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Elucidating the 3D nanoscale structure of tissues and cells is essential for understanding the complexity of biological processes. Electron microscopy (EM) offers the resolution needed for reliable interpretation, but the limited throughput of electron microscopes has hindered its ability to effectively image large volumes. We report a workflow for volume EM with FAST-EM, a novel multibeam scanning transmission electron microscope that speeds up acquisition by scanning the sample in parallel with 64 electron beams. FAST-EM makes use of optical detection to separate the signals of the individual beams. The acquisition and 3D reconstruction of ultrastructural data from multiple biological samples is demonstrated. The results show that the workflow is capable of producing large reconstructed volumes with high resolution and contrast to address biological research questions within feasible acquisition time frames.

Introduction: Systemic inflammation and endothelial dysfunction are potentially modifiable factors implicated in Alzheimer’s disease (AD), which offer potential therapeutic targets to slow disease progression. Methods: We investigated the relationship between baseline circulating levels of inflammatory (TNF-α, IL-1ß) and endothelial cell markers (VCAM-1, ICAM-1, E-selectin) and 18-month cognitive decline (ADAS-cog12) in 266 mild-to-moderate AD patients from the NILVAD study. We employed individual growth models to examine associations, potential mediation, and interaction effects while adjusting for confounders. Results: The average increase in ADAS-cog12 scores over all patients was 8.1 points in 18 months. No significant association was found between the markers and the rate of cognitive decline. Mediation analysis revealed no mediating role for endothelial cell markers, and interaction effects were not observed. Discussion: Our results do not support the role of systemic inflammation or endothelial dysfunction in progression in persons with AD.

Background: Despite previous efforts, medication safety in paediatrics remains a major concern. To inform improvement strategies and further research especially in outpatient care, we systematically reviewed the literature on the frequency and nature of drug-related hospital admissions in children. Methods: Searches covered Embase, Medline, Web of Science, grey literature sources and relevant article citations. Studies reporting epidemiological data on paediatric drug-related hospital admissions published between 01/2000 and 01/2024 were eligible. Study identification, data extraction, and critical appraisal were conducted independently in duplicate using templates based on the ’Joanna Briggs Institute’ recommendations. Results: The review included data from 45 studies reporting > 24,000 hospitalisations for adverse drug events (ADEs) or adverse drug reactions (ADRs). Due to different reference groups, a total of 52 relative frequency values were provided. We stratified these results by study characteristics. As a percentage of inpatients, the highest frequency of drug-related hospitalisation was found with ‘intensive ADE monitoring’, ranging from 3.1% to 5.8% (5 values), whereas with ‘routine ADE monitoring’, it ranged from 0.2% to 1.0% (3 values). The relative frequencies of ‘ADR-related hospitalisations’ ranged from 0.2% to 6.9% for ‘intensive monitoring’ (23 values) and from 0.04% to 3.8% for ‘routine monitoring’ (8 values). Per emergency department visits, five relative frequency values ranged from 0.1% to 3.8% in studies with ‘intensive ADE monitoring’, while all other eight values were ≤ 0.1%. Heterogeneity prevented pooled estimates. Studies rarely reported on the nature of the problems, or studies with broader objectives lacked disaggregated data. Limited data indicated that one in three (median) drug-related admissions could have been prevented, especially by more attentive prescribing. Besides polypharmacy and oncological therapy, no other risk factors could be clearly identified. Insufficient information and a high risk of bias, especially in retrospective and routine observational studies, hampered the assessment. Conclusion: Given the high frequency of drug-related hospitalisations, medication safety in paediatrics needs to be further improved. As routine identification appears unreliable, clinical awareness needs to be raised. To gain more profound insights especially for generating improvement strategies, we have to address under-reporting and methodological issues in future research. Trial registration: PROSPERO (CRD42021296986)

Orthostatic hypotension (OH) is highly prevalent in older adults and associated with dizziness, falls, lower physical and cognitive function, cardiovascular disease, and mortality. OH is currently diagnosed in a clinical setting with single-time point cuff measurements. Continuous blood pressure (BP) devices can measure OH dynamics but cannot be used for daily life monitoring. Near-infrared spectroscopy (NIRS) has potential diagnostic value in measuring cerebral oxygenation continuously over a longer time period, but this needs further validation. This study aimed to compare NIRS-measured (cerebral) oxygenation with continuous BP and transcranial Doppler-measured cerebral blood velocity (CBv) during postural changes. This cross-sectional study included 41 participants between 20 and 88 years old. BP, CBv, and cerebral (long channels) and superficial (short channels) oxygenated hemoglobin (O2Hb) were measured continuously during various postural changes. Pearson correlations between BP, CBv, and O2Hb were calculated over curves and specific characteristics (maximum drop amplitude and recovery). BP and O2Hb only showed good curve-based correlations (0.58–0.75) in the initial 30 s after standing up. Early (30–40 s) and 1-min BP recovery associated significantly with O2Hb, but no consistent associations were found for maximum drop amplitude and late (60–175 s) recovery values. Associations between CBv and O2Hb were poor, but stronger for long-channel than short-channel measurements. BP associated well with NIRS-measured O2Hb in the first 30 s after postural change. Stronger associations for CBv with long-channel O2Hb suggest that long-channel NIRS specifically reflects cerebral blood flow during postural transitions, necessary to better understand the consequences of OH such as intolerance symptoms.

