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Effective science communication is challenging when scientific messages are informed by a continually updating evidence base and must often compete against misinformation. We argue that we need a new program of science communication as collective intelligence-a collaborative approach, supported by technology. This would have four key advantages over the typical model where scientists communicate as individuals: scientific messages would be informed by (a) a wider base of aggregated knowledge, (b) contributions from a diverse scientific community, (c) participatory input from stakeholders, and (d) better responsiveness to ongoing changes in the state of knowledge., Competing Interests: Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Late-onset Alzheimer disease (LOAD) is the most frequent neurodegenerative disease, and the APOE ε4 allele is the most prominent risk factor for LOAD. Four human induced pluripotent stem cell (iPSC) lines MLUi007-J, MLUi008-B, MLUi009-A, and MLUi010-B were generated from LOAD patients and healthy matched donors by reprogramming of B-lymphoblastoid cells (B-LCLs) with episomal plasmids. The application of B-LCLs holds a great promise to model LOAD and other diseases because they can easily be generated from primary peripheral blood mononuclear cells (PBMCs) by infection with the Epstein-Barr virus (EBV)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Medical Faculty of the Martin Luther University Halle-Wittenberg. Published by Elsevier B.V. All rights reserved.)

Since the description of some peculiar symptoms by James Parkinson in 1817, attempts have been made to define its cause or at least to enlighten the pathology of "Parkinson's disease (PD)." The vast majority of PD subtypes and most cases of sporadic PD share Lewy bodies (LBs) as a characteristic pathological hallmark. However, the processes underlying LBs generation and its causal triggers are still unknown. ɑ-Synuclein (ɑ-syn, encoded by the SNCA gene) is a major component of LBs, and SNCA missense mutations or duplications/triplications are causal for rare hereditary forms of PD. Thus, it is imperative to study ɑ-syn protein and its pathology, including oligomerization, fibril formation, aggregation, and spreading mechanisms. Furthermore, there are synergistic effects in the underlying pathogenic mechanisms of PD, and multiple factors-contributing with different ratios-appear to be causal pathological triggers and progression factors. For example, oxidative stress, reduced antioxidative capacity, mitochondrial dysfunction, and proteasomal disturbances have each been suggested to be causal for ɑ-syn fibril formation and aggregation and to contribute to neuroinflammation and neural cell death. Aging is also a major risk factor for PD. Iron, as well as neuromelanin (NM), show age-dependent increases, and iron is significantly increased in the Parkinsonian substantia nigra (SN). Iron-induced pathological mechanisms include changes of the molecular structure of ɑ-syn. However, more recent PD research demonstrates that (i) LBs are detected not only in dopaminergic neurons and glia but in various neurotransmitter systems, (ii) sympathetic nerve fibres degenerate first, and (iii) at least in "brain-first" cases dopaminergic deficiency is evident before pathology induced by iron and NM. These recent findings support that the ɑ-syn/LBs pathology as well as iron- and NM-induced pathology in "brain-first" cases are important facts of PD pathology and via their interaction potentiate the disease process in the SN. As such, multifactorial toxic processes posted on a personal genetic risk are assumed to be causal for the neurodegenerative processes underlying PD. Differences in ratios of multiple factors and their spatiotemporal development, and the fact that common triggers of PD are hard to identify, imply the existence of several phenotypical subtypes, which is supported by arguments from both the "bottom-up/dual-hit" and "brain-first" models. Therapeutic strategies are necessary to avoid single initiation triggers leading to PD., (© 2023. The Author(s).)

The dark pigment neuromelanin (NM) is abundant in cell bodies of dopamine (DA) neurons in the substantia nigra (SN) and norepinephrine (NE) neurons in the locus coeruleus (LC) in the human brain. During the progression of Parkinson's disease (PD), together with the degeneration of the respective catecholamine (CA) neurons, the NM levels in the SN and LC markedly decrease. However, questions remain among others on how NM is associated with PD and how it is synthesized. The biosynthesis pathway of NM in the human brain has been controversial because the presence of tyrosinase in CA neurons in the SN and LC has been elusive. We propose the following NM synthesis pathway in these CA neurons: (1) Tyrosine is converted by tyrosine hydroxylase (TH) to L-3,4-dihydroxyphenylalanine (L-DOPA), which is converted by aromatic L-amino acid decarboxylase to DA, which in LC neurons is converted by dopamine β-hydroxylase to NE; (2) DA or NE is autoxidized to dopamine quinone (DAQ) or norepinephrine quinone (NEQ); and (3) DAQ or NEQ is converted to eumelanic NM (euNM) and pheomelanic NM (pheoNM) in the absence and presence of cysteine, respectively. This process involves proteins as cysteine source and iron. We also discuss whether the NM amounts per neuromelanin-positive (NM + ) CA neuron are higher in PD brain, whether NM quantitatively correlates with neurodegeneration, and whether an active lifestyle may reduce NM formation., (© 2023. The Author(s).)

Ungulate populations are increasing across Europe with important implications for forest plant communities. Concurrently, atmospheric nitrogen (N) deposition continues to eutrophicate forests, threatening many rare, often more nutrient-efficient, plant species. These pressures may critically interact to shape biodiversity as in grassland and tundra systems, yet any potential interactions in forests remain poorly understood. Here, we combined vegetation resurveys from 52 sites across 13 European countries to test how changes in ungulate herbivory and eutrophication drive long-term changes in forest understorey communities. Increases in herbivory were associated with elevated temporal species turnover, however, identities of winner and loser species depended on N levels. Under low levels of N-deposition, herbivory favored threatened and small-ranged species while reducing the proportion of non-native and nutrient-demanding species. Yet all these trends were reversed under high levels of N-deposition. Herbivores also reduced shrub cover, likely exacerbating N effects by increasing light levels in the understorey. Eutrophication levels may therefore determine whether herbivory acts as a catalyst for the "N time bomb" or as a conservation tool in temperate forests., (© 2022. The Author(s).)

Neuromelanin granules (NMGs) are organelle-like structures present in the human substantia nigra pars compacta . In addition to neuromelanin, NMGs contain proteins, lipids and metals. As NMG-containing dopaminergic neurons are preferentially lost in Parkinson's disease and dementia with Lewy bodies (DLB), it is assumed that NMGs may play a role in neurodegenerative processes. Until now, this role is not completely understood and needs further investigation. We therefore set up an exploratory proteomic study to identify differences in the proteomic profile of NMGs from DLB patients (n = 5) compared to healthy controls (CTRL, n = 5). We applied a laser microdissection and mass-spectrometry-based approach, in which we used targeted mass spectrometric experiments for validation. In NMG-surrounding (SN Surr. ) tissue of DLB patients, we found evidence for ongoing oxidative damage and an impairment of protein degradation. As a potentially disease-related mechanism, we found α-synuclein and protein S100A9 to be enriched in NMGs of DLB cases, while the abundance of several ribosomal proteins was significantly decreased. As S100A9 is known to be able to enhance the formation of toxic α-synuclein fibrils, this finding points towards an involvement of NMGs in pathogenesis, however the exact role of NMGs as either neuroprotective or neurotoxic needs to be further investigated. Nevertheless, our study provides evidence for an impairment of protein degradation, ongoing oxidative damage and accumulation of potentially neurotoxic protein aggregates to be central mechanisms of neurodegeneration in DLB.

