Analysis of differential gene regulation in porcine cell cultures after infection with porcine Circovirus type 1 and type 2

Deckblatt-Impressum persönlicher Dank Inhaltsverzeichnis Abkürzungsverzeichnis Einleitung Material und Methoden Ergebnisse Diskussion Zusammenfassung Summary Anhang Literatur Danksagung Lebenslauf SelbständigkeitserklärungDiese Studie befasst sich mit der Untersuchung der porcinen Circoviren Typ 1 (PCV1) und Typ 2 (PCV2), die auf Nukleinsäure- und Proteinebene eine Homologie zwischen 60% und 80% besitzen. Trotz dieser hohen Übereinstimmungen verfügen diese Viren über eine distinkte Pathogenität: PCV1 erwies sich als apathogen, wohingegen PCV2 als auslösendes Agens des PMW Syndroms charakterisiert wurde. PMWS ist eine Erkrankung des Schweins, die mit mangelnder Gewichtszunahme, Atemschwierigkeiten, Ikterus, Lymphadenopathie und Lymphozytendepletion einhergeht. Die unterschiedliche Pathogenität der beiden Virustypen sollte sich in einem abweichenden Transkriptionsmuster in verschiedenen Zellkulturlinien nach Infektion mit PCV1 oder PCV2 widerspiegeln. Die vorliegende Arbeit charakterisiert differentiell regulierte Gene, deren Beteiligung bei der Auslösung des Postweaning Multisystemic Wasting Syndrome (PMWS) nach Infektion mit PCV in Betracht gezogen werden sollte. Mit dem Differential Display Verfahren wurden differentiell regulierte porcine Transkriptfragmente amplifiziert, die mittels BLAST-Vergleich charakterisierten Genen zugeordnet wurden. Die Transkripte wurden durch einen Northern Blot analysiert und mittels SYBR Green und TaqMan® real-time RT-PCR in infizierten und nicht-infizierten porcinen Zellen quantifiziert. Die Durchflusszytometrie korrelierte die Transkriptkonzentration mit der tatsächlichen Proteinexpression. Die untersuchten Zelllinien (L23, L35, L52, PS, PK15, WSH, 293) wiesen viele Gene auf, die nach Infektion mit PCV1 und PCV2 einer virusbedingten Hoch- oder Herabregulation unterlagen und sowohl vom jeweiligen Zelltyp als auch von der Infektionsdauer abhängig waren. Diese ließen sich anhand ihrer Funktion in Gruppen wie z.B. der Immunantwort einteilen. Zu dieser Gruppe zählen das Cytokin Interleukin 18 (IL18) und der Haupthistokompatibilitätskomplex Klasse I (MHC I). Eine veränderte Transkriptionsaktivität konnte weiterhin für Vesikel- und Membran-assoziierte Proteine wie EHD3 und Lyncein festgestellt werden; dies gilt auch für Transkriptions- und Translationsfaktoren wie Caspase3, DAP5/eIF42 (Death associated protein5/Elongations Initiations Faktor 4gamma2), NSAP1 (NS1 associated protein) und StIP1 (STAT3 interacting protein1). Ein möglicher PMWS-assoziierter Faktor könnte das Fragment 40J darstellen, ein bislang unbeschriebenes, ausschließlich in Lymphozyten transkribiertes Gen, dessen Transkription vor allem durch PCV2-Infektion gesteigert wurde. In der vorliegenden Studie wurde nicht ein einzelner Faktor identifiziert, der als der alleinige Auslöser von PMWS betrachtet werden könnte, sondern viele Faktoren, die möglicherweise zur Ausprägung dieser Krankheit beitragen. Dieser Befund wird auch durch Feldstudien unterstützt, die zeigten, dass PMWS eine multifaktorielle Erkrankung ist, die neben der Infektion mit PCV2 auch eine Aktivierung des Immunsystems voraussetzt. Diese Arbeit macht deutlich, dass der Interaktion von PCV2 mit dem Immunsystem vermehrte Aufmerksamkeit geschenkt werden sollte. Des Weiteren liegen diese identifizierten Faktoren im Säugetier hoch konserviert vor und könnten somit für die Xenotransplantation im Falle einer Zoonose nach einer PCV Infektion relevant sein.Porcine Circovirus type 1 and type 2 are closely related and share homologies on nucleotide and protein level between 60% and 80%. In spite of these high consistencies circoviruses differ in their pathogenicity. While PCV1 is a non- pathogenic virus, PCV2 is the etiological agent for the postweaning multisystemic wasting syndrome (PMWS) in young piglets. The aim of this study was to characterise viral and cellular genes that are involved in the initiation of the PMWS after infection. Cells infected either with PCV1 or PCV2 should differ in their transcription profile of genes that respond to the infection. The formation of viral transcripts or proteins might therefore correlate with the regulation of cellular genes. Using Differential Display analysis differentially regulated cellular transcripts were identified through sequencing and comparison with the public database using the BLAST-algorithm. The transcripts were further characterised in northern blot analysis and absolute transcript concentrations were measured with SYBR Green and TaqMan® real-time PCR in infected and non-infected samples. Flowcytometry analysis compared the transcription level with protein expression. Different tested cell lines (L23, L35, L52, PS, PK15, WSH, 293) displayed a distinct regulation of several genes after infection with porcine Circoviruses. These genes were divided into functional groups e.g. genes that are immune activated after viral infection like the cytokine Interleukin 18 (IL18) and the major histocompatibility complex class I (MHC I). A virus induced modulation of vesicle- and membrane-associated proteins like EHD3 and Lyncein was observed as well as a viral effect on transcription and translation factors like caspase3, DAP5/eIF42 (Death associated protein5/elongation initiation factor 4gamma2), NSAP1 (NS1 associated protein) and StIP1 (STAT3 interacting protein1). One PMWS relevant factor could be the new identified fragment 40J, a gene that was transcribed only in lymphocytes and showed up-regulation mainly after PCV2 infection. Concerning the different pathogenicity between PCV1 and PCV2, all investigated genes showed either up- or down-regulation after infection dependent from the cell line. No single gene could be characterised as causative factor of PMWS, but some proteins of the viral influenced transcripts are related to each other and induce mediators that cause PMWS-like symptoms. The PMW Syndrome could therefore be initiated by a shifted regulation of many factors. The fact that these genes are highly conserved among mammals could be relevant for the xenotransplantation in case of a PCV infection in humans

