How to Couple LC-IRMS with HRMS─A Proof-of-Concept Study.

Compound-specific stable isotope analysis (CSIA) is a unique analytical technique for determining small variations in isotope ratios of light isotopes in analytes from complex mixtures. A problem of CSIA using gas chromatography (GC) and liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) is that any structural information of the analytes is lost due to the processes involved in determining the isotope ratio. To obtain the isotopic composition of, for example, carbon from organic compounds, all carbon in each analyte is quantitatively converted to CO2. For GC-IRMS, open split GC-IRMS-MS couplings have been described that allow additional acquisition of structural information of analytes and interferences. Structural analysis using LC-IRMS is more difficult and requires additional technical and instrumental efforts. In this study, LC was combined for the first time with simultaneous analysis by IRMS and high-resolution mass spectrometry (HRMS), enabling the direct identification of unknown or coeluting species. We have thoroughly investigated and optimized the coupling and showed how technical problems, arising from instrumental conditions, can be overcome. To this end, it was successfully demonstrated that a consistent split ratio between IRMS and HRMS could be obtained using a variable postcolumn flow splitter. This coupling provided reproducible results in terms of resulting peak areas, isotope values, and retention time differences for the two mass spectrometer systems. To demonstrate the applicability of the coupling, we chose to address an important question regarding the purity of international isotope standards. In this context, we were able to confirm that the USGS41 reference material indeed contains substantial amounts of pyroglutamic acid as suggested previously in the literature. Moreover, the replacement material, USGS41a, still has significant amounts of pyroglutamic acid as impurity, rendering some caution necessary when using this material for isotopic calibration.

Multimodal MRI cerebral correlates of verbal fluency switching and its impairment in women with depression.

BACKGROUND: The search of biomarkers in the field of depression requires easy implementable tests that are biologically rooted. Qualitative analysis of verbal fluency tests (VFT) are good candidates, but its cerebral correlates are unknown. METHODS: We collected qualitative semantic and phonemic VFT scores along with grey and white matter anatomical MRI of depressed (n = 26) and healthy controls (HC, n = 25) women. Qualitative VFT variables are the "clustering score" (i.e. the ability to produce words within subcategories) and the "switching score" (i.e. the ability to switch between clusters). The clustering and switching scores were automatically calculated using a data-driven approach. Brain measures were cortical thickness (CT) and fractional anisotropy (FA). We tested for associations between CT, FA and qualitative VFT variables within each group. RESULTS: Patients had reduced switching VFT scores compared to HC. Thicker cortex was associated with better switching score in semantic VFT bilaterally in the frontal (superior, rostral middle and inferior gyri), parietal (inferior parietal lobule including the supramarginal gyri), temporal (transverse and fusiform gyri) and occipital (lingual gyri) lobes in the depressed group. Positive association between FA and the switching score in semantic VFT was retrieved in depressed patients within the corpus callosum, right inferior fronto-occipital fasciculus, right superior longitudinal fasciculus extending to the anterior thalamic radiation (all p < 0.05, corrected). CONCLUSION: Together, these results suggest that automatic qualitative VFT scores are associated with brain anatomy and reinforce its potential use as a surrogate for depression cerebral bases.

Video recording and vegetation classification elucidate sheep foraging ecology in species-rich grassland.

