Publications by authors named "Stefan Krebs"

132 Publications

Guaranteed State Estimation Using a Bundle of Interval Observers with Adaptive Gains Applied to the Induction Machine.

Sensors (Basel) 2021 Apr 7;21(8). Epub 2021 Apr 7.

Institute of Control Systems, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany.

The scope of this paper is the design of an interval observer bundle for the guaranteed state estimation of an uncertain induction machine with linear, time-varying dynamics. These guarantees are of particular interest in the case of safety-critical systems. In many cases, interval observers provide large intervals for which the usability becomes impractical. Hence, based on a reduced-order hybrid interval observer structure, the guaranteed enclosure within intervals of the magnetizing current's estimates is improved using a bundle of interval observers. One advantage of such an interval observer bundle is the possibility to reinitialize the interval observers at specified timesteps during runtime with smaller initial intervals, based on previously observed system states, resulting in decreasing interval widths. Thus, unstable observer dynamics are considered so as to take advantage of their transient behavior, whereby the overall stability of the interval estimation is maintained. An algorithm is presented to determine the parametrization of reduced-order interval observers. To this, an adaptive observer gain is introduced with which the system states are observed optimally by considering a minimal interval width at variable operating points. Furthermore, real-time capability and validation of the proposed methods are shown. The results are discussed with simulations as well as experimental data obtained with a test bench.
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http://dx.doi.org/10.3390/s21082584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067717PMC
April 2021

ONT-based draft genome assembly and annotation of Alternaria atra.

Mol Plant Microbe Interact 2021 Mar 29. Epub 2021 Mar 29.

Technical University of Munich, 9184, Phytopathology, Emil Ramann Str, Freising, Germany, 85354;

Species of Alternaria (phylum Ascomycota, family Pleosporaceae) are known as serious plant pathogens, causing major losses on a wide range of crops. Alternaria atra (Preuss) Woudenb. & Crous (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 a.o. 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 Seq-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 understand the mechanisms defining its role as pathogen or biocontrol agent.
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http://dx.doi.org/10.1094/MPMI-01-21-0016-ADOI Listing
March 2021

Capture Sequencing to Explore and Map Rare Casein Variants in Goats.

Front Genet 2021 23;12:620253. Epub 2021 Feb 23.

Animal Breeding Biology and Molecular Genetics, Albrecht Daniel Thaer-Institute of Agricultural and Horticultural Sciences, Humboldt University of Berlin, Berlin, Germany.

Genetic variations in the four casein genes , , , and 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 [ (Bezoar ibex), (Nubian ibex), and (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 gene, the new non-synonymous SNP ss7213522403 segregated in Alpine ibex. In the gene, the new non-synonymous SNPs ss7213522526, ss7213522558, and ss7213522487 were found exclusively in Nubian and Alpine ibex. In the gene, the new non-synonymous SNPs ss7213522477, ss7213522549, and ss7213522575 were found in Nubian ibex only. In the 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 (C1), Bezoar ibex (J), and Alpine ibex (K), for beta and kappa caseins in Alpine ibex (F and X), and for alpha S2 casein in all domesticated and wild goats (H), in Nubian and Desert goats (I), or in Nubian ibex only (J and 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.
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http://dx.doi.org/10.3389/fgene.2021.620253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940697PMC
February 2021

Human ORC/MCM density is low in active genes and correlates with replication time but does not delimit initiation zones.

Elife 2021 Mar 8;10. Epub 2021 Mar 8.

Research Unit Gene Vectors, Helmholtz Zentrum München (GmbH), German Research Center for Environmental Health, Munich, Germany.

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.
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http://dx.doi.org/10.7554/eLife.62161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7993996PMC
March 2021

Epstein-Barr virus inactivates the transcriptome and disrupts the chromatin architecture of its host cell in the first phase of lytic reactivation.

Nucleic Acids Res 2021 04;49(6):3217-3241

Research Unit Gene Vectors, Helmholtz Zentrum München, German Research Center for Environmental Health and German Center for Infection Research (DZIF), Partner site Munich, Germany, Feodor-Lynen-Str. 21, D-81377 Munich, Germany.

Epstein-Barr virus (EBV), a herpes virus also termed HHV 4 and the first identified human tumor virus, establishes a stable, long-term latent infection in human B cells, its preferred host. Upon induction of EBV's lytic phase, the latently infected cells turn into a virus factory, a process that is governed by EBV. In the lytic, productive phase, all herpes viruses ensure the efficient induction of all lytic viral genes to produce progeny, but certain of these genes also repress the ensuing antiviral responses of the virally infected host cells, regulate their apoptotic death or control the cellular transcriptome. We now find that EBV causes previously unknown massive and global alterations in the chromatin of its host cell upon induction of the viral lytic phase and prior to the onset of viral DNA replication. The viral initiator protein of the lytic cycle, BZLF1, binds to >105 binding sites with different sequence motifs in cellular chromatin in a concentration dependent manner implementing a binary molar switch probably to prevent noise-induced erroneous induction of EBV's lytic phase. Concomitant with DNA binding of BZLF1, silent chromatin opens locally as shown by ATAC-seq experiments, while previously wide-open cellular chromatin becomes inaccessible on a global scale within hours. While viral transcripts increase drastically, the induction of the lytic phase results in a massive reduction of cellular transcripts and a loss of chromatin-chromatin interactions of cellular promoters with their distal regulatory elements as shown in Capture-C experiments. Our data document that EBV's lytic cycle induces discrete early processes that disrupt the architecture of host cellular chromatin and repress the cellular epigenome and transcriptome likely supporting the efficient de novo synthesis of this herpes virus.
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http://dx.doi.org/10.1093/nar/gkab099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034645PMC
April 2021

Intravenous thrombolysis in stroke with admission NIHSS score 0 or 1.

