Low-field H-1 NMR spectroscopy for distinguishing between arabica and robusta ground roast coffees

This work reports a new screening protocol for addressing issues of coffee authenticity using low-field (60 MHz) bench-top H-1 NMR spectroscopy. Using a simple chloroform-based extraction, useful spectra were obtained from the lipophilic fraction of ground roast coffees. It was found that 16-O-methylcafestol (16-OMC, a recognized marker compound for robusta beans) gives rise to an isolated peak in the 60 MHz spectrum, which can be used as an indicator of the presence of robusta beans in the sample. A total of 81 extracts from authenticated coffees and mixtures were analysed, from which the detection limit of robusta in arabica was estimated to be between 10% and 20% w/w. Using the established protocol, a surveillance exercise was conducted of 27 retail samples of ground roast coffees which were labelled as "100% arabica". None were found to contain undeclared robusta content above the estimated detection limit. (C) 2016 Published by Elsevier Ltd

Combining nanoindentation with complementary techniques for mechanical and structural characterization of ultra uow-k (ULK) thin films

Nano-porous dielectrics used as insulating materials between on-chip interconnects are an important component in metallization stacks of leading-edge microelectronic products to reduce electrical signal delay and power loss. The main drawbacks of these porous dielectrics are their weak mechanical properties. Therefore, new types of porous organosilicate glasses (OSGs) exhibiting a pore arrangement with a high degree of intermittency were developed to improve their mechanical properties. In this study, we will show the relationship between porosity, pore topology, and elastic modulus based on simulations as well as experimental studies using several OSG films. The main part of this study are the experimental techniques used for mechanical and structural analysis of the OSGs. Mechanical characterization is done using nanoindentation (NI) and is complemented by atomic force acoustic microscopy (AFAM), see Figure 1, as well as surface acoustic wave (SAW) measurements. Hereby, the possibilities and limits of measuring surface gradients in the mechanical properties of thin OSG films using these techniques will be discussed. The structural properties are assessed using positron annihilation lifetime spectroscopy (PALS) and transmission electron microscopy (TEM)

16-O-methylcafestol is present in ground roast Arabica coffees: Implications for authenticity testing

High-field and low-field proton NMR spectroscopy were used to analyse lipophilic extracts from ground roast coffees. Using a sample preparation method that produced concentrated extracts, a small marker peak at 3.16 ppm was observed in 30 Arabica coffees of assured origin. This signal has previously been believed absent from Arabicas, and has been used as a marker for detecting adulteration with robusta. Via 2D 600 MHz NMR and LC-MS, 16-O-methylcafestol and 16-O-methylkahweol were detected for the first time in Arabica roast coffee and shown to be responsible for the marker peak. Using low-field NMR, robusta in Arabica could be detected at levels of the order of 1-2% w/w. A surveillance study of retail purchased "100% Arabica" coffees found that 6 out of 60 samples displayed the 3.16 ppm marker signal to a degree commensurate with adulteration at levels of 3-30% w/w

Telomerecat: A ploidy-agnostic method for estimating telomere length from whole genome sequencing data.

Telomere length is a risk factor in disease and the dynamics of telomere length are crucial to our understanding of cell replication and vitality. The proliferation of whole genome sequencing represents an unprecedented opportunity to glean new insights into telomere biology on a previously unimaginable scale. To this end, a number of approaches for estimating telomere length from whole-genome sequencing data have been proposed. Here we present Telomerecat, a novel approach to the estimation of telomere length. Previous methods have been dependent on the number of telomeres present in a cell being known, which may be problematic when analysing aneuploid cancer data and non-human samples. Telomerecat is designed to be agnostic to the number of telomeres present, making it suited for the purpose of estimating telomere length in cancer studies. Telomerecat also accounts for interstitial telomeric reads and presents a novel approach to dealing with sequencing errors. We show that Telomerecat performs well at telomere length estimation when compared to leading experimental and computational methods. Furthermore, we show that it detects expected patterns in longitudinal data, repeated measurements, and cross-species comparisons. We also apply the method to a cancer cell data, uncovering an interesting relationship with the underlying telomerase genotype

Biological heterogeneity in idiopathic pulmonary arterial hypertension identified through unsupervised transcriptomic profiling of whole blood