Long-term analyses of biodiversity data highlight a 'biodiversity conservation paradox': biological communities show substantial species turnover over the past century 1,2 , but changes in species richness are marginal 1,3-5 . Most studies, however, have focused only on the incidence of species, and have not considered changes in local abundance. Here we asked whether analysing changes in the cover of plant species could reveal previously unrecognized patterns of biodiversity change and provide insights into the underlying mechanisms. We compiled and analysed a dataset of 7,738 permanent and semi-permanent vegetation plots from Germany that were surveyed between 2 and 54 times from 1927 to 2020, in total comprising 1,794 species of vascular plants. We found that decrements in cover, averaged across all species and plots, occurred more often than increments; that the number of species that decreased in cover was higher than the number of species that increased; and that decrements were more equally distributed among losers than were gains among winners. Null model simulations confirmed that these trends do not emerge by chance, but are the consequence of species-specific negative effects of environmental changes. In the long run, these trends might result in substantial losses of species at both local and regional scales. Summarizing the changes by decade shows that the inequality in the mean change in species cover of losers and winners diverged as early as the 1960s. We conclude that changes in species cover in communities represent an important but understudied dimension of biodiversity change that should more routinely be considered in time-series analyses., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Vegetation-plot resurvey data are a main source of information on terrestrial biodiversity change, with records reaching back more than one century. Although more and more data from re-sampled plots have been published, there is not yet a comprehensive open-access dataset available for analysis. Here, we compiled and harmonised vegetation-plot resurvey data from Germany covering almost 100 years. We show the distribution of the plot data in space, time and across habitat types of the European Nature Information System (EUNIS). In addition, we include metadata on geographic location, plot size and vegetation structure. The data allow temporal biodiversity change to be assessed at the community scale, reaching back further into the past than most comparable data yet available. They also enable tracking changes in the incidence and distribution of individual species across Germany. In summary, the data come at a level of detail that holds promise for broadening our understanding of the mechanisms and drivers behind plant diversity change over the last century., (© 2022. The Author(s).)

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in the cell bodies of dopaminergic neurons in the substantia nigra (SN) pars compacta. These neurons are lost in neurodegenerative diseases, such as Parkinson's disease and dementia with Lewy bodies. Although it is known that lipids, proteins, and environmental toxins accumulate in NMGs, the function of NMGs has not yet been finally clarified as well as their origin and the synthesis of neuromelanin. We, therefore, isolated NMGs and surrounding SN tissue from control patients by laser microdissection and analyzed the proteomic profile by tandem mass spectrometry. With our improved workflow, we were able to (1) strengthen the regularly reported link between NMGs and lysosomes, (2) detect tyrosine hydroxylase to be highly abundant in NMGs, which may be related to neuromelanin synthesis and (3) indicate a yet undescribed link between stress granules (SGs) and NMGs. Based on our findings, we cautiously hypothesize, that SGs may be the origin of NMGs or form in close proximity to them, potentially due to the oxidative stress caused by neuromelanin-bound metals., (© 2022. The Author(s).)

Presently, two Chlorella sorokiniana strains sampled during summer (CS-S) and winter (CS-W) from a maturation pond and isolated by dominance were studied on their behavior on temperature and light extremes in batch experiments. Although both strains showed no differences in their tolerance of temperatures up to 45 °C, the growth rates, pigment contents and fatty acid compositions in response to PAR at 700 and 1,500 µmol m -2 sec -1 differed. CS-W was less affected by photoinhibition and maintained constantly high growth rates. High radiation resulted in both strains in an equivalent decrease of chlorophyll a and accessory pigments indicating that the latter did not function as a light filter. PUFAS (18:3 and 16:3) increased in CS-W at high radiation by > 60% and decreased in CS-S by 8 %. Results indicate that CS-W is highly favorable for mass cultivation particularly in outdoors, in which diurnal variations of solar radiation occur., (Copyright © 2022. Published by Elsevier Ltd.)

We have identified GpsA, a predicted glycerol-3-phosphate dehydrogenase, as a virulence factor in the Lyme disease spirochete Borrelia (Borreliella) burgdorferi: GpsA is essential for murine infection and crucial for persistence of the spirochete in the tick. B. burgdorferi has a limited biosynthetic and metabolic capacity; the linchpin connecting central carbohydrate and lipid metabolism is at the interconversion of glycerol-3-phosphate and dihydroxyacetone phosphate, catalyzed by GpsA and another glycerol-3-phosphate dehydrogenase, GlpD. Using a broad metabolomics approach, we found that GpsA serves as a dominant regulator of NADH and glycerol-3-phosphate levels in vitro, metabolic intermediates that reflect the cellular redox potential and serve as a precursor for lipid and lipoprotein biosynthesis, respectively. Additionally, GpsA was required for survival under nutrient stress, regulated overall reductase activity and controlled B. burgdorferi morphology in vitro. Furthermore, during in vitro nutrient stress, both glycerol and N-acetylglucosamine were bactericidal to B. burgdorferi in a GlpD-dependent manner. This study is also the first to identify a suppressor mutation in B. burgdorferi: a glpD deletion restored the wild-type phenotype to the pleiotropic gpsA mutant, including murine infectivity by needle inoculation at high doses, survival under nutrient stress, morphological changes and the metabolic imbalance of NADH and glycerol-3-phosphate. These results illustrate how basic metabolic functions that are dispensable for in vitro growth can be essential for in vivo infectivity of B. burgdorferi and may serve as attractive therapeutic targets., Competing Interests: The authors have declared that no competing interests exist.

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. Besides neuromelanin, NMGs contain a variety of proteins, lipids, and metals. Although NMGs-containing dopaminergic neurons are preferentially lost in neurodegenerative diseases like Parkinson's disease and dementia with Lewy bodies, only little is known about the mechanism of NMG formation and the role of NMGs in health and disease. Thus, further research on the molecular characterization of NMGs is essential. Unfortunately, standard protocols for the isolation of proteins are based on density gradient ultracentrifugation and therefore require high amounts of human tissue. Thus, an automated laser microdissection (LMD)-based protocol is established here which allows the collection of NMGs and surrounding substantia nigra (SN) tissue using minimal amounts of tissue in an unbiased, automatized way. Excised samples are subsequently analyzed by mass spectrometry to decipher their proteomic composition. With this workflow, 2,079 proteins were identified of which 514 proteins were exclusively identified in NMGs and 181 in SN. The present results have been compared with a previous study using a similar LMD-based approach reaching an overlap of 87.6% for both proteomes, verifying the applicability of the revised and optimized protocol presented here. To validate current findings, proteins of interest were analyzed by targeted mass spectrometry, e.g., parallel reaction monitoring (PRM)-experiments.

The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium ratio ∼10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). This discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs, and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we used conventional and noncanonical amino acid-based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in hENaC show that these regions contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work suggests that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. These results further highlight how M1 and pre-M1 are likely to differentially affect pore structure in these related channels., (© 2021 Sheikh et al.)

Many woody and herbaceous plants in temperate forests cannot establish and survive in the absence of mycorrhizal associations. Most temperate forests are dominated by ectomycorrhizal woody plant species, which implies that the carrying capacity of the habitat for arbuscular mycorrhizal fungi (AMF) is relatively low and AMF could in some cases experience a limitation of propagules. Here we address how the AMF community composition varied in a small temperate forest site in Germany in relation to time, space, two plant host species, and also with regard to the degree to which plots were covered with AMF-associating woody species. The AMF communities in our study were non-random. We observed that space had a greater impact on fungal community composition than either time, mycorrhizal state of the close-by woody species, or the identity of the host plant. The identity of the host plant was the only parameter that modified AMF richness in the roots. The set of parameters which we addressed has rarely been studied together, and the resulting ranking could ease prioritizing some of them to be included in future surveys. AMF are crucial for the establishment of understory plants in temperate forests, making it desirable to further explore how they vary in time and space., (© 2021. The Author(s).)

