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Abstract Innate antiviral factors are essential for effective defense against viral pathogens. However, the identity of major restriction mechanisms remains elusive. Current approaches to discover antiviral factors usually focus on the initial steps of viral replication and are limited to a single round of infection. Here, we engineered libraries of >1500 replication-competent HIV-1 constructs each expressing a single gRNAs to target >500 cellular genes for virus-driven discovery of antiviral factors. Passaging in CD4+ T cells robustly enriched HIV-1 encoding sgRNAs against GRN, CIITA, EHMT2, CEACAM3, CC2D1B and RHOA by >50-fold. Using an HIV-1 library lacking the accessory nef gene, we identified IFI16 as a Nef target. Functional analyses in cell lines and primary CD4+ T cells support that the HIV-driven CRISPR screen identified restriction factors targeting virus entry, transcription, release and infectivity. Our HIV-guided CRISPR technique enables sensitive discovery of physiologically relevant cellular defense factors throughout the entire viral replication cycle.

Abstract Under-reporting of COVID-19 and the limited information about circulating SARS-CoV-2 variants remain major challenges for many African countries. We analyzed SARS-CoV-2 infection dynamics in Addis Ababa and Jimma, Ethiopia, focusing on reinfection, immunity, and vaccination effects. We conducted an antibody serology study spanning August 2020 to July 2022 with five rounds of data collection across a population of 4723, sequenced PCR-test positive samples, used available test positivity rates, and constructed two mathematical models integrating this data. A multivariant model explores variant dynamics identifying wildtype, alpha, delta, and omicron BA.4/5 as key variants in the study population, and cross-immunity between variants, revealing risk reductions between 24% and 69%. An antibody-level model predicts slow decay leading to sustained high antibody levels. Retrospectively, increased early vaccination might have substantially reduced infections during the delta and omicron waves in the considered group of individuals, though further vaccination now seems less impactful.

Sponges (Phylum Porifera) are aquatic sessile metazoans found worldwide in marine and freshwater environments. They are significant in the animal tree of life as one of the earliest-branching metazoan lineages and as filter feeders play crucial ecological roles, particularly in coral reefs, but are susceptible to the effects of climate change. In the face of the current biodiversity crisis, genomic data is crucial for species conservation efforts and predicting their evolutionary potential in response to environmental changes. However, there is a limited availability of culturable sponge species with annotated high-quality genomes to further comprehensive insights into animal evolution, function, and their response to the ongoing global change. Despite the publication of a few high-quality annotated sponge genomes, there remains a gap in resources for culturable sponge species. To address this gap, we provide high quality draft genomes of the two congeneric aquarium species Tethya wilhelma and Tethya minuta, small ball-shaped demosponges that are easily maintained long-term in ex situ culture. As such, they offer promising opportunities as laboratory models to contribute to advancing our understanding of sponge biology and provide valuable resources for studying animal evolution, function, and responses to environmental challenges.

Sponges (Phylum Porifera) are aquatic sessile metazoans found worldwide in marine and freshwater environments. They are significant in the animal tree of life as one of the earliest-branching metazoan lineages and as filter feeders play crucial ecological roles, particularly in coral reefs, but are susceptible to the effects of climate change. In the face of the current biodiversity crisis, genomic data is crucial for species conservation efforts and predicting their evolutionary potential in response to environmental changes. However, there is a limited availability of culturable sponge species with annotated high-quality genomes to further comprehensive insights into animal evolution, function, and their response to the ongoing global change. Despite the publication of a few high-quality annotated sponge genomes, there remains a gap in resources for culturable sponge species. To address this gap, we provide high quality draft genomes of the two congeneric aquarium species Tethya wilhelma and Tethya minuta, small ball-shaped demosponges that are easily maintained long-term in ex situ culture. As such, they offer promising opportunities as laboratory models to contribute to advancing our understanding of sponge biology and provide valuable resources for studying animal evolution, function, and responses to environmental challenges.

Sponges (Phylum Porifera) are aquatic sessile metazoans found worldwide in marine and freshwater environments. They are significant in the animal tree of life as one of the earliest-branching metazoan lineages and as filter feeders play crucial ecological roles, particularly in coral reefs, but are susceptible to the effects of climate change. In the face of the current biodiversity crisis, genomic data is crucial for species conservation efforts and predicting their evolutionary potential in response to environmental changes. However, there is a limited availability of culturable sponge species with annotated high-quality genomes to further comprehensive insights into animal evolution, function, and their response to the ongoing global change. Despite the publication of a few high-quality annotated sponge genomes, there remains a gap in resources for culturable sponge species. To address this gap, we provide high quality draft genomes of the two congeneric aquarium species Tethya wilhelma and Tethya minuta, small ball-shaped demosponges that are easily maintained long-term in ex situ culture. As such, they offer promising opportunities as laboratory models to contribute to advancing our understanding of sponge biology and provide valuable resources for studying animal evolution, function, and responses to environmental challenges.

