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Motivation: Analysis of alternative splicing using short-read RNA-seq data is a complex process that involves several steps: alignment of reads to the reference genome, identification of alternatively spliced features, motif discovery, analysis of RNA-protein binding near donor and acceptor splice sites, and exploratory data visualization. To the best of our knowledge, there is currently no integrative open-source software dedicated to this task., Results: Here, we introduce splicekit , a Python package that provides and integrates a set of existing and novel splicing analysis tools for conducting splicing analysis., Availability and Implementation: The software splicekit is open-source and available at Github (https://github.com/bedapub/splicekit) and via the Python Package Index., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press.)

At large scales, the mechanisms underpinning stability in natural communities may vary in importance due to changes in species composition, mean abundance, and species richness. Here we link species characteristics (niche positions) and community characteristics (richness and abundance) to evaluate the importance of stability mechanisms in 156 butterfly communities monitored across three European countries and spanning five bioclimatic regions. We construct niche-based hierarchical structural Bayesian models to explain first differences in abundance, population stability, and species richness between the countries, and then explore how these factors impact community stability both directly and indirectly (via synchrony and population stability). Species richness was partially explained by the position of a site relative to the niches of the species pool, and species near the centre of their niche had higher average population stability. The differences in mean abundance, population stability, and species richness then influenced how much variation in community stability they explained across the countries. We found, using variance partitioning, that community stability in Finnish communities was most influenced by community abundance, whereas this aspect was unimportant in Spain with species synchrony explaining most variation; the UK was somewhat intermediate with both factors explaining variation. Across all countries, the diversity-stability relationship was indirect with species richness reducing synchrony which increased community stability, with no direct effects of species richness. Our results suggest that in natural communities, biogeographical variation observed in key drivers of stability, such as population abundance and species richness, leads to community stability being limited by different factors and that this can partially be explained due to the niche characteristics of the European butterfly assemblage., (© 2023 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

The safety of most human recombinant proteins can be evaluated in transgenic mice tolerant to specific human proteins. However, owing to insufficient genetic diversity and to fundamental differences in immune mechanisms, small-animal models of human diseases are often unsuitable for immunogenicity testing and for predicting adverse outcomes in human patients. Most human therapeutic antibodies trigger xenogeneic responses in wild-type animals and thus rapid clearance of the drugs, which makes in vivo toxicological testing of human antibodies challenging. Here we report the generation of Göttingen minipigs carrying a mini-repertoire of human genes for the immunoglobulin heavy chains γ1 and γ4 and the immunoglobulin light chain κ. In line with observations in human patients, the genetically modified minipigs tolerated the clinically non-immunogenic IgG1κ-isotype monoclonal antibodies daratumumab and bevacizumab, and elicited antibodies against the checkpoint inhibitor atezolizumab and the engineered interleukin cergutuzumab amunaleukin. The humanized minipigs can facilitate the safety and efficacy testing of therapeutic antibodies., (© 2022. The Author(s).)

Background: Patients undergoing extracorporeal membrane oxygenation (ECMO) are critically ill and show high mortality. Inter-hospital transfer of these patients has to be safe, with high survival rates during transport without potentially serious and life-threatening adverse events. The Swiss Air-Rescue provides 24-h/7-days per week inter-hospital helicopter transfers that include on-site ECMO cannulation if needed. This retrospective observational study describes adverse events of patients on ECMO transported by helicopter, and their associated survival., Methods: All patients on ECMO with inter-hospital transfer by helicopter from start of service in February 2009 until May 2021 were included. Patients not transported by helicopter or with missing medical records were excluded. Patient demographics (age, sex) and medical history (type of and reason for ECMO), mission details (flight distance, times, primary or secondary transport), adverse events during the inter-hospital transfer, and survival of transferred patients were recorded. The primary endpoint was patient survival during transfer. Secondary endpoints were adverse events during transfer and 28-day survival., Results: We screened 214 ECMO-related missions and included 191 in this analysis. Median age was 54.6 [IQR 46.1-62.0] years, 70.7% were male, and most patients had veno-arterial ECMO (56.5%). The main reasons for ECMO were pulmonary (46.1%) or cardiac (44.0%) failure. Most were daytime (69.8%) and primary missions (n = 100), median total mission time was 182.0 [143.0-254.0] min, and median transfer distance was 52.7 [33.2-71.1] km. All patients survived the transfer. Forty-four adverse events were recorded during 37 missions (19.4%), where 31 (70.5%) were medical and none resulted in patient harm. Adverse events occurred more frequently during night-time missions (59.9%, p = 0.047). Data for 28-day survival were available for 157 patients, of which 86 (54.8%) were alive., Conclusion: All patients under ECMO survived the helicopter transport. Adverse events were observed for about 20% of the flight missions, with a tendency during the night-time flights, none harmed the patients. Inter-hospital transfer for patients undergoing ECMO provided by 24-h/7-d per week helicopter emergency medical service teams can be considered as feasible and safe. The majority of the patients (54.8%) were still alive after 28 days., (© 2022. The Author(s).)

Climatic anomalies are increasing in intensity and frequency due to rapid rates of global change, leading to increased extinction risk for many species. The impacts of anomalies are likely to vary between species due to different degrees of sensitivity and extents of local adaptation. Here, we used long-term butterfly monitoring data of 143 species across six European bioclimatic regions to show how species' population dynamics have responded to local or globally-calculated climatic anomalies, and how species attributes mediate these responses. Contrary to expectations, degree of apparent local adaptation, estimated from the relative population sensitivity to local versus global anomalies, showed no associations with species mobility or reproductive rate but did contain a strong phylogenetic signal. The existence of phylogenetically-patterned local adaptation to climate has important implications for forecasting species responses to current and future climatic conditions and for developing appropriate conservation practices., (© 2022. The Author(s).)

