Differenzierung von Weizensorten basierend auf Proteinmustern mittels Kapillargelelektrophorese-am-Chip

Im Rahmen dieser Diplomarbeit sollte die Übertragbarkeit der SDS-PAGE Methodik von Weizenextrakten zwecks Sortenerkennung und Homogenitätsprüfung auf die CGE-am-Chip Methode untersucht werden. Trotz der Miniaturisierung der Analysenstrecke sind HMW-GUE mittels CGE-am-Chip elektrophoretisch trenn- und detektierbar. Der Umstieg auf CGE-am-Chip ist jedoch nicht trivial, da sich das Migrationsmuster und die Migrationsreihenfolge der HMW-GUE bei beiden Methoden signifikant unterscheiden. Die Robustheit der Methode wurde untersucht – die Intra- und Interchipreproduzierbarkeit hat sich bezüglich der Migrationszeit als sehr hoch erwiesen. Die Signalintensität zeigt jedochkeine hohe Reproduzierbarkeit. Weizenextrakte sind auch nach vierwöchiger Lagerung bei 4°C mit Reproduzierbarkeiten im Bereich der Methodenreproduzierbarkeit unproblematisch vermessbar. Auch nach achtwöchiger Lagerung bei 4°C lässt sich für die meisten Proben ein aussagekräftiges CGE-am-Chip-Elektropherogramm erzeugen. Weizenextrakte können bis zu zwei Mal aufgetaut und wieder eingefroren werden, ohne dass sich negative Auswirkung auf die Vermessbarkeit mittels CGE-am-Chip zeigen. Um trotz der Veränderung des Migrationsmusters zweifelsfreie Zuordnungen der HMW-GUE-Banden zu erlauben, wurde versucht, mittels SDS-PAGE aufgetrennte Weizenproteine aus dem Gel zu extrahieren und erneut durch CGE-am-Chip zu vermessen. Die gewählten Extraktionsmethoden waren Schütteln in 5 unterschiedlichen Extraktionsmittel, Elektroelution und Elektroblott mit anschließender Extraktion von der Membran. Neben zwei unterschiedlichen SDS-PAGE-Gelzusammensetzungen wurde auch eine SDS-PAGE Technik, die teilweise aus einem hydrolytisch spaltbaren Quervernetzter besteht, untersucht inwieweit diese eine Extrahierbarkeit der Weizenproteine zulassen. Mit keiner der gewählten Methoden war es aber möglich, nachweisbare Mengen an HMW-GUE zu extrahieren – als zusätzliche Nachweismethoden wurden MALDI-TOF-MS, SDS-PAGE mit Silberfärbung und ein hochempfindlicher CGE-am-Chip-Assay mit vorangeschalteter Fluoreszenzderivatisierung gewählt. Dies hängt auch mit den speziellen physiko-chemischen Eigenschaften der HMW-GUE Protein zusammen. Zusätzlich wurde die Möglichkeit der CGE-am-Chip-Banden(Peak)zuordnung mittels statistischer Methoden untersucht. Zwei unüberwachte statistische Methoden (Hierarchische Clusteranalyse und PCA) und eine überwachte Methode (visuelle Zuordnung) wurden angewendet. Der zur Verfügung stehenden Datensatz (38 unterschiedliche Proben, 15 verschiedene Weizensorten, 9 unterschiedliche HMW-GUE-Sätze) war zu uneinheitlich, um mittels unüberwachten statistischen Methodeneine eindeutige Zuordnung treffen können. Durch Vermessen größerer bzw. einheitlicherer Datensätze könnte in Zukunft jedoch auch mittels dieser Strategie und den aufgezeigten Methoden eine Zuordnung gelingen. Mit der überwachten statistischen Methode ist die Zuordnung jedoch gelungen. Aus der visuellen Zuordnung ergeben sich einige überraschende Erkenntnisse für die CGE-am-Chip von Weizenextrakten. So liefern manche Allele nicht zwei, sondern drei Signale. Eine Aufspaltung der als 2* bezeichnete SDS-PAGE-Bande in zwei Untergruppen mit sehr unterschiedlichen Migrationszeiten wurde für zwei verschiedene Weizensorten beobachtet (jeweils nur eine der Varianten kommt bei einer Sorte vor). Für die Weizensorte Alora konnte ein zusätzliches Signal im CGE-am-Chip-Elektropherogramm beobachtet werden, das bisher noch nicht beschrieben war.The aim of this master thesis was to investigate whether the SDS-PAGE method for wheat grain extracts for variety and homogeneity control is transferable to the highthroughput CGE-on-a-chip method. Despite the miniaturization of the separating segment, HMW-GUE can be separated and detected in the lab-on-a-chip system. Due to changing migration patterns and migration sequences of the HMW-GUE, however, a change of method is nontrivial. The validity of the method was tested – the intra- and inter-chip reproducibility of the migration time is rather high, the signal intensity is however not very reproducible. After storage at 4°C for four weeks wheat extracts can still be measured with good reproducibility close to the method’s technical reproducibility. Even after storage at 4°C for eight weeks conclusive CGE-on-a-chip electropherograms could be obtained. Extracts of wheat can undergo up to two thaw-freeze cycles without causing degrading results obtained by means of the CGE-on-a-chip technique. In order to assign protein bands despite the changed migration sequence it was tried to extract wheat proteins which had been separated via SDS-PAGE from those gels and measure them on the chip. The chosen methods of extraction were shaking with five different eluants, electroelution and electroblotting with subsequent elution from the membrane. In addition to two different compositons of SDS-PAGE-gels, a SDS-PAGE-gel with partly hydrolytically cleavable crosslinker was investigated in terms of its extractability of wheat proteins. None of these methods allowed extracting detectable amounts of HMW-GUE. Additional methods of detection were MALDI-TOF-MS, SDS-PAGE with silver staining and a high sensitivity CGE-on-a-chip assay with direct fluorescence dye labeling. The difficulties are closely associated with the special physic-chemical quality of the HMW-GUE proteins. The possibilities of protein band assignment by means of statistical methods were examined, too. Two unsupervised statistical methods (hierarchical cluster analysis and PCA) and one supervised method (visual assignment) were evaluated. The available data set (38 different samples, 15 different wheat varieties, 9 different HMW-GUE-sets) were too heterogeneous in order to obtain a clear assignment through unsupervised statistical methods. Larger and more homogeneous data sets could however allow a wheat assignment through unsupervised statistical methods in the future.On the other hand an assignment was obtained successfully through a supervised statistical method (visual assignment). Based on this method some surprising results could be obtained for certain wheat protein bands. Some alleles showed not two, but three signals. Splitting of the SDS-PAGE band called 2* to two signals with very different migration times could be observed for two different wheat varieties (for each wheat variety, respectively, only one 2* signal). An additional signal could be observed for the CGE-on-a-chip electropherogram of the wheat variety Alora which has not been described so far