Keeping track of worm trackers

C. elegans is used extensively as a model system in the neurosciences due to its well defined nervous system. However, the seeming simplicity of this nervous system in anatomical structure and neuronal connectivity, at least compared to higher animals, underlies a rich diversity of behaviors. The usefulness of the worm in genome-wide mutagenesis or RNAi screens, where thousands of strains are assessed for phenotype, emphasizes the need for computational methods for automated parameterization of generated behaviors. In addition, behaviors can be modulated upon external cues like temperature, O2 and CO2 concentrations, mechanosensory and chemosensory inputs. Different machine vision tools have been developed to aid researchers in their efforts to inventory and characterize defined behavioral “outputs”. Here we aim at providing an overview of different worm-tracking packages or video analysis tools designed to quantify different aspects of locomotion such as the occurrence of directional changes (turns, omega bends), curvature of the sinusoidal shape (amplitude, body bend angles) and velocity (speed, backward or forward movement)

Levetiracetam versus Phenobarbital for Neonatal Seizures: A Retrospective Cohort Study

BACKGROUND Although phenobarbital (PB) is commonly used as a first-line antiseizure medication (ASM) for neonatal seizures, in 2015 we chose to replace it with levetiracetam (LEV), a third-generation ASM. Here, we compared the safety and efficacy of LEV and PB as first-line ASM, considering the years before and after modifying our treatment protocol. METHODS We conducted a retrospective cohort study of 108 neonates with electroencephalography (EEG)-confirmed seizures treated with first-line LEV or PB in 2012 to 2020. RESULTS First-line ASM was LEV in 33 (31%) and PB in 75 (69%) neonates. The etiology included acute symptomatic seizures in 69% of cases (30% hypoxic-ischemic encephalopathy, 32% structural vascular, 6% infectious, otherwise metabolic) and neonatal epilepsy in 22% (5% structural due to brain malformation, 17% genetic). Forty-two of 108 (39%) neonates reached seizure freedom following first-line therapy. Treatment response did not vary by first-line ASM among all neonates, those with acute symptomatic seizures, or those with neonatal-onset epilepsy. Treatment response was lowest for neonates with a higher seizure frequency, particularly for those with status epilepticus versus rare seizures (P < 0.001), irrespective of gestational age, etiology, or EEG findings. Adverse events were noted in 22 neonates treated with PB and in only one treated with LEV (P < 0.001). CONCLUSIONS Our study suggests a potential noninferiority and a more acceptable safety profile for LEV, which may thus be a reasonable option as first-line ASM for neonatal seizures in place of PB. Treatment should be initiated as early as possible since higher seizure frequencies predispose to less favorable responses