Single-cell RNA sequencing (scRNA-seq) revolutionized our understanding of disease biology. The promise it presents to also transform translational research requires highly standardized and robust software workflows. Here, we present the toolkit Besca , which streamlines scRNA-seq analyses and their use to deconvolute bulk RNA-seq data according to current best practices. Beyond a standard workflow covering quality control, filtering, and clustering, two complementary Besca modules, utilizing hierarchical cell signatures and supervised machine learning, automate cell annotation and provide harmonized nomenclatures. Subsequently, the gene expression profiles can be employed to estimate cell type proportions in bulk transcriptomics data. Using multiple, diverse scRNA-seq datasets, some stemming from highly heterogeneous tumor tissue, we show how Besca aids acceleration, interoperability, reusability and interpretability of scRNA-seq data analyses, meeting crucial demands in translational research and beyond., (© The Author(s) 2021. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

Age-related macular degeneration (AMD), one of the leading causes of blindness worldwide, causes personal suffering and high socioeconomic costs. While there has been progress in the treatments for the neovascular form of AMD, no therapy is yet available for the more common dry form, also known as geographic atrophy. We analysed the retinal tissue in a mouse model of retinal degeneration caused by sodium iodate (NaIO 3 )-induced retinal pigment epithelium (RPE) atrophy to understand the underlying pathology. RNA sequencing (RNA-seq), qRT-PCR, Western blot, immunohistochemistry of the retinas and multiplex ELISA of the mouse serum were applied to find the pathways involved in the degeneration. NaIO 3 caused patchy RPE loss and thinning of the photoreceptor layer. This was accompanied by the increased retinal expression of complement components c1s, c3 , c4 , cfb and cfh . C1s, C3, CFH and CFB were complement proteins, with enhanced deposition at day 3. C4 was upregulated in retinal degeneration at day 10. Consistently, the transcript levels of proinflammatory ccl-2 , -3 , -5 , il-1β , il-33 and tgf-β were increased in the retinas of NaIO 3 mice, but vegf-a mRNA was reduced. Macrophages, microglia and gliotic Müller cells could be a cellular source for local retinal inflammatory changes in the NaIO 3 retina. Systemic complement and cytokines/chemokines remained unaltered in this model of NaIO 3 -dependent retinal degeneration. In conclusion, systemically administered NaIO 3 promotes degenerative and inflammatory processes in the retina, which can mimic the hallmarks of geographic atrophy.

Ecological networks are valuable for ecosystem analysis but their use is often limited by a lack of data because many types of ecological interaction, for example, predation, are short-lived and difficult to observe or detect. While there are different methods for inferring the presence of interactions, they have rarely been used to predict the interaction strengths that are required to construct weighted, or quantitative, ecological networks.Here, we develop a trait-based approach suitable for inferring weighted networks, that is, with varying interaction strengths. We developed the method for seed-feeding carabid ground beetles (Coleoptera: Carabidae) although the principles can be applied to other species and types of interaction.Using existing literature data from experimental seed-feeding trials, we predicted a per-individual interaction cost index based on carabid and seed size. This was scaled up to the population level to create inferred weighted networks using the abundance of carabids and seeds from empirical samples and energetic intake rates of carabids from the literature. From these weighted networks, we also derived a novel measure of expected predation pressure per seed type per network.This method was applied to existing ecological survey data from 255 arable fields with carabid data from pitfall traps and plant seeds from seed rain traps. Analysis of these inferred networks led to testable hypotheses about how network structure and predation pressure varied among fields.Inferred networks are valuable because (a) they provide null models for the structuring of food webs to test against empirical species interaction data, for example, DNA analysis of carabid gut regurgitates and (b) they allow weighted networks to be constructed whenever we can estimate interactions between species and have ecological census data available. This permits ecological network analysis even at times and in places when interactions were not directly assessed., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Microglia, the innate immune cells of the brain, are essential for maintaining homeostasis by their ramified, highly motile processes and for orchestrating the immune response to pathological stimuli. They are implicated in several neurodegenerative diseases like Alzheimer's and Parkinson's disease. One commonality of these diseases is their strong correlation with aging as the highest risk factor and studying age-related alterations in microglia physiology and associated signaling mechanism is indispensable for a better understanding of age-related pathomechanisms. CD22 has been identified as a modifier of microglia phagocytosis in a recent study, but not much is known about the function of CD22 in microglia. Here we show that CD22 surface levels are upregulated in aged versus adult microglia. Furthermore, in the amyloid mouse model PS2APP, Aβ-containing microglia also exhibit increased CD22 signal. To assess the impact of CD22 blockage on microglia morphology and dynamics, we have established a protocol to image microglia process motility in acutely prepared brain slices from CX3CR1-GFP reporter mice. We observed a significant reduction of microglial ramification and surveillance capacity in brain slices from aged versus adult mice. The age-related decrease in surveillance can be restored by antibody-mediated CD22 blockage in aged mice, whereas surveillance in adult mice is not affected by CD22 inhibition. Moreover to complement the results obtained in mice, we show that human iPSC-derived macrophages exhibit an increased phagocytic capacity upon CD22 blockage. Downstream analysis of antibody-mediated CD22 inhibition revealed an influence on BMP and TGFβ associated gene networks. Our results demonstrate CD22 as a broad age-associated modulator of microglia functionality with potential implications for neurodegenerative disorders., Competing Interests: During the course of this study, all authors are or were full time employees or trainees at Roche and they may additionally hold Roche stock/stock options. The reviewer DE declared a shared affiliation with one of the authors VA to the handling editor at the time of the review., (Copyright © 2021 Aires, Coulon-Bainier, Pavlovic, Ebeling, Schmucki, Schweitzer, Kueng, Gutbier and Harde.)

