Numération bactérienne en épifluorescence par la méthode couplée DAPI-INT : application à un cas concret

Un protocole simple de coloration par la méthode couplée DAPI-INT en microscopie à épifluorescence, associé à une lecture des préparations par caméra et vidéo-Imprimante, permet la mesure de paramètres biologiques impliqués directement dans les processus de dégradation de la matière organique en rivière : numérations de microflore totale, nombre de cellules physiologiquement actives (réduisant l'INT), nombre de cellules avec réserves de PHB, dimensions cellulaires et répartition en classes de taille aboutissant à l'évaluation de la biomasse carbonée.Tous les comptages et mesures sont réalisés à partir de clichés d'imprimante thermique, ce qui assure une reproductibilité et une comparaison entre échantillons plus objective qu'à partir de seules observations directes.Cette méthode permet de différer soit les comptages (échantillons fixés au formol après incubation à l'INT), soit surtout les mesures de dimensions cellulaires (archivage des clichés pour une exploitation ultérieure).Son application au cas de la rivière Charente montre une évolution particulière de la biomasse bactérienne en aval des rejets de l'agglomération d'Angoulême. La mise en évidence de cellules de grande taille (biovolume moyen de 0,3 µm3) et d'une population plus active pourrait traduire une modification physiologique de micro-organismes autochtones réagissant à des conditions de milieu particulières (rejets d'effluents carencés en azote).The increasing appreciation of heterotrophic bacterioplankton as an important and dynamic compartment in the carbon cycle of aquatic ecosystems has led to the development of methods allowing reproducible and reliable direct bacterial counts. Thus, since the 1970's, enumeration of bacteria by epifluorescence microscopy has become a widely used method for the estimation of cell number and biovolume. Cell counts are made using either the acridine orange (Hobbie et al. 1977) or the DAPI (Porter 1980) procedure on fixed samples. A tracer of cellular respiratory activity (INT) can be linked to either of these two methods before the samples are killed by formalin. Two observations of the same field, one in transmitted light, the other by epifluorescence, allow a determination of the percentage of active bacterioplankton (Tabor and Neihof 1982, Dufour 1990).However, the various authors working with these procedures have modified the protocol and up to now there has not been a standard method. Thus, before using it for the first time, numerous verifications about filters, concentration of dye solution and methods for estimating cell volume are necessary.In this study a simultaneous comparison, between three brands of filters, two dye solutions and three methods for numeration and cell volume estimation (by direct count, by photographs and by video graphic printer) has been performed.All the tests have been clone with samples collected from rivera or lakes. In comparison to laboratory cultures, natural bacteria are observed with difficulty on account of their small size and especially because natural organic matter absorbs dye solution and so interferes with counts. We prefer to use DAPI rather than acridine orange, because DAPI is colorless in direct light, so it doesn't interfere with enumeration of respiring bacteria (red crystal inside cell). To obtain the best contrast for observation in epifluorescence microscopy, a very fast drying in the open air and a structure less troublesome for the observation in direct light, we have chosen Millipore black filters. A camera associated with a video printer gives better results compared to the photographs : no fluorescent "halo" around the tell image and no waste of time waiting for development of the film. All counts and measures were made from video graphic printer photographs, which assures reproductibility and more objective comparisons between samples, compared to direct counts (with eyes) that are tiring and subjective. Finally, we can defer counting (samples fixed in formol after INT incubation) or cell size measurements (analysis of recorded photos) to a later time.On the basis of this tests, the DAPI-INT method in fluorescence microscopy, associated with the reading of slides with a camera and a video-printer, allows one to measure biological parameters directly implicated in the degradation of river organic matter : abundance of total microflora, number of physiologically active cells (reducing INT) and cells with PHB reserves, cell sizes and size range distribution used to evaluate carbon biomass.The application of this method to the river Charente (April to November 1991) shows a particular fate of bacterial biomass downstream from the city of Angouleme. A multifactor analysis of variance with a multiple range analyses (Tukey) shows that bacterial cells collected downstream from the effluent discharge of the Angouleme wastewater treatment plant are the largest (average cell volume up to 0.3 µm3) and the more active cells. Similar trends have also been observed by Servais and Garnier (1989) downstream from the Paris wastewater treatment plant (Achères) on the Seine river. These authors suggested that allochtonous populations associated with the urban sewage were different from the autochtonous populations (higher biovolume). To verify this hypothesis, a sampling campaign (June 92) was performed on five stations along an eight kilometers river reach. There weren't large size bacteria downstream from the effluent discharge of Frégeneuil, Fléac and St Michel wastewater treatment plants. We Pound the largest bacteria at Nersac. Thus it seems that these large bacteria don't came from wastewater treatment plant. The increase of autochtonous bacterial size could be interpretated as a nutritional stress, the increase of the C/N ratio preventing cellular division (nitrogen deficient sewages)

