, a major concern of drinking water producers. In this context, a good knowledge of the factors controlling bacterial development is required; the aim of the present study is to understand the rote of biodegradable dissolved organic carbon (BDOC) in the bacterial dynamics of the distribution system.This paper discusses the results obtained in a study carried out in order to assess the dynamics of biodegradable dissolved organic carbon and suspended bacteria in the water distribution system of the Northern Parisian suburbs lad by the Méry-sur-Oise treatment plant.The results show clearly that a significant decrease in BDOC occurs within the smallest pipes, when the BDOC level in the finished water is higher than about 0.20 mgC.L-1. However, no decrease in BDOC is observed when the BDOC in the finished water is lower than 0.16 mgC.L-1. The bacterial abundance in the distribution system is primarily linked to the absence of free chicane. Temperature and BDOC concentration in the finished water are also major controlling factors of bacterial numbers. Bacterial growth rates are in the range 0.005 to 0.1 h-1 in the absence of free chlorine, the highest of these values are in the same range as the growth rates measured for bacteria in natural aquatic ecosystems. Fixed biomass to the inner pipes surface are in the range 0.25 to 0.65 µgC.cm-2 and the average growth rate of fixed bacteria seems to be roughly in the same order of magnitude as the average growth rate of the suspended bacteria.A model of the dynamics of BDOC and bacteria in distribution network, incorporating the knowledge gained from this and previous studies concerning the control of bacterial activity by dissolved organic matter, is presented. It involves a mathematical representation of the kinetics of bacterial adsorption-desorption processes, bacterial attachment, bacterial utilization of biodegradable dissolved organic matter and impact of chlorine on free and fixed bacteria. It allows simulation of the impact of reducing the BDOC in the finished water on processes associated with bacterial regrowth in the distribution network.

BK Channels Control Cerebellar Purkinje and Golgi Cell Rhythmicity In Vivo

Calcium signaling plays a central role in normal CNS functioning and dysfunction. As cerebellar Purkinje cells express the major regulatory elements of calcium control and represent the sole integrative output of the cerebellar cortex, changes in neural activity- and calcium-mediated membrane properties of these cells are expected to provide important insights into both intrinsic and network physiology of the cerebellum. We studied the electrophysiological behavior of Purkinje cells in genetically engineered alert mice that do not express BK calcium-activated potassium channels and in wild-type mice with pharmacological BK inactivation. We confirmed BK expression in Purkinje cells and also demonstrated it in Golgi cells. We demonstrated that either genetic or pharmacological BK inactivation leads to ataxia and to the emergence of a beta oscillatory field potential in the cerebellar cortex. This oscillation is correlated with enhanced rhythmicity and synchronicity of both Purkinje and Golgi cells. We hypothesize that the temporal coding modification of the spike firing of both Purkinje and Golgi cells leads to the pharmacologically or genetically induced ataxia

Clinical Trial Readiness for Spinal Muscular Atrophy: Experience of an International Educational-Training Initiative

Several successful clinical trials have been conducted in spinal muscular atrophy (SMA) over recent years which have led to the approval of splicing modifiers and gene transfer therapies. With an increasing number of other agents progressing through pre-clinical and clinical development, increasing worldwide clinical trial readiness is becoming essential.SMA Europe initiated a clinical trial readiness project, which included the development of a pilot face-to-face educational-training initiative for clinical specialists and physiotherapists involved in SMA, with an emphasis on the patient perspective. Participants were selected through two surveys and, ahead of the meeting, a mock protocol with specific questions was provided. The initiative involved a series of presentations, role-play and interactive exercises. We describe here our experience and evaluation of this educational-training initiative, emphasising scientific aspects, psychosocial implications and level of satisfaction.From a participant, patient and industry perspective, such training was considered successful and met the objective, which was to improve clinical trial readiness in emerging sites. Resource planning, ethical considerations and communication with patients were identified as three important topics for future training. This initiative highlights the need to develop a training programme to achieve clinical trial readiness across Europe and showcases a collaborative effort with different stakeholders, clinicians, patient advocacy groups and sponsors to address an important issue

SMA – OUTCOME MEASURES AND REGISTRIES

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Response of plasma microRNAs to nusinersen treatment in patients with SMA.