Genome-wide sequencing technologies have greatly contributed to our understanding of the genetic basis of neurodevelopmental disorders such as autism spectrum disorder (ASD). Interestingly, a number of ASD-related genes express natural antisense transcripts (NATs). In some cases, these NATs have been shown to play a regulatory role in sense strand gene expression and thus contribute to brain function. However, a detailed study examining the transcriptional relationship between ASD-related genes and their NAT partners is lacking. We performed strand-specific, deep RNA sequencing to profile expression of sense and antisense reads with a focus on 100 ASD-related genes in medial prefrontal cortex (mPFC) and striatum across mouse post-natal development (P7, P14, and P56). Using de novo transcriptome assembly, we generated a comprehensive long non-coding RNA (lncRNA) transcriptome. We conducted BLAST analyses to compare the resultant transcripts with the human genome and identified transcripts with high sequence similarity and coverage. We assembled 32861 de novo antisense transcripts mapped to 12182 genes, of which 1018 are annotated by Ensembl as lncRNA. We validated the expression of a subset of selected ASD-related transcripts by PCR, including Syngap1 and Cntnap2 . Our analyses revealed that more than 70% (72/100) of the examined ASD-related genes have one or more expressed antisense transcripts, suggesting more ASD-related genes than previously thought could be subject to NAT-mediated regulation in mice. We found that expression levels of antisense contigs were mostly positively correlated with their cognate coding sense strand RNA transcripts across developmental age. A small fraction of the examined transcripts showed brain region specific enrichment, indicating possible circuit-specific roles. Our BLAST analyses identified 110 of 271 ASD-related de novo transcripts with >90% identity to the human genome at >90% coverage. These findings, which include an assembled de novo antisense transcriptome, contribute to the understanding of NAT regulation of ASD-related genes in mice and can guide NAT-mediated gene regulation strategies in preclinical investigations toward the ultimate goal of developing novel therapeutic targets for ASD., Competing Interests: BK, RS, NG, TB, and BJH were full time employees of F. Hoffmann-La Roche Ltd. of Basel, Switzerland during the course of studies. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Koç, Fucile, Schmucki, Giroud, Bergauer and Hall.)

As the pressure to take action against global warming is growing in urgency, scenarios that incorporate multiple social, economic and environmental drivers become increasingly critical to support governments and other stakeholders in planning climate change mitigation or adaptation actions. This has led to the recent explosion of future scenario analyses at multiple scales, further accelerated since the development of the Intergovernmental Panel on Climate Change (IPCC) research community Shared Socioeconomic Pathways (SSPs) and Representative Concentration Pathways (RCPs). While RCPs have been widely applied to climate models to produce climate scenarios at multiple scales for investigating climate change impacts, adaptation and vulnerabilities (CCIAV), SSPs are only recently being scaled for different geographical and sectoral applications. This is seen in the UK where significant investment has produced the RCP-based UK Climate Projections (UKCP18), but no equivalent UK version of the SSPs exists. We address this need by developing a set of multi-driver qualitative and quantitative UK-SSPs, following a state-of-the-art scenario methodology that integrates national stakeholder knowledge on locally-relevant drivers and indicators with higher level information from European and global SSPs. This was achieved through an intensive participatory process that facilitated the combination of bottom-up and top-down approaches to develop a set of UK-specific SSPs that are locally comprehensive, yet consistent with the global and European SSPs. The resulting scenarios balance the importance of consistency and legitimacy, demonstrating that divergence is not necessarily the result of inconsistency, nor comes as a choice to contextualise narratives at the appropriate scale., 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 © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Microglia are key in the homeostatic well-being of the brain and microglial dysfunction has been implicated in neurodegenerative disorders such as Alzheimer's disease (AD). Due to the many limitations to study microglia in situ or isolated for large scale drug discovery applications, there is a high need to develop robust and scalable human cellular models of microglia with reliable translatability to the disease. Here, we describe the generation of microglia-like cells from human induced pluripotent stem cells (iPSC) with distinct phenotypes for mechanistic studies in AD. We started out from an established differentiation protocol to generate primitive macrophage precursors mimicking the yolk sac ontogeny of microglia. Subsequently, we tested 36 differentiation conditions for the cells in monoculture where we exposed them to various combinations of media, morphogens, and extracellular matrices. The optimized protocol generated robustly ramified cells expressing key microglial markers. Bulk mRNA sequencing expression profiles revealed that compared to cells obtained in co-culture with neurons, microglia-like cells derived from a monoculture condition upregulate mRNA levels for Triggering Receptor Expressed On Myeloid Cells 2 (TREM2), which is reminiscent to the previously described disease-associated microglia. TREM2 is a risk gene for AD and an important regulator of microglia. The regulatory function of TREM2 in these cells was confirmed by comparing wild type with isogenic TREM2 knock-out iPSC microglia. The TREM2-deficient cells presented with stronger increase in free cytosolic calcium upon stimulation with ATP and ADP, as well as stronger migration towards complement C5a, compared to TREM2 expressing cells. The functional differences were associated with gene expression modulation of key regulators of microglia. In conclusion, we have established and validated a work stream to generate functional human iPSC-derived microglia-like cells by applying a directed and neuronal co-culture independent differentiation towards functional phenotypes in the context of AD. These cells can now be applied to study AD-related disease settings and to perform compound screening and testing for drug discovery., Competing Interests: During the course of this study, MR, IP, ND, CS, DR, RS, ME, JZ, CP, SG, and MB are or were full time employees or trainees at Roche and they may additionally hold Roche stock/stock options. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Reich, Paris, Ebeling, Dahm, Schweitzer, Reinhardt, Schmucki, Prasad, Köchl, Leist, Cowley, Zhang, Patsch, Gutbier and Britschgi.)

Trait-based analyses explaining the different responses of species and communities to environmental changes are increasing in frequency. European butterflies are an indicator group that responds rapidly to environmental changes with extensive citizen science contributions to documenting changes of abundance and distribution. Species traits have been used to explain long- and short-term responses to climate, land-use and vegetation changes. Studies are often characterised by limited trait sets being used, with risks that the relative roles of different traits are not fully explored. Butterfly trait information is dispersed amongst various sources and descriptions sometimes differ between sources. We have therefore drawn together multiple information sets to provide a comprehensive trait database covering 542 taxa and 25 traits described by 217 variables and sub-states of the butterflies of Europe and Maghreb (northwest Africa) which should serve for improved trait-based ecological, conservation-related, phylogeographic and evolutionary studies of this group of insects. We provide this data in two forms; the basic data and as processed continuous and multinomial data, to enhance its potential usage.