Cerebrospinal fluid (CSF) diagnostics has emerged as a valid tool for a variety of neurological diseases. However, CSF diagnostics has been playing a subordinate role in the diagnosis of many neurological conditions. Thus, in the multitude of neuromuscular diseases in which motor neurons are affected, a CSF sample is rarely taken routinely. However, CSF diagnostics has the potential to specify the diagnosis and monitor the treatment of neuromuscular disorders. In this review, we therefore focused on a variety of neuromuscular diseases, among them amyotrophic lateral sclerosis (ALS), peripheral neuropathies, and spinal muscular atrophy (SMA), for which CSF diagnostics has emerged as a promising option for determining the disease itself and its progression. We focus on potentially valuable biomarkers among different disorders, such as neurofilaments, cytokines, other proteins, and lipids to determine their suitability, differentiating between different neurological disorders and their potential to determine early disease onset, disease progression, and treatment outcome. We further recommend novel approaches, e.g., the use of mass spectrometry as a promising alternative techniques to standard ELISA assays, potentially enhancing biomarker significance in clinical applications.

Purpose: Genetic diagnostics of neurodevelopmental disorders with epilepsy (NDDE) are predominantly applied in children, thus limited information is available regarding adults or elderly., Methods: We investigated 150 adult/elderly individuals with NDDE by conventional karyotyping, FMR1 testing, chromosomal microarray, panel sequencing, and for unresolved cases, also by exome sequencing (n single  = 71, n trios  = 24)., Results: We identified (likely) pathogenic variants in 71 cases (47.3%) comprising fragile X syndrome (n = 1), disease-causing copy number (n = 23), and single-nucleotide variants (n = 49). Seven individuals displayed multiple independent genetic diagnoses. The diagnostic yield correlated with the severity of intellectual disability. Individuals with anecdotal evidence of exogenic early-life events (e.g., nuchal cord, complications at delivery) with alleged/unproven association to the disorder had a particularly high yield of 58.3%. Screening for disease-specific comorbidities was indicated in 45.1% and direct treatment consequences arose in 11.8% of diagnosed individuals., Conclusion: Panel/exome sequencing displayed the highest yield and should be considered as first-tier diagnostics in NDDE. This high yield and the numerous indications for additional screening or treatment modifications arising from genetic diagnoses indicate a current medical undersupply of genetically undiagnosed adult/elderly individuals with NDDE. Moreover, knowledge of the course of elderly individuals will ultimately help in counseling newly diagnosed individuals with NDDE., (© 2021. The Author(s).)

Protein sequence databases play a crucial role in the majority of the currently applied mass-spectrometry-based proteomics workflows. Here UniProtKB serves as one of the major sources, as it combines the information of several smaller databases and enriches the entries with additional biological information. For the identification of peptides in a sample by tandem mass spectra, as generated by data-dependent acquisition, protein sequence databases provide the basis for most spectrum identification search engines. In addition, for targeted proteomics approaches like selected reaction monitoring (SRM) and parallel reaction monitoring (PRM), knowledge of the peptide sequences, their masses, and whether they are unique for a protein is essential. Because most bottom-up proteomics approaches use trypsin to cleave the proteins in a sample, the tryptic peptides contained in a protein database are of great interest. We present a database, called MaCPepDB (mass-centric peptide database), that consists of the complete tryptic digest of the Swiss-Prot and TrEMBL parts of UniProtKB. This database is especially designed to not only allow queries of peptide sequences and return the respective information about connected proteins and thus whether a peptide is unique but also allow queries of specific masses of peptides or precursors of MS/MS spectra. Furthermore, posttranslational modifications can be considered in a query as well as different mass deviations for posttranslational modifications. Hence the database can be used by a sequence query not only to, for example, check in which proteins of the UniProt database a tryptic peptide can be found but also to find possibly interfering peptides in PRM/SRM experiments using the mass query. The complete database contains currently 5 939 244 990 peptides from 185 561 610 proteins (UniProt version 2020_03), for which a single query usually takes less than 1 s. For easy exploration of the data, a web interface was developed. A REST application programming interface (API) for programmatic and workflow access is also available at https://macpepdb.mpc.rub.de.

Schall and Heinrichs question our interpretation that the climatic debt in understory plant communities is locally modulated by canopy buffering. However, our results clearly show that the discrepancy between microclimate warming rates and thermophilization rates is highest in forests where canopy cover was reduced, which suggests that the need for communities to respond to warming is highest in those forests., (Copyright © 2020, American Association for the Advancement of Science.)

This paper is the continuation (Part 2) of an extensive, critical reappraisal of the international historiography on modern anesthesia and its technology. The first paper of this series provided general definitions, backgrounds and an update on recent research on one aspect of this topic: the history of professionalization / specialization (Part 1). 1 This paper goes on to provide a first, international comparison of entire anesthesia devices and on the history of nitrous-oxide-based anesthesia (c. 1900-1930s). Results: A comparative chronology of internationally recognized milestones of entire anesthesia machines does not suggest significant differences between the nations of continental Europe on one side, or the USA and Britain on the other. The international historiography on one of the key techniques for which these devices were designed (nitrous-oxide-based anesthesia), is likewise demonstrably biased. These findings are further evidence that a frequently held hypothesis, which suggests national dominances in these fields, is incorrect. Contributing factors and wider contexts of this phenomenon can be further confirmed: These are an under-recognition of non-Anglo-American (particularly continental-European) and of primarily surgical contributions; contemporary international conflicts and inter-professional demarcation disputes. In addition, it can be shown that these phenomena had already started around the same time (c. 1900s-1930s). There also is evidence to suggest that they were at times reciprocal and quite deliberate. The author illustrates and argues that the currently prevalent historiography on modern anesthesia requires a thorough reassessment. This should be based on a perspective of internationalism and transdisciplinary reciprocity and should recognize much broader historical contexts., Competing Interests: Declaration of Competing Interest The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

This paper is the first in a series of publications. These investigate, whether important elements of the historiography of anesthesia require a critical reappraisal. A systematic, combined presentation, contextualization and assessment of recent European research is provided. This includes the author's own findings. These emanate from two extensive projects. They combine very recent findings with results of earlier research, conducted by the author and numerous collaborators over the last 18 years. The findings represent an ever increasing and ever more robust body of evidence. They add an important new element to our international historiography. As an introduction, several definitions will be given for criteria, which designate "modern" anesthesia and its technology. On one of these criteria, the history of professionalization and specialization, a short overview will be given. This will be followed by an overview of general contexts, key features and early achievements of anesthesia-related technology. All results will be compared with a currently dominating narrative: This alleges "dominance" of US-American and British pioneers and developments. Apparent biases and inconsistencies are identified. These suggest that our current, international historiography of anesthesia may require a critical reassessment. Three subsequent articles will focus on specific aspects of anesthesia technique and technology. Their results likewise suggest a history of internationalism and trans-disciplinary reciprocity, rather than of national dominances. Further investigations will aim to ascertain the nature and extent of potential interactions, which may nowadays be underrecognized., (Copyright © 2020 Anesthesia History Association. Published by Elsevier Inc. All rights reserved.)