AbstractBackgroundVarious methods exist for the induction of labor (IOL), and there is limited consensus as to optimal methods. Off-label misoprostol is recommended by the World Health Organization (WHO) for IOL but preparing it into doses suitable for IOL lacks precision, with potential adverse outcomes if dosing is inaccurate. This study explores potential outcomes and costs associated with increased uptake of a low-dose (25 µg) oral misoprostol formulation (Angusta; Norgine BV, Amsterdam) approved for IOL, in France, Belgium, and the Netherlands.MethodsA literature review was undertaken to derive probabilities of delivery outcomes (vaginal, instrumental, and cesarean sections) for IOL methods, from published meta-analyses. Outcomes for oral misoprostol tablets (25 µg) were unavailable in the meta-analyses, so were estimated using data from two published retrospective cohort studies. A model was developed to predict the frequency of IOL outcomes and associated costs at the national level, across multiple scenarios. Scenarios were tested using a moderate, medium, and high increase in oral misoprostol tablet (25 µg) uptake. Market shares, costs, and induction rates were defined for each country using multiple data sources.ResultsIncreased uptake of oral misoprostol tablets (25 µg) was estimated to be associated with a slightly increased rate of routine vaginal deliveries, and concurrent decreases in instrumental vaginal deliveries and cesarean sections. Since routine vaginal deliveries are less costly than other delivery outcomes, increased uptake of oral misoprostol tablets (25 µg) within the IOL market has the potential to be cost-saving. These trends were predicted using 25 µg oral misoprostol tablet outcomes informed by both retrospective studies.ConclusionPreliminary outcomes suggest that oral misoprostol tablets at 25 µg per dose may improve outcomes in IOL and be cost-saving. Further study is required to validate these findings and assess the comparative efficacy of IOL methods, including oral misoprostol tablets (25 µg).

ABSTRACTMAP1LC3/LC3 (microtubule associated protein 1 light chain 3) is widely used as marker of autophagic compartments at different stages of maturation. Electron microscopy (EM) combined with immunolabeling is the only technique that can reveal the ultrastructural identity of LC3-labeled compartments. However, immuno-EM of endogenous LC3 proteins has proven difficult. Here, we test a panel of commercially available antibodies and apply different labeling conditions to present an optimized procedure for LC3 immuno-EM. Using ultrathin cryosections and protein A-colloidal gold or gold enhancement labeling, we localize endogenous LC3 in starved cells or tissues in the presence or absence of the proton pump inhibitor bafilomycin A1. We localize LC3 to early and late stage autophagic compartments that can be classified by their morphology. By on-section correlative light-electron microscopy (CLEM) we show that comparable fluorescent LC3-positive puncta can represent different autophagic intermediates. We also show that our approach is sufficiently robust to label endogenous LC3 simultaneously with other lysosomal and autophagy markers, LAMP1 or SQSTM1/p62, and can be used for quantitative approaches. Thus, we demonstrate that bafilomycin A1treatment from 2.5 up to 24 h does not inhibit fusion between autophagosomes and lysosomes, but leads to the accumulation of LC3-positive material inside autolysosomes. Together, this is the first study presenting an extensive overview of endogenous LC3 localization at ultrastructural resolution without the need for cell permeabilization and using a commercially available antibody. This provides researchers with a tool to study canonical and non-canonical roles of LC3 in native conditions.Abbreviations:BafA1: bafilomycin A1; BSA: bovine serum albumin; BSA-c: acetylated BSA; BSA5: BSA conjugated to 5-nm gold particles; CLEM: correlative light-electron microscopy; EGFP: enhanced green fluorescent protein; EM: electron microscopy; FBS: fetal bovine serum; FSG: fish skin gelatin; GA: glutaraldehyde; IF: immunofluorescence; LAMP1: lysosomal associated membrane protein 1; LC3s: LC3 proteins; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ON: overnight; PAG: protein A-conjugated gold particles; PAG1-3: PAG5, PAG10, PAG15, protein A conjugated to 1-3-, 5-, 10-, or 15-nm gold particles; PB: Sorensen’s phosphate buffer; PBS: phosphate-buffered saline; PFA: paraformaldehyde; RT: room temperature.

BackgroundPathogenic germline variants in Transient Receptor Potential Vanilloid 4 Cation Channel (TRPV4) lead to channelopathies, which are phenotypically diverse and heterogeneous disorders grossly divided in neuromuscular disorders and skeletal dysplasia. We recently reported in sporadic giant cell lesions of the jaws (GCLJs) novel, somatic, heterozygous, gain-of-function mutations in TRPV4, at Met713.MethodsHere we report two unrelated women with a de novo germline p.Leu619Pro TRPV4variant and an overlapping systemic disorder affecting all organs individually described in TRPV4 channelopathies.ResultsFrom an early age, both patients had several lesions of the nervous system including progressive polyneuropathy, and multiple aggressive giant cell-rich lesions of the jaws and craniofacial/skull bones, and other skeletal lesions. One patient had a relatively milder disease phenotype possibly due to postzygotic somatic mosaicism. Indeed, the TRPV4p.Leu619Pro variant was present at a lower frequency (variant allele frequency (VAF)=21.6%) than expected for a heterozygous variant as seen in the other proband, and showed variable regional frequency in the GCLJ (VAF ranging from 42% to 10%). In silico structural analysis suggests that the gain-of-function p.Leu619Pro alters the ion channel activity leading to constitutive ion leakage.ConclusionOur findings define a novel polysystemic syndrome due to germline TRPV4p.Leu619Pro and further extend the spectrum of TRPV4channelopathies. They further highlight the convergence of TRPV4mutations on different organ systems leading to complex phenotypes which are further mitigated by possible post-zygotic mosaicism. Treatment of this disorder is challenging, and surgical intervention of the GCLJ worsens the lesions, suggesting the future use of MEK inhibitors and TRPV4 antagonists as therapeutic modalities for unmet clinical needs.