Int J Stroke 2021 Feb 10:1747493021991969. Epub 2021 Feb 10.

Medical Faculty, Sigmund Freud University Vienna, Austria.

Background: Up to 30% of stroke patients initially presenting with non-disabling or mild deficits may experience poor functional outcome. Despite, intravenous thrombolysis remains controversial in this subgroup of stroke patients due to its uncertain risk benefit ratio.

Aim: We aimed to analyze the real-world experience with intravenous thrombolysis in stroke patients presenting with very low NIHSS.

Methods: Data of stroke patients presenting with mild initial stroke severity (NIHSS 0-5) including vascular risk factors, stroke syndrome and etiology, early neurological deterioration, symptomatic intracerebral haemorrhage (sICH), and functional outcome by modified Rankin Scale were extracted from a large nationwide stroke registry and analysed. Patients were categorized and compared according to admission severity NIHSS 0-1 versus NIHSS 2-5 and intravenous thrombolysis use.

Results: Seven hundred and three (2%) of 35,113 patients presenting with NIHSS 0-1 and 6316 (13.9%) of 45,521 of patients presenting with NIHSS 2-5 underwent intravenous thrombolysis. In the NIHSS 0-1 group, intravenous thrombolysis was associated with early neurological deterioration (adjusted OR 8.84, CI 6.61-11.83), sICH (adjusted OR 9.32, CI 4.53-19.15) and lower rate of excellent outcome (mRS 0-1) at three months (adjusted OR 0.67, CI 0.5-0.9). In stroke patients with NIHSS 2-5, intravenous thrombolysis was associated with early neurological deterioration (adjusted OR 1.7, 1.47-1.98), sICH (adjusted OR 5.75, CI 4.45-7.45), and higher rate of excellent outcome (mRS 0-1) at three months (adjusted OR 1.21, CI 1.08-1.34).

Conclusions: Among patients with NIHSS 0-1, intravenous thrombolysis did not increase the likelihood of excellent outcome. Moreover, potential signals of harm were observed. Further research seems to be warranted.
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http://dx.doi.org/10.1177/1747493021991969DOI Listing
February 2021

Prospective Longitudinal Serosurvey of Health Care Workers in the First Wave of the SARS-CoV-2 Pandemic in a Quaternary Care Hospital in Munich, Germany.

Clin Infect Dis 2021 Jan 3. Epub 2021 Jan 3.

Emergency Department, University Hospital, LMU Munich, Munich, Germany.

Background: High infection rates among health care personnel in an uncontained pandemic can paralyze health systems due to staff shortages. Risk constellations and rates of seroconversion for health care workers during the first wave of the SARS-CoV-2 pandemic are still largely unclear.

Methods: Health care personnel (n=300) on different organizational units in the LMU Munich University Hospital were included and followed in this prospective longitudinal study in the period of March 24 until July 7, 2020. Participants were monitored in intervals of two to six weeks using different antibody assays for serological testing and questionnaires to evaluate risk contacts. In a subgroup of infected participants, we obtained nasopharyngeal swabs to perform whole genome sequencing for outbreak characterization.

Results: Health care workers involved in patient care on dedicated COVID-19 wards or on regular non-COVID-19 wards showed a higher rate of SARS-CoV-2 seroconversion compared to staff in the emergency department and non-frontline personnel. The landscape of risk contacts in these units was dynamic, with a decrease of unprotected risk contacts in the emergency department and an increase on non-COVID-19 wards. Both, the intensity and number of risk contacts, were associated with higher rates of seroconversion. On regular wards, staff infections tended to occur in clusters, while infections on COVID-19 wards were less frequent and apparently independent of each other.

Conclusion: The risk of SARS-CoV-2 infection for front-line health care workers was increased during the first pandemic wave in Southern Germany. Stringent measures for infection control are essential to protect all patient-facing staff during the ongoing pandemic.
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http://dx.doi.org/10.1093/cid/ciaa1935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7799305PMC
January 2021

Complete mitochondrial genomes of Karchaev goat ().

Mitochondrial DNA B Resour 2020 Oct 27;5(3):3645-3646. Epub 2020 Oct 27.

L.K. Ernst Federal Science Center for Animal Husbandry, Podolsk, Russia.