The biology of parasitoids in natural ecosystems remains very poorly studied, though they are key species for their functioning. Here we focused on Phobocampe confusa , a Nymphalini specialist, responsible for high mortality rates in charismatic butterfly species in Europe (genus Aglais ). We studied its ecology and genetic structure in connection with those of its host butterflies in Sweden. To this aim, we gathered data from 428 P. confusa individuals reared from 6094 butterfly larvae (of A. urticae , A. io , and in two occasions of Araschnia levana ) collected over two years (2017 and 2018) and across 19 sites distributed along a 500 km latitudinal gradient. We found that P. confusa is widely distributed along the latitudinal gradient. Its distribution seems constrained over time by the phenology of its hosts. The large variation in climatic conditions between sampling years explains the decrease in phenological overlap between P. confusa and its hosts in 2018 and the 33.5% decrease in the number of butterfly larvae infected. At least in this study, P. confusa seems to favour A. urticae as host. While it parasitized nests of A. urticae and A. io equally, the proportion of larvae parasitized is significantly higher for A. urticae . At the landscape scale, P. confusa is almost exclusively found in vegetated open land and near deciduous forests, whereas artificial habitats are negatively correlated with the likelihood of a nest to be parasitized. The genetic analyses on 89 adult P. confusa and 87 adult A. urticae using CO1 and AFLP markers reveal a low genetic diversity in P. confusa and a lack of genetic structure in both species, at the scale of our sampling. Further genetic studies using high-resolution genomics tools will be required to better understand the population genetic structure of P. confusa , its biotic interactions with its hosts, and ultimately the stability and the functioning of natural ecosystems.

The European Union's Natura 2000 (N2000) is among the largest international networks of protected areas. One of its aims is to secure the status of a predetermined set of (targeted) bird and butterfly species. However, nontarget species may also benefit from N2000. We evaluated how the terrestrial component of this network affects the abundance of nontargeted, more common bird and butterfly species based on data from long-term volunteer-based monitoring programs in 9602 sites for birds and 2001 sites for butterflies. In almost half of the 155 bird species assessed, and particularly among woodland specialists, abundance increased (slope estimates ranged from 0.101 [SD 0.042] to 3.51 [SD 1.30]) as the proportion of landscape covered by N2000 sites increased. This positive relationship existed for 27 of the 104 butterfly species (estimates ranged from 0.382 [SD 0.163] to 4.28 [SD 0.768]), although most butterflies were generalists. For most species, when land-cover covariates were accounted for these positive relationships were not evident, meaning land cover may be a determinant of positive effects of the N2000 network. The increase in abundance as N2000 coverage increased correlated with the specialization index for birds, but not for butterflies. Although the N2000 network supports high abundance of a large spectrum of species, the low number of specialist butterflies with a positive association with the N2000 network shows the need to improve the habitat quality of N2000 sites that could harbor open-land butterfly specialists. For a better understanding of the processes involved, we advocate for standardized collection of data at N2000 sites., (© 2019 Society for Conservation Biology.)

Endothelial cells (ECs) display remarkable plasticity during development before becoming quiescent and functionally mature. EC maturation is directed by several known transcription factors (TFs), but the specific set of TFs responsible for promoting high-resistance barriers, such as the blood-brain barrier (BBB), have not yet been fully defined. Using expression mRNA data from published studies on ex vivo ECs from the central nervous system (CNS), we predicted TFs that induce high-resistance barrier properties of ECs as in the BBB. We used our previously established method to  generate ECs from human pluripotent stem cells (hPSCs), and then we overexpressed the candidate TFs in hPSC-ECs and measured barrier resistance and integrity using electric cell-substrate impedance sensing, trans-endothelial electrical resistance and FITC-dextran permeability assays. SOX18 and TAL1 were the strongest EC barrier-inducing TFs, upregulating Wnt-related signaling and EC junctional gene expression, respectively, and downregulating EC proliferation-related genes. These TFs were combined with SOX7 and ETS1 that together effectively induced EC barrier resistance, decreased paracellular transport and increased protein expression of tight junctions and induce mRNA expression of several genes involved in the formation of EC barrier and transport. Our data shows identification of a transcriptional network that controls barrier resistance in ECs. Collectively this data may lead to novel approaches for generation of in vitro models of the BBB.

With the expansion in the quantity and types of biodiversity data being collected, there is a need to find ways to combine these different sources to provide cohesive summaries of species' potential and realized distributions in space and time. Recently, model-based data integration has emerged as a means to achieve this by combining datasets in ways that retain the strengths of each. We describe a flexible approach to data integration using point process models, which provide a convenient way to translate across ecological currencies. We highlight recent examples of large-scale ecological models based on data integration and outline the conceptual and technical challenges and opportunities that arise., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Eastern woodchuck ( Marmota monax ) has been extensively used in research of chronic hepatitis B and liver cancer because its infection with the woodchuck hepatitis virus closely resembles a human hepatitis B virus infection. Development of novel immunotherapeutic approaches requires genetic information on immune pathway genes in this animal model. The woodchuck genome was assembled with a combination of high-coverage whole-genome shotgun sequencing of Illumina paired-end, mate-pair libraries and fosmid pool sequencing. The result is a 2.63 Gigabase (Gb) assembly with a contig N50 of 74.5 kilobases (kb), scaffold N50 of 892 kb, and genome completeness of 99.2%. RNA sequencing (RNA-seq) from seven different tissues aided in the annotation of 30,873 protein-coding genes, which in turn encode 41,826 unique protein products. More than 90% of the genes have been functionally annotated, with 82% of them containing open reading frames. This genome sequence and its annotation will enable further research in chronic hepatitis B and hepatocellular carcinoma and contribute to the understanding of immunological responses in the woodchuck., (Copyright © 2019 Alioto et al.)

Identification of novel antibiotics remains a major challenge for drug discovery. The present study explores use of phenotypic readouts beyond classical antibacterial growth inhibition adopting a combined multiparametric high content screening and genomic approach. Deployment of the semi-automated bacterial phenotypic fingerprint (BPF) profiling platform in conjunction with a machine learning-powered dataset analysis, effectively allowed us to narrow down, compare and predict compound mode of action (MoA). The method identifies weak antibacterial hits allowing full exploitation of low potency hits frequently discovered by routine antibacterial screening. We demonstrate that BPF classification tool can be successfully used to guide chemical structure activity relationship optimization, enabling antibiotic development and that this approach can be fruitfully applied across species. The BPF classification tool could be potentially applied in primary screening, effectively enabling identification of novel antibacterial compound hits and differentiating their MoA, hence widening the known antibacterial chemical space of existing pharmaceutical compound libraries. More generally, beyond the specific objective of the present work, the proposed approach could be profitably applied to a broader range of diseases amenable to phenotypic drug discovery.

