Synthese und enzymatische Tests von reversiblen Terminatoren für die Sequenzierung durch Synthese (SBS)

Based on the commonly used and well-established state-of-the-art DNA sequencing method, i. e. Sanger sequencing, the major target of future research is to develop a fast, cost-effective and gelelectrophoresis-free sequencing method. The aim of the new sequencing technologies is to detect DNA mutations faster and more accurate in order to develop individual therapies for patients (personalized medicine). For this purpose, a lot of novel sequencing techniques like pyrosequencing, mass-spectrometry-assisted sequencing, sequencing by hybridization etc. have been put into practice and already led to commercialized sequencers. The sequencing technology we were mostly interested in is the so-called sequencing-by-synthesis method (SBS). This PhD thesis covers the synthesis of modified nucleosides – the so-called reversible terminators – and their evaluation as reversible terminators. These 3′-modified and dye-labeled nucleotides are incorporated by the polymerase into the DNA-template, then the DNA-synthesis is stopped. After detection of the fluorescent signal, the reversible terminator has to be cleavable in a way (i. e. the polymerase-blocking modification) that the DNA-synthesis can continue. As a result of the polymerase-acceptance tests that have been carried out with the two triphosphates cyanoethoxymethyl(CEM)-dTTP and cyanoethyl(CE)-dTTP as substrates it became clear that the latter one was better incorporated than the first one. Based on this knowledge all four key compounds for the whole reversible terminators possessing the cyanoethyl (CE) group where synthesized within this PhD thesis. Additionally to the synthesis of the modified key compounds, the cleavability of the cyanoethyl function had to be evaluated which is an essential requirement of a reversible terminator for SBS. For addressing this issue, three different CE- and CEM modified monophosphates were created. For each of these three monophosphates an individual synthetic strategy has been developed within this PhD work, each of these strategies and subsequent phosphorylation led to the desired modification. These previously unknown model compounds mimicking the solubility of short oligonucleotides were employed the for qualitative cleavage experiments after their purification and spectroscopical characterization. With these three monophosphates suitable cleavage conditions for a quantitative removal of the CE and the CEM group were examined. In case of the CE function we selectively improved the cleavage conditions while varying the solvent, the reaction temperature as well as the amount of cleaving agent used, in order to make the conditions applicable for an SBS experiment. Due to the fact that the CE function was the most important modification for our SBS experiment, we could even optimize the cleavage efficiency by employing co-solvents like DMSO or DMF. An additional cleavage experiment was carried out by using a short CE-modified oligomer which led to further results that were comparable to the ones obtained from the cleavage experiments of the monomers. One big difference is the required amount of TBAF as cleaving agent for the quantitative removal of the CE-modification from the oligomer. In this case, 7500 equivalents of TBAF are needed for complete CE cleavage at 45 °C compared to the amount of 40 to 80 equivalents TBAF for the monomer (monophosphate). As a conclusion of this result we assume that the amount of cleaving agent and the solubility of the oligomer plays an important role in the CE cleavage efficiency. This assumption was already supported by Saneyoshi et al. who demonstrated for CE-modified RNA oligonucleotides that the CE-cleavage rate is strongly lowered with the increasing of the oligomer length. Thus we could demonstrate that the CE function is quantitatively removable from an oligomer without destroying it. With these results in hands we could prove that the CEM and the CE group are quantitatively cleavable and therefore applicable as blocking groups for reversible terminators. The conditions for the CE cleavage are used for the ArraySBS-“proof-of-principle” which is currently under investigation.Basierend auf der weitverbreiteten und etablierten DNA-Sequenzierungs-methode