Digital archives, e-books and narrative space

In this paper we are concerned with the capacity of digital media to enable publics to tell their own environmental stories using digital broadcast archives (DBAs). We consider how digital media afford different ways of telling stories in relation to digital media archives. Central to this discussion is our experience of writing e‐books as part of the AHRC‐funded project “Earth in Vision: BBC coverage of environmental change 1960–2010”. The e‐book format has been adopted in order to explore some of the possibilities for writing environmental history and politics using DBAs

Developmental Acquisition of a Rapid Calcium-Regulated Vesicle Supply Allows Sustained High Rates of Exocytosis in Auditory Hair Cells

Auditory hair cells (HCs) have the remarkable property to indefinitely sustain high rates of synaptic vesicle release during ongoing sound stimulation. The mechanisms of vesicle supply that allow such indefatigable exocytosis at the ribbon active zone remain largely unknown. To address this issue, we characterized the kinetics of vesicle recruitment and release in developing chick auditory HCs. Experiments were done using the intact chick basilar papilla from E10 (embryonic day 10) to P2 (two days post-hatch) by monitoring changes in membrane capacitance and Ca2+ currents during various voltage stimulations. Compared to immature pre-hearing HCs (E10-E12), mature post-hearing HCs (E18-P2) can steadily mobilize a larger readily releasable pool (RRP) of vesicles with faster kinetics and higher Ca2+ efficiency. As assessed by varying the inter-pulse interval of a 100 ms paired-pulse depolarization protocol, the kinetics of RRP replenishment were found much faster in mature HCs. Unlike mature HCs, exocytosis in immature HCs showed large depression during repetitive stimulations. Remarkably, when the intracellular concentration of EGTA was raised from 0.5 to 2 mM, the paired-pulse depression level remained unchanged in immature HCs but was drastically increased in mature HCs, indicating that the Ca2+ sensitivity of the vesicle replenishment process increases during maturation. Concomitantly, the immunoreactivity of the calcium sensor otoferlin and the number of ribbons at the HC plasma membrane largely increased, reaching a maximum level at E18-P2. Our results suggest that the efficient Ca2+-dependent vesicle release and supply in mature HCs essentially rely on the concomitant engagement of synaptic ribbons and otoferlin at the plasma membrane

Quasiprojectile breakup and isospin equilibration at Fermi energies: an indication of longer projectile-target contact times?

An investigation of the quasiprojectile breakup channel in semiperipheral and peripheral collisions of $^{58,64}$Ni+$^{58,64}$Ni at 32 and 52 MeV/nucleon is presented. Data have been acquired in the first experimental campaign of the INDRA-FAZIA apparatus in GANIL. The effect of isospin diffusion between projectile and target in the two asymmetric reactions has been highlighted by means of the isospin transport ratio technique, exploiting the neutron-to-proton ratio of the quasiprojectile reconstructed from the two breakup fragments. We found evidence that, for the same reaction centrality, a higher degree of relaxation of the initial isospin imbalance is achieved in the breakup channel with respect to the more populated binary output, possibly indicating the indirect selection of specific dynamical features. We have proposed an interpretation based on different average projectile-target contact times related to the two exit channels under investigation, with a longer interaction for the breakup channel. The time information has been extracted from AMD simulations of the studied systems coupled to GEMINI++: the model calculations support the hypothesis hereby presented

Adverse Events of Extracorporeal Ultrasound-Guided High Intensity Focused Ultrasound Therapy

High-intensity focused ultrasound (HIFU) is considered to be an alternative to surgery. Extracorporeal ultrasound-guided HIFU (USgFU) has been clinically used to treat solid tumors. Preliminary trials in a small sample of a Western population suggested that this modality was safe. Most trials are performed in China thereby providing comprehensive data for understanding the safety profile. The aim of this study was to evaluate adverse events of USgFU therapy.Clinical data were searched in 2 Chinese databases. Adverse events of USgFU were summarized and compared with those of magnetic resonance-guided HIFU (MRgFU; for uterine, bone or breast tumor) and transrectal ultrasound-guided HIFU (for prostate cancer or benign prostate hyperplasia). USgFU treatment was performed using 7 types of device. Side effects were evaluated in 13262 cases. There were fewer adverse events in benign lesions than in malignant lesions (11.81% vs. 21.65%, p<0.0001). Rates of adverse events greatly varied between the disease types (0-280%, p<0.0001) and between the applied HIFU devices in both malignant (10.58-44.38%, p<0.0001) and benign lesions (1.67-17.57%, p<0.0001). Chronological analysis did not demonstrate a decrease in the rate of adverse events. Based upon evaluable adverse events, incidences in USgFU were consistent with those in MRgFU or transrectal HIFU. Some side effects frequently occurred following transrectal HIFU were not reported in USgFU. Several events including intrahepatic metastasis, intraoperative high fever, and occlusions of the superior mesenteric artery should be of particular concern because they have not been previously noted. The types of adverse events suggested that they were ultrasonic lesions.The frequency of adverse events depended on the location of the lesion and the type of HIFU device; however, side effects of USgFU were not yet understood. USgFU did not decrease the incidence of adverse events compared with MRgFU