Karachaev goat () is a local breed from North-Caucasus region, Russia. Here we present complete mitochondrial genome of Karachaev goat from the republic of Karachaevo-Cherkessia, Russia. The length of the studied sequence was 16,624 bp in size. It was shown that the studied specimen belonged to haplogroup A.
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http://dx.doi.org/10.1080/23802359.2020.1831988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594705PMC
October 2020

Full genome sequence of bovine alphaherpesvirus 2 (BoHV-2).

Arch Virol 2021 Feb 14;166(2):639-643. Epub 2020 Dec 14.

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, 17493, Greifswald - Insel Riems, Deutschland.

We present the complete genome sequence of bovine alphaherpesvirus 2 (BoHV-2), a member of the family Herpesviridae, subfamily Alphaherpesvirinae, genus Simplexvirus. BoHV-2 is the causative agent of bovine ulcerative mammillitis (bovine herpes mammillitis) and pseudo-lumpy skin disease. The genomic architecture of BoHV-2 is typical of most simplexvirus genomes and congruent with that of human alphaherpesvirus 1 (HHV-1). The genome comprises a total of 131,245 base pairs and has an overall G+C content of 64.9 mol%. A total of 75 open reading frames are predicted. The gene repertoire of BoHV-2 is analogous to that of HHV-1, although the coding region of US12 is missing. A phylogenetic analysis supported BoHV-2 as a member of the genus Simplexvirus.
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http://dx.doi.org/10.1007/s00705-020-04895-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7850993PMC
February 2021

Design and Derivation of the Austrian Prehospital Stroke Scale (APSS) to Predict Severe Stroke with Large Vessel Occlusion.

Prehosp Emerg Care 2021 Jan 12:1-9. Epub 2021 Jan 12.

Prediction of large vessel occlusion (LVO) is highly relevant for accurate prehospital transportation triage. The Austrian Prehospital Stroke Scale (APSS) score for LVO prediction was developed using critical synthesis of previously published LVO-scores. The aim of this study was to investigate the accuracy of the APSS and compare it to other LVO-scores. APSS consists of 5 items: "facial palsy," "motor arm," "language," "motor leg" and "gaze deviation." The score ranges from 0 to 9 points. Data from 741 consecutive stroke patients with acute vessel imaging admitted to an independent comprehensive stroke center was used to test the predictive performance of the APSS in context of other LVO-scores (CPSS, FAST-ED, G-FAST, sNIHSS-EMS and RACE). In the prediction of treatable LVO the APSS showed the highest area under the curve (0.834) with significant difference to CPSS (p = 0.010) and G-FAST (p = 0.006) and showed highest sensitivity (69%) as compared to other LVO scores. Specificity (85%), positive predictive value (75%), negative predictive value (81%) and accuracy (79%) were comparable to other LVO scores. Receiver operating curve analysis revealed an optimal cutoff for LVO prediction at APSS equal to 4 points. The easy assessable 5-item APSS score tended to outperform other LVO scores. Real-life prospective evaluation in prehospital setting is ongoing.
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http://dx.doi.org/10.1080/10903127.2020.1851329DOI Listing
January 2021

A de novo frameshift mutation in ZEB2 causes polledness, abnormal skull shape, small body stature and subfertility in Fleckvieh cattle.

Sci Rep 2020 10 12;10(1):17032. Epub 2020 Oct 12.

Population Genomics Group, Department of Veterinary Sciences, Ludwig-Maximilians-University Munich, 80539, Munich, Germany.

Polledness in cattle is an autosomal dominant trait. Previous studies have revealed allelic heterogeneity at the polled locus and four different variants were identified, all in intergenic regions. In this study, we report a case of polled bull (FV-Polled1) born to horned parents, indicating a de novo origin of this polled condition. Using 50K genotyping and whole genome sequencing data, we identified on chromosome 2 an 11-bp deletion (AC_000159.1:g.52364063_52364073del; Del11) in the second exon of ZEB2 gene as the causal mutation for this de novo polled condition. We predicted that the deletion would shorten the protein product of ZEB2 by almost 91%. Moreover, we showed that all animals carrying Del11 mutation displayed symptoms similar to Mowat-Wilson syndrome (MWS) in humans, which is also associated with genetic variations in ZEB2. The symptoms in cattle include delayed maturity, small body stature and abnormal shape of skull. This is the first report of a de novo dominant mutation affecting only ZEB2 and associated with a genetic absence of horns. Therefore our results demonstrate undoubtedly that ZEB2 plays an important role in the process of horn ontogenesis as well as in the regulation of overall development and growth of animals.
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http://dx.doi.org/10.1038/s41598-020-73807-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7550345PMC
October 2020

High conservation combined with high plasticity: genomics and evolution of Borrelia bavariensis.

BMC Genomics 2020 Oct 8;21(1):702. Epub 2020 Oct 8.

National Reference Centre for Borrelia at the Bavarian Health and Food Safety Authority, Veterinärstr 2, 85764, Oberschleissheim, Germany.

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.
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http://dx.doi.org/10.1186/s12864-020-07054-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7542741PMC
October 2020

STROKE-CARD care to prevent cardiovascular events and improve quality of life after acute ischaemic stroke or TIA: A randomised clinical trial.