In the age of big data, it is time to ensure that units are routinely documented for easy, unambiguous exchange of information.

The multisubunit HOPS tethering complex is a well-established regulator of lysosome fusion with late endosomes and autophagosomes. However, the role of the HOPS complex in other stages of endo-lysosomal trafficking is not well understood. To address this, we made HeLa cells knocked out for the HOPS-specific subunits Vps39 or Vps41, or the HOPS-CORVET-core subunits Vps18 or Vps11. In all four knockout cells, we found that endocytosed cargos were trapped in enlarged endosomes that clustered in the perinuclear area. By correlative light-electron microscopy, these endosomes showed a complex ultrastructure and hybrid molecular composition, displaying markers for early (Rab5, PtdIns3P, EEA1) as well as late (Rab7, CD63, LAMP1) endosomes. These “HOPS bodies” were not acidified, contained enzymatically inactive cathepsins and accumulated endocytosed cargo and cation-independent mannose-6-phosphate receptor (CI-MPR). Consequently, CI-MPR was depleted from the TGN, and secretion of lysosomal enzymes to the extracellular space was enhanced. Strikingly, HOPS bodies also contained the autophagy proteins p62 and LC3, defining them as amphisomes. Together, these findings show that depletion of the lysosomal HOPS complex has a profound impact on the functional organization of the entire endosomal system and suggest the existence of a HOPS-independent mechanism for amphisome formation.

In previous work, the similarity relationship for the water vapor variance in the interfacial layer (IL) at the top of the convective boundary layer (CBL) was proposed to be proportional to the convective velocity scale and the gradients of the water vapor mixing ratio and the Brunt‐Vaisala frequency in the entrainment zone. In the presence of wind shear in the IL, the similarity relationship was hypothesized to also include a dependence on the gradient Richardson number. Simultaneous measurements of the surface buoyancy flux, wind‐shear profiles from a radar wind profiler, water vapor mixing ratio and temperature measurements and their gradients from a Raman lidar provide a unique opportunity to thoroughly examine the function used in defining the variance and validate it. These observations were made over the Atmospheric Radiation Measurement Southern Great Plains site. We identified 19 cases from 2016 during which the CBL was quasi‐stationary and well mixed for at least 2 hr in the afternoon. Furthermore, we simulated the CBL using a large‐eddy simulation (LES) model for these cases and derived the water vapor variance and other profiles to test the similarity function. Utilizing this unique combination of observations and LES, we demonstrate that the water vapor variance in the IL has little‐to‐no dependence on wind shear. Furthermore, we demonstrate that the predicted variance using the original similarity function matches the observed and LES‐modeled variance very well, with linear correlations between the two variances of 0.82 and 0.95, respectively. Numerical weather prediction and global circulation models need to be able to predict the variance in water vapor in the atmosphere, as this is an important signature of turbulent mixing. However, the variance is something that is not directly resolved by the model and must be approximated using variables that the models actually resolve such as gradients in water vapor, temperature, and wind. This study evaluates a commonly used approximation approach, illustrates its shortcomings, and suggests how the approximation can be improved. A similarity relationship is used to predict water vapor variance in the interfacial layer from other variablesWind shear is shown to be unimportant for the prediction of water vapor variance in the interfacial layerThere is very high correlation between the true and predicted variance in both the observations and LES data

Dietary lipids are taken up as FAs by the intestinal epithelium and converted by diacylglycerol acyltransferase (DGAT) enzymes into triglycerides, which are packaged in chylomicrons or stored in cytoplasmic lipid droplets (LDs). DGAT1-deficient patients suffer from vomiting, diarrhea, and protein losing enteropathy, illustrating the importance of this process to intestinal homeostasis. Previously, we have shown that DGAT1 deficiency causes decreased LD formation and resistance to unsaturated FA lipotoxicity in patient-derived intestinal organoids. However, LD formation was not completely abolished in patient-derived organoids, suggesting the presence of an alternative mechanism for LD formation. Here, we show an unexpected role for DGAT2 in lipid metabolism, as DGAT2 partially compensates for LD formation and lipotoxicity in DGAT1-deficient intestinal stem cells. Furthermore, we show that (un)saturated FA-induced lipotoxicity is mediated by ER stress. More importantly, we demonstrate that overexpression of DGAT2 fully compensates for the loss of DGAT1 in organoids, indicating that induced DGAT2 expression in patient cells may serve as a therapeutic target in the future.

ABSTRACTMacroautophagy (hereafter autophagy) is a lysosomal degradation pathway critical for maintaining cellular homeostasis and viability, and is predominantly regarded as a rapid and dynamic cytoplasmic process. To increase our understanding of the transcriptional and epigenetic events associated with autophagy, we performed extensive genome-wide transcriptomic and epigenomic profiling after nutrient deprivation in human autophagy-proficient and autophagy-deficient cells. We observed that nutrient deprivation leads to the transcriptional induction of numerous autophagy-associated genes. These transcriptional changes are reflected at the epigenetic level (H3K4me3, H3K27ac, and H3K56ac) and are independent of autophagic flux. As a proof of principle that this resource can be used to identify novel autophagy regulators, we followed up on one identified target: EGR1 (early growth response 1), which indeed appears to be a central transcriptional regulator of autophagy by affecting autophagy-associated gene expression and autophagic flux. Taken together, these data stress the relevance of transcriptional and epigenetic regulation of autophagy and can be used as a resource to identify (novel) factors involved in autophagy regulation.