Idiopathic pulmonary arterial hypertension (IPAH) is a rare but fatal disease diagnosed by right heart catheterisation and the exclusion of other forms of pulmonary arterial hypertension, producing a heterogeneous population with varied treatment response. Here we show unsupervised machine learning identification of three major patient subgroups that account for 92% of the cohort, each with unique whole blood transcriptomic and clinical feature signatures. These subgroups are associated with poor, moderate, and good prognosis. The poor prognosis subgroup is associated with upregulation of the ALAS2 and downregulation of several immunoglobulin genes, while the good prognosis subgroup is defined by upregulation of the bone morphogenetic protein signalling regulator NOG, and the C/C variant of HLA-DPA1/DPB1 (independently associated with survival). These findings independently validated provide evidence for the existence of 3 major subgroups (endophenotypes) within the IPAH classification, could improve risk stratification and provide molecular insights into the pathogenesis of IPAH

Publisher Correction: Telomerecat: A ploidy-agnostic method for estimating telomere length from whole genome sequencing data.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper

PCR and DNA probe hybridization to monitor the efficacy of drug treatment in cattle naturally infected with Trypanosoma spp. in the province of Kénédougou in Burkina Faso, West Africa.

Titelblatt, Inhaltsverzeichnis, Lebenslauf 1\. Einleitung 2\. Literaturübersicht 3\. Material und Methoden 4\. Ergebnisse 5\. Diskussion 6\. Zusammenfassung / Summary / Résumé 7\. Literaturverzeichnis 8\. AnhangDie durch Tsetsefliegen übertragene Trypanosomose der Nutztiere (Nagana) verursacht in den afrikanischen Ländern südlich der Sahara erhebliche ökonomische Verluste. Bei der Bekämpfung dieser Tierseuche steht der Einsatz von Trypanoziden im Vordergrund. Aufgrund ihres nunmehr über 40-jährigen Einsatzes haben sich Resistenzen gegenüber den beiden bedeutendsten Wirkstoffen Isometamidiumchlorid (ISMM) und Diminazenazeturat (DIM) gebildet. Vorkommen und Verbreitung von resistenten Trypanosomenpopulationen wurden in Rinderherden in der Provinz Kénédougou im Südwesten von Burkina Faso (Westafrika) mittels parasitologischer Methoden im Rahmen eines durch das BMZ geförderten Projektes (1998 - 1999) bestimmt. Ziel der vorliegenden Dissertation war es, die Eignung der Polymerasekettenreaktion (PCR) zur Überprüfung der prophylaktischen Wirkung von ISMM und des therapeutischen Erfolgs von DIM zu testen. Die mit der PCR analysierten Blutproben stammten von Rindern, die im Rahmen der Blockbehandlungsstudie des BMZ-Projektes untersucht wurden. Eine Gesamtpopulation von 738 Rindern aus 10 Dörfern mit einer erhöhten Trypanosomenprävalenz von über 10% wurde zu Beginn der Studie mit ISMM (1 mg/kg KGW) behandelt. Erneut parasitologisch positive Rinder wurden bei den 14-tägig stattfindenden Nachuntersuchungen zusätzlich mit DIM (3,5 mg/kg KGW) behandelt. Vor der ISMM-Behandlung wurden parasitologisch mit der sogenannten "Buffy Coat Technik" (BCT) bei 90 der 738 untersuchten Rinder (12,2%) Trypanosomen diagnostiziert. Mit der PCR wurden Blutproben der 90 BCT-positiven Rinder und dieselbe Anzahl Blutproben BCT-negativer Rinder, welche zufällig aus den 648 Blutproben BCT-negativer Rinder ausgewählt wurden, untersucht. Mit den Primerpaaren für Trypanosoma brucei, T. vivax und T. congolense savannah wurden in drei Simplex-PCR-Läufen insgesamt 92,2% (83) der 90 parasitologisch positiven Rinder bestätigt. Von den 90 parasitologisch negativen Rindern reagierten 37,8% (34) positiv. Vierzehn Tage nach der ISMM-Applikation wurde der prophylaktische Erfolg mit beiden Methoden überprüft. Dabei wurden wiederum Trypanosomeninfektionen diagnostiziert. Von den parasitologisch positiven Rindern reagierten 97,1% (34/35) und von den parasitologisch negativen Rindern reagierten 28,8% (41/142) PCR-positiv. Auch 14 Tage nach einer zusätzlichen DIM-Therapie von 34 Rindern, die nach der ISMM-Prophylaxe parasitämisch waren, wurden mit beiden Methoden Trypanosomeninfektionen