Tagging Complex Non-Verbal German Chunks with Conditional Random Fields

We report on chunk tagging methods for German that recognize complex non-verbal phrases using structural chunk tags with Conditional Random Fields (CRFs). This state-of-the-art method for sequence classification achieves 93.5% accuracy on newspaper text. For the same task, a classical trigram tagger approach based on Hidden Markov Models reaches a baseline of 88.1%. CRFs allow for a clean and principled integration of linguistic knowledge such as part-of-speech tags, morphological constraints and lemmas. The structural chunk tags encode phrase structures up to a depth of 3 syntactic nodes. They include complex prenominal and postnominal modifiers that occur frequently in German noun phrases

Oxidative folding of proteins in the mitochondrial intermembrane space of Leishmania tarentolae

Mitochondrial genes encode for a few proteins. Thus, the majority of proteins has to be imported to the organelles, which is only possible in the unfolded state. The subsequent folding guarantees functionality. One of the proteins responsible for folding in the intermembrane space is Mia40, which is known in opisthokonts. No ortholog for Mia40 is known in kinetoplastida such as Leishmania tarentolae. First, already known candidates for Mia40 orthologs were investigated. In previous work Mic20 had been identified in Trypanosomes.1–4 Gene editing cassettes to knock-out or modify the gene LtaPh_3313851, which encodes the Mic20 ortholog, could not be inserted homozygously. Thus, the gene is assumed to be essential. Another protein that plays an important role in mitochondrial protein import is Erv, a known interactor of Mia40. Erv is also found in Leishmania. Two proteins of so far unknown function had been identified as potential interactors of Erv and could be candidates for Mia40 orthologs.5 Potential knock-out strains of one protein-encoding gene each were investigated. The knock-out of LtaP32.0380 was assumed to be complete and the gene dispensable. The knock-out cassette for LtaP07.0980 could be shown to be inserted heterozygously, which could indicate the essentiality of the gene. To identify new candidates for Mia40 orthologs in Leishmania tarentolae, potential substrates6 of the Mia40/Erv pathway were used as baits in the present work. Gene editing via CRISPR/Cas9 included attempts to insert knock-out or tagging cassettes to five different genes. Homozygous insertion succeeded for the C-terminal His8-tagging cassettes for LtaP19.1110, and for the N-terminal His8-tagging cassettes for LtaP25.1620 and LtaP09.1390. No homozygous gene editing could be observed for LtaP35.0210. The knock-out of LtaP04.0060 was assumed to be complete. The presence of the N-terminal His8-tagged substrate 4 (LtaP09.1390) could be shown in cell lysates. The correct position of tagged substrate 4 in the cell was confirmed. Further cell lysates were purified in pull-downs on Ni-NTA to obtain tagged substrate 4 with its interaction partners. The presence of tagged proteins in the eluates could be confirmed. To identify interacting proteins, mass spectrometry analysis was performed. In further experiments, DTT and TMAD were used to alter the redox conditions in the cells before lysis and purification. The evaluation of the data included the comparison of the proteins identified in different experiments and the comparison with potential interactors of Erv.5,7 Also, properties of Mia40 that might be conserved were considered. Two characteristic motifs of known Mia40 orthologs are a CPC and a twin CX9C motif. Thus, proteins with these or similar motifs were specifically searched for. Different candidates for Mia40 orthologs were identified and discussed.Mitochondriale Gene codieren für wenige Proteine. Daher muss die Mehrheit der Proteine in diese Organellen importiert werden. Dies ist nur in ungefaltetem Zustand möglich. Nachfolgendes Falten garantiert die Funktionalität der Proteine. Ein Protein, das für die Faltung im Intermembranraum verantwortlich ist, ist Mia40, das aus Opisthokonta bekannt ist. Für Mia40 ist kein Ortholog in Kinetoplastida, wie beispielsweise Leishmanien bekannt. Zunächst wurden aus der Literatur bekannte Kandidaten für Orthologe untersucht. Als solches wurde Mic20 in Trypanosoma identifiziert.1–4 Kassetten für den Knock-out oder die Modifizierung des Gens LtaPh_3313851, das für das Mic20 Ortholog kodiert, konnten nicht homozygot eingeführt werden. Daher wurde angenommen, dass das Gen unverzichtbar ist. Ein anderes Protein, das eine wichtige Rolle im mitochondrialen Proteinimport spielt ist Erv, von dem bekannt ist, dass es sich um einen Interaktionspartner von Mia40 handelt. Erv wurde auch in Leishmanien identifiziert. Zwei Proteine mit bisher unbekannter Funktion wurden als Interaktoren von Erv identifiziert und könnten Kandidaten für Mia40 Orthologe sein.5 Stämme in denen je eines der beiden Proteine potentiell ausgeknockt werden sollte wurden untersucht. Der Knock-out des Gens LtaP32.0380 wurde als vollständig und das Gen als entbehrlich interpretiert. Es konnte gezeigt werden, dass die Knock-out-Kassette für LtaP07.0980 heterozygot eingefügt war. Dies deutet auf eine Unverzichtbarkeit des Gens hin. Um neue Kandidaten für Orthologe von Mia40 in Leishmania tarentolae zu identifizieren wurden in der vorliegenden Arbeit potentielle Substrate6 des Mia40/Erv-Importwegs als Köder eingesetzt. Die genetische Manipulation mittels CRISPR/Cas9 umfasste das Einfügen von Knock-out- oder Tagging-Kassetten an fünf verschiedenen Genen. Homozygote Insertion gelang für die C-terminalen His8-Tagging-Kassetten für LtaP19.1110 und für die N-terminalen His8-Tagging-Kassetten für LtaP25.1620 und LtaP09.1390. Keine homozygote Genmanipulation konnte für LtaP35.0210 beobachtet werden. Als vollständig wurde der Knock-out von LtaP04.0660 angesehen. Das N-terminal His8-getaggte Substrat 4 (LtaP09.1390) konnte in Zelllysaten nachgewiesen werden. Die korrekte Lokalisierung von getaggtem Substrat 4 in der Zelle wurde bestätigt. Weitere Zelllysate wurden aufgereinigt in Pull-downs an Ni-NTA, um das getaggte Substrat 4 mit seinen Interaktionspartnern zu erhalten. Getaggte Proteine konnten in den Eluaten nachgewiesen werden. Um die Interaktoren zu identifizieren wurden massenspektrometrische Analysen durchgeführt. In weiteren Experimenten wurden DTT und TMAD verwendet um die Redoxbedingungen der Zellen zu manipulieren, bevor die Zellen lysiert und aufgereinigt wurden. Die Auswertung der identifizierten Proteine beinhaltete den Vergleich verschiedener Experimente untereinander und mit den Interaktoren von Erv.5,7 Auch Eigenschaften von Mia40, die konserviert sein könnten wurden berücksichtigt. Zwei charakteristische Motive in den bekannten Mia40 Orthologen sind ein CPC- und ein doppeltes CX9C-Motiv. Daher wurde gezielt nach Proteinen mit diesen oder ähnlichen Motiven gesucht. Verschiedene Kandidaten für Mia40 Orthologe wurden identifiziert und diskutiert