peer reviewedOBJECTIVE: Spinal muscular atrophy (SMA) is a common genetic cause of infant mortality. Nusinersen treatment ameliorates the clinical outcome of SMA, however, some patients respond well, while others have limited response. We investigated microRNAs in blood samples from SMA patients and their response to nusinersen treatment evaluating the potential of circulating microRNAs as biomarkers for SMA. METHODS: In a discovery cohort study, microRNA next-generation sequencing was performed in blood samples from SMA patients (SMA type 2, n = 10; SMA type 3, n = 10) and controls (n = 7). The dysregulated microRNAs were further analysed in the therapeutic response cohort comprised of SMA type 1 patients (n = 22) who had received nusinersen treatment, at three time points along the treatment course (baseline, 2 and 6 months of treatment). The levels of the studied microRNAs were correlated to the SMA clinical outcome measures. RESULTS: In the discovery cohort, 69 microRNAs were dysregulated between SMA patients and controls. In the therapeutic response cohort, the baseline plasma levels of miR-107, miR-142-5p, miR-335-5p, miR-423-3p, miR-660-5p, miR-378a-3p and miR-23a-3p were associated with the 2 and 6 months response to nusinersen treatment. Furthermore, the levels of miR-107, miR-142-5p, miR-335-5p, miR-423-3p, miR-660-5p and miR-378-3p at 2 months of treatment were associated with the response after 6 months of nusinersen treatment. INTERPRETATION: Blood microRNAs could be used as biomarkers to indicate SMA patients' response to nusinersen and to monitor the efficacy of the therapeutic intervention. In addition, some of these microRNAs provide insight into processes involved in SMA that could be exploited as novel therapeutic targets

Mild clinical presentation in KLHL40-related nemaline myopathy (NEM 8).

Nemaline myopathies are clinically and genetically heterogeneous muscle diseases characterized by the presence of nemaline bodies (rods) in muscle fibers. Mutations in the KLHL40 (kelch-like family member 40) gene (NEM 8) are common cause of severe/lethal nemaline myopathy. We report an 8-year-old girl born to consanguineous Moroccan parents, who presented with hypotonia and poor sucking at birth, delayed motor development, and further mild difficulties in walking and fatigability. A muscle biopsy revealed the presence of nemaline bodies. KLHL40 gene Sanger sequencing disclosed a never before reported pathogenic homozygous mutation which resulted in absent KLHL40 protein expression in the muscle. This further expands the phenotypical spectrum of KLHL40 related nemaline myopathy

DMD Genotypes and Motor Function in Duchenne Muscular Dystrophy: A Multi-institution Meta-analysis With Implications for Clinical Trials

BACKGROUND AND OBJECTIVES: Clinical trials of genotype-targeted treatments in Duchenne muscular dystrophy (DMD) traditionally compare treated patients to untreated patients with the same DMD genotype class. This avoids confounding of drug efficacy by genotype effects but also shrinks the pool of eligible controls, increasing challenges for trial enrollment in this already rare disease. To evaluate the suitability of genotypically unmatched controls in DMD, we quantified effects of genotype class on 1-year changes in motor function endpoints used in clinical trials. METHODS: Over 1,600 patient-years of follow-up (>700 patients) were studied from six real-world/natural history data sources (UZ Leuven, PRO-DMD-01 shared by CureDuchenne, iMDEX, North Star UK, Cincinnati Children's Hospital Medical Center, and the DMD Italian Group), with genotypes classified as amenable to skipping exons 44, 45, 51 or 53, other skippable, nonsense, and other mutations. Associations between genotype class and 1-year changes in North Star Ambulatory Assessment total score (ΔNSAA) and in 10-meter walk/run velocity (Δ10MWR) were studied in each data source with and without adjustment for baseline prognostic factors. RESULTS: The studied genotype classes accounted for approximately 2% of variation in ΔNSAA outcomes after 12 months, whereas other prognostic factors explained >30% of variation in large data sources. Based on a meta-analysis across all data sources, pooled effect estimates for the studied skip-amenable mutation classes were all small in magnitude (<2 units in ΔNSAA total score in 1-year follow up), smaller than clinically important differences in NSAA, and were precisely estimated with standard errors <1 unit after adjusting for non-genotypic prognostic factors. DISCUSSION: These findings suggest viability of trial designs incorporating genotypically mixed or unmatched controls for up to 12 months in duration for motor function outcomes, which would ease recruitment challenges and reduce numbers of patients assigned to placebos. Such trial designs, including multi-genotype platform trials and hybrid designs, should ensure baseline balance between treatment and control groups for the most important prognostic factors, while accounting for small remaining genotype effects quantified in the present study

Analytical validation of innovative magneto-inertial outcomes: a controlled environment study.

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