Out-of-equilibrium self-assembly of proteins such as actin and tubulin is a key regulatory process controlling cell shape, motion and division. The design of functional nanosystems based on dissipative self-assembly has proven to be remarkably difficult due to a complete lack of control over the spatial and temporal characteristics of the assembly process. Here, we show the dissipative self-assembly of FtsZ protein (a bacterial homologue of tubulin) within coacervate droplets. More specifically, we show how such barrier-free compartments govern the local availability of the energy-rich building block guanosine triphosphate, yielding highly dynamic fibrils. The increased flux of FtsZ monomers at the tips of the fibrils results in localized FtsZ assembly, elongation of the coacervate compartments, followed by division of the fibrils into two. We rationalize the directional growth and division of the fibrils using dissipative reaction–diffusion kinetics and capillary action of the filaments as main inputs. The principle presented here, in which open compartments are used to modulate the rates of dissipative self-assembly by restricting the absorption of energy from the environment, may provide a general route to dissipatively adapting nanosystems exhibiting life-like behaviour. Protein fibrils grow and divide inside a coacervate compartment in response to the local concentration of a fuel.

This study explores water vapor turbulence in the convective boundary layer (CBL) using the Raman lidar observations from the Atmospheric Radiation Measurement site located at Darwin, Australia. An autocovariance technique was used to separate out the random instrument error from the atmospheric variability during time periods when the CBL is cloud‐free, quasi‐stationary, and well mixed. We identified 45 cases, comprising of 8 wet and 37 dry seasons events, over the 5‐year data record period. The dry season in Darwin is known by warm and dry sunny days, while the wet season is characterized by high humidity and monsoonal rains. The inherent variability of the latter resulted in a more limited number of cases during the wet season. Profiles of the integral scale, variance, coefficient of the structure function, and skewness were analyzed and compared with similar observations from the Raman lidar at the Atmospheric Radiation Measurement Southern Great Plains (SGP) site. The wet season shows larger median variance profiles than the dry season, while the median profile of the variance from the dry season and the SGP site are found to be more comparable particularly between 0.4 and 0.75 zi. The variance and coefficient of the structure function show qualitatively the same vertical pattern. Furthermore, deeper CBL, larger gradient of water vapor mixing ratio at zi, and the strong correlation with the water vapor variance at ziare seen during the dry season. The median value in the skewness is mostly positive below 0.6 ziunlike the SGP site. Analysis of water vapor turbulence profiles was performed for the wet and dry season cases separatelyComparisons of turbulent statistics revealed that the dry season is more comparable to observations in Oklahoma than to the wet seasonThe dry season showed deeper CBL, larger gradient of water vapor mixing at zi, and smaller variance

Large‐eddy simulations are used to study the influence of drizzle on stratocumulus organization, based on measurements made as part of the Second Dynamics and Chemistry of the Marine Stratocumulus field study‐II. Cloud droplet number concentration (Nc) is prescribed and considered as the proxy for different aerosol loadings. Our study shows that the amount of cloudiness does not decrease linearly with precipitation rate. An Ncthreshold is observed below which the removal of cloud water via precipitation efficiently reduces cloud depth, allowing evaporation to become efficient and quickly remove the remaining thin clouds, facilitating a fast transition from closed cells to open cells. Using Fourier analysis, stratocumulus length scales are found to increase with drizzle rates. Raindrop evaporation below 300 m lowers the cloud bases and amplifies moisture variances in the subcloud layer, while it does not alter the horizontal scales in the cloud layer, suggesting that moist cold pool dynamic forcings are not essential for mesoscale organization of stratocumulus. The cloud scales are greatly increased when the boundary layer is too deep to maintain well mixed. Raindrop evaporation below 300 m amplifies subcloud variances and forms cumulus‐like clouds below stratocumulus, while it does not alter the mesoscale organization of cloud layer; no significant moist cold pool dynamic forcing is observedAn Ncthreshold is observed below which removal of cloud water via precipitation efficiently reduces cloud depth, allowing evaporation to become efficient and quickly remove the remaining thin clouds, leading to a fast transition from closed cells to open cellsMesoscale organization on an insufficient scale biases the mean properties toward less cloud water as a result of additional entrainment via forced circulations

Large-eddy simulations of shallow convection are used to evaluate the role of convective mixing and evaporative cooling in the vertical transport of mass, heat, and moisture in nonprecipitating shallow convection. Evaporative cooling is found to increase mass flux and the magnitude of heat and moisture fluxes, comparing twin large-eddy simulations with either suppressed or active evaporative cooling. Nonetheless, subsiding shells transport mass downward even when evaporative cooling is suppressed, emphasizing that evaporative cooling is not the primary cause of existence of subsiding shells and accompanied buoyancy reversal. Instead, vertical convective mixing is found to be the primary reason of buoyancy reversal. Evaporative cooling yet accelerates downdrafts (updrafts) in the shell (cloudy) regions as well as increases the cloud cover in the lower cloud layer. The cloudy regions are more humid, and the liquid water potential temperature is lower compared to the evaporative-cooling-suppressed experiment. The primary effect of evaporative cooling is thus to increase the updraft core anomalies, thus enhancing vertical turbulent fluxes. Cumulus subsiding shells exist even when evaporative cooling is suppressedVertical convective mixing and condensation induce buoyancy reversal in shallow convectionEvaporative cooling increases the mass flux and amplitude of the vertical moisture and heat fluxes