der Wahl, i. e. Sanger-Sequenzierung, ist die Entwicklung neuartiger Methoden im Sinne eines schnelleren, gelelektrophoresefreiem und kostengünstigeren Sequenzierungsverfahren derzeit eine große Herausforderung in der Forschung. Die Zielsetzung dieser neuartigen Sequenzierungsverfahren ist es, schneller und präziser genbasierte Krankheiten detektieren und daraus resultierend individuelle Therapien für den Menschen entwickeln zu können. Hierzu wurden schon grundlegende und neuartige Sequenzierungsprinzipien, wie z. B. Pyrosequenzierung, Massenspektrometrie-basiertes Sequenzieren, Sequenzieren durch Hybridisierung etc. praktisch umgesetzt, d. h. neuartige Sequenzierungsgeräte werden heute schon kommerziell vermarktet. Ein für uns besonders interessantes Sequenzierungsverfahren der derzeit erforschten ist „sequencing-by-synthesis“, zu Deutsch die Sequenzierung durch Synthese (kurz SBS). Im Rahmen dieser Doktorarbeit wurden modifizierte Nukleoside - sogenannte reversible Terminatoren – für die Sequenzierung-durch-Synthese hergestellt und auf ihre Eigenschaft als Terminatoren bei der Polymerase-Reaktion geprüft. Reversible Terminatoren sind für die SBS-Methode unabdingbar: Es handelt sich hierbei um modifizierte Nukleotide in Form von farbstoffgelabelten Triphosphaten, die von einer Polymerase eingebaut werden und die DNA-Synthese temporär stoppen. Im selben Maße muss dieser Stopp aber reversibel sein, d. h. der Terminator muss derart spaltbar sein (in diesem Falle an der blockierenden Gruppe), dass er anschließend nach Detektion des Farbstoffes wieder die weitere DNA-Synthese erlaubt. In den Polymerase-Akzeptanztests mit den 3′-modifizierten Nukleosiden, die im Rahmen eines EU-Projektes sowohl jeweils mit cyanoethoxymethyl(CEM)-dTTP als auch mit cyanoethyl(CE)-dTTP als Substrat durchgeführt wurden, zeigte sich, dass letzteres der beiden Nukleotide von der Polymerase besser eingebaut wurde als ersteres. Auf diesem Wissen basierend wurden alle vier Schlüsselverbindungen der reversiblen Terminatoren, welche die Cyanoethyl-Gruppe besitzen, im Rahmen dieser Doktorarbeit synthetisiert. Darüber hinaus war auch die quantitative Spaltbarkeit der CE- und der CEM-Funktion zu prüfen. Dazu wurden im Rahmen dieser Arbeit modifizierte Monophosphate über eine jeweilige individuelle Synthesestrategie hergestellt. Diese bislang unbekannten Modellverbindungen, welche die Löslichkeit kurzer Oligonukleotide nachahmen sollten, wurden nach ihrer Synthese und spektroskopischer Charakterisierung für qualitative Spaltungsexperimente eingesetzt. Somit wurden geeignete Spaltungsbedingungen für die Cyanoethoxymethyl (CEM)- als auch für die Cyanoethyl (CE)-Funktion gefunden. Für die Cyanoethyl-Gruppe wurden die Spaltungsbedingungen gezielt optimiert, indem das Lösungsmittel, die Reaktionstemperatur sowie die eingesetzte Menge an Spaltungsreagenz variiert wurde, um die Bedingungen für ein SBS-Experiment anwendbar zu machen. Ein zusätzliches Cyanoethyl-Spaltungsexperiment, das mit einem kurzen Oligomer durchgeführt wurde, lieferte weitere Resultate, die mit den aus den Spaltungstests der Monomere gewonnenen übereinstimmen. Ein großer Unterschied zwischen den Monomeren und dem Oligomer bezüglich der Spaltungseffizienz ist die benötigte Menge an Spaltungsreagenz. Im Falle des Oligomers werden 7500 Äquivalente benötigt, um die Cyanoethyl-Gruppe bei 45 °C vollständig zu entfernen. Im Gegensatz dazu werden im Falle des Monophosphats nur 40 bis 80 Äquivalente benötigt. Basierend auf dieser Beobachtung kann man annehmen, dass sowohl die Löslichkeit des Oligomers als auch die TBAF-Konzentration eine entscheidende Rolle in der Effizienz der CE-Spaltung spielt. Dies wurde auch schon von Saneyoshi et al. demonstriert, der postulierte, dass im Falle der RNA die Abspaltung der Cyanoethyl-Gruppe in Abhängigkeit von der Größe des Oligomers verzögert wird. Wir haben demnach zeigen können, dass die CE-Gruppe mit TBAF in THF quantitativ abspaltbar ist, ohne dass das Oligonukleotid zerstört wird. Darüber hinaus wurde die Spaltbarkeit und damit die Reversibilität der CEM- und der CE-Gruppe als blockierende Gruppen bestätigt. Die Bedingungen aus dem CE-Oligomer-Spaltungstest werden derzeit im SBS-„Proof-of-Principle“, welches mit Hairpin-Templaten auf einem DNA-Chip Array durchgeführt wird, angewandt und optimiert