ABSTRACT: Although the quantitative trait locus (QTL) on chromosome 18 (BTA18) associated with paternal calving ease and stillbirth in Holstein Friesian cattle and its cross has been known for over 20 years, to our knowledge, the exact causal genetic sequence has yet escaped identification. The aim of this study was to re-examine the region of the published QTL on BTA18 and to investigate the possible reasons behind this elusiveness. For this purpose, we carried out a combined linkage disequilibrium and linkage analysis using genotyping data of 2,697 German Holstein Friesian (HF) animals and subsequent whole-genome sequencing (WGS) data analyses and genome assembly of HF samples. We confirmed the known QTL in the 95% confidence interval of 1.089 Mbp between 58.34 and 59.43 Mbp on BTA18. Additionally, these 4 SNPs in the near-perfect linkage disequilibrium with the QTL haplotype were identified: rs381577268 (on 57,816,137 bp, C/T), rs381878735 (on 59,574,329 bp, A/T), rs464221818 (on 59,329,176 bp, C/T), and rs472502785 (on 59,345,689 bp, T/C). Search for the causal mutation using short and long-read sequences, and methylation data of the BTA18 QTL region did not reveal any candidates though. The assembly showed problems in the region, as well as an abundance of segmental duplications within and around the region. Taking the QTL of BTA18 in Holstein cattle as an example, the data presented in this study comprehensively characterize the genomic features that could also be relevant for other such elusive QTL in various other cattle breeds and livestock species as well.

Activating colony-stimulating factor-3 receptor gene (CSF3R) mutations are recurrent in acute myeloid leukemia (AML) with t(8;21) translocation. However, the nature of oncogenic collaboration between alterations of CSF3R and the t(8;21) associated RUNX1-RUNX1T1 fusion remains unclear. In CD34+ hematopoietic stem and progenitor cells from healthy donors, double oncogene expression led to a clonal advantage, increased self-renewal potential, and blast-like morphology and distinct immunophenotype. Gene expression profiling revealed hedgehog signaling as a potential mechanism, with upregulation of GLI2 constituting a putative pharmacological target. Both primary hematopoietic cells and the t(8;21) positive AML cell line SKNO-1 showed increased sensitivity to the GLI inhibitor GANT61 when expressing CSF3R T618I. Our findings suggest that during leukemogenesis, the RUNX1-RUNXT1 fusion and CSF3R mutation act in a synergistic manner to alter hedgehog signaling, which can be exploited therapeutically.

Abnormalities are indispensable for studying normal biological processes and mechanisms. In the present work, we draw attention to the remarkable phenomenon of a perpetually and robustly upregulated gene, the thyroglobulin gene (Tg). The gene is expressed in the thyroid gland and, as it has been recently demonstrated, forms so-called transcription loops, easily observable by light microscopy. Using this feature, we show that Tg is expressed at a high level from the moment a thyroid cell acquires its identity and both alleles remain highly active over the entire life of the cell, i.e., for months or years depending on the species. We demonstrate that this high upregulation is characteristic of thyroglobulin genes in all major vertebrate groups. We provide evidence that Tg is not influenced by the thyroid hormone status, does not oscillate round the clock and is expressed during both the exocrine and endocrine phases of thyrocyte activity. We conclude that the thyroglobulin gene represents a unique and valuable model to study the maintenance of a high transcriptional upregulation.

Abstract Understanding the sequence-dependent DNA damage formation requires probing a complete pool of sequences over a wide dose range of the damage-causing exposure. We used high throughput sequencing to simultaneously obtain the dose dependence and quantum yields for oligonucleotide damages for all possible 4096 DNA sequences with hexamer length. We exposed the DNA to ultraviolet radiation at 266 nm and doses of up to 500 absorbed photons per base. At the dimer level, our results confirm existing literature values of photodamage, whereas we now quantified the susceptibility of sequence motifs to UV irradiation up to previously inaccessible polymer lengths. This revealed the protective effect of the sequence context in preventing the formation of UV-lesions. For example, the rate to form dipyrimidine lesions is strongly reduced by nearby guanine bases. Our results provide a complete picture of the sensitivity of oligonucleotides to UV irradiation and allow us to predict their abundance in high-UV environments.

Abstract Background The efficacy of recanalization treatment in patients with ischemic stroke due to large vessel occlusion (LVO) is highly time dependent. We aimed to investigate the effects of an optimization of prehospital and intrahospital pathways on time metrics and efficacy of endovascular treatment in ischemic stroke due to LVO. Methods Patients treated with mechanical thrombectomy (MT) at the Hospital of St. John of God Vienna, Austria, between 2013 and 2020 were extracted from the Austrian Stroke Unit Registry. Study endpoints including time metrics, early neurological improvement and functional outcome measured by modified Rankin Scale (mRS) at 3 months were compared before and after optimization of prehospital and intrahospital pathways. Results Two hundred ninety-nine patients were treated with MT during the study period, 94 before and 205 after the workflow optimization. Workflow optimization was significantly associated with time metrics improvement (door to groin puncture time 45 versus 31 min; p

Schlafen 11 serves as an antiviral restriction factor and a predictive biomarker in cancer. Here, the authors use cryoelectron microscopy and biochemical assays to understand tRNA endoribonuclease and DNA binding functions of human Schlafen 11.