Safety and efficacy of therapeutic antibodies are often dependent on their interaction with Fc receptors for IgG (FcγRs). The Göttingen minipig represents a valuable species for biomedical research but its use in preclinical studies with therapeutic antibodies is hampered by the lack of knowledge about the porcine FcγRs. Genome analysis and sequencing now enabled the localization of the previously described FcγRIIIa in the orthologous location to human FCGR3A. In addition, we identified nearby the gene coding for the hitherto undescribed putative porcine FcγRIIa. The 1'241 bp long FCGR2A cDNA translates to a 274aa transmembrane protein containing an extracellular region with high similarity to human and cattle FcγRIIa. Like in cattle, the intracellular part does not contain an immunoreceptor tyrosine-based activation motif (ITAM) as in human FcγRIIa. Flow cytometry of the whole blood and single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs) of Göttingen minipigs revealed the expression profile of all porcine FcγRs which is compared to human and mouse. The new FcγRIIa is mainly expressed on platelets making the minipig a good model to study IgG-mediated platelet activation and aggregation. In contrast to humans, minipig blood monocytes were found to express inhibitory FcγRIIb that could lead to the underestimation of FcγR-mediated effects of monocytes observed in minipig studies with therapeutic antibodies.

Modification of SMN2 exon 7 (E7) splicing is a validated therapeutic strategy against spinal muscular atrophy (SMA). However, a target-based approach to identify small-molecule E7 splicing modifiers has not been attempted, which could reveal novel therapies with improved mechanistic insight. Here, we chose as a target the stem-loop RNA structure TSL2, which overlaps with the 5' splicing site of E7. A small-molecule TSL2-binding compound, homocarbonyltopsentin (PK4C9), was identified that increases E7 splicing to therapeutic levels and rescues downstream molecular alterations in SMA cells. High-resolution NMR combined with molecular modelling revealed that PK4C9 binds to pentaloop conformations of TSL2 and promotes a shift to triloop conformations that display enhanced E7 splicing. Collectively, our study validates TSL2 as a target for small-molecule drug discovery in SMA, identifies a novel mechanism of action for an E7 splicing modifier, and sets a precedent for other splicing-mediated diseases where RNA structure could be similarly targeted.

Background & Aims: The hallmarks of chronic HBV infection are a high viral load (HBV DNA) and even higher levels (>100-fold in excess of virions) of non-infectious membranous particles containing the tolerogenic viral S antigen (HBsAg). Currently, standard treatment effectively reduces viremia but only rarely results in a functional cure (defined as sustained HBsAg loss). There is an urgent need to identify novel therapies that reduce HBsAg levels and restore virus-specific immune responsiveness in patients. We report the discovery of a novel, potent and orally bioavailable small molecule inhibitor of HBV gene expression (RG7834)., Methods: RG7834 antiviral characteristics and selectivity against HBV were evaluated in HBV natural infection assays and in a urokinase-type plasminogen activator/severe combined immunodeficiency humanized mouse model of HBV infection, either alone or in combination with entecavir., Results: Unlike nucleos(t)ide therapies, which reduce viremia but do not lead to an effective reduction in HBV antigen expression, RG7834 significantly reduced the levels of viral proteins (including HBsAg), as well as lowering viremia. Consistent with its proposed mechanism of action, time course RNA-seq analysis revealed a fast and selective reduction in HBV mRNAs in response to RG7834 treatment. Furthermore, oral treatment of HBV-infected humanized mice with RG7834 led to a mean HBsAg reduction of 1.09 log 10 compared to entecavir, which had no significant effect on HBsAg levels. Combination of RG7834, entecavir and pegylated interferon α-2a led to significant reductions of both HBV DNA and HBsAg levels in humanized mice., Conclusion: We have identified a novel oral HBV viral gene expression inhibitor that blocks viral antigen and virion production, that is highly selective for HBV, and has a unique antiviral profile that is clearly differentiated from nucleos(t)ide analogues., Lay Summary: We discovered a novel small molecule viral expression inhibitor that is highly selective for HBV and unlike current therapy inhibits the expression of viral proteins by specifically reducing HBV mRNAs. RG7834 can therefore potentially provide anti-HBV benefits and increase HBV cure rates, by direct reduction of viral agents needed to complete the viral life cycle, as well as a reduction of viral agents involved in evasion of the host immune responses., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The importance of Cultural Ecosystem Services (CES) to human wellbeing is widely recognised. However, quantifying these non-material benefits is challenging and consequently they are often not assessed. Mapping approaches are increasingly being used to understand the spatial distribution of different CES and how this relates to landscape characteristics. This study uses an online Public Participation Geographic Information System (PPGIS) to elicit information on outdoor locations important to respondents in Wiltshire, a dynamic lowland landscape in southern England. We analysed these locations in a GIS with spatial datasets representing potential influential factors, including protected areas, land use, landform, and accessibility. We assess these characteristics at different spatial and visual scales for different types of cultural engagement. We find that areas that are accessible, near to urban centres, with larger views, and a high diversity of protected habitats, are important for the delivery of CES. Other characteristics including a larger area of woodland and the presence of sites of historic interest in the surrounding landscape were also influential. These findings have implications for land-use planning and the management of ecosystems, by demonstrating the benefits of high quality ecological sites near to towns. The importance of maintaining and restoring landscape features, such as woodlands, to enhance the delivery of CES were also highlighted., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss. The protein HtrA1 is enriched in retinal pigment epithelial (RPE) cells isolated from AMD patients and in drusen deposits. However, it is poorly understood how increased levels of HtrA1 affect the physiological function of the RPE at the intracellular level. Here, we developed hfRPE (human fetal retinal pigment epithelial) cell culture model where cells fully differentiated into a polarized functional monolayer. In this model, we fine-tuned the cellular levels of HtrA1 by targeted overexpression. Our data show that HtrA1 enzymatic activity leads to intracellular degradation of tubulin with a corresponding reduction in the number of microtubules, and consequently to an altered mechanical cell phenotype. HtrA1 overexpression further leads to impaired apical processes and decreased phagocytosis, an essential function for photoreceptor survival. These cellular alterations correlate with the AMD phenotype and thus highlight HtrA1 as an intracellular target for therapeutic interventions towards AMD treatment., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Small molecule splicing modifiers have been previously described that target the general splicing machinery and thus have low specificity for individual genes. Several potent molecules correcting the splicing deficit of the SMN2 (survival of motor neuron 2) gene have been identified and these molecules are moving towards a potential therapy for spinal muscular atrophy (SMA). Here by using a combination of RNA splicing, transcription, and protein chemistry techniques, we show that these molecules directly bind to two distinct sites of the SMN2 pre-mRNA, thereby stabilizing a yet unidentified ribonucleoprotein (RNP) complex that is critical to the specificity of these small molecules for SMN2 over other genes. In addition to the therapeutic potential of these molecules for treatment of SMA, our work has wide-ranging implications in understanding how small molecules can interact with specific quaternary RNA structures.