Atrial Natriuretic Peptide, a Regulator of Nuclear Factor-κB Activation in Vivo

Natriuretic peptides (NPs) comprise a family of vasoactive hormones that play important roles in the regulation of cardiovascular and renal homeostasis. Along this line, atrial NP (ANP) (international non-proprietary name: carperitide, HANP) is an approved drug for the treatment of acute heart failure. In recent years, evidence has been given that the NP system possesses a far broader biological spectrum than the regulation of blood pressure and volume homeostasis. In fact, a substantial amount of in vitro work indicates that ANP affects important inflammatory processes and signaling pathways. Quite surprisingly, however, no information exists on the in vivo antiinflammatory potential and signaling of ANP. We show here that pretreatment of lipopolysaccharide (Salmonella abortus equi, 2.5 mg/kg)-challenged mice with ANP (5μg/kg iv, 15 min) rapidly inhibits nuclear factor-κB activation via inhibition of phosphorylation and degradation of the IκB-α protein. ANP also reduces Akt activation upon lipopolysaccharide injection. In ANP-pretreated mice, the increase of TNF-α serum concentration is markedly prevented; most importantly, the survival of these animals improved. These findings demonstrate both in vitro and in vivo an antiinflammatory profile of ANP that deserves to be further investigated in a therapeutic perspective

Comparing costs for different conservation strategies of garlic (Allium sativum L.) germplasm in genebanks

The maintenance of plant genetic resources in living plant collections (genebanks) causes costs due to employment of staff, usage of buildings, equipment and consumables. Since this is especially challenging in vegetatively propagated material, studies were performed for the case of garlic, which is one of the major vegetatively maintained crops in the genebank of IPK Gatersleben. Data were recorded to compare various scenarios of the main strategies field maintenance and cryopreservation. A spreadsheet tool was developed to be used for cost assessment and for drawing conclusions concerning the most effective way of maintenance. Field culture is cheaper in the short term, whereas after a break-even point cryopreservation becomes the more efficient storage method in the long term. This break-even point depends on the particular scenario, which is determined by various factors such as field and in vitro multiplication rates of various genotypes, presence of bulbils in a part of the genepool, the sample size of the accessions as well as the number of stored accessions in cryopreservation. The comparative discussion is exemplified for a 1-year field rotation versus cryopreservation using either in vitro plantlets or a combination of bulbils and unripe inflorescence bases as organ sources. For the more expensive use of in vitro plants cryopreservation becomes less costly than field culture only after 13 years, whereas this is the case already after 8-9 years when using a combination of bulbils in winter and inflorescence bases in summer

„Energie-Kinder“ lassen Drachen steigen

Wie aus dem Satz „Manchmal habe ich zu viel Energie!“ das Projekt vom „Energie-Kind“ entstand. Drei Ergotherapiestudentinnen der ZHAW starteten im Oktober 2020 im Rahmen der Projektwerkstatt ein besonderes Unterfangen: ein Projekt mit der Schule Schachen, welches die Schüler und Schülerinnen einer 2. Klasse maßgebend mitgestalten konnten

New charge tranfer salts containing BEDT-TTF and structurally related transition metal complexes

The two-dimensional behaviour of BEDT-TTF salts is normally explained with the typical geometry of this donor and its arrangement in the crystal lattice. In order to extend the range of physical properties produced by BEDT-TIF like compounds, we synthesized sulfur containing transition metal complexes with a molecular structure very similar to BEDT-TTF. The bis(5,6-dihydro-1,4-dithiin-2,3-dithiolate)metallates of the nickel triad can be prepared in different oxidation states as anions and in a fully oxidized neutral form

11C-acetate PET in the early evaluation of prostate cancer recurrence

Purpose: The first aim of the study was to investigate the diagnostic potential of 11C-acetate PET in the early detection of prostate cancer recurrence. A second aim was the evaluation of early and late PET in this context. Methods: The study population comprised 32 prostate cancer patients with early evidence of relapse after initial radiotherapy (group A) or radical surgery (group B). The median PSA of group A (n=17) patients was 6ng/ml (range 2.6-30.2) while that of group B (n=15) was 0.4ng/ml (range 0.08-4.8). Pelvic-abdominal-thoracic PET was started 2min after injection of 11C-acetate and evaluated after fusion with CT. Results: Group A: Taking a SUVmax≥2 as the cut-off, PET showed local recurrences in 14/17 patients and two equivocal results. Distant disease was observed in six patients and an equivocal result was obtained in one. Endorectal MRI was positive in 12/12 patients. Biopsy confirmed local recurrence in six of six (100%) patients. PET was positive in five of the six patients with biopsy-proven recurrences, the result in the remaining patient being equivocal. Group B: Among the 15 patients, visual interpretation was positive for local recurrences in five patients and equivocal in four. One obturator lymph node was positive. Endorectal MRI was positive in 11/15 patients and equivocal in two. Positional correlation of positive/equivocal results on PET and endorectal MRI was observed in seven of nine patients. PSA decreased significantly after salvage radiotherapy in 8/14 patients, providing strong evidence for local recurrence. PET of the eight patients responding to RT was positive in three and equivocal in two. Conclusion: 11C-acetate PET was found to be valuable in the early evaluation of prostate cancer relapse. Optimising scanning time and use of modern PET-CT equipment might allow further improvemen