Background and Purpose It is unclear whether a particular stroke imaging modality offers an advantage for the acute stroke treatment. The aim of this study was to compare procedure times, efficacy and safety of thrombolysis and/or thrombectomy based on computed tomography (CT) versus magnetic resonance imaging (MRI) acute stroke imaging. Methods Data of stroke patients who received intravenous thrombolysis (IVT) and/or mechanical thrombectomy (MT) were extracted from a nationwide, prospective stroke unit registry and categorized according to initial imaging modality. Study endpoints included procedure times, symptomatic intracerebral hemorrhage (sICH), early neurological improvement, 3-month functional outcome by modified Rankin Scale (mRS) and mortality. Results Stroke patients (n=16,799) treated with IVT and 2,248 treated with MT were included. MRI-guided patients (n=2,599) were younger, had less comorbidities and higher rates of strokes with unknown onset as compared to CT-guided patients. In patients treated with IVT, no differences were observed regarding the rates of functional outcome by mRS 0–1 (adjusted odds ratio [OR], 0.87; 95% confidence interval [CI], 0.71 to 1.05), sICH (adjusted OR, 0.82; 95% CI, 0.61 to 1.08), and mortality (adjusted OR, 0.88; 95% CI, 0.63 to 1.22). Patients undergoing MT selected by MRI as compared to CT showed equal rates of functional outcome by mRS 0–2 (adjusted OR, 0.87; 95% CI, 0.65 to 1.16), sICH (adjusted OR, 0.9; 95% CI, 0.51 to 1.69), and mortality (adjusted OR, 0.62; 95% CI, 0.35 to 1.09). MRI-guided patients showed a significant intrahospital delay of about 20 minutes in both the IVT and the MT group. Conclusions This large non-randomized comparison study indicates that CT- and MRI-guided patient selection for IVT/MT may perform equally well in terms of functional outcome and safety.

Background and Purpose Studies on mechanical thrombectomy (MT) in acute ischemic stroke (AIS) patients with preexisting disability are limited. We aimed to compare the outcomes of MT versus best medical treatment (BMT) in these patients. Methods In the nationwide Austrian registry and Swiss monocentric registry, we identified 462 AIS patients with pre-stroke disability (modified Rankin Scale [mRS] score ≥3) and acute large vessel occlusion. The primary outcome was returning to pre-stroke mRS or better at 3 months. Secondary outcomes were early neurological improvement (National Institutes of Health Stroke Scale score improvement ≥8 at 24 to 48 hours), 3-month mortality, and symptomatic intracerebral hemorrhage (sICH). Multivariable regression models and propensity score matching (PSM) were used for statistical analyses. Results Compared with the BMT group (n=175), the MT group (n=175) had younger age, more severe strokes, and lower pre-stroke mRS, but similar proportion of receiving intravenous thrombolysis. MT was associated with higher odds of returning to baseline mRS or better at 3 months (adjusted odds ratio [aOR], 2.5; 95% confidence interval [CI], 1.39 to 4.47), early neurological improvement (aOR, 2.62; 95% CI, 1.41 to 4.88), and lower risk of 3-month mortality (aOR, 0.29; 95% CI, 0.18 to 0.49). PSM analysis showed similar findings. MT was not associated with an increased risk of sICH (4.0% vs. 2.1% in all patients; 4.2% vs. 2.4% in the PSM cohort). Conclusions MT in patients with pre-stroke mRS ≥3 might improve the 3-month outcomes and short-term neurological impairment, suggesting that pre-stroke disability alone should not be a reason to withhold MT, but that individual case-by-case decisions may be more appropriate.

SARS-CoV-2 mutations associated with the escape from antibody-mediated neutralization have been widely reported. Here, in a patient with defective antibody responses, the authors find a potential association between SARS-CoV-2 mutations and CD8 T alterations to implicate possible contributions of CD8 T cells in evasion of SARS-CoV-2 from host immunity.

Using SNP-association analysis and genetic mapping, a SNP and an insertion in and close to HOXB13 associated with short tail length is identified in Merino sheep, which may be a target for safely selecting shorter tails and improving sheep welfare.