A seed-like proteome in oil-rich tubers

There are numerous examples of plant organs or developmental stages that are desiccation-tolerant and can withstand extended periods of severe water loss. One prime example are seeds and pollen of many spermatophytes. However, in some plants, also vegetative organs can be desiccation-tolerant. One example are the tubers of yellow nutsedge (Cyperus esculentus), which also store large amounts of lipids similar to seeds. Interestingly, the closest known relative, purple nutsedge (Cyperus rotundus), generates tubers that do not accumulate oil and are not desiccation-tolerant. We generated nanoLC-MS/MS-based proteomes of yellow nutsedge in five replicates of four stages of tuber development and compared them to the proteomes of roots and leaves, yielding 2257 distinct protein groups. Our data reveal a striking upregulation of hallmark proteins of seeds in the tubers. A deeper comparison to the tuber proteome of the close relative purple nutsedge (C. rotundus) and a previously published proteome of Arabidopsis seeds and seedlings indicates that indeed a seed-like proteome was found in yellow but not purple nutsedge. This was further supported by an analysis of the proteome of a lipid droplet-enriched fraction of yellow nutsedge, which also displayed seed-like characteristics. One reason for the differences between the two nutsedge species might be the expression of certain transcription factors homologous to ABSCISIC ACID INSENSITIVE3, WRINKLED1, and LEAFY COTYLEDON1 that drive gene expression in Arabidopsis seed embryos

Postoperative complications are associated with long-term changes in the gut microbiota following colorectal cancer surgery

Changes in the gut microbiome have already been associated with postoperative complications in major abdominal surgery. However, it is still unclear whether these changes are transient or a long-lasting effect. Therefore, the aim of this prospective clinical pilot study was to examine long-term changes in the gut microbiota and to correlate these changes with the clinical course of the patient. Methods: In total, stool samples of 62 newly diagnosed colorectal cancer patients undergoing primary tumor resection were analyzed by 16S-rDNA next-generation sequencing. Stool samples were collected preoperatively in order to determine the gut microbiome at baseline as well as at 6, 12, and 24 months thereafter to observe longitudinal changes. Postoperatively, the study patients were separated into two groups-patients who suffered from postoperative complications

High β-1,4-Galactosyltransferase-I expression in peripheral T-lymphocytes is associated with a low risk of relapse in germ-cell cancer patients receiving high-dose chemotherapy with autologous stem cell reinfusion.

Survival of patients with germ-cell cancer (GCC) and primary progression or relapse after cisplatin-based first-line chemotherapy is highly heterogeneous, ranging from close to zero to more than 70%. We investigated β-1,4-Galactosyltransferase-I () expression levels in peripheral lymphocytes in a cohort of 46 testicular cancer patients. enhances immune cell crosstalk via glycosylation of surface molecules. A high expression level of in T-lymphocytes, but not in monocytes, was associated with a lower risk of relapse with a hazard ratio (HR) of 0.66 (95% confidence interval (CI) of HR: 0.45-0.97; = 0.02) upon multivariate Cox regression analysis. Correspondingly, interleukin 10 (IL10), a cytokine released by cytotoxic T-cells, was likewise significantly elevated in T-lymphocytes of non-relapse GCC patients (HR: 0.3; 95% CI of HR: 0.14-0.65; = 0.002). Our data indicate that glycosylation and activation of T-lymphocytes may play a pivotal role in disease control in GCC patients with primary progressive or relapsed disease

Extraordinary rocks from the peak ring of the Chicxulub impact crater: P-wave velocity, density, and porosity measurements from IODP/ICDP Expedition 364

Joint International Ocean Discovery Program and International Continental Scientific Drilling Program Expedition 364 drilled into the peak ring of the Chicxulub impact crater. We present P-wave velocity, density, and porosity measurements from Hole M0077A that reveal unusual physical properties of the peak-ring rocks. Across the boundary between post-impact sedimentary rock and suevite (impact melt-bearing breccia) we measure a sharp decrease in velocity and density, and an increase in porosity. Velocity, density, and porosity values for the suevite are 2900–3700 m/s, 2.06–2.37 g/cm3, and 20–35%, respectively. The thin (25 m) impact melt rock unit below the suevite has velocity measurements of 3650–4350 m/s, density measurements of 2.26–2.37 g/cm3, and porosity measurements of 19–22%. We associate the low velocity, low density, and high porosity of suevite and impact melt rock with rapid emplacement, hydrothermal alteration products, and observations of pore space, vugs, and vesicles. The uplifted granitic peak ring materials have values of 4000–4200 m/s, 2.39–2.44 g/cm3, and 8–13% for velocity, density, and porosity, respectively; these values differ significantly from typical unaltered granite which has higher velocity and density, and lower porosity. The majority of Hole M0077A peak-ring velocity, density, and porosity measurements indicate considerable rock damage, and are consistent with numerical model predictions for peak-ring formation where the lithologies present within the peak ring represent some of the most shocked and damaged rocks in an impact basin. We integrate our results with previous seismic datasets to map the suevite near the borehole. We map suevite below the Paleogene sedimentary rock in the annular trough, on the peak ring, and in the central basin, implying that, post impact, suevite covered the entire floor of the impact basin. Suevite thickness is 100–165 m on the top of the peak ring but 200 m in the central basin, suggesting that suevite flowed downslope from the collapsing central uplift during and after peak-ring formation, accumulating preferentially within the central basin

Cohesin SMC1β protects telomeres in meiocytes

Telomeres fail to attach to the nuclear envelope and lose structural integrity in cells lacking SMC1β