Preparation, structure and physical properties of BEDT-TTF nitrates

Electrocrystallization of BEDT-TIF in THF with tetrabutylammonium nitrate as supporting electrolyte leads to the deposition of at least three different phases. Sheets of BEDT-TTF radical cations with short intermolecular S···S contacts (>3.315(4) A) are separated by anion layers. Two more BEDT-TrF nitrates have been characterized by their unit cell data. Resuhs of temperature dependent electrical conductivity and thermopower measurements on crystals of the a-phase are presented. They are metallic down to about 30 K

Glycolytic flux control by drugging phosphoglycolate phosphatase

Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates

First Experience and the Effectiveness of Immunomodulating Treatment in Inflammatory Demyelinating CNS Diseases : Analysis of Nine Patients

Publisher Copyright: © 2015 by Lana Vainšteine.Therapeutic plasma exchange (TPE) is used in many neurological disorders to remove immunoglobulin and other immunologically active substances. We observed patients that were admitted in Riga East Clinical University Hospital "Gaiezers", Clinic of Neurology and Neurosurgery, Multiple Sclerosis Unit, and were diagnosed with relapsing remitting multiple sclerosis (MS), according to McDonald criteria 2010 (five patients), Neuromyelitis optica (NMO) spectrum disorders (three patients) and one with NMO, according to Wingerchuk 2006 criteria. All relapses were confirmed according to clinical criteria. Visual acuity was assessed by an ophthalmologist, and neurological status by a neurologist. All patients received at least 1 cycle of 1000 mg methylprednisolone intravenous for five to seven days. The expanded disability status scale score in the MS patient group was in range 4.0-9.0 before TPE and 3.5-6.5 range after TPE. Best improvement was observed in the MS group: mean symptom reduction of 20%. Patients with NMO spectrum disorder had an EDSS score of 8.0-8.5 range on admission and 6.5-8.0 range after TPE. After one month, one patient in the NMO spectrum disorder group had good response to TPE and EDSS was 3.5, two patients had only slight improvement (EDSS scores 8.0 and 7.5). Condition of patients with NMO did not improve even after a month.publishersversionPeer reviewe

Diagnostic discrimination of a novel high-sensitivity cardiac troponin I assay and derivation/validation of an assay-specific 0/1h-algorithm

BACKGROUND We aimed to assess the diagnostic utility of the Dimension EXL LOCI High-Sensitivity Troponin I (hs-cTnI-EXL) assay. METHODS This multicenter study included patients with chest discomfort presenting to the emergency department. Diagnoses were centrally and independently adjudicated by two cardiologists using all available clinical information. Adjudication was performed twice including serial measurements of high-sensitivity cardiac troponin (hs-cTn) I-Architect (primary analysis) and serial measurements of hs-cTnT-Elecsys (secondary analysis) in addition to the clinically used (hs)-cTn. The primary objective was to assess and compare the discriminatory performance of hs-cTnI-EXL, hs-cTnI-Architect and hs-cTnT-Elecsys for acute myocardial infarction (MI). Furthermore, we derived and validated a hs-cTnI-EXL-specific 0/1h-algorithm. RESULTS Adjudicated MI was the diagnosis in 204/1454 (14%) patients. The area under the receiver operating characteristics curve for hs-cTnI-EXL was 0.94 (95%CI, 0.93-0.96), and comparable to hs-cTnI-Architect (0.95; 95%CI, 0.93-0.96) and hs-cTnT-Elecsys (0.93; 95%CI, 0.91-0.95). In the derivation cohort (n = 813), optimal criteria for rule-out of MI were 3h) or <9ng/L and 0h-1h-change <5ng/L, and for rule-in ≥160ng/L at presentation or 0h-1h-change ≥100ng/L. In the validation cohort (n = 345), these cut-offs ruled-out 56% of patients (negative predictive value 99.5% (95%CI, 97.1-99.9), sensitivity 97.8% (95%CI, 88.7-99.6)), and ruled-in 9% (positive predictive value 83.3% (95%CI, 66.4-92.7), specificity 98.3% (95%CI, 96.1-99.3)). Secondary analyses using adjudication based on hs-cTnT measurements confirmed the findings. CONCLUSIONS The overall performance of the hs-cTnI-EXL was comparable to best-validated hs-cTnT/I assays and an assay-specific 0/1h-algorithm safely rules out and accurately rules in acute MI. CLINICAL TRIAL REGISTRATION ClinicalTrials.gov number, NCT00470587