ObjectiveTo determine the longitudinal changes in viral load of hepatitis B virus (HBV)–infected healthcare workers (HCWs) and its consequences for exclusion of infected HCWs performing exposure-prone procedures, various HBV DNA safety thresholds, and the frequency of monitoring.DesignRetrospective cohort study June 1, 1996–January 31, 2013.ParticipantsIn the Netherlands, chronically HBV-infected HCWs performing exposure-prone procedures are notified to the Committee for Prevention of Iatrogenic Hepatitis B. Of the 126 notified HCWs, 45 had 2 or more HBV DNA levels determined without antiviral therapy.MethodsA time-to-event analysis for HBV-infected HCWs categorized in various viremia levels surpassing a HBV DNA threshold level of 1×105copies/mL, above which exposure-prone procedures are not allowed in the Netherlands.ResultsFluctuations of HBV DNA in follow-up samples ranged from −5.4 to +2.2 log10copies/mL. A high correlation was seen for each HBV DNA level with the 3 previous levels. In a time-to-event analysis, after 6 months 7.2%, 6.5%, and 14.3% of individuals had surpassed the threshold of 1×105copies/mL for viral load categories 4.8×103to 1.5×104; 1.5×104to 4.0×104; and 4.0×104to 1.0×105, respectively.ConclusionsWe propose standard retesting every 6 months, with more frequent retesting just below the high threshold value (1×105copies/mL), and prolonging this standard interval to 1 year after 3 consecutive levels below the threshold in policies with lower safety thresholds (1×103or 1×104copies/mL).Infect Control Hosp Epidemiol2016;37:655–660

Understanding the dynamics of complex enzymatic reactions in highly crowded small volumes is crucial for the development of synthetic minimal cells. Compartmentalized biochemical reactions in cell-sized containers exhibit a degree of randomness due to the small number of molecules involved. However, it is unknown how the physical environment contributes to the stochastic nature of multistep enzymatic processes. Here, we present a robust method to quantify gene expression noise in vitro using droplet microfluidics. We study the changes in stochasticity in the cell-free gene expression of two genes compartmentalized within droplets as a function of DNA copy number and macromolecular crowding. We find that decreased diffusion caused by a crowded environment leads to the spontaneous formation of heterogeneous microenvironments of mRNA as local production rates exceed the diffusion rates of macromolecules. This heterogeneity leads to a higher probability of the molecular machinery staying in the same microenvironment, directly increasing the system's stochasticity.

ABSTRACTMethicillin-resistant Staphylococcus pseudintermedius(MRSP), which is often multidrug resistant (MDR), has recently emerged as a threat to canine health worldwide. Knowledge of the temporal distribution of specific MRSP lineages, their antimicrobial resistance phenotypes, and their association with clinical conditions may help us to understand the emergence and spread of MRSP in dogs. The aim of this study was to determine the yearly proportions of MRSP lineages and their antimicrobial-resistant phenotypes in the Netherlands and to examine possible associations with clinical conditions. MRSP was first isolated from a canine specimen submitted for diagnostics to the Faculty of Veterinary Medicine of Utrecht University in 2004. The annual cumulative incidence of MRSP among S. pseudintermediusincreased from 0.9% in 2004 to 7% in 2013. MRSP was significantly associated with pyoderma and, to a lesser extent, with wound infections and otitis externa. Multilocus sequence typing (MLST) of 478 MRSP isolates yielded 39 sequence types (ST) belonging to 4 clonal complexes (CC) and 15 singletons. CC71 was the dominant lineage that emerged since 2004, and CC258, CC45, and several unlinked isolates became more frequent during the following years. All but two strains conferred an MDR phenotype, but strains belonging to CC258 or singletons were less resistant. In conclusion, our study showed that MDR CC71 emerged as the dominant lineage from 2004 and onward and that less-resistant lineages were partly replacing CC71.

High‐resolution ground‐based measurements are used to assess the realism of fine‐scale numerical simulations of shallow cumulus cloud fields. The overlap statistics of cumuli as produced by large‐eddy simulations (LES) are confronted with Cloudnet data sets at the Jülich Observatory for Cloud Evolution. The Cloudnet pixel is small enough to detect cumuliform cloud overlap. Cloud fraction masks are derived for five different cases, using gridded time‐height data sets at various temporal and vertical resolutions. The overlap ratio (R), i.e., the ratio between cloud fraction by volume and by area, is studied as a function of the vertical resolution. Good agreement is found between Rderived from observations and simulations. An inverse linear function is found to best describe the observed overlap behavior, confirming previous LES results. Simulated and observed decorrelation lengths are smaller (∼300 m) than previously reported (>1 km). A similar diurnal variation in the overlap efficiency is found in observations and simulations. Inefficient cloud overlap at small scales is now supported by observationsInverse linear functions best describe cloud overlapLES and observations both indicate a diurnal cycle in the overlap parameter

Blind and visually impaired persons face many challenges due to isolation, the most important is lacking education due to social immobility. Yet, since the development of the well-known white-red cane, only few advances have been made to increase the mobility of blind people. GPS systems are often used for pedestrian navigation but lack precision, foremost in rural areas where navigation is most often needed. The aim of the IMAGO project is therefore the development of an inexpensive and unobtrusive navigation method for blind and visually impaired persons. Navigation is performed using structure from motion and image-based localization techniques. Route models are created as 3D point clouds through several steps: (i) image acquisition along the routes; (ii) bulk-transfer of images to a server; (iii) feature extraction; (iv) feature matching between images; (v) creation of 3D point cloud with structure from motion. Similarly, the navigation chains seven steps: (a) image acquisition while walking a route; (b) immediate transfer of image to a server; (c) feature extraction; (d) feature matching between image and 3D route model; (e) localization/camera matrix calculation; (f) navigation/calculation of direction based on localization; (g) transfer of direction to user. Current smartphones are used as devices both for recording of routes as well as navigation. Thereby, a high level of dissemination without additional costs is possible, both, within blind people for navigation, as well as seeing people for route creation. Additionally, haptic feedback can be used via a smart cane to reduce auditive feedback. The proposed system yields a high positioning accuracy of 80% of samples being located within 1.6 m. Thus, the system is usable for pedestrian navigation, especially for visually impaired persons.