Factors influencing grazing behavior in species-rich grasslands have been little studied. Methodologies have mostly had a primary focus on grasslands with lower floristic diversity.We test the hypothesis that grazing behavior is influenced by both animal and plant factors and investigate the relative importance of these factors, using a novel combination of video technology and vegetation classification to analyze bite and step rates.In a semi-natural, partially wooded grassland in northern Estonia, images of the vegetation being grazed and records of steps and bites were obtained from four video cameras, each mounted on the sternum of a sheep, during 41 animal-hours of observation over five days. Plant species lists for the immediate field of view were compiled. Images were partnered by direct observation of the nearest-neighbor relationships of the sheep. TWINSPAN, a standard vegetation classification technique allocating species lists to objectively defined classes by a principal components procedure, was applied to the species lists and 25 vegetation classes (15 open pasture and 10 woodland) were identified from the images.Taking bite and step rates as dependent variables, relative importance of animal factors (sheep identity), relative importance of day, and relative importance of plant factors (vegetation class) were investigated. The strongest effect on bite rates was of vegetation class. Sheep identity was less influential. When the data from woodland were excluded, sheep identity was more important than vegetation class as a source of variability in bite rate on open pasture.The original hypothesis is therefore supported, and we further propose that, at least with sheep in species-rich open pastures, animal factors will be more important in determining grazing behavior than plant factors. We predict quantifiable within-breed and between-breed differences, which could be exploited to optimize conservation grazing practices and contribute to the sustainability of extensive grazing systems.

Sheep in Species-Rich Temperate Grassland: Combining Behavioral Observations with Vegetation Characterization.

Foraging behavior of livestock in species-rich, less intensively managed grassland communities will require different methodologies from those appropriate in floristically simple environments. In this pilot study on sheep in species-rich grassland in northern Estonia, foraging behavior and the plant species of the immediate area grazed by the sheep were registered by continually-recording Go-Pro cameras. From three days of observation of five sheep (706 animal-minutes), foraging behavior was documented. Five hundred and thirty-six still images were sampled, and a plant species list was compiled for each. Each plant species was assigned a score indicating its location, in the ecophysiological sense, on the main environmental gradient. The scores of the plant species present were averaged for each image. Thus, the fine structure of foraging behavior could be studied in parallel with the vegetation of the precise area being grazed. As expected, there was considerable individual variation, and we characterized foraging behavior by quantifying the patterns of interspersion of grazing and non-grazing behaviors. This combination of behavior recording and vegetation classification could enable a numerical analysis of the responses of grazing livestock to vegetation conditions.

Changes and drivers in Spanish landscapes at the Rural-Urban Interface between 1956 and 2018.

This paper presents unique comprehensive quantitative measures of the changes in Spanish rural landscapes, specifically in relation to the Rural-Urban Interface (RUI). Our objective was to investigate the urbanisation process in rural areas of Spain, thus the expansion of the RUI in an environmentally complex study area (it includes arid regions, continental areas or high mountains). The data were produced by the Spanish Rural Landscapes Monitoring System project (SISPARES), which has carried out five separate surveys for 1956, 1984, 1998, 2008 and 2018. SISPARES data provide RUI quantification by identifying urban patches, using aerial images, in a permanent network of 206 stratified random landscape sampling units (4 × 4 km each) deliberately located in rural areas. The cost-effective and spatio-temporal SISPARES approach enables the production of landscape geographical models at each survey date and the modelling of its evolution over each time period. We hypothesised that the RUI expansion did not follow a homogeneous spatio-temporal pattern, and therefore neither did the drivers responsible for it. Building on SISPARES data, we evaluated through CART analyses the relationship of RUI expansion with relevant environmental and anthropogenic driving factors, such as climate, bedrock, landscape composition and human population density. The historical narrative perspective was also used to aid discussion of the results. Historically, Spain contained few areas of RUI, but major changes were determined in the initial study period (1956-1984), which conformed a seminal geographic pattern for subsequent RUI increment. In contrast, the RUI expansion did not increase greatly during the following periods, between 1984 and 2018. The RUI expansion has been primarily driven by anthropogenic factors, constrained by the environmental characteristics of Spain. The expansion pattern is likely to has shifted from a national scale in the 1950s, to a continental scale at later dates as the result of the changes in socio-political scenarios.

The use of cattle Bos taurus for restoring and maintaining holarctic landscapes: Conclusions from a long-term study (1946-2017) in northern England.