The mechanism by which the content of the major groups of seminal plasma proteins in stallion semen changes between the breeding and non-breeding seasons remains unknown. Here, we investigated the proportions of non-heparin-binding, phosphorylcholine-binding, and heparin-binding proteins in seminal plasma with the aim of relating them to sperm quality and testosterone levels in good and bad freezer stallions. Only minor variations in the major protein groups were found between the breeding and non-breeding seasons. In the non-breeding season, a higher content of a subset of non-heparin binding proteins as well as of heparin-binding proteins was found. Analysis of semen characteristics revealed a somewhat contrasting picture. While only minor variations in sperm kinematics and sperm morphology were found between seasons, the flow-cytometric measurements of mitochondrial membrane potential and also, to some extent, reactive oxygen species production indicated lower sperm quality in the breeding season. Chromatin integrity and testosterone levels were unchanged between seasons. The results suggest that stallion ejaculates could be used year-round for freezing, since only minor differences in protein composition exist between the breeding and non-breeding seasons, as well as between good and bad freezers. In addition, sperm quality is not impaired during the non-breeding season.

The aim of the present study was to characterise key enzymes involved in polyunsaturated fatty acid (PUFA) synthesis in the testis and epididymis collected from 2-year-old healthy warmblood stallions (n=10). The mRNA expression of fatty acid synthase, the Δ9-, Δ6-, Δ5- and Δ4-desaturases and elongases 6, 5 and 2 (encoded by the fatty acid synthase (FASN), the stearoyl-CoA desaturase (SCD), the fatty acid desaturase 2 (FADS2), the fatty acid desaturase 1 (FADS1), the delta 4-desaturase, sphingolipid 1 (DEGS1), ELOVL fatty acid elongase 6(ELOVL6), ELOVL fatty acid elongase 5 (ELOVL5), ELOVL fatty acid elongase 2 (ELOVL2) genes respectively) was determined in equine testis and epididymis. All enzymes were present in testicular tissue and along the epididymis, but mRNA expression differed among localisations. The protein localisation of FADS1, FADS2 and ELOVL5 was determined by immunohistochemistry. In the testes, FADS1 was expressed in the germinal cells and ELOVL5 was expressed in germinal and Leydig cells; FADS2 was not detected. In the epididymis, FADS1 and FADS2 were expressed in the principal and basal cells, whereas ELOVL5 was found only in the principal cells of the caput. All three enzymes were present in epididymal vesicles secreted by an apocrine mechanism. These results suggest active PUFA metabolism during spermatogenesis and epididymal sperm maturation in stallions.

Biodiversity time series reveal global losses and accelerated redistributions of species, but no net loss in local species richness. To better understand how these patterns are linked, we quantify how individual species trajectories scale up to diversity changes using data from 68 vegetation resurvey studies of seminatural forests in Europe. Herb-layer species with small geographic ranges are being replaced by more widely distributed species, and our results suggest that this is due less to species abundances than to species nitrogen niches. Nitrogen deposition accelerates the extinctions of small-ranged, nitrogen-efficient plants and colonization by broadly distributed, nitrogen-demanding plants (including non-natives). Despite no net change in species richness at the spatial scale of a study site, the losses of small-ranged species reduce biome-scale (gamma) diversity. These results provide one mechanism to explain the directional replacement of small-ranged species within sites and thus explain patterns of biodiversity change across spatial scales.

Climate warming is causing a shift in biological communities in favor of warm-affinity species (i.e., thermophilization). Species responses often lag behind climate warming, but the reasons for such lags remain largely unknown. Here, we analyzed multidecadal understory microclimate dynamics in European forests and show that thermophilization and the climatic lag in forest plant communities are primarily controlled by microclimate. Increasing tree canopy cover reduces warming rates inside forests, but loss of canopy cover leads to increased local heat that exacerbates the disequilibrium between community responses and climate change. Reciprocal effects between plants and microclimates are key to understanding the response of forest biodiversity and functioning to climate and land-use changes., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Manipulation of proteins by chemical modification is a powerful way to decipher their function. However, most ribosome-dependent and semi-synthetic methods have limitations in the number and type of modifications that can be introduced, especially in live cells. Here, we present an approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into proteins expressed in eukaryotic cells. We insert synthetic peptides into GFP, Na V 1.5 and P2X2 receptors via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating protein function. We anticipate the approach will overcome some drawbacks of existing protein enigineering methods.

Functional traits respond to environmental drivers, hence evaluating trait-environment relationships across spatial environmental gradients can help to understand how multiple drivers influence plant communities. Global-change drivers such as changes in atmospheric nitrogen deposition occur worldwide, but affect community trait distributions at the local scale, where resources (e.g. light availability) and conditions (e.g. soil pH) also influence plant communities. We investigate how multiple environmental drivers affect community trait responses related to resource acquisition (plant height, specific leaf area (SLA), woodiness, and mycorrhizal status) and regeneration (seed mass, lateral spread) of European temperate deciduous forest understoreys. We sampled understorey communities and derived trait responses across spatial gradients of global-change drivers (temperature, precipitation, nitrogen deposition, and past land use), while integrating in-situ plot measurements on resources and conditions (soil type, Olsen phosphorus (P), Ellenberg soil moisture, light, litter mass, and litter quality). Among the global-change drivers, mean annual temperature strongly influenced traits related to resource acquisition. Higher temperatures were associated with taller understoreys producing leaves with lower SLA, and a higher proportional cover of woody and obligate mycorrhizal (OM) species. Communities in plots with higher Ellenberg soil moisture content had smaller seeds and lower proportional cover of woody and OM species. Finally, plots with thicker litter layers hosted taller understoreys with larger seeds and a higher proportional cover of OM species. Our findings suggest potential community shifts in temperate forest understoreys with global warming, and highlight the importance of local resources and conditions as well as global-change drivers for community trait variation., (© 2019 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

The sodium leak channel (NALCN) is essential for survival in mammals: NALCN mutations are life-threatening in humans and knockout is lethal in mice. However, the basic functional and pharmacological properties of NALCN have remained elusive. Here, we found that robust function of NALCN in heterologous systems requires co-expression of UNC79, UNC80, and FAM155A. The resulting NALCN channel complex is constitutively active and conducts monovalent cations but is blocked by physiological concentrations of extracellular divalent cations. Our data support the notion that NALCN is directly responsible for the increased excitability observed in a variety of neurons in reduced extracellular Ca 2+ . Despite the smaller number of voltage-sensing residues in NALCN, the constitutive activity is modulated by voltage, suggesting that voltage-sensing domains can give rise to a broader range of gating phenotypes than previously anticipated. Our work points toward formerly unknown contributions of NALCN to neuronal excitability and opens avenues for pharmacological targeting., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

In previous studies, cold atmospheric plasma (CAP) was explored as an antibacterial and antiviral agent for the treatment of chronic wounds. The aim of the present study was to investigate whether CAP may also be suitable as an antiviral therapy against herpes simplex virus type 1 (HSV-1). HSV-1 most frequently manifests as recurrent herpes labialis, but it can also cause encephalitis, conjunctivitis or herpes neonatorum as a perinatal infection. HSV-1 encoding the reporter gene GFP was propagated. The CAP dose for HSV-1 treatment was gradually increased, ranging from 0-150 s, and aciclovir was used as a positive control. After CAP treatment, the virus suspension was applied to a standard HSV research cell line (Vero cells) and the neuroblastoma cell line SH-SY5Y as a model for neuronal infection. The results showed that plasma treatment had a negligible antiviral effect on HSV-1 in both Vero- and SH-SY5Y cells at high dose. However, when we lowered the viral load 100-fold, we observed a significantly decreased number of internalized HSV-1 genomes 3 h post-infection for CAP-treated viruses. This difference was less pronounced with respect to GFP expression levels 24 h post-infection, which was in sharp contrast to the acyclovir-treated positive control, for which the viral load was reduced from 95 to 25%. In summary, we observed a low but measurable antiviral effect of CAP on HSV-1.