La préservation nerveuse lors de la prostatectomie totale a été associée à un risque supérieur de marges positives. Nous présentons les Résultats préliminaires et difficultés rencontrées au cours d’une étude translationnelle ex-vivo visant à caractériser le tissu résiduel comme prostatique ou non-prostatique, à l’aide d’une sonde innovante et d’un anticorps anti-PSMA conjugué à un fluorophore.

The aim of this randomized controlled equivalence trial was to demonstrate that, in diabetic patients, dilating the pupils before as compared with after refraction and visual acuity assessment would not lead to different advice given to patients.

Pseudotriloop structures in RNA are recognized features involved in RNA folding but it has been difficult to predict their formation based on sequence alone. This study using NMR and UV-melting analyses reveals RNA features that contribute to formation and stability of pseudotriloop structures.

This study describes the evaluation, validation, and use of contactless postcolumn fractionation of bioactive mixtures with acetylcholine binding protein (AChBP) affinity analysis with help of a spotter technology. The high-resolution fractionation tailors the fractionation frequency to the chromatographic peaks. Postcolumn reagents for AChBP bioaffinity profiling are mixed prior to droplet ejection into 1536-well plates. After an incubation step, microplate reader analysis is used to determine bioactive compounds in a mixture. For ligands tested, a good correlation was found for IC50s determined in flow injection analysis mode when compared with traditional radioligand binding assays. After the evaluation and validation, bioaffinity profiling of actual mixtures was performed. The advantage of this “atline” technology using postcolumn bioaffinity analysis when compared to continuous flow online postcolumn bioaffinity profiling is the possibility to choose postcolumn incubation times freely without compromising resolution due to diffusion effects.

This review critically evaluates the efficacy of different tocolytics in reducing uterine pressure and contractions in term labor. The available evidence supports the use of beta-adrenergic-receptor agonists such as terbutaline or ritodrine; they appear to have an immediate and comparable profound effect on uterine activity in term labor. However, the preferred type of beta-adrenergic receptor agonist and dosage are unclear. The oxytocin receptor antagonist atosiban has a high specificity for the uterus with limited or no systemic effects and could therefore be an attractive alternative for use in term labor. The evidence on the tocolytic potency of a single bolus of atosiban for tocolysis in term labor is encouraging but limited and needs further research. Moreover, atosiban lacks United States Food and Drug Administration approval. Literature documenting efficacy and safety of nitroglycerin or magnesium sulfate in term labor is far from convincing. The theoretical basis for the use of tocolytics for nonreassuring intrapartum fetal heart rate patterns is to reduce the aggravating influence of uterine contractions. However, the clinical evidence that tocolytics in term active labor are actually beneficial in improving neonatal outcome is very limited.

All known guanine-sensing riboswitches regulate gene expression by specifically binding to guanine (G) or related analogs with high affinity to switch off transcription. The aptamers of this class of riboswitches are characterized by three helices (P1-P3), surrounding a central core of phylogenetically conserved nucleotides and a long-range loop-loop interaction. To gain more insight into the switching mechanism, we present here a comparison between the solution-state structures of the G-free and G-bound forms of the guanine aptamer from the xpt-pbuX operon of Bacillus subtilis, as derived from NMR chemical shifts and magnetic-field-induced residual dipolar couplings. The high-resolution NMR analysis shows the G-free aptamer is highly structured with parallel P2 and P3 helices and the long-range loop-loop interaction already present, implying that the structure is largely preformed to bind the ligand. Structural changes upon guanine binding are found to be localized to the central core. In the free state, the G-quadruple interaction and two base pairs of the P1 stem flanking the central core appear to be largely disordered. The ligand thus binds via a combined predetermined-induced fit mechanism, involving a previously unstructured five-residue loop of the J2-3 junction that folds over the ligand. These limited additional interactions within a preorganized setting possibly explain how the aptamer rapidly responds to ligand binding, which is necessary to switch the structural state of the expression platform within a narrow time frame before the RNA polymerase escapes the 5'-UTR.

The previously described NMR structure of a 5'-CU-3'/5'-UU-3' motif, which is highly conserved within the 3'-UTR Y-stem of poliovirus-like enteroviruses, revealed striking regularities of the local helix geometry, thus retaining the pseudo-twofold symmetry of the RNA helix. A mutant virus with both pyrimidine base pairs changed into Watson-Crick replicated as wild type, indicating the functional importance of this symmetry relation in viral RNA replication. Here we investigated the effect of changing only one of the two pyrimidine base pairs to Watson-Crick. We determined the NMR structures of two Y-stem variants: one containing the 5'-CU-3'/5'-AU-3' motif, which has been found in wild-type virus isolates as well, and the other containing a 5'-CU-3'/5'-UG-3' motif, which is not present in any enterovirus sequenced to date. Both structures show single pyrimidine mismatches with intercalated bases. In the 5'-CU-3'/5'-AU-3' motif a C-U Watson-Crick-type base pair is formed that retains the pseudo-twofold symmetry, while in the 5'-CU-3'/5'-UG-3' motif a single asymmetric U-U mismatch breaks the twofold symmetry. Surprisingly, for the nonnatural variant no effect of the single base-pair replacement was observed on polioviral RNA replication using an in vitro replicon assay.

