SMART users guides: SMART_NL & SMS_NL

Instructions to install and to use the model SMART_NL are given, and how to use the model SMART_NL with SUMO included. Besides, a user manual for configuration management is given

Aminoglycoside-Induced Phosphatidylserine Externalization in Sensory Hair Cells Is Regionally Restricted, Rapid, and Reversible

The aminophospholipid phosphatidylserine (PS) is normally restricted to the inner leaflet of the plasma membrane. During certain cellular processes, including apoptosis, PS translocates to the outer leaflet and can be labeled with externally applied annexin V, a calcium-dependent PS-binding protein. In mouse cochlear cultures, annexin V labeling reveals that the aminoglycoside antibiotic neomycin induces rapid PS externalization, specifically on the apical surface of hair cells. PS externalization is observed within ~75 s of neomycin perfusion, first on the hair bundle and then on membrane blebs forming around the apical surface. Whole-cell capacitance also increases significantly within minutes of neomycin application, indicating that blebbing is accompanied by membrane addition to the hair cell surface. PS externalization and membrane blebbing can, nonetheless, occur independently. Pretreating hair cells with calcium chelators, a procedure that blocks mechanotransduction, or overexpressing a phosphatidylinositol 4,5-biphosphate (PIP2)-binding pleckstrin homology domain, can reduce neomycin-induced PS externalization, suggesting that neomycin enters hair cells via transduction channels, clusters PIP2, and thereby activates lipid scrambling. The effects of short-term neomycin treatment are reversible. After neomycin washout, PS is no longer detected on the apical surface, apical membrane blebs disappear, and surface-bound annexin V is internalized, distributing throughout the supranuclear cytoplasm of the hair cell. Hair cells can therefore repair, and recover from, neomycin-induced surface damage. Hair cells lacking myosin VI, a minus-end directed actin-based motor implicated in endocytosis, can also recover from brief neomycin treatment. Internalized annexin V, however, remains below the apical surface, thereby pinpointing a critical role for myosin VI in the transport of endocytosed material away from the periphery of the hair cell

Environmental monitoring in heterogeneous soil-landscapes; A Dutch case study

The spatial heterogeneity of agricultural soil-landscapes is mostly not taken into account in environmental policies. Most environmental goals have been defined at national level or farm level but not at the landscape level. The potential for setting up a regional environmental monitoring network that supports self governance was explored. The research was performed in the Northern Friesian Woodland

Myosin VIIA is required for aminoglycoside accumulation in cochlear hair cells.

Myosin VIIA is expressed by sensory hair cells and has a primary structure predicting a role in membrane trafficking and turnover, processes that may underlie the susceptibility of hair cells to aminoglycoside antibiotics. [3H]Gentamicin accumulation and the effects of aminoglycosides were therefore examined in cochlear cultures of mice with different missense mutations in the myosin VIIA gene, Myo7a, to see whether myosin VIIA plays a role in aminoglycoside ototoxicity. Hair cells from homozygous mutant Myo7a(sh1) mice, with a mutation in a non-conserved region of the myosin VIIA head, respond rapidly to aminoglycoside treatment and accumulate high levels of gentamicin. Hair cells from homozygous mutant Myo7a(6J) mice, with a mutation at a highly conserved residue close to the ATP binding site of the myosin VIIA head, do not accumulate [3H]gentamicin and are protected from aminoglycoside ototoxicity. Hair cells from heterozygotes of both alleles accumulate [3H]gentamicin and respond to aminoglycosides. Although aminoglycoside uptake is thought to be via apical surface-associated endocytosis, coated pit numbers on the apical membrane of heterozygous and homozygous Myo7a(6J) hair cells are similar. Pulse-chase experiments with cationic ferritin confirm that the apical endocytotic pathway is functional in homozygous Myo7a(6J) hair cells. Transduction currents can be recorded from both heterozygous and homozygous Myo7a(6J) hair cells, suggesting it is unlikely that the drug enters via diffusion through the mechanotransducer channel. The results show that myosin VIIA is required for aminoglycoside accumulation in hair cells. Myosin VIIA may transport a putative aminoglycoside receptor to the hair cell surface, indirectly translocate it to sites of membrane retrieval, or retain it in the endocytotic pathway