Well-annotated and contiguous genomes are an indispensable resource for understanding the evolution, development, and metabolic capacities of organisms. Sponges, an ecologically important non-bilaterian group of primarily filter-feeding sessile aquatic organisms, are underrepresented with respect to available genomic resources. Here we provide a high-quality and well-annotated genome of Aphrocallistes vastus, a glass sponge (Porifera: Hexactinellida) that forms large reef structures off the coast of British Columbia (Canada). We show that its genome is approximately 80 Mb, small compared to most other metazoans, and contains nearly 2500 nested genes, more than other genomes. Hexactinellida is characterized by a unique skeletal architecture made of amorphous silicon dioxide (SiO2), and we identified 419 differentially expressed genes between the osculum, i.e. the vertical growth zone of the sponge, and the main body. Among the upregulated ones, mineralization-related genes such as glassin, as well as collagens and actins, dominate the expression profile during growth. Silicateins, suggested being involved in silica mineralization, especially in demosponges, were not found at all in the A. vastus genome and suggests that the underlying mechanisms of SiO2 deposition in the Silicea sensu stricto (Hexactinellida + Demospongiae) may not be homologous.

Abstract Background Patients with ischaemic stroke or transient ischaemic attack (TIA) are at high risk of incident cardiovascular events and recurrent stroke. Despite compelling evidence about the efficacy of secondary prevention, a substantial gap exists between risk factor management in real life and that recommended by international guidelines. We conducted the STROKE-CARD trial (NCT02156778), a multifaceted pragmatic disease management program between 2014 and 2018 with follow-up until 2019. This program successfully reduced cardiovascular risk and improved health-related quality of life and functional outcome in patients with acute ischaemic stroke or TIA within 12 months after the index event. To investigate potential long-term effects of STROKE-CARD care compared to standard care, an extension of follow-up is warranted. Methods We aim to include all patients from the STROKE-CARD trial (n = 2149) for long-term follow-up between 2019 and 2021 with the study visit scheduled 3–6 years after the stroke/TIA event. The co-primary endpoint is the composite of major recurrent cardiovascular events (nonfatal stroke, nonfatal myocardial infarction, and vascular death) from hospital discharge until the long-term follow-up visit and health-related quality of life measured with the European Quality of Life-5 Dimensions (EQ-5D-3L) at the final visit. Secondary endpoints include overall mortality, long-term functional outcome, and target-level achievement in risk factor management. Discussion This long-term follow-up will provide evidence on whether the pragmatic post-stroke/TIA intervention program STROKE-CARD is capable of preventing recurrent cardiovascular events and improving quality-of-life in the long run. Trial registration clinicaltrials.gov: NCT04205006 on 19 December 2019.

Abstract The contribution of domestic cattle in human societies is enormous, making cattle, along with other essential benefits, the economically most important domestic animal in the world today. To expand existing knowledge on cattle domestication and mitogenome diversity, we performed a comprehensive complete mitogenome analysis of the species (802 sequences, 114 breeds). A large sample was collected in South‐east Europe, an important agricultural gateway to Europe during Neolithization and a region rich in cattle biodiversity. We found 1725 polymorphic sites (810 singletons, 853 parsimony‐informative sites and 57 indels), 701 unique haplotypes, a haplotype diversity of 0.9995 and a nucleotide diversity of 0.0015. In addition to the dominant T3 and several rare haplogroups (Q, T5, T4, T2 and T1), we have identified maternal line in Austrian Murbodner cattle that possess surviving aurochs’ mitochondria haplotype P1 that diverged prior to the Neolithization process. This is convincing evidence for rare female‐mediated adaptive introgression of wild aurochs into domesticated cattle in Europe. We revalidated the existing haplogroup classification and provided Bayesian phylogenetic inference with a more precise estimated divergence time than previously available. Occasionally, classification based on partial mitogenomes was not reliable; for example, some individuals with haplogroups P and T5 were not recognized based on D‐loop information. Bayesian skyline plot estimates (median) show that the earliest population growth began before domestication in cattle with haplogroup T2, followed by Q (~10.0–9.5 kyBP), whereas cattle with T3 (~7.5 kyBP) and T1 (~3.0–2.5 kyBP) expanded later. Overall, our results support the existence of interactions between aurochs and cattle during domestication and dispersal of cattle in the past, contribute to the conservation of maternal cattle diversity and enable functional analyses of the surviving aurochs P1 mitogenome.