Supramolecular fibre-like aggregates of different poly(ethylene glycol) modified ?-sheet block copolymers have been adsorbed on a highly ordered pyrolytic graphite surface, and studied by tapping-mode atomic force microscopy. High-resolution images provided detailed information on stability, adaptability, and internal structure of the fibres. Dynamic processes observed in real space and time included fibre rearrangement, and even unprecedented fibre growth.

The cloverleaf structure in the 5'-untranslated region of enterovirus RNA that regulates viral RNA replication contains an evolutionarily conserved YNMG tetraloop closed by a Y-G base pair. This loop is believed to interact specifically with the viral protease 3C. To further characterize the specificity of this interaction, the tetraloop and two flanking base pairs of the poliovirus RNA were randomized, and viable viral clones were obtained using in vivo SELEX. Among many different mutants with the canonical YNMG sequences to be described elsewhere, a large-plaque-forming clone contained a deviating uGCUAg sequence. The NMR structure of a small hairpin capped with uGCUAg that we present here shows that the GCUA tetraloop adopts a novel fold, which is highly similar to that of the YNMG tetraloop with common stacking properties and hydrogen-bond interactions including an unusual syn conformation of the adenosine. Thermodynamic studies show moderate stabilities of hairpins with canonical YNMG and the novel GCUA loops, which, together with the similarity of spatial structures, illustrates that the tetraloop structure itself is crucial for the RNA-protein interaction required for the viral replication. A re-evaluation of the ribosomal secondary structure database reveals a hairpin containing a GCUA loop, which covaries with YNMG and is involved in a tertiary interaction, and in the 50S ribosomal subunit from Haloarcula marismortui the structurally comparable apex of stem-loop 35a is a recognition site for protein L2. These observations show a more general occurrence and importance of the so-far unrecognized GYYA hairpin loops.

RNA pseudoknots play important roles in many biological processes. In the simian retrovirus type-1 (SRV-1) a pseudoknot together with a heptanucleotide slippery sequence are responsible for programmed ribosomal frameshifting, a translational recoding mechanism used to control expression of the Gag-Pol polyprotein from overlapping gagand polopen reading frames. Here we present the three-dimensional structure of the SRV-1 pseudoknot determined by NMR. The structure has a classical H-type fold and forms a triple helix by interactions between loop 2 and the minor groove of stem 1 involving base-base and base-sugar interactions and a ribose zipper motif, not identified in pseudoknots so far. Further stabilization is provided by a stack of five adenine bases and a uracil in loop 2, enforcing a cytidine to bulge. The two stems of the pseudoknot stack upon each other, demonstrating that a pseudoknot without an intercalated base at the junction can induce efficient frameshifting. Results of mutagenesis data are explained in context with the present three-dimensional structure. The two base-pairs at the junction of stem 1 and 2 have a helical twist of approximately 49°, allowing proper alignment and close approach of the three different strands at the junction. In addition to the overwound junction the structure is somewhat kinked between stem 1 and 2, assisting the single adenosine in spanning the major groove of stem 2. Geometrical models are presented that reveal the importance of the magnitude of the helical twist at the junction in determining the overall architecture of classical pseudoknots, in particular related to the opening of the minor groove of stem 1 and the orientation of stem 2, which determines the number of loop 1 nucleotides that span its major groove.

A phase II study was performed to investigate the efficacy and tolerability of gemcitabine as third-line chemotherapy for patients with metastatic breast cancer, previously treated with both an anthracycline - and taxane-containing regimen. Twenty-three patients were treated with gemcitabine 1200 mg/m2in a 30-min infusion on day 1, 8 and 15 of a 28 day cycle. Seventy-four percent of the patients had visceral metastases. No complete or partial responses were observed. Six patients (26%) had stable disease with a median duration of 4.0 months. The median time to progression was 1.9 months and the median survival time was 7.8 months. Neutropenia grade 3 and 4 was observed in four patients (18%). Non-hematological toxicity grade 3 included nausea and vomiting in 14%, skin toxicity in 9% and elevation of transaminases in 23% of the patients. Gemcitabine is ineffective as third-line single agent therapy in patients failing anthracycline and taxane treatment for metastatic breast cancer.

Chelate complexes of Fe^II were investigated with respect to their reactivity against nitric oxide and dioxygen. Through a systematic variation of the structure of the polyaminocarboxylate EDTA, a series of 38 potential chelate ligands were selected for Fe^II. The nitrosyl complexes were prepared from the Fe^II chelates with NO gas and examined spectroscopically by UV/Vis and ATR-IR techniques, and themodynamically by determining the overall binding constants for NO. In addition, the reversibility of NO binding to these Fe^II chelates and the rate of the competing oxidation by dioxygen were studied qualitatively. Whereas the studied complexes all form more or less stable nitrosyl complexes with a characteristic band pattern in the UV/Vis spectra and only slightly diverging frequencies for the NO stretching vibration in the IR spectra, they differ considerably in the reversibility of NO binding, the overall NO binding constants and the sensitivity towards dioxygen. It was found that an increasing number of donor groups on the chelate ligand causes a stronger coordination to Fe^II, and increases the tendency of the Fe^II chelates to transfer electron density from iron to substrates like dioxygen or nitric oxide. This results in an accelerated oxidation of Fe^II to Fe^III by dioxygen and a more pronounced tendency of the corresponding nitrosyl complexes to slowly decompose to Fe^III and N2O. In addition, the overall binding constant for NO (KNO), which spans a range from 1·10³ to 2·10⁷ M⁻¹, increases in the same direction as a result of the inductive effect of the chelate ligand.