festgestellt. Dabei waren alle parasitologisch positiven Rinder (5) sowie 55,2% (16) der parasitologisch negativen Rinder PCR-positiv. Während vor der ISMM- Prophylaxe ca. 30% mehr positive Rinder mit der PCR im Vergleich zur BCT nachgewiesen wurden, waren es nach der ISMM-Prophylaxe 114% bzw. nach der zusätzlichen DIM-Therapie 320%. Die Zunahme der diagnostischen Differenz zwischen den beiden Methoden ist sehr wahrscheinlich auf ein häufigeres Unterschreiten der parasitologischen Nachweisgrenze nach den Trypanozidbehandlungen zurückzuführen. Die Abtötung der sensiblen Trypanosomenpopulationen führte zu allgemein niedrigeren Parasitämien, wenngleich resistente Populationen überlebten. Auf der Ebene der beteiligten Dörfer hatte dieser Unterschied zur Folge, dass mit der BCT im Gegensatz zur PCR das Auftreten trypanozidresistenter Infektionen mancherorts nicht erkannt wurde. Die mit beiden Methoden am häufigsten diagnostizierte Trypanosomenspezies war T. congolense gefolgt von T. vivax und T. brucei. Vor der ISMM- Prophylaxe wurden mit der PCR im Vergleich zur BCT insbesondere mehr Infektionen mit T. brucei und mehr Mischinfektionen identifiziert, die zumeist in Verbindung auftraten. Nach der ISMM- Prophylaxe wurden wiederum alle Spezies parasitologisch diagnostiziert, während mit der PCR nur noch T. congolense savannah und T. vivax nachweisbar waren. Die beiden Spezies waren mit der PCR auch noch nach der DIM-Therapie nachweisbar, wogegen parasitologisch nur noch T. congolense identifiziert wurde. Die Identität der PCR-Amplifikate wurde mit spezifischen DNA-Sonden verifiziert. Von den bei der Elektrophorese negativ beurteilten PCR-Ansätzen erzeugten außerdem bis zu 10,7% Hybridisierungssignale, wodurch die Sensitivität der PCR noch gesteigert wurde. Zur Vereinfachung und Kostenreduzierung der Methode wurde eine Multiplex-PCR mit den drei verwendeten Primerpaaren getestet. Eine wiederholte Analyse von Proben mit Einfachinfektionen erbrachte dieselben Ergebnisse wie mit der Simplex-PCR. Proben mit Mischinfektionen wurden hingegen nur etwa zur Hälfte als solche identifiziert, während bei den übrigen Proben Einfachinfektionen nachgewiesen wurden. Bei manchen Proben entstand ein unspezifisches DNA-Produkt. Für einen Einsatz bei Feldstudien müsste die Multiplex-PCR optimiert werden, um die kompetitive Hemmung der Primerpaare bei Mischinfektionen zu minimieren und die Entstehung unspezifischer Produkte zu vermeiden. Die Simplex-PCR wird aufgrund der Untersuchungsergebnisse dieser Arbeit als geeignete Methode zur Überprüfung des Behandlungserfolges von T. congolense- und T. vivax-Infektionen bei Rindern unter Feldbedingungen beurteilt. In Verbindung mit der DNA-Sondenhybridisierung konnte damit die Existenz von Isometamidium- und Diminazen-resistenten T. congolense- und T. vivax-Populationen bei Rindern in der Provinz Kénédougou in Burkina Faso bestätigt werden. Bei T. brucei- Infektionen ist die Eignung der Methode aufgrund der Tendenz dieser Spezies, in das ZNS auszuwandern und sich somit der Trypanozidwirkung und dem Nachweis im Blut zu entziehen, eingeschränkt.The Tsetse transmitted African animal trypanosomosis (Nagana) causes considerable economic losses in sub-Saharan Africa. The use of trypanocidal drugs is the most widely accepted means of controlling the disease. Since they have been on the market for more than 40 years, resistance has developed against the two most important compounds isometamidium chloride (ISMM) and diminazene aceturate (DIM). Under a BMZ- funded project (1998 - 1999), the occurrence and distribution of drug resistant trypanosome populations in cattle were investigated with parasitological methods in the province of Kénédougou in southwest Burkina Faso. The objective of the present study was to assess the polymerase chain reaction (PCR) as a tool to monitor the efficacy of prophylactic treatment with ISMM and curative treatment with DIM. The PCR blood samples originated from cattle, which were examined during the block treatment study of the BMZ-project. The study involved 10 villages with a high trypanoso-mosis prevalence (>10%) where a total of 738 cattle were