Species of Alternaria (phylum Ascomycota, family Pleosporaceae) are known as serious plant pathogens, causing major losses on a wide range of crops. Alternaria atra (previously known as Ulocladium atrum) can grow as a saprophyte on many hosts and causes Ulocladium blight on potato. It has been reported that it can also be used as a biocontrol agent against Botrytis cinerea. Here, we present a scaffold-level reference genome assembly for A. atra. The assembly contains 43 scaffolds with a total length of 39.62 Mbp, with scaffold N50 of 3,893,166 bp, L50 of 4, and the longest 10 scaffolds containing 89.9% of the assembled data. RNA-sequencing-guided gene prediction using BRAKER resulted in 12,173 protein-coding genes with their functional annotation. This first high-quality reference genome assembly and annotation for A. atra can be used as a resource for studying evolution in the highly complicated Alternaria genus and might help in understanding the mechanisms defining its role as pathogen or biocontrol agent.[Graphic: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Abstract Background Borrelia bavariensis is one of the agents of Lyme Borreliosis (or Lyme disease) in Eurasia. The genome of the Borrelia burgdorferi sensu lato species complex, that includes B. bavariensis, is known to be very complex and fragmented making the assembly of whole genomes with next-generation sequencing data a challenge. Results We present a genome reconstruction for 33 B. bavariensis isolates from Eurasia based on long-read (Pacific Bioscience, for three isolates) and short-read (Illumina) data. We show that the combination of both sequencing techniques allows proper genome reconstruction of all plasmids in most cases but use of a very close reference is necessary when only short-read sequencing data is available. B. bavariensis genomes combine a high degree of genetic conservation with high plasticity: all isolates share the main chromosome and five plasmids, but the repertoire of other plasmids is highly variable. In addition to plasmid losses and gains through horizontal transfer, we also observe several fusions between plasmids. Although European isolates of B. bavariensis have little diversity in genome content, there is some geographic structure to this variation. In contrast, each Asian isolate has a unique plasmid repertoire and we observe no geographically based differences between Japanese and Russian isolates. Comparing the genomes of Asian and European populations of B. bavariensis suggests that some genes which are markedly different between the two populations may be good candidates for adaptation to the tick vector, (Ixodes ricinus in Europe and I. persulcatus in Asia). Conclusions We present the characterization of genomes of a large sample of B. bavariensis isolates and show that their plasmid content is highly variable. This study opens the way for genomic studies seeking to understand host and vector adaptation as well as human pathogenicity in Eurasian Lyme Borreliosis agents.

ObjectivesEndoprosthetic loosening still plays a major role in orthopaedic and dental surgery and includes various cellular immune processes within peri-implant tissues. Although the dental and orthopaedic processes vary in certain parts, the clinical question arises whether there are common immune regulators of implant loosening. Analyzing the key gene expressions common to both processes reveals the mechanisms of osteoclastogenesis within periprosthetic tissues of orthopaedic and dental origin.MethodsDonor peripheral blood mononuclear cells (PBMCs) and intraoperatively obtained periprosthetic fibroblast-like cells (PPFs) were (co-)cultured with [± macrophage-colony stimulating factor (MCSF) and Receptor Activator of NF-κB ligand (RANKL)] in transwell and monolayer culture systems and examined for osteoclastogenic regulations [MCSF, RANKL, osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα)] as well as the ability of bone resorption. Sequencing analysis compared dental and orthopaedic (co-)cultures.ResultsMonolayer co-cultures of both origins expressed high levels of OPG, resulting in inhibition of osteolysis shown by resorption assay on dentin. The high OPG-expression, low RANKL/OPG ratios and a resulting inhibition of osteolysis were displayed by dental and orthopaedic PPFs in monolayer even in the presence of MCSF and RANKL, acting as osteoprotective and immunoregulatory cells. The osteoprotective function was only observed in monolayer cultures of dental and orthopaedic periprosthetic cells and downregulated in the transwell system. In transwell co-cultures of PBMCs/PPFs profound changes of gene expression, with a significant decrease of OPG (20-fold dental versus 100 fold orthopaedic), were identified. Within transwell cultures, which offer more in vivo like conditions, RANKL/OPG ratios displayed similar high levels to the original periprosthetic tissue. For dental and orthopaedic implant loosening, overlapping findings in principal component and heatmap analysis were identified.ConclusionsThus, periprosthetic osteoclastogenesis may be a correlating immune process in orthopaedic and dental implant failure leading to comparable reactions with regard to osteoclast formation. The transwell cultures system may provide an in vivo like model for the exploration of orthopaedic and dental implant loosening.

Abstract The C9orf72 repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD). Non‐canonical translation of the expanded repeat results in abundant poly‐GA inclusion pathology throughout the CNS. (GA)149‐CFP expression in mice triggers motor deficits and neuroinflammation. Since poly‐GA is transmitted between cells, we investigated the therapeutic potential of anti‐GA antibodies by vaccinating (GA)149‐CFP mice. To overcome poor immunogenicity, we compared the antibody response of multivalent ovalbumin‐(GA)10 conjugates and pre‐aggregated carrier‐free (GA)15. Only ovalbumin‐(GA)10 immunization induced a strong anti‐GA response. The resulting antisera detected poly‐GA aggregates in cell culture and patient tissue. Ovalbumin‐(GA)10 immunization largely rescued the motor function in (GA)149‐CFP transgenic mice and reduced poly‐GA inclusions. Transcriptome analysis showed less neuroinflammation in ovalbumin‐(GA)10‐immunized poly‐GA mice, which was corroborated by semiquantitative and morphological analysis of microglia/macrophages. Moreover, cytoplasmic TDP‐43 mislocalization and levels of the neurofilament light chain in the CSF were reduced, suggesting neuroaxonal damage is reduced. Our data suggest that immunotherapy may be a viable primary prevention strategy for ALS/FTD in C9orf72 mutation carriers.