Aim: Forest understorey microclimates are often buffered against extreme heat or cold, with important implications for the organisms living in these environments. We quantified seasonal effects of understorey microclimate predictors describing canopy structure, canopy composition and topography (i.e., local factors) and the forest patch size and distance to the coast (i.e., landscape factors)., Location: Temperate forests in Europe., Time Period: 2017-2018., Major Taxa Studied: Woody plants., Methods: We combined data from a microclimate sensor network with weather-station records to calculate the difference, or offset, between temperatures measured inside and outside forests. We used regression analysis to study the effects of local and landscape factors on the seasonal offset of minimum, mean and maximum temperatures., Results: The maximum temperature during the summer was on average cooler by 2.1 °C inside than outside forests, and the minimum temperatures during the winter and spring were 0.4 and 0.9 °C warmer. The local canopy cover was a strong nonlinear driver of the maximum temperature offset during summer, and we found increased cooling beneath tree species that cast the deepest shade. Seasonal offsets of minimum temperature were mainly regulated by landscape and topographic features, such as the distance to the coast and topographic position., Main Conclusions: Forest organisms experience less severe temperature extremes than suggested by currently available macroclimate data; therefore, climate-species relationships and the responses of species to anthropogenic global warming cannot be modelled accurately in forests using macroclimate data alone. Changes in canopy cover and composition will strongly modulate the warming of maximum temperatures in forest understories, with important implications for understanding the responses of forest biodiversity and functioning to the combined threats of land-use change and climate change. Our predictive models are generally applicable across lowland temperate deciduous forests, providing ecologically important microclimate data for forest understories., (© 2019 The Authors. Global Ecology and Biogeography published by John Wiley & Sons Ltd.)

Mechanical systems facilitate the development of a hybrid quantum technology comprising electrical, optical, atomic and acoustic degrees of freedom 1 , and entanglement is essential to realize quantum-enabled devices. Continuous-variable entangled fields-known as Einstein-Podolsky-Rosen (EPR) states-are spatially separated two-mode squeezed states that can be used for quantum teleportation and quantum communication 2 . In the optical domain, EPR states are typically generated using nondegenerate optical amplifiers 3 , and at microwave frequencies Josephson circuits can serve as a nonlinear medium 4-6 . An outstanding goal is to deterministically generate and distribute entangled states with a mechanical oscillator, which requires a carefully arranged balance between excitation, cooling and dissipation in an ultralow noise environment. Here we observe stationary emission of path-entangled microwave radiation from a parametrically driven 30-micrometre-long silicon nanostring oscillator, squeezing the joint field operators of two thermal modes by 3.40 decibels below the vacuum level. The motion of this micromechanical system correlates up to 50 photons per second per hertz, giving rise to a quantum discord that is robust with respect to microwave noise 7 . Such generalized quantum correlations of separable states are important for quantum-enhanced detection 8 and provide direct evidence of the non-classical nature of the mechanical oscillator without directly measuring its state 9 . This noninvasive measurement scheme allows to infer information about otherwise inaccessible objects, with potential implications for sensing, open-system dynamics and fundamental tests of quantum gravity. In the future, similar on-chip devices could be used to entangle subsystems on very different energy scales, such as microwave and optical photons.

Soil bacteria and understorey plants interact and drive forest ecosystem functioning. Yet, knowledge about biotic and abiotic factors that affect the composition of the bacterial community in the rhizosphere of understorey plants is largely lacking. Here, we assessed the effects of plant species identity (Milium effusum vs. Stachys sylvatica), rhizospheric soil characteristics, large-scale environmental conditions (temperature, precipitation and nitrogen (N) deposition), and land-use history (ancient vs. recent forests) on bacterial community composition in rhizosphere soil in temperate forests along a 1700 km latitudinal gradient in Europe. The dominant bacterial phyla in the rhizosphere soil of both plant species were Acidobacteria, Actinobacteria and Proteobacteria. Bacterial community composition differed significantly between the two plant species. Within plant species, soil chemistry was the most important factor determining soil bacterial community composition. More precisely, soil acidity correlated with the presence of multiple phyla, e.g. Acidobacteria (negatively), Chlamydiae (negatively) and Nitrospirae (positively), in both plant species. Large-scale environmental conditions were only important in S. sylvatica and land-use history was not important in either of the plant species. The observed role of understorey plant species identity and rhizosphere soil characteristics in determining soil bacterial community composition extends our understanding of plant-soil bacteria interactions in forest ecosystem functioning., (© FEMS 2019.)

The importance of the amino acid L-arginine (ARG) for conceptus growth and litter size has been demonstrated in various species. L-arginine is part of embryo-derived polyamines, a substrate for nitric oxide synthase and stimulates protein synthesis by the embryo. In the present study, we have investigated whether dietary L-arginine supplementation stimulates early conceptus growth in mares. Warmblood mares with singleton pregnancies received either an arginine-supplemented diet (approximately 0.0125% of body weight, n = 12) or a control diet (n = 11) from days 15 to 45 after ovulation. Diameter of the embryonic vesicle (days 14, 17, 20 of pregnancy) and size of the embryo respective foetus (length and maximal diameter, days 25-45 of pregnancy at 5-day intervals) were determined by transrectal ultrasound. At foaling, weight and size of the foal and the placenta were determined. Blood for determination of equine chorionic gonadotrophin (eCG) and progestin concentrations was collected repeatedly. Neither eCG nor progestin concentration in plasma of mares differed between groups at any time. No effects of arginine treatment on diameter of the embryonic vesicle between days 14 and 20 of pregnancy were detected. Diameter of the embryo/foetus on days 40 to 45 of pregnancy strongly tended to be enhanced by arginine supplementation (p = 0.06). Weight and size of neither the foal nor placenta at birth differed between groups. In conclusion, L-arginine supplementation was without negative effects on early equine embryos and may support embryonic growth at the beginning of placentation., (© 2019 The Authors. Reproduction in Domestic Animals Published by Blackwell Verlag GmbH.)

Light is a union of electric and magnetic fields, and nowhere is the complex relationship between these fields more evident than in the near fields of nanophotonic structures. There, complicated electric and magnetic fields varying over subwavelength scales are generally present, which results in photonic phenomena such as extraordinary optical momentum, superchiral fields, and a complex spatial evolution of optical singularities. An understanding of such phenomena requires nanoscale measurements of the complete optical field vector. Although the sensitivity of near-field scanning optical microscopy to the complete electromagnetic field was recently demonstrated, a separation of different components required a priori knowledge of the sample. Here, we introduce a robust algorithm that can disentangle all six electric and magnetic field components from a single near-field measurement without any numerical modeling of the structure. As examples, we unravel the fields of two prototypical nanophotonic structures: a photonic crystal waveguide and a plasmonic nanowire. These results pave the way for new studies of complex photonic phenomena at the nanoscale and for the design of structures that optimize their optical behavior., Competing Interests: The authors declare no competing financial interests.