Prevention of SIV Rectal Transmission and Priming of T Cell Responses in Macaques after Local Pre-exposure Application of Tenofovir Gel

Martin Cranage and colleagues find that topical tenofovir gel can protect against rectal challenge with SIV in a macaque model, and can permit the induction of SIV-specific T cell responses

Functional Implications of Novel Human Acid Sphingomyelinase Splice Variants

BACKGROUND: Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity. METHODOLOGY/PRINCIPAL FINDINGS: We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models. CONCLUSIONS/SIGNIFICANCE: These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity

Production of He-4 and (4) in Pb-Pb collisions at root(NN)-N-S=2.76 TeV at the LHC

Results on the production of He-4 and (4) nuclei in Pb-Pb collisions at root(NN)-N-S = 2.76 TeV in the rapidity range vertical bar y vertical bar <1, using the ALICE detector, are presented in this paper. The rapidity densities corresponding to 0-10% central events are found to be dN/dy4(He) = (0.8 +/- 0.4 (stat) +/- 0.3 (syst)) x 10(-6) and dN/dy4 = (1.1 +/- 0.4 (stat) +/- 0.2 (syst)) x 10(-6), respectively. This is in agreement with the statistical thermal model expectation assuming the same chemical freeze-out temperature (T-chem = 156 MeV) as for light hadrons. The measured ratio of (4)/He-4 is 1.4 +/- 0.8 (stat) +/- 0.5 (syst). (C) 2018 Published by Elsevier B.V.Peer reviewe