Uncertain models, e.g., due to component variations and measurement errors during system identification, in combination with the desire to be able to provide guarantees regarding system performances a priori, represent major challenges for users when designing controllers on the basis of models. The aim of this publication is to mitigate this obstacle in the design. For this purpose, criteria of classical control engineering are combined with interval arithmetic in general and the SIVIA algorithm in particular. Thereby, the approach is valid for linear dead time systems. The algorithm evaluates performance criteria for closed interval-bounded sets of parameter values in a single iteration. When overestimation prevents a definite result, the size of the parameter set is recursively reduced by bisecting the evaluated set. The application of the methodology to the controller design for an electromechanical linear actuator shows that the approach not only contributes to theory but can also simplify the practical controller design, especially in the area of electric drives.

Identification of fusion genes in clinical routine is mostly based on cytogenetics and targeted molecular genetics, such as metaphase karyotyping, fluorescence in situ hybridization and reverse-transcriptase polymerase chain reaction. However, sequencing technologies are becoming more important in clinical routine as processing time and costs per sample decrease. To evaluate the performance of fusion gene detection by RNAsequencing compared to standard diagnostic techniques, we analyzed 806 RNA-sequencing samples from patients with acute myeloid leukemia using two state-of-the-art software tools, namely Arriba and FusionCatcher. RNA-sequencing detected 90% of fusion events that were reported by routine with high evidence, while samples in which RNA-sequencing failed to detect fusion genes had overall lower and inhomogeneous sequence coverage. Based on properties of known and unknown fusion events, we developed a workflow with integrated filtering strategies for the identification of robust fusion gene candidates by RNA-sequencing. Thereby, we detected known recurrent fusion events in 26 cases that were not reported by routine and found discrepancies in evidence for known fusion events between routine and RNA-sequencing in three cases. Moreover, we identified 157 fusion genes as novel robust candidates and comparison to entries from ChimerDB or Mitelman Database showed novel recurrence of fusion genes in 14 cases. Finally, we detected the novel recurrent fusion gene NRIP1- MIR99AHG resulting from inv(21)(q11.2;q21.1) in nine patients (1.1%) and LTN1-MX1 resulting from inv(21)(q21.3;q22.3) in two patients (0.25%). We demonstrated that NRIP1-MIR99AHG results in overexpression of the 3' region of MIR99AHG and the disruption of the tricistronic miRNA cluster miR-99a/let-7c/miR-125b-2. Interestingly, upregulation of MIR99AHG and deregulation of the miRNA cluster, residing in the MIR99AHG locus, are known mechanisms of leukemogenesis in acute megakaryoblastic leukemia. Our findings demonstrate that RNA-sequencing has a strong potential to improve the systematic detection of fusion genes in clinical applications and provides a valuable tool for fusion discovery.

Objective: The liver regulates the availability of insulin to other tissues and is the first line insulin response organ physiologically exposed to higher insulin concentrations than the periphery. Basal insulin during fasting inhibits hepatic gluconeogenesis and glycogenolysis, whereas postprandial insulin peaks stimulate glycogen synthesis. The molecular consequences of chronic insulin deficiency for the liver have not been studied systematically. Methods: We analyzed liver samples of a genetically diabetic pig model (MIDY) and of wild-type (WT) littermate controls by RNA sequencing, proteomics, and targeted metabolomics/lipidomics. Results: Cross-omics analyses revealed increased activities in amino acid metabolism, oxidation of fatty acids, ketogenesis, and gluconeogenesis in the MIDY samples. In particular, the concentrations of the ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) and of retinol dehydrogenase 16 (RDH16), which catalyzes the first step in retinoic acid biogenesis, were highly increased. Accordingly, elevated levels of retinoic acid, which stimulates the expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK1), were measured in the MIDY samples. In contrast, pathways related to extracellular matrix and inflammation/pathogen defense response were less active than in the WT samples. Conclusions: The first multi-omics study of a clinically relevant diabetic large animal model revealed molecular signatures and key drivers of functional alterations of the liver in insulin-deficient diabetes mellitus. The multi-omics data set provides a valuable resource for comparative analyses with other experimental or clinical data sets. Keywords: Liver, Insulin deficiency, Transcriptome, Proteome, Metabolome, Lipidome

Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing, and fork directionality profiles obtained by RNA-seq, Repli-seq, and OK-seq. Both, the origin recognition complex (ORC) and the minichromosome maintenance complex (MCM) are significantly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early- than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating regions, and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late-replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms specifying when and where replication initiates in human cells.