Background: In the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development., Results: Here we present a new chromosome level based version of the Göttingen minipig genome together with a comparative transcriptional analysis of tissues with pharmaceutical relevance as basis for translational research. We relied on mapping and assembly of WGS (whole-genome-shotgun sequencing) derived reads to the reference genome of the Duroc pig and predict 19,228 human orthologous protein-coding genes. Genome-based prediction of the sequence of human drug targets enables the prediction of drug cross-reactivity based on conservation of binding sites. We further support the finding that the genome of Sus scrofa contains about ten-times less pseudogenized genes compared to other vertebrates. Among the functional human orthologs of these minipig pseudogenes we found HEPN1, a putative tumor suppressor gene. The genomes of Sus scrofa, the Tibetan boar, the African Bushpig, and the Warthog show sequence conservation of all inactivating HEPN1 mutations suggesting disruption before the evolutionary split of these pig species. We identify 133 Sus scrofa specific, conserved long non-coding RNAs (lncRNAs) in the minipig genome and show that these transcripts are highly conserved in the African pigs and the Tibetan boar suggesting functional significance. Using a new minipig specific microarray we show high conservation of gene expression signatures in 13 tissues with biomedical relevance between humans and adult minipigs. We underline this relationship for minipig and human liver where we could demonstrate similar expression levels for most phase I drug-metabolizing enzymes. Higher expression levels and metabolic activities were found for FMO1, AKR/CRs and for phase II drug metabolizing enzymes in minipig as compared to human. The variability of gene expression in equivalent human and minipig tissues is considerably higher in minipig organs, which is important for study design in case a human target belongs to this variable category in the minipig. The first analysis of gene expression in multiple tissues during development from young to adult shows that the majority of transcriptional programs are concluded four weeks after birth. This finding is in line with the advanced state of human postnatal organ development at comparative age categories and further supports the minipig as model for pediatric drug safety studies., Conclusions: Genome based assessment of sequence conservation combined with gene expression data in several tissues improves the translational value of the minipig for human drug development. The genome and gene expression data presented here are important resources for researchers using the minipig as model for biomedical research or commercial breeding. Potential impact of our data for comparative genomics, translational research, and experimental medicine are discussed.

The blood-brain barrier and the blood-cerebrospinal fluid barrier prevent access of biotherapeutics to their targets in the central nervous system and therefore prohibit the effective treatment of neurological disorders. In an attempt to discover novel brain transport vectors in vivo, we injected a T7 phage peptide library and continuously collected blood and cerebrospinal fluid (CSF) using a cisterna magna cannulated conscious rat model. Specific phage clones were highly enriched in the CSF after four rounds of selection. Validation of individual peptide candidates showed CSF enrichments of greater than 1000-fold. The biological activity of peptide-mediated delivery to the brain was confirmed using a BACE1 peptide inhibitor linked to an identified novel transport peptide which led to a 40% reduction of Amyloid-β in CSF. These results indicate that the peptides identified by the in vivo phage selection approach could be useful transporters for systemically administrated large molecules into the brain with therapeutic benefits.

Recombinant interferon-alpha (IFN-α) is an approved therapy for chronic hepatitis B (CHB), but the molecular basis of treatment response remains to be determined. The woodchuck model of chronic hepatitis B virus (HBV) infection displays many characteristics of human disease and has been extensively used to evaluate antiviral therapeutics. In this study, woodchucks with chronic woodchuck hepatitis virus (WHV) infection were treated with recombinant woodchuck IFN-α (wIFN-α) or placebo (n = 12/group) for 15 weeks. Treatment with wIFN-α strongly reduced viral markers in the serum and liver in a subset of animals, with viral rebound typically being observed following cessation of treatment. To define the intrahepatic cellular and molecular characteristics of the antiviral response to wIFN-α, we characterized the transcriptional profiles of liver biopsies taken from animals (n = 8-12/group) at various times during the study. Unexpectedly, this revealed that the antiviral response to treatment did not correlate with intrahepatic induction of the majority of IFN-stimulated genes (ISGs) by wIFN-α. Instead, treatment response was associated with the induction of an NK/T cell signature in the liver, as well as an intrahepatic IFN-γ transcriptional response and elevation of liver injury biomarkers. Collectively, these data suggest that NK/T cell cytolytic and non-cytolytic mechanisms mediate the antiviral response to wIFN-α treatment. In summary, by studying recombinant IFN-α in a fully immunocompetent animal model of CHB, we determined that the immunomodulatory effects, but not the direct antiviral activity, of this pleiotropic cytokine are most closely correlated with treatment response. This has important implications for the rational design of new therapeutics for the treatment of CHB.