Forecasting the growth of tree species to future environmental changes requires a better understanding of its determinants. Tree growth is known to respond to global-change drivers such as climate change or atmospheric deposition, as well as to local land-use drivers such as forest management. Yet, large geographical scale studies examining interactive growth responses to multiple global-change drivers are relatively scarce and rarely consider management effects. Here, we assessed the interactive effects of three global-change drivers (temperature, precipitation and nitrogen deposition) on individual tree growth of three study species (Quercus robur/petraea, Fagus sylvatica and Fraxinus excelsior). We sampled trees along spatial environmental gradients across Europe and accounted for the effects of management for Quercus. We collected increment cores from 267 trees distributed over 151 plots in 19 forest regions and characterized their neighbouring environment to take into account potentially confounding factors such as tree size, competition, soil conditions and elevation. We demonstrate that growth responds interactively to global-change drivers, with species-specific sensitivities to the combined factors. Simultaneously high levels of precipitation and deposition benefited Fraxinus, but negatively affected Quercus' growth, highlighting species-specific interactive tree growth responses to combined drivers. For Fagus, a stronger growth response to higher temperatures was found when precipitation was also higher, illustrating the potential negative effects of drought stress under warming for this species. Furthermore, we show that past forest management can modulate the effects of changing temperatures on Quercus' growth; individuals in plots with a coppicing history showed stronger growth responses to higher temperatures. Overall, our findings highlight how tree growth can be interactively determined by global-change drivers, and how these growth responses might be modulated by past forest management. By showing future growth changes for scenarios of environmental change, we stress the importance of considering multiple drivers, including past management and their interactions, when predicting tree growth., (© 2018 John Wiley & Sons Ltd.)

Anti-Müllerian hormone (AMH) has gained increasing interest as a biomarker for assessment of gonadal activity. The ability to predict the ovarian follicular reserve of prepubertal female horses (fillies) or to identify stallions with testicular pathologies already during their prepubertal life has not been analyzed so far. Both would help to select fertile horses and reduce costs associated with keeping animals. The objectives of the present study were to (1) assess AMH, LH, FSH, progesterone (females) and testosterone (males) dynamics in prepubertal horses from birth onwards and (2) determine whether AMH concentrations detected in plasma of prepubertal female and male horses are correlated with postpubertal gonadal development. Warmblood foals (n = 30, 14 females, 10 normal males and 6 males with abnormal testicular development) born between February and May of two consecutive years (n = 28 in the first year and n = 2 the next year), were included in the study. Information on gestational length, parity of the dam and placental weight was collected for all foals. Blood samples for hormone analysis were collected from birth onwards every four weeks up to the age of one year. At two years, blood samples were collected on the day when antral follicle count (AFC) and total testicular volume (TTV) were assessed. AMH was detectable in the plasma of all animals from birth onwards and its concentration was significantly higher (P < .001) in males than in females, regardless of testicular development. In males, AMH and testosterone concentration were similar for all animals during the first year of life, regardless of testicular development. At two years, AMH concentration was higher (P < .05) in males with abnormal testicular development than in those with normal testes. In females, AMH concentration at two years was correlated with AMH concentration at birth (P < .05) and with AFC (P < .001). At birth, LH concentration was lower (P < .05) in stallions with abnormal testes (0.3 ± 0.2 ng/ml) than in controls (0.6 ± 0.2 ng/ml). A high negative correlation between AMH concentration and gestation length was observed in males during the first eight weeks of life (P < .01, r = -0.64 to -0.71). Elevated progesterone concentrations over 1 ng/ml were observed in several females starting with 20 weeks of age. This was paralleled by an increase in AMH concentration and was preceded by FSH and LH increases. In conclusion, AMH determination can be reliably used from two years onwards to identify stallions with abnormal testicular development, but it is inconclusive before puberty. In female horses, determination of AMH concentration at a prepubertal age allows for prediction of AMH and AFC after puberty. We suggest that premature luteinisation occurs before the onset of puberty in female horses and that LH secretion in the perinatal period is involved in testicular development and descent in the horse., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Freezing and thawing processes induce structural and functional damage to sperm plasma membranes and internal organelles. Adding seminal plasma (SP) has been found to minimize or repair the cryoinjuries in some species. The objective of this study was to investigate whether adding SP from stallions of known freezability after thawing could repair cryoinjuries. Semen was collected from warmblood stallions (n = 8, three ejaculates/stallion) and processed by Single Layer Centrifugation (SLC) to remove SP prior to freezing. Pooled SP (5%) from bad freezer (BF) or good freezer (GF) stallions was added after thawing. Post-thaw sperm quality was assessed by flow cytometry in terms of chromatin integrity (%DFI), membrane integrity, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and MitoSOX. Sperm kinematics were also assessed by computer-assisted sperm analysis. The %DFI was lower in SLC control (C) than in BF or GF (P < 0.0001, P < 0.0003 respectively). The proportion of viable spermatozoa with intact cell membranes was higher in C than in SP treated groups (C vs. BF, P = 0.02; C vs GF, P = 0.05). There were fewer spermatozoa with low MMP and more with high MMP for C than GF (P = 0.006). The spermatozoa treated with SP from good freezers produced more ROS than when treated with SP from bad freezers (P = 0.007). Motility parameters were not affected by adding SP. In conclusion, adding SP after thawing does not have a beneficial effect on sperm quality, suggesting an inability to repair stallion sperm cryoinjuries, regardless of whether the SP originated from stallions semen, which has good or bad quality after thawing., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Seminal plasma (SP) contains proteins that may influence cryosurvival and prevent capacitation-like changes due to freezing and thawing. The objective of this study was to investigate the effect of adding pooled SP from "good" (GF) or "bad" (BF) freezer stallions on sperm cells' fertilizing ability. "Good freezers" refers to stallions that usually produce ejaculates which can withstand cryopreservation, whilst "bad freezer" stallions produce ejaculates which cannot tolerate the freezing process. A heterologous zona binding assay with in vitro matured bovine oocytes was used to assess the binding ability of equine sperm cells as a possible alternative to artificial insemination trials. The effect of adding SP i) prior to cryopreservation; ii) after thawing of sperm cells selected by single layer centrifugation (SLC); iii) to capacitation medium, was evaluated. Adding SP from GF stallions prior to cryopreservation reduced the mean number of sperm cells bound to the zona pellucida (ZP) compared to control (P = 0.0003), SP-free sperm cells and group received SP from BF stallions (P ≤ 0.0001 for both). After thawing SLC-selected sperm cells treated with 5% SP showed a decrease in binding ability compared with SP-free sperm cells (P ≤ 0.0001). The binding affinity of sperm cells was higher in the group treated with SP from GF than with SP from BF stallions (P ≤ 0.05). Prolonged exposure to SP impaired the ability of stallion sperm cells to undergo capacitation and bind to ZP, regardless of the source of SP (P ≤ 0.0001). The response of equine sperm cells to SP is influenced by the ability of the sperm cells to withstand cryopreservation and is affected by the timing of exposure and the origin of SP. Customization of the protocol for individual stallions is recommended to optimize the effect., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: This cross-sectional study used transmission-mode ultrasound to evaluate dynamic tendon properties during walking in surgically repaired and contralateral Achilles tendon (AT), with a median (range) post-operative period of 22 (4-58) months. It was hypothesised that the axial transmission speed of ultrasound (TSOU) during walking would be slower, indicating lower material stiffness in repaired compared with contralateral AT., Methods: Ten patients [median (range) age 47 (37-69) years; height 180 (170-189) cm; weight 93 (62-119) kg], who had undergone open surgical repair of the AT and were clinically recovered according to their treating clinicians, walked barefoot on a treadmill at self-selected speed (1.0 ± 0.2 m/s). Synchronous measures of TSOU, sagittal ankle motion, vertical ground reaction force (GRF), and spatiotemporal gait parameters were recorded during 20 s of steady-state walking. Paired t tests were used to evaluate potential between-limb differences in TSOU, GRF, ankle motion, and spatiotemporal gait parameters., Results: TSOU was significantly lower (≈175 m/s) in the repaired than in the contralateral AT over the entire gait cycle (P < 0.05). Sagittal ankle motion was significantly greater (≈3°) in the repaired than in the contralateral limb (P = 0.036). There were no significant differences in GRF or spatiotemporal parameters between limbs., Conclusions: Repaired AT was characterised by a lower TSOU, reflecting a lower material stiffness in the repaired tendon than in the contralateral tendon. A lower material stiffness may underpin greater ankle joint motion of the repaired limb during walking and long-term deficits in the muscle-tendon unit reported with AT repair. Treatment and rehabilitation approaches that focus on increasing the material stiffness of the repaired AT may be clinically beneficial. Transmission-mode ultrasound would seem useful for quantifying tendon properties post AT rupture repair and may have the potential to individually guide rehabilitation programmes, thereby aiding safer return to physical activity., Level of Evidence: II.