EClinicalMedicine 2020 Aug 28;25:100476. Epub 2020 Jul 28.

Department of Neurology, Medical University of Innsbruck, Innsbruck, Austria.

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 (ABCD 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<0.001). Among pre-specified secondary outcomes, STROKE-CARD care improved all individual EQ-5D-3L dimensions and functional outcome on the modified Rankin Scale at 12 months. Post hoc explanatory analyses identified considerable demands for additional rehabilitation and refinement of preventive therapy regimes at the 3-month visit and high proportions of post-stroke complications and warning signs of imminent cardiovascular diseases within the first three months.

Interpretation: The pragmatic and easily implementable STROKE-CARD care programme reduced cardiovascular risk and improved health-related quality of life and functional outcome in patients with acute ischaemic stroke or TIA.

Funding: Tirol Kliniken, Tyrolean Health Insurance Company, Tyrol Health Care Funds, Boehringer Ingelheim, Nstim Services, Sanofi, Bayer Healthcare.
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http://dx.doi.org/10.1016/j.eclinm.2020.100476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486330PMC
August 2020

Linkage of genetic drivers and strain-specific germline variants confound mouse cancer genome analyses.

Nat Commun 2020 09 8;11(1):4474. Epub 2020 Sep 8.

Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675, Munich, Germany.

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http://dx.doi.org/10.1038/s41467-020-18095-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7479137PMC
September 2020

Heart rate entropy is associated with mortality after intracereberal hemorrhage.

J Neurol Sci 2020 Nov 12;418:117033. Epub 2020 Jul 12.

Division of Neurosurgery, Department of Clinical Neurosciences, Cambridge University, Cambridge, United Kingdom.

Background: Autonomic nervous system changes have been associated with outcome after intracerebral hemorrhage (ICH) previously. We aimed to investigate the association of heart rate entropy (HRE) with mortality after ICH.

Methods: Sample HRE, heart rate variability and baroreflex sensitivity were examined in consecutive ICH patients. Hematoma volume, intraventricular hemorrhage, infratentorial origin, consciousness impairment and age were combined into standard ICH score.

Results: In 47 patients suffering ICH (mean age 61 years, median hemorrhage volume 38 mL) the areas under the curve (AUC) for mortality were 0.86, 0.83, 0.76, 0.74, 0.72 and 0.7 for HRE, ICH-score, normalized low frequency powers, low frequency/high frequency powers ratio, normalized high frequency powers and BRS, respectively. HRE and ICH score were associated with mortality independently (adjusted odd ratio (aOR) 0.09, 95% confidence interval (CI) 0.1-0.8, p = .03 and aOR 2.6, CI 1.03-6.6, p = .04). Combining ICH score with HRE into a novel score resulted in an AUC of 0.94, CI 0.88-0.99, p < .001.

Conclusion: Compared to several autonomic markers HRE seems to bear the largest amount of information on death probability after ICH. Moreover, HRE may predict mortality comparable to ICH score. Combining HRE with ICH score may increase the predictive performance for mortality after ICH.
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http://dx.doi.org/10.1016/j.jns.2020.117033DOI Listing
November 2020

An improved method for high-throughput quantification of autophagy in mammalian cells.

Sci Rep 2020 07 22;10(1):12241. Epub 2020 Jul 22.

Institute of Molecular Virology, Ulm University Medical Center, 89081, Ulm, Germany.

Autophagy is a cellular homeostatic pathway with functions ranging from cytoplasmic protein turnover to immune defense. Therapeutic modulation of autophagy has been demonstrated to positively impact the outcome of autophagy-dysregulated diseases such as cancer or microbial infections. However, currently available agents lack specificity, and new candidates for drug development or potential cellular targets need to be identified. Here, we present an improved method to robustly detect changes in autophagy in a high-throughput manner on a single cell level, allowing effective screening. This method quantifies eGFP-LC3B positive vesicles to accurately monitor autophagy. We have significantly streamlined the protocol and optimized it for rapid quantification of large numbers of cells in little time, while retaining accuracy and sensitivity. Z scores up to 0.91 without a loss of sensitivity demonstrate the robustness and aptness of this approach. Three exemplary applications outline the value of our protocols and cell lines: (I) Examining autophagy modulating compounds on four different cell types. (II) Monitoring of autophagy upon infection with e.g. measles or influenza A virus. (III) CRISPR/Cas9 screening for autophagy modulating factors in T cells. In summary, we offer ready-to-use protocols to generate sensitive autophagy reporter cells and quantify autophagy in high-throughput assays.
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http://dx.doi.org/10.1038/s41598-020-68607-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376206PMC
July 2020

Hematopoietic stem-cell senescence and myocardial repair - Coronary artery disease genotype/phenotype analysis of post-MI myocardial regeneration response induced by CABG/CD133+ bone marrow hematopoietic stem cell treatment in RCT PERFECT Phase 3.

EBioMedicine 2020 Jul 4;57:102862. Epub 2020 Jul 4.