Functional Implications of Novel Human Acid Sphingomyelinase Splice Variants

BACKGROUND: Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity. METHODOLOGY/PRINCIPAL FINDINGS: We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models. CONCLUSIONS/SIGNIFICANCE: These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity

17β-Estradiol Enhances Breast Cancer Cell Motility and Invasion via Extra-Nuclear Activation of Actin-Binding Protein Ezrin

Estrogen promotes breast cancer metastasis. However, the detailed mechanism remains largely unknown. The actin binding protein ezrin is a key component in tumor metastasis and its over-expression is positively correlated to the poor outcome of breast cancer. In this study, we investigate the effects of 17β-estradiol (E2) on the activation of ezrin and its role in estrogen-dependent breast cancer cell movement. In T47-D breast cancer cells, E2 rapidly enhances ezrin phosphorylation at Thr567 in a time- and concentration-dependent manner. The signalling cascade implicated in this action involves estrogen receptor (ER) interaction with the non-receptor tyrosine kinase c-Src, which activates the phosphatidylinositol-3 kinase/Akt pathway and the small GTPase RhoA/Rho-associated kinase (ROCK-2) complex. E2 enhances the horizontal cell migration and invasion of T47-D breast cancer cells in three-dimensional matrices, which is reversed by transfection of cells with specific ezrin siRNAs. In conclusion, E2 promotes breast cancer cell movement and invasion by the activation of ezrin. These results provide novel insights into the effects of estrogen on breast cancer progression and highlight potential targets to treat endocrine-sensitive breast cancers

Clinical practice guidelines for BRCA1 and BRCA2 genetic testing

BRCA1 and BRCA2 gene pathogenic variants account for most hereditary breast cancer and are increasingly used to determine eligibility for PARP inhibitor (PARPi) therapy of BRCA-related cancer. Because issues of BRCA testing in clinical practice now overlap with both preventive and therapeutic management, updated and comprehensive practice guidelines for BRCA genotyping are needed. The integrative recommendations for BRCA testing presented here aim to (1) identify individuals who may benefit from genetic counselling and risk-reducing strategies; (2) update germline and tumour-testing indications for PARPi-approved therapies; (3) provide testing recommendations for personalised management of early and metastatic breast cancer; and (4) address the issues of rapid process and tumour analysis. An international group of experts, including geneticists, medical and surgical oncologists, pathologists, ethicists and patient representatives, was commissioned by the French Society of Predictive and Personalised Medicine (SFMPP). The group followed a methodology based on specific formal guidelines development, including (1) evaluating the likelihood of BRCAm from a combined systematic review of the literature, risk assessment models and expert quotations, and (2) therapeutic values of BRCAm status for PARPi therapy in BRCA-related cancer and for management of early and advanced breast cancer. These international guidelines may help clinicians comprehensively update and standardise BRCA testing practices

Identification of Novel Functional Inhibitors of Acid Sphingomyelinase

We describe a hitherto unknown feature for 27 small drug-like molecules, namely functional inhibition of acid sphingomyelinase (ASM). These entities named FIASMAs (Functional Inhibitors of Acid SphingoMyelinAse), therefore, can be potentially used to treat diseases associated with enhanced activity of ASM, such as Alzheimer's disease, major depression, radiation- and chemotherapy-induced apoptosis and endotoxic shock syndrome. Residual activity of ASM measured in the presence of 10 µM drug concentration shows a bimodal distribution; thus the tested drugs can be classified into two groups with lower and higher inhibitory activity. All FIASMAs share distinct physicochemical properties in showing lipophilic and weakly basic properties. Hierarchical clustering of Tanimoto coefficients revealed that FIASMAs occur among drugs of various chemical scaffolds. Moreover, FIASMAs more frequently violate Lipinski's Rule-of-Five than compounds without effect on ASM. Inhibition of ASM appears to be associated with good permeability across the blood-brain barrier. In the present investigation, we developed a novel structure-property-activity relationship by using a random forest-based binary classification learner. Virtual screening revealed that only six out of 768 (0.78%) compounds of natural products functionally inhibit ASM, whereas this inhibitory activity occurs in 135 out of 2028 (6.66%) drugs licensed for medical use in humans

Structure and Function of the Hair Cell Ribbon Synapse

Faithful information transfer at the hair cell afferent synapse requires synaptic transmission to be both reliable and temporally precise. The release of neurotransmitter must exhibit both rapid on and off kinetics to accurately follow acoustic stimuli with a periodicity of 1 ms or less. To ensure such remarkable temporal fidelity, the cochlear hair cell afferent synapse undoubtedly relies on unique cellular and molecular specializations. While the electron microscopy hallmark of the hair cell afferent synapse — the electron-dense synaptic ribbon or synaptic body — has been recognized for decades, dissection of the synapse’s molecular make-up has only just begun. Recent cell physiology studies have added important insights into the synaptic mechanisms underlying fidelity and reliability of sound coding. The presence of the synaptic ribbon links afferent synapses of cochlear and vestibular hair cells to photoreceptors and bipolar neurons of the retina. This review focuses on major advances in understanding the hair cell afferent synapse molecular anatomy and function that have been achieved during the past years