Circulating tumor DNA (ctDNA) is a new circulating tumor biomarker which might be used as a prognostic biomarker in a way similar to circulating tumor cells (CTCs). Here, we used the high prevalence of TP53 mutations in triple negative breast cancer (TNBC) to compare ctDNA and CTC detection rates and prognostic value in metastatic TNBC patients. Forty patients were enrolled before starting a new line of treatment. TP53 mutations were characterized in archived tumor tissues and in plasma DNA using two next generation sequencing (NGS) platforms in parallel. Archived tumor tissue was sequenced successfully for 31/40 patients. TP53 mutations were found in 26/31 (84%) of tumor samples. The same mutation was detected in the matched plasma of 21/26 (81%) patients with an additional mutation found only in the plasma for one patient. Mutated allele fractions ranged from 2 to 70% (median 5%). The observed correlation between the two NGS approaches (R(2) = 0.903) suggested that ctDNA levels data were quantitative. Among the 27 patients with TP53 mutations, CTC count was ≥1 in 19 patients (70%) and ≥5 in 14 patients (52%). ctDNA levels had no prognostic impact on time to progression (TTP) or overall survival (OS), whereas CTC numbers were correlated with OS (p = 0.04) and marginally with TTP (p = 0.06). Performance status and elevated LDH also had significant prognostic impact. Here, absence of prognostic impact of baseline ctDNA level suggests that mechanisms of ctDNA release in metastatic TNBC may involve, beyond tumor burden, biological features that do not dramatically affect patient outcome., (© 2014 UICC.)

Spinal muscular atrophy (SMA) is a genetic disease caused by mutation or deletion of the survival of motor neuron 1 (SMN1) gene. A paralogous gene in humans, SMN2, produces low, insufficient levels of functional SMN protein due to alternative splicing that truncates the transcript. The decreased levels of SMN protein lead to progressive neuromuscular degeneration and high rates of mortality. Through chemical screening and optimization, we identified orally available small molecules that shift the balance of SMN2 splicing toward the production of full-length SMN2 messenger RNA with high selectivity. Administration of these compounds to Δ7 mice, a model of severe SMA, led to an increase in SMN protein levels, improvement of motor function, and protection of the neuromuscular circuit. These compounds also extended the life span of the mice. Selective SMN2 splicing modifiers may have therapeutic potential for patients with SMA., (Copyright © 2014, American Association for the Advancement of Science.)

Peak overlap is one of the major factors complicating the analysis of biomolecular NMR spectra. We present a general method for predicting the extent of peak overlap in multidimensional NMR spectra and its validation using both, experimental data sets and Monte Carlo simulation. The method is based on knowledge of the magnetization transfer pathways of the NMR experiments and chemical shift statistics from the Biological Magnetic Resonance Data Bank. Assuming a normal distribution with characteristic mean value and standard deviation for the chemical shift of each observable atom, an analytic expression was derived for the expected overlap probability of the cross peaks. The analytical approach was verified to agree with the average peak overlap in a large number of individual peak lists simulated using the same chemical shift statistics. The method was applied to eight proteins, including an intrinsically disordered one, for which the prediction results could be compared with the actual overlap based on the experimentally measured chemical shifts. The extent of overlap predicted using only statistical chemical shift information was in good agreement with the overlap that was observed when the measured shifts were used in the virtual spectrum, except for the intrinsically disordered protein. Since the spectral complexity of a protein NMR spectrum is a crucial factor for protein structure determination, analytical overlap prediction can be used to identify potentially difficult proteins before conducting NMR experiments. Overlap predictions can be tailored to particular classes of proteins by preparing statistics from corresponding protein databases. The method is also suitable for optimizing recording parameters and labeling schemes for NMR experiments and improving the reliability of automated spectra analysis and protein structure determination.

Background: Whole transcriptome analyses are an essential tool for understanding disease mechanisms. Approaches based on next-generation sequencing provide fast and affordable data but rely on the availability of annotated genomes. However, there are many areas in biomedical research that require non-standard animal models for which genome information is not available. This includes the Syrian hamster Mesocricetus auratus as an important model for dyslipidaemia because it mirrors many aspects of human disease and pharmacological responses. We show that complementary use of two independent next generation sequencing technologies combined with mapping to multiple genome databases allows unambiguous transcript annotation and quantitative transcript imaging. We refer to this approach as "triple match sequencing" (TMS)., Results: Contigs assembled from a normalized Roche 454 hamster liver library comprising 1.2 million long reads were used to identify 10'800 unique transcripts based on homology to RefSeq database entries from human, mouse, and rat. For mRNA quantification we mapped 82 million SAGE tags (SOLiD) from the same RNA source to the annotated hamster liver transcriptome contigs. We compared the liver transcriptome of hamster with equivalent data from human, rat, minipig, and cynomolgus monkeys to highlight differential gene expression with focus on lipid metabolism. We identify a cluster of five genes functionally related to HDL metabolism that is expressed in human, cynomolgus, minipig, and hamster but lacking in rat as a non-responder species for lipid lowering drugs., Conclusions: The TMS approach is suited for fast and inexpensive transcript profiling in cells or tissues of species where a fully annotated genome is not available. The continuously growing number of well annotated reference genomes will further empower reliable transcript identification and thereby raise the utility of the method for any species of interest.

The long-tailed macaque, also referred to as cynomolgus monkey (Macaca fascicularis), is one of the most important nonhuman primate animal models in basic and applied biomedical research. To improve the predictive power of primate experiments for humans, we determined the genome sequence of a Macaca fascicularis female of Mauritian origin using a whole-genome shotgun sequencing approach. We applied a template switch strategy that uses either the rhesus or the human genome to assemble sequence reads. The sixfold sequence coverage of the draft genome sequence enabled discovery of about 2.1 million potential single-nucleotide polymorphisms based on occurrence of a dimorphic nucleotide at a given position in the genome sequence. Homology-based annotation allowed us to identify 17,387 orthologs of human protein-coding genes in the M. fascicularis draft genome, and the predicted transcripts enabled the design of a M. fascicularis-specific gene expression microarray. Using liver samples from 36 individuals of different geographic origin we identified 718 genes with highly variable expression in liver, whereas the majority of the transcriptome shows relatively stable and comparable expression. Knowledge of the M. fascicularis draft genome is an important contribution to both the use of this animal in disease models and the safety assessment of drugs and their metabolites. In particular, this information allows high-resolution genotyping and microarray-based gene-expression profiling for animal stratification, thereby allowing the use of well-characterized animals for safety testing. Finally, the genome sequence presented here is a significant contribution to the global "3R" animal welfare initiative, which has the goal to reduce, refine, and replace animal experiments.