Cattle Bos taurus can perform valuable ecological functions in the maintenance of high nature value (HNV) pastoral systems. They have also attracted attention as potentially filling the ecological niches of megaherbivores, notably the extinct aurochs Bos primigenius, in rewilding initiatives. Native cattle breeds are recognized under the 1992 Rio Convention as components of biodiversity. They are used in HNV settings, but their conservation as breeds has rarely been an important consideration for their management in these contexts.The Chillingham herd has been kept under minimal management in Chillingham Park (northern England) for several centuries. Chillingham Park is not a rewilding scenario, but the long-term study of the cattle can be informative for the design of rewilding schemes that involve cattle as megaherbivores. The pastures of the park are species-rich seminatural grasslands.To 2004, pasture management was influenced by the need to provide herbage for a flock of sheep that was under separate ownership, as well as for the cattle. Surveys of the vegetation conducted in 1979 and 2006-2008 showed a decline of plant species richness (species per 100 m2 quadrat) from 33.8 in 1979 to 22.6 in 2006-2008. This was acceptable as the conservation priority has always been the cattle herd. With removal of the sheep from 2004, it became possible to include recovery of plant diversity as a management goal.In 2017, the cattle numbered 111 (64 in 1979). Plant species richness in 2017 had increased to 26.3 species per quadrat. It has therefore been possible at Chillingham both to conserve the cattle herd and to improve plant diversity. While providing basic information of relevance to the management of cattle in free-ranging situations, this study also suggests a general principle, that the management of pastoral landscapes by native breeds of cattle, can deliver multiple conservation benefits.

Country-scale mapping of ecosystem services provided by semi-natural grasslands.

Semi-natural grasslands harbour high biodiversity and play a key role in the supply of ecosystem services (ES). However, abandonment, changes in traditional management practices and agricultural intensification constitute a major threat to these grasslands worldwide and these practices have led to declines in species diversity. In this paper the multi-functionality of semi natural-grasslands is assessed from the ES perspective, within a range of common semi-natural grassland types throughout Estonia. The analysis follows a stepwise approach based on the ES cascade model. Firstly, analyses of the relationships between plant species distribution patterns and environmental factors are described. Secondly, the effect of grassland abandonment on plant species diversity, as well as on the presence of rare and protected plant species is tested. In order to overcome the lack of data on ES at the national scale, plant species diversity and soil organic carbon are tested as surrogate indicators for five ESS: pollination, herbs for traditional medicinal use, nutrient cycling, nutrient retention and biomass production. In the final step, the spatial distribution of ES is assessed, based on an ES hotspots map obtained by detecting areas where high levels of plant species diversity and soil organic carbon overlap. The results show that the majority of ES hotspots are present in wooded meadows and pastures. However, there is an important threat to these hotspots because 45% are not eligible for agri-environmental support.

[The Conundrum of Relationships in the Multivariate Dataset of nvAMD Treatment]. / Die Frage nach Zusammenhängen in multivariaten Datensätzen zur Behandlung der nvAMD.

Background The evaluation of relationships in clinical case series is complicated by statistical pitfalls, one of which is spurious correlation. In this case, one or more potential factors are actually not related to a target measure, yet it may be wrongly inferred that they are. This may be due to either coincidence or the presence of a one or more confounding unregarded factors. In randomised clinical trials, it is attempted to eliminate such confounding factors or to control them by randomisation. However, this approach is not applicable in retrospective case series, and other statistical methods must be applied, such as multivariate analyses, taking into account all known variables. Patients/Material and Methods Statistical evaluation of the impact of different variables on the change in visual acuity after year 2 in a cohort of 1300 cases of nvAMD. The investigated variables are: visual acuity (VA) at baseline, central retinal thickness at baseline, age at baseline, mean interval between visits, number of injections, initial gain in VA after upload, and a change of the anti-VEGF compound. The target outcome measure was the change in VA as letter score at the end of year 2. Results All investigated variables have a marked impact on VA outcome when only single regression analysis is performed. Initial analysis of a switch in therapy gives a seemingly large effect on VA. However, multivariate analysis to avoid spurious correlations shows that only the following variables influence VA outcome with statistical significance: VA at baseline, initial gain in VA after upload, number of injections, mean interval between visits, and central retinal thickness at baseline. A switch in therapy had no statistically significant effect on VA outcome. Conclusion A switch in anti-VEGF therapy is not causally related to better VA outcome. In the statistical evaluation of non-randomised, retrospective case series, special attention must be given to potential confounding variables. When subgroups under investigation are not well balanced for confounding factors, misleading spurious correlations can result, leading to incorrect inferences.