Estimating nitrogen fluxes at the European scale by upscaling INTEGRATOR model outputs from selected sites

A comparison was made between upscaled model results of nitrogen (N) fluxes to air and water from 450 sites within the EU-27 and results derived for the entire EU-27 area using the model INTEGRATOR. The 450 sites were selected using stratified random sampling, dividing the EU-27 into 150 strata and selecting three sites at random within each stratum. The strata were based on important environmental factors influencing N fluxes. Hierarchical divisive cluster analysis was used to reduce the numerous combinations of environmental factors to the required total of 150, such that the heterogeneity of environmental factors within strata was as small as possible. Modelled NH<sub>3</sub>, N<sub>2</sub>O and NO<sub>x</sub> emissions and N leaching/runoff obtained were scaled up from the 450 sites to the entire EU-27 and were within 10% of results obtained by running the model for the whole of the EU-27 using about 36 500 sites. This implies that a reliable estimate of N fluxes for EU-27 can be made by upscaling results of the 450 selected sites suggesting that dramatic reduction in computation time can be achieved without substantial deterioration of result

Uncertainties in model predictions of nitrogen fluxes from agro-ecosystems in Europe

To assess the responses of nitrogen and greenhouse gas emissions to pan-European changes in land cover, land management and climate, an integrated dynamic model, INTEGRATOR, has been developed. This model includes both simple process-based descriptions and empirical relationships and uses detailed GIS-based environmental and farming data in combination with various downscaling methods. This paper analyses the propagation of uncertainties in model inputs and parameters to outputs of INTEGRATOR, using a Monte Carlo analysis. Uncertain model inputs and parameters were represented by probability distributions, while spatial correlation in these uncertainties was taken into account by assigning correlation coefficients at various spatial scales. The uncertainty propagation was analysed for the emissions of NH<sub>3</sub>, N<sub>2</sub>O and NO<sub>x</sub>, N leaching to groundwater and N runoff to surface water for the entire EU27 and for individual countries. Results show large uncertainties for N leaching and runoff (relative errors of ∼ 19% for Europe as a whole), and smaller uncertainties for emission of N<sub>2</sub>O, NH<sub>3</sub> and NO<sub>x</sub> (relative errors of ∼ 12%). Uncertainties for Europe as a whole were much smaller compared to uncertainties at country level, because errors partly cancelled out due to spatial aggregation

d-Tubocurarine and Berbamine: Alkaloids That Are Permeant Blockers of the Hair Cell's Mechano-Electrical Transducer Channel and Protect from Aminoglycoside Toxicity

Aminoglycoside antibiotics are widely used for the treatment of life-threatening bacterial infections, but cause permanent hearing loss in a substantial proportion of treated patients. The sensory hair cells of the inner ear are damaged following entry of these antibiotics via the mechano-electrical transducer (MET) channels located at the tips of the hair cell’s stereocilia. d-Tubocurarine (dTC) is a MET channel blocker that reduces the loading of gentamicin-Texas Red (GTTR) into rat cochlear hair cells and protects them from gentamicin treatment. Berbamine is a structurally related alkaloid that reduces GTTR labeling of zebrafish lateral-line hair cells and protects them from aminoglycoside-induced cell death. Both compounds are thought to reduce aminoglycoside entry into hair cells through the MET channels. Here we show that dTC (≥6.25 µM) or berbamine (≥1.55 µM) protect zebrafish hair cells in vivo from neomycin (6.25 µM, 1 h). Protection of zebrafish hair cells against gentamicin (10 µM, 6 h) was provided by ≥25 µM dTC or ≥12.5 µM berbamine. Hair cells in mouse cochlear cultures are protected from longer-term exposure to gentamicin (5 µM, 48 h) by 20 µM berbamine or 25 µM dTC. Berbamine is, however, highly toxic to mouse cochlear hair cells at higher concentrations (≥30 µM) whilst dTC is not. The absence of toxicity in the zebrafish assays prompts caution in extrapolating results from zebrafish neuromasts to mammalian cochlear hair cells. MET current recordings from mouse outer hair cells (OHCs) show that both compounds are permeant open-channel blockers, rapidly and reversibly blocking the MET channel with half-blocking concentrations of 2.2 µM (dTC) and 2.8 µM (berbamine) in the presence of 1.3 mM Ca2+ at −104 mV. Berbamine, but not dTC, also blocks the hair cell’s basolateral K + current, IK,neo, and modeling studies indicate that berbamine permeates the MET channel more readily than dTC. These studies reveal key properties of MET-channel blockers required for the future design of successful otoprotectants