Department of Advanced Medicine Science, Tokai University School of Medicine, Shimokasuya 143, Isehara, Kanagawa 259-1143, Japan. Electronic address:

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<0•05) and 872 genes in coexpression analysis (n=23, q<0•05). Machine Learning clustering analysis revealed distinct RvsNR preoperative gene-expression signatures in peripheral blood acorrelated to SH2B3 (p<0.05). Mutation analysis revealed increased specific variants in RvsNR. (R: 48 genes; NR: 224 genes). 2. Preclinical:SH2B3/LNK-silenced hematopoietic stem cell (HSC) clones displayed significant overgrowth of myeloid and immune cells in bone marrow, peripheral blood, and tissue at day 160 after competitive bone-marrow transplantation into mice. SH2B3/LNK mice demonstrated enhanced cardiac repair through augmenting the kinetics of bone marrow-derived endothelial progenitor cells, increased capillary density in ischemic myocardium, and reduced left ventricular fibrosis with preserved cardiac function. 3.

Validation: Evaluation analysis in 14 additional patients revealed 85% RvsNR (12/14 patients) prediction accuracy for the identified biomarker signature.

Interpretation: Myocardial repair is affected by HSC gene response and somatic mutation. Machine Learning can be utilized to identify and predict pathological HSC response.

Funding: German Ministry of Research and Education (BMBF): Reference and Translation Center for Cardiac Stem Cell Therapy - FKZ0312138A and FKZ031L0106C, German Ministry of Research and Education (BMBF): Collaborative research center - DFG:SFB738 and Center of Excellence - DFG:EC-REBIRTH), European Social Fonds: ESF/IV-WM-B34-0011/08, ESF/IV-WM-B34-0030/10, and Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany. Japanese Ministry of Health : Health and Labour Sciences Research Grant (H14-trans-001, H17-trans-002) TRIAL REGISTRATION: ClinicalTrials.gov NCT00950274.
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http://dx.doi.org/10.1016/j.ebiom.2020.102862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7339012PMC
July 2020

The First Draft Genome Assembly of Snow Sheep (Ovis nivicola).

Genome Biol Evol 2020 08;12(8):1330-1336

Population Genomics Group, Department of Veterinary Sciences, Ludwig Maximilian University of Munich, Munich, Germany.

The snow sheep, Ovis nivicola, which is endemic to the mountain ranges of northeastern Siberia, are well adapted to the harsh cold climatic conditions of their habitat. In this study, using long reads of Nanopore sequencing technology, whole-genome sequencing, assembly, and gene annotation of a snow sheep were carried out. Additionally, RNA-seq reads from several tissues were also generated to supplement the gene prediction in snow sheep genome. The assembled genome was ∼2.62 Gb in length and was represented by 7,157 scaffolds with N50 of about 2 Mb. The repetitive sequences comprised of 41% of the total genome. BUSCO analysis revealed that the snow sheep assembly contained full-length or partial fragments of 97% of mammalian universal single-copy orthologs (n = 4,104), illustrating the completeness of the assembly. In addition, a total of 20,045 protein-coding sequences were identified using comprehensive gene prediction pipeline. Of which 19,240 (∼96%) sequences were annotated using protein databases. Moreover, homology-based searches and de novo identification detected 1,484 tRNAs; 243 rRNAs; 1,931 snRNAs; and 782 miRNAs in the snow sheep genome. To conclude, we generated the first de novo genome of the snow sheep using long reads; these data are expected to contribute significantly to our understanding related to evolution and adaptation within the Ovis genus.
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http://dx.doi.org/10.1093/gbe/evaa124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7487135PMC
August 2020

Multicentre comparison of quantitative PCR-based assays to detect SARS-CoV-2, Germany, March 2020.

Euro Surveill 2020 06;25(24)

German Center for Infection Research, Partner Site Munich and Associated Partner Site Charité, Berlin and Associated Partner Site Frankfurt, Germany.

Containment strategies and clinical management of coronavirus disease (COVID-19) patients during the current pandemic depend on reliable diagnostic PCR assays for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we compare 11 different RT-PCR test systems used in seven diagnostic laboratories in Germany in March 2020. While most assays performed well, we identified detection problems in a commonly used assay that may have resulted in false-negative test results during the first weeks of the pandemic.
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http://dx.doi.org/10.2807/1560-7917.ES.2020.25.24.2001057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7315722PMC
June 2020

Clinical presentation and differential splicing of SRSF2, U2AF1 and SF3B1 mutations in patients with acute myeloid leukemia.

Leukemia 2020 10 1;34(10):2621-2634. Epub 2020 May 1.

Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, LMU Munich, Munich, Germany.