Genetic variations in the four casein genes CSN1S1, CSN2, CSN1S2, and CSN3 have obtained substantial attention since they affect the milk protein yield, milk composition, cheese processing properties, and digestibility as well as tolerance in human nutrition. Furthermore, milk protein variants are used for breed characterization, biodiversity, and phylogenetic studies. The current study aimed at the identification of casein protein variants in five domestic goat breeds from Sudan (Nubian, Desert, Nilotic, Taggar, and Saanen) and three wild goat species [Capra aegagrus aegagrus (Bezoar ibex), Capra nubiana (Nubian ibex), and Capra ibex (Alpine ibex)]. High-density capture sequencing of 33 goats identified in total 22 non-synonymous and 13 synonymous single nucleotide polymorphisms (SNPs), of which nine non-synonymous and seven synonymous SNPs are new. In the CSN1S1 gene, the new non-synonymous SNP ss7213522403 segregated in Alpine ibex. In the CSN2 gene, the new non-synonymous SNPs ss7213522526, ss7213522558, and ss7213522487 were found exclusively in Nubian and Alpine ibex. In the CSN1S2 gene, the new non-synonymous SNPs ss7213522477, ss7213522549, and ss7213522575 were found in Nubian ibex only. In the CSN3 gene, the non-synonymous SNPs ss7213522604 and ss7213522610 were found in Alpine ibex. The identified DNA sequence variants led to the detection of nine new casein protein variants. New variants were detected for alpha S1 casein in Saanen goats (CSN1S1∗C1), Bezoar ibex (CSN1S1∗J), and Alpine ibex (CSN1S1∗K), for beta and kappa caseins in Alpine ibex (CSN2∗F and CSN3∗X), and for alpha S2 casein in all domesticated and wild goats (CSN1S2∗H), in Nubian and Desert goats (CSN1S2∗I), or in Nubian ibex only (CSN1S2∗J and CSN1S2∗K). The results show that most novel SNPs and protein variants occur in the critically endangered Nubian ibex. This highlights the importance of the preservation of this endangered breed. Furthermore, we suggest validating and further characterizing the new casein protein variants.

Background: Patients with ischaemic stroke or transient ischaemic attack (TIA) are at high risk of recurrent stroke and other cardiovascular diseases and commonly suffer from reduced quality of life. We aimed to determine whether the disease management programme STROKE-CARD can prevent cardiovascular diseases and improve quality of life in these patients. Methods: In this pragmatic open-label two-centre randomised controlled trial with blinded outcome assessment, we randomly assigned patients with acute ischaemic stroke or TIA (ABCD2 score ≥3) in a 2:1 ratio to receive STROKE-CARD care or standard care. STROKE-CARD care is a disease management programme by a multidisciplinary stroke team that comprises a standardised 3-month visit and access to a web-based patient portal targeting risk factor management, post-stroke complications, comorbidities and cardiovascular warning signs, rehabilitation demands, and patient education, counselling, and self-empowerment. Co-primary outcomes were analysed on an intention-to-treat basis and were: (i) major cardiovascular disease events defined as nonfatal ischaemic or haemorrhagic stroke, nonfatal myocardial infarction, or vascular death occurring between hospital discharge and 12 months; and (ii) health-related quality of life at 12 months quantified with the EuroQol-5-Dimensions-3-Levels (EQ-5D-3L) overall utility score. This trial is registered with ClinicalTrials.gov, number NCT02156778. Findings: Of 2149 patients enrolled between January 2014 and December 2017 (mean age 69 years, 41% female, 83% with ischaemic stroke, 17% with TIA), 1438 were assigned to STROKE-CARD care and 711 to standard care. Major cardiovascular disease events occurred in 78 patients in the STROKE-CARD care group (5.4%) and in 59 patients in the standard care group (8.3%) (hazard ratio, 0.63; 95% confidence interval: 0.45-0.88; P=0.007). STROKE-CARD care also led to a better EQ-5D-3L overall utility score at 12 months (P

Background: Bone marrow stem cell clonal dysfunction by somatic mutation is suspected to affect post-infarction myocardial regeneration after coronary bypass surgery (CABG). Methods: Transcriptome and variant expression analysis was studied in the phase 3 PERFECT trial post myocardial infarction CABG and CD133+ bone marrow derived hematopoetic stem cells showing difference in left ventricular ejection fraction (∆LVEF) myocardial regeneration Responders (n=14; ∆LVEF +16% day 180/0) and Non-responders (n=9; ∆LVEF -1.1% day 180/0). Subsequently, the findings have been validated in an independent patient cohort (n=14) as well as in two preclinical mouse models investigating SH2B3/LNK antisense or knockout deficient conditions. Findings: 1. Clinical: R differed from NR in a total of 161 genes in differential expression (n=23, q