[Real-life Data on the Treatment of Diabetic Macular Oedema in Germany]. / Daten zur Behandlungsrealität des diabetischen Makulaödems mit Ranibizumab.

Background Controlled prospective clinical trials on the treatment of diabetic macular oedema (DME) using anti-VEGF compounds show very good results in visual acuity gain over several years. To date, only limited data are available from comparable studies under real-life settings in clinical routine. However, real-life data from other indications for anti-VEGF treatment suggest that, in clinical routine, gain in visual acuity is less pronounced and cannot be maintained over a longer period of time, which is related to the significantly lower number of injections administered in clinical routine. Here we report a case series from our clinical routine of patients treated with ranibizumab for visual significant DME. Patients/Material and Methods Retrospective case series of 335 cases with follow-up of up to 3 years. All cases in our clinic treated for visual significant DME with at least one injection of ranibizumab were evaluated for the course of visual acuity gain and number of injections received. Results A mean gain in visual acuity (VA) of + 5.6 and + 3.7 letters was found in years one and two, respectively, with a median VA of logMAR 0.52 (decimal 0.3) at baseline. The steepest increase in VA is found during the first 4 months, with a less pronounced increase up to month 16. The mean number of injections was 5.7, 3.2 and 1.1 for years one, two, and three, respectively. Conclusions Results of real life treatment in clinical routine are found to fall behind controlled, prospective trials, as found to the findings in other anti-VEGF indications: mean gain in VA is lower than in prospective trials, and the initial gain cannot be fully maintained over a prolonged time period. Similarly, the number of injections received is markedly lower than in controlled trials. This can partially be explained by differences in study populations and a negative selection bias in longer term results. However, additional barriers that hamper timely treatment tailored to disease activity requirements must be identified and circumvented where possible.

[Time Course of Changes in Visual Acuity After a Single Injection of Aflibercept or Ranibizumab in Neovascular Age-related Macular Degeneration - Analysis of Aggregated Real Life Data]. / Der zeitliche Verlauf der Visusentwicklung nach einer Injektion von Aflibercept oder Ranibizumab bei der neovaskulären altersbedingten Makuladegeneration.

Background Treatment of neovascular age-related macular degeneration (nvAMD) under real life conditions may differ from controlled prospective trials with respect to the number of injections and long term preservation of visual acuity. In many instances, intervals for controls and re-injection cannot be maintained as frequently as required. This case series examines actual intervals for control and re-injection visits, in order to estimate how prolonged intervals have an impact on momentary visual acuity and how long term visual outcome is affected. Patients/Material and Methods Retrospective case series of 1,324 eyes with nvAMD, treated with a total of 8,150 ranibizumab injections (according to the PRN regimen) or 1,725 aflibercept injections (according to a fixed regimen), during the observation period of up to 3 years. The evaluation covered the time interval between visits, impact of this on the course of visual acuity, as well as the number of injections throughout treatment. Results Planned intervals of 4 or 8 weeks between visits were more often exceeded in the PRN regimen than with the fixed regimen. Visual acuity does not peak after 4 weeks, but only between 6 and 8 weeks. No statistically significant difference between aflibercept and ranibizumab was found. If the mean interval for re-injection was maintained at 4 - 6 weeks, this gave the greatest gains in visual acuity at end of years 1 and 2, respectively. Any prolongation of these intervals was accompanied by worse long term visual acuity. The fixed regimen is associated with consistently briefer re-treatment intervals during years 2 and 3, than with the PRN regimen. Conclusion Our data point to the importance of frequent controls accompanied by timely retreatment, as these have a major impact on visual outcome. It therefore appears to be more important to choose a treatment plan that facilitates frequent re-injections than to select either of the two compounds.