Previous studies demonstrated that splicing factor mutations are recurrent events in hematopoietic malignancies with both clinical and functional implications. However, their aberrant splicing patterns in acute myeloid leukemia remain largely unexplored. In this study, we characterized mutations in SRSF2, U2AF1, and SF3B1, the most commonly mutated splicing factors. In our clinical analysis of 2678 patients, splicing factor mutations showed inferior relapse-free and overall survival, however, these mutations did not represent independent prognostic markers. RNA-sequencing of 246 and independent validation in 177 patients revealed an isoform expression profile which is highly characteristic for each individual mutation, with several isoforms showing a strong dysregulation. By establishing a custom differential splice junction usage pipeline, we accurately detected aberrant splicing in splicing factor mutated samples. A large proportion of differentially used junctions were novel, including several junctions in leukemia-associated genes. In SRSF2(P95H) mutants, we further explored the possibility of a cascading effect through the dysregulation of the splicing pathway. Furthermore, we observed a validated impact on overall survival for two junctions overused in SRSF2(P95H) mutants. We conclude that splicing factor mutations do not represent independent prognostic markers. However, they do have genome-wide consequences on gene splicing leading to dysregulated isoform expression of several genes.
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http://dx.doi.org/10.1038/s41375-020-0839-4DOI Listing
October 2020

The secretome of skin cancer cells activates the mTOR/MYC pathway in healthy keratinocytes and induces tumorigenic properties.

Biochim Biophys Acta Mol Cell Res 2020 08 10;1867(8):118717. Epub 2020 Apr 10.

Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU, München, Germany. Electronic address:

Cutaneous squamous cell carcinoma (cSCC) is the most prominent tumor of non-melanoma skin cancers and the most aggressive tumor among keratinocyte carcinoma of the skin, showing a high potential for local invasion and metastasis. The cSCC incidences increased dramatically in recent years and the disease occurs more commonly than any other malignancy. The secretome of cancer cells is currently the focus of many studies in order to identify new marker proteins for different types of cancer and to investigate its influence on the tumor microenvironment. In our study we evaluated whether the secretome of cSCC cells has an impact on keratinocytes, the surrounding tissue cells of cSCC. Therefore, we analyzed and compared the secretome of human A431 cancer cells and of HaCaT keratinocytes by mass spectrometry. In a second experiment, keratinocytes were exposed to the secretome of A431 cells and vice versa and the transcriptome was analyzed by next-generation sequencing. HaCaT cells incubated with A431 conditioned medium revealed a significantly activated mammalian target of rapamycin pathway with a concomitant increase in proliferation and migration. In conclusion, our data demonstrate the impact of the secretome of cancer cells on the transcription machinery of the cells surrounding the tumor, leading to a tumorigenic cell fate.
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http://dx.doi.org/10.1016/j.bbamcr.2020.118717DOI Listing
August 2020

A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol.

Nature 2020 03 4;579(7799):433-437. Epub 2020 Mar 4.

Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

Mitochondrial fidelity is tightly linked to overall cellular homeostasis and is compromised in ageing and various pathologies. Mitochondrial malfunction needs to be relayed to the cytosol, where an integrated stress response is triggered by the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) in mammalian cells. eIF2α phosphorylation is mediated by the four eIF2α kinases GCN2, HRI, PERK and PKR, which are activated by diverse types of cellular stress. However, the machinery that communicates mitochondrial perturbation to the cytosol to trigger the integrated stress response remains unknown. Here we combine genome engineering and haploid genetics to unbiasedly identify genes that affect the induction of C/EBP homologous protein (CHOP), a key factor in the integrated stress response. We show that the mitochondrial protease OMA1 and the poorly characterized protein DELE1, together with HRI, constitute the missing pathway that is triggered by mitochondrial stress. Mechanistically, stress-induced activation of OMA1 causes DELE1 to be cleaved into a short form that accumulates in the cytosol, where it binds to and activates HRI via its C-terminal portion. Obstruction of this pathway can be beneficial or adverse depending on the type of mitochondrial perturbation. In addition to the core pathway components, our comparative genetic screening strategy identifies a suite of additional regulators. Together, these findings could be used to inform future strategies to modulate the cellular response to mitochondrial dysfunction in the context of human disease.
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http://dx.doi.org/10.1038/s41586-020-2076-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116715PMC
March 2020

ZBTB7A prevents RUNX1-RUNX1T1-dependent clonal expansion of human hematopoietic stem and progenitor cells.

Oncogene 2020 04 2;39(15):3195-3205. Epub 2020 Mar 2.

Department of Medicine III, University Hospital, LMU Munich, 81377, Munich, Germany.

ZBTB7A is frequently mutated in acute myeloid leukemia (AML) with t(8;21) translocation. However, the oncogenic collaboration between mutated ZBTB7A and the RUNX1-RUNX1T1 fusion gene in AML t(8;21) remains unclear. Here, we investigate the role of ZBTB7A and its mutations in the context of normal and malignant hematopoiesis. We demonstrate that clinically relevant ZBTB7A mutations in AML t(8;21) lead to loss of function and result in perturbed myeloid differentiation with block of the granulocytic lineage in favor of monocytic commitment. In addition, loss of ZBTB7A increases glycolysis and hence sensitizes leukemic blasts to metabolic inhibition with 2-deoxy-D-glucose. We observed that ectopic expression of wild-type ZBTB7A prevents RUNX1-RUNX1T1-mediated clonal expansion of human CD34+ cells, whereas the outgrowth of progenitors is enabled by ZBTB7A mutation. Finally, ZBTB7A expression in t(8;21) cells lead to a cell cycle arrest that could be mimicked by inhibition of glycolysis. Our findings suggest that loss of ZBTB7A may facilitate the onset of AML t(8;21), and that RUNX1-RUNX1T1-rearranged leukemia might be treated with glycolytic inhibitors.
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http://dx.doi.org/10.1038/s41388-020-1209-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7142018PMC
April 2020

Thrombolysis in Stroke With Unknown Onset Based on Non-Contrast Computerized Tomography (TRUST CT).