Objective: Managing type 1 diabetes (T1D) requires the ability to make complex and critical decisions regarding treatment, to execute complex tasks accurately, and to make adjustments when problems arise. This requires effective neuropsychological competences of patients and their families, especially in the domain of executive functioning (EF): the ability to self-monitor, plan, solve problems, and set priorities. Previous research focused mainly on child EF, neglecting the impact of parental EF. This study included both mothers and fathers and examined associations between child and parental EF and treatment adherence to T1D in a broad age range of patients., Methods: Parents of 270 patients (6-18 years) with T1D (mean age 12.7 years; 52.6% female) were included. Mothers (N = 232) and fathers (N = 168) completed questionnaires on child and parental EF and on treatment adherence. Analyses examined the associations linking child and parental EF to treatment adherence and glycemic control (and potential moderation effects in these associations) using hierarchical linear regression., Results: Child EF problems were negatively associated with treatment adherence. As an indication of moderation, this effect was stronger in older children. Better treatment adherence and glycemic control were reported when both child and parent showed less EF problems. Effects were more pronounced in mothers than in fathers., Conclusions: This study demonstrated a significant interplay between child and parental EF in the association with treatment adherence and glycemic control. Researchers and clinicians should remain attentive toward the role of neuropsychological concepts such as EF. Implementation in clinical practice seems meaningful., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The contemporary state of functional traits and species richness in plant communities depends on legacy effects of past disturbances. Whether temporal responses of community properties to current environmental changes are altered by such legacies is, however, unknown. We expect global environmental changes to interact with land-use legacies given different community trajectories initiated by prior management, and subsequent responses to altered resources and conditions. We tested this expectation for species richness and functional traits using 1814 survey-resurvey plot pairs of understorey communities from 40 European temperate forest datasets, syntheses of management transitions since the year 1800, and a trait database. We also examined how plant community indicators of resources and conditions changed in response to management legacies and environmental change. Community trajectories were clearly influenced by interactions between management legacies from over 200 years ago and environmental change. Importantly, higher rates of nitrogen deposition led to increased species richness and plant height in forests managed less intensively in 1800 (i.e., high forests), and to decreases in forests with a more intensive historical management in 1800 (i.e., coppiced forests). There was evidence that these declines in community variables in formerly coppiced forests were ameliorated by increased rates of temperature change between surveys. Responses were generally apparent regardless of sites' contemporary management classifications, although sometimes the management transition itself, rather than historic or contemporary management types, better explained understorey responses. Main effects of environmental change were rare, although higher rates of precipitation change increased plant height, accompanied by increases in fertility indicator values. Analysis of indicator values suggested the importance of directly characterising resources and conditions to better understand legacy and environmental change effects. Accounting for legacies of past disturbance can reconcile contradictory literature results and appears crucial to anticipating future responses to global environmental change., (© 2017 John Wiley & Sons Ltd.)

Addition of seminal plasma (SP) prior to cryopreservation may influence stallion sperm cryosurvival. The objective of this study was to investigate the addition of pooled SP from "good" or "bad" freezer stallions to spermatozoa selected by single layer centrifugation (SLC) prior to cryopreservation on post-thaw sperm quality. Semen from 12 stallions was collected; 5 mL was frozen as control (C) and the remainder was processed by SLC to remove SP and was divided into three aliquots: i) SLC sample without SP (SLC); ii) SLC plus pooled SP from "good freezer" stallions (SLC-GF); iii) SLC plus pooled SP from "bad freezer" stallions (SLC-BF). After thawing, the following parameters were evaluated: chromatin integrity (DNA fragmentation index; %DFI), mitochondrial membrane potential (MMP), membrane integrity (MI), reactive oxygen species (ROS) and sperm kinematics. The %DFI was reduced (P < 0.0001) in SLC samples compared to controls. The SLC group showed a lower proportion of spermatozoa with low MMP and a higher proportion of spermatozoa with high MMP than other groups (P < 0.0001), and had lower hydrogen peroxide content than control. Sperm kinematics were not different. In conclusion, selection by SLC prior to cryopreservation improved post-thaw sperm quality; inclusion of SP from "good" and "bad" freezer stallions did not have an additional beneficial effect., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Fluorescent labels offer the capability to follow conformational dynamics of membrane proteins, but signal detection in such recordings is inherently difficult to achieve in a cell membrane and lacks sufficient time resolution to follow physiologically relevant transitions. Here, we develop high-sensitivity patch-clamp fluorometry (hsPCF), a fluorescence-based approach that results in up to 10-fold increased signals and affords 50-fold faster fluorescence recordings than previous methods. The increased time resolution is paired with a very high versatility in terms of the choice of fluorescent dye, cell type, and protein of interest. We highlight this versatility by providing insight into the conformational dynamics of both ligand- and voltage-gated ion channels using fluorescent labels introduced in extracellular or transmembrane positions while changing either the extra- or intracellular solutions. Together, hsPCF will thus enable the future study of membrane-embedded proteins with sufficient temporal resolution to resolve conformational dynamics., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The tick Ixodes ricinus has considerable impact on the health of humans and other terrestrial animals because it transmits several tick-borne pathogens (TBPs) such as B. burgdorferi (sensu lato), which causes Lyme borreliosis (LB). Small forest patches of agricultural landscapes provide many ecosystem services and also the disservice of LB risk. Biotic interactions and environmental filtering shape tick host communities distinctively between specific regions of Europe, which makes evaluating the dilution effect hypothesis and its influence across various scales challenging. Latitude, macroclimate, landscape and habitat properties drive both hosts and ticks and are comparable metrics across Europe. Therefore, we instead assess these environmental drivers as indicators and determine their respective roles for the prevalence of B. burgdorferi in I. ricinus., Methods: We sampled I. ricinus and measured environmental properties of macroclimate, landscape and habitat quality of forest patches in agricultural landscapes along a European macroclimatic gradient. We used linear mixed models to determine significant drivers and their relative importance for nymphal and adult B. burgdorferi prevalence. We suggest a new prevalence index, which is pool-size independent., Results: During summer months, our prevalence index varied between 0 and 0.4 per forest patch, indicating a low to moderate disservice. Habitat properties exerted a fourfold larger influence on B. burgdorferi prevalence than macroclimate and landscape properties combined. Increasingly available ecotone habitat of focal forest patches diluted and edge density at landscape scale amplified B. burgdorferi prevalence. Indicators of habitat attractiveness for tick hosts (food resources and shelter) were the most important predictors within habitat patches. More diverse and abundant macro- and microhabitat had a diluting effect, as it presumably diversifies the niches for tick-hosts and decreases the probability of contact between ticks and their hosts and hence the transmission likelihood., Conclusions: Diluting effects of more diverse habitat patches would pose another reason to maintain or restore high biodiversity in forest patches of rural landscapes. We suggest classifying habitat patches by their regulating services as dilution and amplification habitat, which predominantly either decrease or increase B. burgdorferi prevalence at local and landscape scale and hence LB risk. Particular emphasis on promoting LB-diluting properties should be put on the management of those habitats that are frequently used by humans. In the light of these findings, climate change may be of little concern for LB risk at local scales, but this should be evaluated further.