Acid α-glucosidase, the product of a housekeeping gene, is a lysosomal enzyme that degrades glycogen. A deficiency of this enzyme is responsible for a recessively inherited myopathy and cardiomyopathy, glycogenesis type II. We have previously demonstrated that the human acid α-glucosidase gene expression is regulated by a silencer within intron 1, which is located in the 5′-untranslated region. In this study, we have used deletion analysis, electrophoretic mobility shift assay, and footprint analysis to further localize the silencer to a 25-base pair element. The repressive effect on the TKpromoter was about 50% in both orientations in expression plasmid, and two transcriptional factors were identified with antibodies binding specifically to the element. Mutagenesis and functional analyses of the element demonstrated that the mammalian homologue 1 of Drosophila hairyandEnhancer of split(Hes-1) binding to an E box (CACGCG) and global transcription factor-YY1 binding to its core site function as a transcriptional repressor. Furthermore, the overexpression of Hes-1 significantly enhanced the repressive effect of the silencer element. The data should be helpful in understanding the expression and regulation of the human acid α-glucosidase gene as well as other lysosomal enzyme genes.

The archaeal transcriptional initiation machinery closely resembles core elements of the eukaryal polymerase II system. However, apart from the established basal archaeal transcription system, little is known about the modulation of gene expression in archaea. At present, no obvious eukaryal-like transcriptional regulators have been identified in archaea. Instead, we have previously isolated an archaeal gene, the Pyrococcus furiosus lrpA, that potentially encodes a bacterial-like transcriptional regulator. In the present study, we have for the first time addressed the actual involvement of an archaeal Lrp homologue in transcription modulation. For that purpose, we have produced LrpA in Escherichia coli. In a cell-free P. furiosus transcription system we used wild-type and mutated lrpA promoter fragments to demonstrate that the purified LrpA negatively regulates its own transcription. In addition, gel retardation analyses revealed a single protein-DNA complex, in which LrpA appeared to be present in (at least) a tetrameric conformation. The location of the LrpA binding site was further identified by DNaseI and hydroxyl radical footprinting, indicating that LrpA binds to a 46-base pair sequence that overlaps the transcriptional start site of its own promoter. The molecular basis of the transcription inhibition by LrpA is discussed.

According to computer simulations by Parks and Komorita (1997), group-based reciprocal strategies (GBRS; i.e., strategies in which players cooperate only when a sufficient number of others do) are more effective than the strategy of unconditional defection (ALL-D) in 100-person social dilemmas. In this article, theoretical arguments and a replication of the Parks and Komorita simulations are presented, which demonstrate that when strategies are analyzed at the individual level (at which they were defined), GBRS are actually less effective than ALL-D. The divergence of this conclusion from that of Parks and Komorita is attributed to specific selection factors in design and analysis of the original simulations. However, when the strategies are analyzed as strategies for subgroups rather than individuals, the claim that GBRS are more effcetive than ALL-D finds more support, most notably because players using these and other conditionally cooperative strategies tend to defect when single or in small subgroups but to cooperate when in large subgroups.

The solution structure of the self-complementary deca-ribonucleotide 5'-r(GCGA*AUUCGC)-3' containing 9-[2-O-(β-D-ribofuranosyl)-β-D-ribofuranosyl]adenine (A*), a modified nucleotide that occurs in lower eukaryotic methionine initiator tRNAs (tRNAsi^Met), was determined by NMR spectroscopy. Unexpectedly, the modification has no effect on the thermal stability of the duplex. However, the extra ribose moiety is in the C(3')-endo conformation and takes up a well-defined position in the minor groove, which is in agreement with its position in tRNAsi^Met as determined by X-ray crystallography. Molecular-dynamics simulations on the RNA duplex in H2O show that the position of the extra ribofuranose moiety seems to be stabilized by bridged H-bonds (mediated by two H2O molecules) to the backbone of the complementary chain.

We have reviewed the long-term results of 22 patients (23 fusions) with fractures of the os calcis, who had subtalar arthrodesis with correction of the deformity between 1975 and 1991. The mean follow-up was nine years (5 to 20).All patients were evaluated according to a modified foot score. A radiological assessment was used in which linear and angular variables were measured including the fibulocalcaneal abutment, the height of the heel and fat pad, the angle of the arch and the lateral talocalcaneal and the lateral talar declination angles.The technique used restores the normal relationship between the hindfoot and midfoot and corrects the height of the heel. This leads to better biomechanical balance of the neighbouring joints and gives a favourable clinical outcome.The modified foot score showed a good or excellent result in 51% of the feet. Residual complaints were mostly due to problems with the soft tissues. Subjectively, an excellent or good score was achieved in 78% of the cases.After statistical analysis, except for the height of the heel and the degenerative changes in the calcaneocuboid joint, no significant difference was found in the measured variables between the operated and the contralateral side.

Preaxial polydactyly is a congenital hand malformation that includes duplicated thumbs, various forms of triphalangeal thumbs, and duplications of the index finger. A locus for preaxial polydactyly has been mapped to a region of 1.9 cM on chromosome 7q36 between polymorphic markers D7S550 and D7S2423. We constructed a detailed physical map of the preaxial polydactyly candidate region. With a combination of methods we identified and positioned 11 transcripts within this map. By recombination analysis on families with preaxial polydactyly, using newly developed polymorphic markers, we were able to reduce the candidate region to approximately 450 kb. The homeobox geneHLXB9,a putative receptor C7orf2, and two transcripts of unknown function, C7orf3 and C7orf4, map in the refined candidate region and have been subjected to mutation analysis in individuals with preaxial polydactyly.