J Am Heart Assoc 2020 02 12;9(4):e014265. Epub 2020 Feb 12.

Department of Neurology with Focus on Neurovascular Diseases and Neurooncology and Hertie Institute for Clinical Brain Research University Hospital Tübingen Germany.

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, =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, <0.001). A months, 39 (33.3%) IVT patients reached a modified Rankin Scale score of 0 or 1 versus 23 (20.5%) controls (adjusted odds ratio 1.94, CI 1.0-3.76, =0.05). Conclusions Non-contrast computed tomography-based thrombolysis in WUS/SUO seems feasible and safe and may be effective. Randomized prospective comparisons are warranted. Clinical Trial Registration URL: https://www.clinicaltrials.gov/. Unique identifier: NCT03634748.
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http://dx.doi.org/10.1161/JAHA.119.014265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070213PMC
February 2020

The clinical mutatome of core binding factor leukemia.

Leukemia 2020 06 2;34(6):1553-1562. Epub 2020 Jan 2.

Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand.

The fusion genes CBFB/MYH11 and RUNX1/RUNX1T1 block differentiation through disruption of the core binding factor (CBF) complex and are found in 10-15% of adult de novo acute myeloid leukemia (AML) cases. This AML subtype is associated with a favorable prognosis; however, nearly half of CBF-rearranged patients cannot be cured with chemotherapy. This divergent outcome might be due to additional mutations, whose spectrum and prognostic relevance remains hardly defined. Here, we identify nonsilent mutations, which may collaborate with CBF-rearrangements during leukemogenesis by targeted sequencing of 129 genes in 292 adult CBF leukemia patients, and thus provide a comprehensive overview of the mutational spectrum ('mutatome') in CBF leukemia. Thereby, we detected fundamental differences between CBFB/MYH11- and RUNX1/RUNX1T1-rearranged patients with ASXL2, JAK2, JAK3, RAD21, TET2, and ZBTB7A being strongly correlated with the latter subgroup. We found prognostic relevance of mutations in genes previously known to be AML-associated such as KIT, SMC1A, and DHX15 and identified novel, recurrent mutations in NFE2 (3%), MN1 (4%), HERC1 (3%), and ZFHX4 (5%). Furthermore, age >60 years, nonprimary AML and loss of the Y-chromosomes are important predictors of survival. These findings are important for refinement of treatment stratification and development of targeted therapy approaches in CBF leukemia.
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http://dx.doi.org/10.1038/s41375-019-0697-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7266744PMC
June 2020

Active poly-GA vaccination prevents microglia activation and motor deficits in a C9orf72 mouse model.

EMBO Mol Med 2020 02 20;12(2):e10919. Epub 2019 Dec 20.

German Center for Neurodegenerative Diseases (DZNE), Munich, Munich, Germany.

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) -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) -CFP mice. To overcome poor immunogenicity, we compared the antibody response of multivalent ovalbumin-(GA) conjugates and pre-aggregated carrier-free (GA) . Only ovalbumin-(GA) immunization induced a strong anti-GA response. The resulting antisera detected poly-GA aggregates in cell culture and patient tissue. Ovalbumin-(GA) immunization largely rescued the motor function in (GA) -CFP transgenic mice and reduced poly-GA inclusions. Transcriptome analysis showed less neuroinflammation in ovalbumin-(GA) -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.
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http://dx.doi.org/10.15252/emmm.201910919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005532PMC
February 2020

sp. nov., a novel species of the complex, occupying a basal position to North American species.

Int J Syst Evol Microbiol 2020 Feb;70(2):849-856

University of California, Department of Environmental Science, Policy and Management, Berkeley, CA, USA.

species are vector-borne parasitic bacteria with unusual, highly fragmented genomes that include a linear chromosome and linear as well as circular plasmids that differ numerically between and within various species. Strain CA690, which was cultivated from a questing nymph in the San Francisco Bay area, CA, was determined to be genetically distinct from all other described species belonging to the complex. The genome, including plasmids, was assembled using a hybrid assembly of short Illumina reads and long reads obtained via Oxford Nanopore Technology. We found that strain CA690 has a main linear chromosome containing 902176 bp with a blast identity ≤91 % compared with other species chromosomes and five linear and two circular plasmids. A phylogeny based on 37 single-copy genes of the main linear chromosome and rooted with the relapsing fever species strain Ly revealed that strain CA690 had a sister-group relationship with, and occupied a basal position to, species occurring in North America. We propose to name this species sp. nov. The type strain, CA690, has been deposited in two national culture collections, DSMZ (=107169) and ATCC (=TSD-160).
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http://dx.doi.org/10.1099/ijsem.0.003833DOI Listing
February 2020

Sex-specific programming effects of parental obesity in pre-implantation embryonic development.