Polytheonamide B (pTB), a highly cytotoxic polypeptide, is one of the most unusual nonribosomal peptides of sponge origin. pTB is a linear 48-residue peptide with alternating D- and L-amino acids and contains a total of eight types of nonproteinogenic amino acids. To investigate the mechanisms underlying its cytotoxic activity, we determined the three-dimensional structure of pTB by NMR spectroscopy, structure calculation, and energy minimization. pTB adopts a single right-handed β(6.3)-helical structure in a 1:1 mixture of methanol/chloroform with a length of approximately 45 A and a hydrophilic pore of ca. 4 A inner diameter. These features indicate that pTB molecules form transmembrane channels that permeate monovalent cations as gramicidin A channels do. The strong cytotoxicity of pTB can be ascribed to its ability to form single molecule channels through biological membranes.

Habitat-corridors are assumed to counteract the negative impacts of habitat loss and fragmentation, but their efficiency in doing so depends on the maintenance of ecological processes in corridor conditions. For plants dispersing in linear habitats, one of these critical processes is the maintenance of adequate pollen transfer to insure seed production within the corridor. This study focuses on a common, self-incompatible forest herb, Trillium grandiflorum, to assess plant-pollinator interactions and the influence of spatial processes on plant reproduction in hedgerow corridors compared to forests. First, using pollen supplementation experiments over 2 years, we quantified the extent of pollen limitation in both habitats, testing the prediction of greater limitation in small hedgerow populations than in forests. While pollen limitation of fruit and seed set was common, its magnitude did not differ between habitats. Variations among sites, however, suggested an influence of landscape context on pollination services. Second, we examined the effect of isolation on plant reproduction by monitoring fruit and seed production, as well as pollinator activity and assemblage, in small flower arrays transplanted in hedgerows at increasing distances from forest and from each other. We detected no difference in the proportion of flowers setting fruit or in pollinator activity with isolation, but we observed some differences in pollinator assemblages. Seed set, on the other hand, declined significantly with increasing isolation in the second year of the study, but not in the first year, suggesting altered pollen transfer with distance. Overall, plants in hedgerow corridors and forests benefited from similar pollination services. In this system, plant-pollinator interactions and reproduction seem to be influenced more by variations in resource distribution over years and landscapes than by local habitat conditions.

Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent metal ions such as Zn(II), Cu(I), and Cd(II) that are involved in metal ion homeostasis and detoxification, as well as protection against reactive oxygen species. Here we show the NMR solution structure of the beta(E)-domain of the early cysteine-labeled protein (E(c)-1) from wheat (beta(E)-E(c)-1), which represents the first three-dimensional structure of a plant MT. The beta(E)-domain comprises the 51 C-terminal residues of E(c)-1 and exhibits a distinctive unprecedented structure with two separate metal-binding centers, a mononuclear Zn(II) binding site constituted by two cysteine and two highly conserved histidine residues as found in certain zinc-finger motifs, and a cluster formed by three Zn(II) ions coordinated by nine Cys residues that resembles the cluster in the beta-domain of vertebrate MTs. Cys-metal ion connectivities were determined by exhaustive structure calculations for all 7560 possible configurations of the three-metal cluster. Backbone dynamics investigated by (15)N relaxation experiments support the results of the structure determination in that beta(E)-E(c)-1 is a rigidly folded polypeptide. To further investigate the influence of metal ion binding on the stability of the structure, we replaced Zn(II) with Cd(II) ions and examined the effects of metal ion release on incubation with a metal ion chelator.

A new algorithm, DYNASSIGN, for the automated assignment of NMR chemical shift resonances was developed in which expected cross peaks in multidimensional NMR spectra are represented by peak-particles and assignment restraints are translated into a potential energy function. Molecular dynamics simulation techniques are used to calculate a trajectory of the system of peak-particles subjected to the potential function in order to find energetically optimal configurations that correspond to correct assignments. Peak-particle dynamics-based simulated annealing was combined with the Hungarian algorithm for local optimization, and a residue-based score was introduced to distinguish between reliable assignments and "unassigned" resonances for which no reliable assignment can be established. The DYNASSIGN algorithm was implemented in the program CYANA and tested with data sets obtained from the experimental NMR data of nine small proteins. With a set of 10 commonly used NMR spectra, on average 82.5% of all backbone and side-chain (1)H, (13)C and (15)N resonances could be assigned with an average error rate of 3.5%.

Noncrop areas such as hedgerows in agricultural landscapes can perform several ecological and agronomic functions (e.g., habitat, movement corridors, windbreak, etc.), but their dynamics and drivers of changes are often poorly known. We conducted a study in three agricultural landscapes of southern Quebec, Canada, to assess and compare the spatial and temporal (1958-1997) dynamics of three hedgerow networks in relation to geomorphic conditions (marine, glacial, and mixed deposit) and land-use changes. Hedgerow networks were mapped and described in terms of their structure (density, degree of connectivity, and presence of trees or shrubs) and their relationship to other components of the landscape (connection to woodland). Relationships were assessed in time and space using nonparametric correlation, Mantel test, and principal components analysis (PCA). Results show significant differences between hedgerow structure for the three landscapes and distinct temporal and spatial dynamics that can be related to changes in management practices and agricultural policies. On marine deposits, increases in hedgerow density did not always correspond to an increase in their degree of connectivity, suggesting a possible reduction in network quality. On glacial deposits, hedgerow density declined following abandonment of agricultural land, but rather than disappearing, these linear structures were integrated into adjacent brush or forested areas. Our analysis reveals the complex spatial and temporal dynamics of the hedgerow networks and highlights the need to take into account spatial attributes such as connectivity and connection to woodland to evaluate more accurately overall network quality.