Background Intravenous thrombolysis (IVT) in wake‐up stroke (WUS) or stroke with unknown onset (SUO) has been recently proven to be safe and effective using advanced neuroimaging (magnetic resonance imaging or computerized tomography‐perfusion) for patient selection. However, in most of the thrombolyzing centers advanced neuroimaging is not instantly available. We hypothesize that pragmatic non‐contrast computed tomography‐based IVT in WUS/SUO may be feasible and safe. Methods and Results TRUST‐CT (Thrombolysis in Stroke With Unknown Onset Based on Non‐Contrast Computerized Tomography) is an international multicenter registry‐based study. WUS/SUO patients undergoing non‐contrast computed tomography‐based IVT with National Institute of Health Stroke Scale ≥4 and initial Alberta Stroke Program Early Computerized Tomography score ≥7 were included and compared with propensity score matched non‐thrombolyzed WUS/SUO controls. Primary end point was the incidence of symptomatic intracranial hemorrhage; secondary end points included 24‐hour National Institute of Health Stroke Scale improvement of ≥4 and modified Rankin Scale at 90 days. One hundred and seventeen WUS/SUO patients treated with non‐contrast computed tomography‐based IVT were included. As compared with 112 controls, the median admission National Institute of Health Stroke Scale was 10 and the median Alberta Stroke Program Early Computerized Tomography score was 10 in both groups. Four (3.4%) IVT patients and one control patient (0.9%) suffered symptomatic intracranial hemorrhage (adjusted odds ratio 7.9, 95% CI 0.65–96, P=0.1). A decrease of ≥4 National Institute of Health Stroke Scale points was observed in 67 (57.3%) of IVT patients as compared with 25 (22.3%) in controls (adjusted odds ratio 5.8, CI 3.0–11.2, P

Abstract Background Patients with ischaemic stroke or transient ischaemic attack (TIA) are at high risk of future cardiovascular events. Despite compelling evidence about the efficacy of secondary prevention, a substantial gap exists between risk factor management in real life and that recommended by international guidelines. Moreover, stroke is a leading cause of disability and morbidity which partly emerges from post-stroke complications. Methods/design We designed a block-randomised (2:1 ratio) open pragmatic trial [NCT02156778] with blinded outcome assessment comparing STROKE-CARD to usual post-stroke-patient care. STROKE-CARD is a multifaceted post-stroke disease management program with the objective of reducing recurrent cardiovascular events and improving quality of life in ischaemic stroke and TIA-patients. It combines intensified multi-domain secondary prevention, systematic detection and treatment of post-stroke complications, and patient self-empowerment. Enrolment of 2160 patients with acute ischaemic stroke or TIA (ABCD2-Score ≥ 3) is planned at two study centres in Austria. The co-primary efficacy endpoints are (i) the composite of major recurrent cardiovascular events (nonfatal stroke, nonfatal myocardial infarction, and vascular death) occurring within 12 months after the index event and (ii) one-year health-related quality-of-life measured with the European Quality of Life-5 Dimensions (EQ-5D-3 L) questionaire. Secondary endpoints include all-cause mortality, functional outcome, and target-level achievement in risk factor management. Discussion This trial will provide evidence on whether the pragmatic post-stroke intervention program STROKE-CARD can help prevent cardiovascular events and improve quality-of-life within the setting of a high-quality acute stroke care system. In case of success, STROKE-CARD may be implemented in daily clinical routine and serve as a model for other disease management initiatives. Trial registration ClinicalTrials.gov: NCT02156778. (June 5, 2014, retrospectively registered).

Abstract Background It has been known for almost a century that the belted phenotype in cattle follows a pattern of dominant inheritance. In 2009, the approximate position of the belt locus in Brown Swiss cattle was mapped to a 922-kb interval on bovine chromosome 3 and, subsequently, assigned to a 336-kb haplotype block based on an animal set that included, Brown Swiss, Dutch Belted (Lakenvelder) and Belted Galloway individuals. A possible candidate gene in this region i.e. HES6 was investigated but the causal mutation remains unknown. Thus, to elucidate the causal mutation of this prominent coat color phenotype, we decided to remap the belted phenotype in an independent animal set of several European bovine breeds, i.e. Gurtenvieh (belted Brown Swiss), Dutch Belted and Belted Galloway and to systematically scan the candidate region. We also checked the presence of the detected causal mutation in the genome of belted individuals from a Siberian cattle breed. Results A combined linkage disequilibrium and linkage analysis based on 110 belted and non-belted animals identified a candidate interval of 2.5 Mb. Manual inspection of the haplotypes in this region identified four candidate haplotypes that consisted of five to eight consecutive SNPs. One of these haplotypes overlapped with the initial 922-kb interval, whereas two were positioned proximal and one was positioned distal to this region. Next-generation sequencing of one heterozygous and two homozygous belted animals identified only one private belted candidate allele, i.e. a multiplication event that is located between 118,608,000 and 118,614,000 bp. Targeted locus amplification and quantitative real-time PCR confirmed an increase in copy number of this region in the genomes of both European (Belted Galloway, Dutch Belted and Gurtenvieh) and Siberian (Yakutian cattle) breeds. Finally, using nanopore sequencing, the exact breakpoints were determined at 118,608,362 and 118,614,132 bp. The closest gene to the candidate causal mutation (16 kb distal) is TWIST2. Conclusions Based on our findings and those of a previously published study that identified the same multiplication event, a quadruplication on bovine chromosome 3 between positions 118,608,362 and 118,614,132 bp is the most likely candidate causal mutation for the belted phenotype in cattle.