Int J Obes (Lond) 2020 05 27;44(5):1185-1190. Epub 2019 Nov 27.

Institute of Molecular Animal Breeding and Biotechnology, Gene Center, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377, Munich, Germany.

Background: Obesity is a global rising problem with epidemiological dimension. Obese parents can have programming effects on their offspring leading to obesity and associated diseases in later life. This constitutes a vicious circle. Epidemiological data and studies in rodents demonstrated differential programming effects in male and female offspring, but the timing of their developmental origin is not known.

Methods: This study investigated if sex-specific programming effects of parental obesity can already be detected in the pre-implantation period. Diet-induced obese male or female mice were mated with normal-weight partners and blastocysts were recovered.

Results: Gene expression profiling revealed sex-specific responses of the blastocyst transcriptome to maternal and paternal obesity. The changes in the transcriptome of male blastocysts were more pronounced than those of female blastocysts, with a stronger impact of paternal than of maternal obesity. The sperm of obese mice revealed an increased abundance of several miRNAs compared with lean mice.

Conclusions: Our study indicates that sex-specific programming effects of parental obesity already start in the pre-implantation period and reveals specific alterations of the sperm miRNA profile as mechanistic link to programming effects of paternal obesity.
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http://dx.doi.org/10.1038/s41366-019-0494-xDOI Listing
May 2020

Temporal trends in intracerebral hemorrhage: Evidence from the Austrian Stroke Unit Registry.

PLoS One 2019 20;14(11):e0225378. Epub 2019 Nov 20.

Department of Neurology, St. John's Hospital, Vienna, Austria.

Background: To assess changes in frequency, severity, complications, therapy and outcome of intracerebral hemorrhage in patients treated in stroke units in Austria, we evaluated data from the Austrian Stroke Unit Registry between 2008 and 2016.

Methods And Findings: Data of 6707 cases of ICH covering a time span of 9 years and including information on age, risk factors, pre-stroke modified Rankin Score (mRS), baseline stroke severity (NIHSS), complications, therapy, functional outcome, and mortality were extracted from the Austrian Stroke Unit Registry. A multivariate regularized logistic regression model and linear models for temporal dependence were computed for analyzing statistical inference and time trends. Bonferroni correction was applied to correct for multiple testing. Between 2008 and 2016, the proportion of ICH admissions to stroke units in Austria declined, with a shift among patients towards older age (>70 years, p = 0.04) and lower admission NIHSS scores. While no significant time trends in risk factors, pre-stroke mRS and medical complications were observed, therapeutic interventions declined significantly (p<0.001). Three-month mortality increased over the years independently (p = 0.003).

Conclusions: Despite declining incidence and clinical severity of ICH we observed a clear increase in three-month mortality. This effect seems to be independent of predictors including age, admission NIHSS, pre-morbid MRS, or medical complications. The observations from this large retrospective database cohort study underline an urgent call for action in the therapy of ICH.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0225378PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6867701PMC
March 2020

Absolute nucleosome occupancy map for the genome.

Genome Res 2019 12 6;29(12):1996-2009. Epub 2019 Nov 6.

Molecular Biology Division, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians-Universität München, 82152 Planegg-Martinsried, Germany.

Mapping of nucleosomes, the basic DNA packaging unit in eukaryotes, is fundamental for understanding genome regulation because nucleosomes modulate DNA access by their positioning along the genome. A cell-population nucleosome map requires two observables: nucleosome positions along the DNA ("Where?") and nucleosome occupancies across the population ("In how many cells?"). All available genome-wide nucleosome mapping techniques are yield methods because they score either nucleosomal (e.g., MNase-seq, chemical cleavage-seq) or nonnucleosomal (e.g., ATAC-seq) DNA but lose track of the total DNA population for each genomic region. Therefore, they only provide nucleosome positions and maybe compare relative occupancies between positions, but cannot measure absolute nucleosome occupancy, which is the fraction of all DNA molecules occupied at a given position and time by a nucleosome. Here, we established two orthogonal and thereby cross-validating approaches to measure absolute nucleosome occupancy across the genome via restriction enzymes and DNA methyltransferases. The resulting high-resolution (9-bp) map shows uniform absolute occupancies. Most nucleosome positions are occupied in most cells: 97% of all nucleosomes called by chemical cleavage-seq have a mean absolute occupancy of 90 ± 6% (±SD). Depending on nucleosome position calling procedures, there are 57,000 to 60,000 nucleosomes per yeast cell. The few low absolute occupancy nucleosomes do not correlate with highly transcribed gene bodies, but correlate with increased presence of the nucleosome-evicting chromatin structure remodeling (RSC) complex, and are enriched upstream of highly transcribed or regulated genes. Our work provides a quantitative method and reference frame in absolute terms for future chromatin studies.
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http://dx.doi.org/10.1101/gr.253419.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886505PMC
December 2019