Mutations in protocadherin 15 and cadherin 23 affect tip links and mechanotransduction in mammalian sensory hair cells

Immunocytochemical studies have shown that protocadherin-15 (PCDH15) and cadherin-23 (CDH23) are associated with tip links, structures thought to gate the mechanotransducer channels of hair cells in the sensory epithelia of the inner ear. The present report describes functional and structural analyses of hair cells from Pcdh15av3J (av3J), Pcdh15av6J (av6J) and Cdh23v2J (v2J) mice. The av3J and v2J mice carry point mutations that are predicted to introduce premature stop codons in the transcripts for Pcdh15 and Cdh23, respectively, and av6J mice have an in-frame deletion predicted to remove most of the 9th cadherin ectodomain from PCDH15. Severe disruption of hair-bundle morphology is observed throughout the early-postnatal cochlea in av3J/av3J and v2J/v2J mice. In contrast, only mild-to-moderate bundle disruption is evident in the av6J/av6J mice. Hair cells from av3J/av3J mice are unaffected by aminoglycosides and fail to load with [3H]-gentamicin or FM1-43, compounds that permeate the hair cell's mechanotransducer channels. In contrast, hair cells from av6J/av6J mice load with both FM1-43 and [3H]-gentamicin, and are aminoglycoside sensitive. Transducer currents can be recorded from hair cells of all three mutants but are reduced in amplitude in all mutants and have abnormal directional sensitivity in the av3J/av3J and v2J/v2J mutants. Scanning electron microscopy of early postnatal cochlear hair cells reveals tip-link like links in av6J/av6J mice, substantially reduced numbers of links in the av3J/av3J mice and virtually none in the v2J/v2J mice. Analysis of mature vestibular hair bundles reveals an absence of tip links in the av3J/av3J and v2J/v2J mice and a reduction in av6J/av6J mice. These results therefore provide genetic evidence consistent with PCDH15 and CDH23 being part of the tip-link complex and necessary for normal mechanotransduction

Predictive and prognostic markers in neurooncology

Abstract Over the past few years molecular assays have been introduced to aid in typing and grading of gliomas. This is the result of improved understanding of these tumors at the molecular level. In particular, the presence or absence of combined 1p/19 loss in oligodendroglial tumors, epidermal growth factor receptor amplification, epidermal growth factor receptor vIII mutations in grade III tumors and glioblastoma multiforme, and MGMT promoter gene methylation in glioblastoma multiforme are now being used to tailor treatment decisions in patients. However, the application of these tests is far from straightforward, and certain standards are required before any test can be introduced in the daily management of patients. Some of these requirements concern inter-and intratest variability, including whether a test gives the same results if repeated in the same or in another laboratory or when different methodologies are used (e.g. loss of heterozygosity vs fluorescence in situ hybridization and a polymerase chain reaction-based test vs immunohistochemistry). The sensitivity and specificity of a test (or negative and positive predictive value) indicate the likelihood that the test results are positive if the disease is present and the likelihood that the disease is present if the test results are positive. Studies on these test characteristics usually require the presence of a gold standard to which new tests should be compared. Last but not least there is the question of what added value the test has; this criterion determines the clinical usefulness of the assay and why some recently introduced molecular assays need to be scrutinized