Background: The castor bean tick (Ixodes ricinus) transmits infectious diseases such as Lyme borreliosis, which constitutes an important ecosystem disservice. Despite many local studies, a comprehensive understanding of the key drivers of tick abundance at the continental scale is still lacking. We analyze a large set of environmental factors as potential drivers of I. ricinus abundance. Our multi-scale study was carried out in deciduous forest fragments dispersed within two contrasting rural landscapes of eight regions, along a macroclimatic gradient stretching from southern France to central Sweden and Estonia. We surveyed the abundance of I. ricinus, plant community composition, forest structure and soil properties and compiled data on landscape structure, macroclimate and habitat properties. We used linear mixed models to analyze patterns and derived the relative importance of the significant drivers., Results: Many drivers had, on their own, either a moderate or small explanatory value for the abundance of I. ricinus, but combined they explained a substantial part of variation. This emphasizes the complex ecology of I. ricinus and the relevance of environmental factors for tick abundance. Macroclimate only explained a small fraction of variation, while properties of macro- and microhabitat, which buffer macroclimate, had a considerable impact on tick abundance. The amount of forest and the composition of the surrounding rural landscape were additionally important drivers of tick abundance. Functional (dispersules) and structural (density of tree and shrub layers) properties of the habitat patch played an important role. Various diversity metrics had only a small relative importance. Ontogenetic tick stages showed pronounced differences in their response. The abundance of nymphs and adults is explained by the preceding stage with a positive relationship, indicating a cumulative effect of drivers., Conclusions: Our findings suggest that the ecosystem disservices of tick-borne diseases, via the abundance of ticks, strongly depends on habitat properties and thus on how humans manage ecosystems from the scale of the microhabitat to the landscape. This study stresses the need to further evaluate the interaction between climate change and ecosystem management on I. ricinus abundance.

In pregnant mares, peripheral insulin antagonism channels glucose preferentially to the foetus. On the other hand, horses reduce their metabolic activity in winter. Taking these aspects of equine pregnancy and metabolism together, we hypothesized that glucose clearance from blood and the insulin response to glucose do not only change throughout gestation but also with season. To test this hypothesis, the glucose and insulin response to an oral glucose test and relative insulin release were analysed in pregnant mares (n=12) and in geldings (n=10) as controls. Animals were tested in June, September, December, and in March (geldings) and on day 320 of gestation (mares). Furthermore, the 6 mares foaling early and 6 foaling later in the year were compared. In mares and geldings, plasma glucose concentration increased after glucose feeding (p<0.001). The increase was more pronounced in mares (p<0.05) and increased from June to December in mares (p<0.001) but not geldings (month x group p<0.05). This indicates constant glucose clearance in geldings but reduced clearance in pregnant mares. A partial insulin resistance is thus induced by pregnancy independent from season. Insulin release increased after glucose feeding (p<0.001) similarly in geldings and mares. The insulin response to glucose and relative insuslin release increased from June to December (p<0.001) indicating seasonal changes in β-cell sensitivity. Glucose and insulin concentration did not differ between early and late foaling mares. In conclusion, in horses, β-cell sensitivity to glucose is affected by season while insulin sensitivity during pregnancy decreases independent from season., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Seasonal changes in metabolic rate have been shown in horses and we hypothesized that this leads to the birth of smaller foals early in the year. Mares and their foals were assigned to three groups by day of foaling within the year (e.g. 1 January = day 1): Group 1 (n = 10) day 40-65, group 2 (n = 8) day 67-92, group 3 (n = 9) day 94-121. Groups did not differ with regard to parity. In foals, height at withers and body weight were determined on days 1-5 and weekly until 12 weeks of age. Chest circumference, distances fetlock to carpus, carpus to elbow, poll to nose and crown-rump length were determined on day 5 and weekly until 12 weeks of age. Placental weight (p < 0.05) and surface (p < 0.01) were lower in mares of group 1 than in groups 2 and 3. Foal weight and length measurements increased over time (p < 0.001). Height at withers was consistently lower in group 1 than in groups 2 and 3 (p < 0.05) while foal weight did not differ among groups. Fetlock to carpus, carpus to elbow (both p < 0.01) and poll to nose length (p < 0.05) were lower in group 1 than in groups 2 and 3. Neither gestation length nor sex ratio of foals differed among groups. In conclusion, foetal size is reduced when the final growth phase coincides with the winter months. This also impacts neonatal growth during the first three months of life., (Copyright © 2017. Published by Elsevier Inc.)

Increased extracellular proton concentrations during neurotransmission are converted to excitatory sodium influx by acid-sensing ion channels (ASICs). 10-fold sodium/potassium selectivity in ASICs has long been attributed to a central constriction in the channel pore, but experimental verification is lacking due to the sensitivity of this structure to conventional manipulations. Here, we explored the basis for ion selectivity by incorporating unnatural amino acids into the channel, engineering channel stoichiometry and performing free energy simulations. We observed no preference for sodium at the "GAS belt" in the central constriction. Instead, we identified a band of glutamate and aspartate side chains at the lower end of the pore that enables preferential sodium conduction.

In mares, foaling is associated with changes in hematology, plasma electrolytes, blood pressure and heart rate and it has been hypothesized that these are induced by oxytocin. To test this hypothesis, mares (n = 8-14/group) were treated with oxytocin (OT; 20 I.U.) or saline (CON) at 1 h (test A) and 12 h after foaling (test B) and during first postpartum diestrus (test C). Heart rate, heart rate variability (HRV), atrioventricular blocks, salivary cortisol concentration, blood pressure, plasma electrolytes and blood count were determined. Heart rate decreased from test A to C (P < 0.001) but at no time differed between groups. The HRV, blood pressure and occurrence of atrioventricular blocks did not change in response to oxytocin. Cortisol concentration decreased from test A to C (P < 0.001). Oxytocin induced a cortisol release in test B (time x treatment P < 0.001, time x test P < 0.001). Plasma sodium and chloride concentrations decreased from test A to C (P < 0.001) but did not differ between groups. In test A, potassium concentration increased in CON but not OT mares (time P < 0.01, time x test P < 0.01, time x treatment P < 0.05). Polymorphnuclear cell (PMN) numbers in blood decreased from test A to C (P < 0.001) while lymphocytes increased (P < 0.05). At no time PMN and lymphocytes differed between groups. Oxytocin treatment had no effect on skin temperature. In conclusion, except for a limited effect on cortisol release, oxytocin was without effect and the hypothesis of oxytocin-induced alterations in cardiac parameters, plasma electrolytes and hematology of foaling mares was not verified., (Copyright © 2016 Elsevier Inc. All rights reserved.)