Screening of healthcare workers for SARS-CoV-2 highlights the role of asymptomatic carriage in COVID-19 transmission

Funder: Addenbrooke's Charitable Trust, Cambridge University Hospitals; FundRef: http://dx.doi.org/10.13039/501100002927Significant differences exist in the availability of healthcare worker (HCW) SARS-CoV-2 testing between countries, and existing programmes focus on screening symptomatic rather than asymptomatic staff. Over a 3 week period (April 2020), 1032 asymptomatic HCWs were screened for SARS-CoV-2 in a large UK teaching hospital. Symptomatic staff and symptomatic household contacts were additionally tested. Real-time RT-PCR was used to detect viral RNA from a throat+nose self-swab. 3% of HCWs in the asymptomatic screening group tested positive for SARS-CoV-2. 17/30 (57%) were truly asymptomatic/pauci-symptomatic. 12/30 (40%) had experienced symptoms compatible with coronavirus disease 2019 (COVID-19)>7 days prior to testing, most self-isolating, returning well. Clusters of HCW infection were discovered on two independent wards. Viral genome sequencing showed that the majority of HCWs had the dominant lineage B∙1. Our data demonstrates the utility of comprehensive screening of HCWs with minimal or no symptoms. This approach will be critical for protecting patients and hospital staff

Genomic epidemiology of SARS-CoV-2 in a UK university identifies dynamics of transmission

AbstractUnderstanding SARS-CoV-2 transmission in higher education settings is important to limit spread between students, and into at-risk populations. In this study, we sequenced 482 SARS-CoV-2 isolates from the University of Cambridge from 5 October to 6 December 2020. We perform a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. We observe limited viral introductions into the university; the majority of student cases were linked to a single genetic cluster, likely following social gatherings at a venue outside the university. We identify considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and following a national lockdown. Transmission clusters were largely segregated within the university or the community. Our study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics.</jats:p

A prenylated dsRNA sensor protects against severe COVID-19

Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that OAS1, through RNase L, potently inhibits SARS-CoV-2. We show that a common splice-acceptor SNP (Rs10774671) governs whether people express prenylated OAS1 isoforms that are membrane-associated and sense specific regions of SARS-CoV-2 RNAs, or only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. Importantly, in hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting this antiviral defense is a major component of a protective antiviral response

Research and Design of a Routing Protocol in Large-Scale Wireless Sensor Networks

无线传感器网络，作为全球未来十大技术之一，集成了传感器技术、嵌入式计算技术、分布式信息处理和自组织网技术，可实时感知、采集、处理、传输网络分布区域内的各种信息数据，在军事国防、生物医疗、环境监测、抢险救灾、防恐反恐、危险区域远程控制等领域具有十分广阔的应用前景。 本文研究分析了无线传感器网络的已有路由协议，并针对大规模的无线传感器网络设计了一种树状路由协议，它根据节点地址信息来形成路由，从而简化了复杂繁冗的路由表查找和维护，节省了不必要的开销，提高了路由效率，实现了快速有效的数据传输。 为支持此路由协议本文提出了一种自适应动态地址分配算——ADAR（AdaptiveDynamicAddre...As one of the ten high technologies in the future, wireless sensor network, which is the integration of micro-sensors, embedded computing, modern network and Ad Hoc technologies, can apperceive, collect, process and transmit various information data within the region. It can be used in military defense, biomedical, environmental monitoring, disaster relief, counter-terrorism, remote control of haz...学位：工学硕士院系专业：信息科学与技术学院通信工程系_通信与信息系统学号：2332007115216

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M&gt;70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0&lt;e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level