Farmland biodiversity and agricultural management on 237 farms in 13 European and two African regions.

Farmland is a major land cover type in Europe and Africa and provides habitat for numerous species. The severe decline in farmland biodiversity of the last decades has been attributed to changes in farming practices, and organic and low-input farming are assumed to mitigate detrimental effects of agricultural intensification on biodiversity. Since the farm enterprise is the primary unit of agricultural decision making, management-related effects at the field scale need to be assessed at the farm level. Therefore, in this study, data were collected on habitat characteristics, vascular plant, earthworm, spider, and bee communities and on the corresponding agricultural management in 237 farms in 13 European and two African regions. In 15 environmental and agricultural homogeneous regions, 6-20 farms with the same farm type (e.g., arable crops, grassland, or specific permanent crops) were selected. If available, an equal number of organic and non-organic farms were randomly selected. Alternatively, farms were sampled along a gradient of management intensity. For all selected farms, the entire farmed area was mapped, which resulted in total in the mapping of 11 338 units attributed to 194 standardized habitat types, provided together with additional descriptors. On each farm, one site per available habitat type was randomly selected for species diversity investigations. Species were sampled on 2115 sites and identified to the species level by expert taxonomists. Species lists and abundance estimates are provided for each site and sampling date (one date for plants and earthworms, three dates for spiders and bees). In addition, farmers provided information about their management practices in face-to-face interviews following a standardized questionnaire. Farm management indicators for each farm are available (e.g., nitrogen input, pesticide applications, or energy input). Analyses revealed a positive effect of unproductive areas and a negative effect of intensive management on biodiversity. Communities of the four taxonomic groups strongly differed in their response to habitat characteristics, agricultural management, and regional circumstances. The data has potential for further insights into interactions of farmland biodiversity and agricultural management at site, farm, and regional scale.

Performance optimization of current focusing and virtual electrode strategies in retinal implants.

The electrode configuration in an implanted visual prosthesis array affects the spatial electric field distribution within the retina, contributing to current focusing and virtual electrode (VE) stimulation strategies. In this paper, a finite element model incorporating various electrode configurations was used to study the interaction between electrode size and electrode-to-cell distance in current focusing and VE stimulation paradigms. The electrode array unit comprises an active electrode, six flanking return electrodes and a distant monopolar return. A quasi-monopolar (QMP) fraction is defined as the proportion of current which can be preferentially returned through the distant return, in comparison with the more adjacent flanking electrodes. The simulation results indicate that current focusing and VE strategies can be optimized by tuning the QMP fraction. The QMP fraction is adjusted to optimize the electric field spread based on retinal ganglion cell (RGC) density in the degenerate retina, thereby offsetting the effect of inhomogeneous distribution of surviving RGCs and leading to a uniform stimulation paradigm across electrodes. Importantly, there is negligible difference in functional performance across electrode configurations for distances less than the electrode diameter, implying that the stimulation mode does not significantly affect activation threshold or activated retinal area for electrode diameters greater than the retinal thickness. Furthermore, the QMP fraction has a significant effect on VE performance, defined by activation threshold and activated retinal area, when threshold current is evenly divided between two adjacent active electrodes.

The management of endodontically treated teeth using a Computer-Aided Design and Computer-Assisted Manufacturing/Computer-Aided Design and Computer-Integrated Manufacturing system.

This article introduces a new approach for restoring endodontically treated posterior teeth. Significantly reduced natural tooth structure often remains not only because of previous restorative measures but also because of endodontic procedures. Cast restorations for these teeth are almost universally recommended. The exception to this is the rare instance in which only conservative endodontic access openings exist in teeth presenting with no former existing restorations. Typically, multiple clinical appointments are required to complete the final cast restoration. This article presents Computer-Aided Design and Computer-Assisted Manufacturing/Computer-Aided Design and Computer-Integrated Manufacturing applications for restorative dentistry that provide the necessary care in an expeditious manner.