treated with ISMM (1mg/kg b.w.). Cattle, parasitaemic during one of the fortnightly follow-up visits, were treated additionally with DIM (3.5 mg/kg b.w.). Before the ISMM treatment was administered, trypanosomes were diagnosed parasitologically with the so-called "buffy coat technique" in 90 out of 738 cattle (12%). Blood samples from the 90 BCT-positive cattle plus another 90 randomly selected blood samples from 648 blood samples of BCT-negative cattle were analysed with the PCR using primers for Trypanosoma brucei, T. vivax and T. congolense savannah. Out of the BCT positive blood samples, 92.2% (83) were identified with the PCR. Out of the 90 BCT- negative samples 37.8% (34) were PCR positive. The efficacy of a prophylactic treatment was assessed a fortnight after the administration of ISMM. Trypanosomes were again detected using either method. Parasitaemia, based on BCT, occurred in 19.8% of the cattle. The detection rate of BCT-positives with the PCR reached 97.1% (34/35) with the post-treatment samples. Furthermore, 28.8% (41/142) of the BCT-negative samples were positive according to the PCR. A fortnight after an additional curative DIM treatment of 34 cattle, which had been parasitologically positive after the prophylactic treatment with ISMM, again trypanosome infections were diagnosed using either method. All BCT-positive cases (5) were confirmed by the PCR. Another 55.2% (16) positive cases were identified using the PCR. Whereas about 30% more positive cases were identified with the PCR compared to the BCT before the prophylactic treatment with ISMM, 114% more positive cases were detected after the ISMM-prophylaxis and 320% after the DIM-therapy, respectively. The increasing discrepancy in the number of positives with either method is almost certainly due to a larger number of cattle with a lower parasitaemia beyond the detection limit of the BCT after trypanocide treatment. The elimination of the sensible trypanosome populations led to generally lower parasitaemias, although resistant populations survived. In consequence, treatment failure remained parasitologically undetected in a number of villages when compared with the PCR. T. congolense was the most frequently diagnosed species with either method followed by T. vivax and T. brucei. Before the prophylactic treatment, particularly mixed infections and infections with T. brucei, which were mostly associated with the former, were identified more frequently by PCR than by BCT. After the ISMM-prophylaxis, all previously identified trypanosome species were detected again with the BCT, whereas only T. congolense savannah and T. vivax were detected with the PCR. Both of the latest were identified again with the PCR after the curative treatment with DIM, whereas the only parasitologically identified species was T. congolense. The PCR results were confirmed by specific DNA probes. Moreover, up to 10.7% signals appeared with electrophoresis-negative PCR samples, leading to a further increase in the PCR sensitivity. In order to facilitate the procedure and reduce costs, a multiplex PCR was tested with the three primer pairs. When samples with single infections were analysed, the multiplex PCR results were exactly the same as the simplex PCR results. However, out of samples with mixed infections only 53% were identified as such, whereas the other samples were diagnosed as single infections. In some cases an unspecific product appeared. For use in field studies, the multiplex PCR should be optimised in order to minimize the competitive inhibition among the primer pairs and to avoid the amplification of unspecific products. The above mentioned results demonstrate that the simplex PCR seems to be ideally suited to monitor the success of drug treatment in trypanosome-infected cattle in the field. In combination with the results of the DNA probes, the existence of isometamidium- and diminazene-resistant trypanosome populations in cattle in the province Kénédougou in Burkina Faso could be confirmed. Since T. brucei might invade the central nervous system where it is inaccessible to the drugs, the test may be curtailed for this species