Toxocara vitulorum & Fasciola gigantica in cattle and buffalo in northern Laos

LUZIA RAST – PhD THESIS… abstract Toxocara vitulorum & Fasciola gigantica in Cattle and Buffalo in Northern Laos In South-East Asia agricultural production including livestock is predominately produced within mixed smallholder farming systems. These mostly operate at subsistence levels. Cattle and buffalo are typically kept as assets rather than for optimal production purposes. Economic growth and urbanisation in the region continues to lead to increased demand for red meat products. This provides opportunities and pressures for smallholder farmers to increase their livestock outputs and supply this market with consistent and high quality products. Laos is well placed to supply this increasing regional demand but constraints inhibiting optimal production outputs from smallholder farming systems need to be addressed. These constraints include low capacity animal health systems, lack of infrastructure, traditional low input/low output farming methods and endemic livestock diseases. There is limited documentation about disease prevalence in large ruminants and basic production benchmarks such as reproductive, morbidity and mortality rates. Anecdotal reports indicate that Fasciola gigantica and Toxocara vitulorum are endemic in Laos and contribute to substantial production losses. This is despite the availability of relative cheap and simple treatment technology for T.vitulorum and substantial past research investment in the region on both parasites. The research presented in this thesis contributes to knowledge on the prevalence of T.vitulorum and F.gigantica in cattle and buffalo and the impact of these parasitic infections on production in mixed smallholder farming systems in northern Laos, which are typical for many other parts of South-East Asia. It further contributes to defining and documenting basic large ruminant production parameters within these production systems and quantifies the financial impact of T.vitulorum treatment of calves. Importantly this research identified a large gap in knowledge and in the sustained adoption of effective control practices for large ruminant internal parasites and explored pathways and gives recommendations to address this. The thesis presents data from seven separate field studies completed in northern Laos between 2009 and 2012. Two cross-sectional field surveys were conducted and results showed that both T.vitulorum in cattle and buffalo calves and F.gigantica in adult cattle and buffalo had high apparent prevalence (22.6% and 17.2% respectively) and were geographically widespread throughout northern Laos. Results further indicated that both parasites affected cattle and buffalo at similar levels and that no specific clinical signs were associated with either parasite. This was in line with past research on F.gigantica infection but not for T.vitulorum with limited past research and anecdotal reports indicating that Toxocariasis causes diarrhoea and rough coats in calves and especially so in buffalo calves. Slaughterhouse surveys were conducted in five main provincial slaughterhouses in northern Laos. Results showed a prevalence of faecal eggs for F.gigantica (34.1%) plus liver lesions consistent with F.gigantica infection in 71.0% of slaughtered cattle and buffalo providing further evidence of the endemic nature of this parasite and its potential clinical impact across northern Laos. Additional findings of the slaughterhouse surveys were that a large proportion of slaughtered female animals were pregnant (44% cattle, 47% buffalo), 9.8% of slaughtered animals had FMD lesions and meat inspectors were rarely present for the entire slaughter process with no condemnation of any products. Two separate farmer surveys on a sample of farmers that had their large ruminants tested for either T.vitulorum or F.gigantica were conducted using face-to-face semi-structured interviews. Results of the T.vitulorum farmer survey (n=273) showed that there was a relative high rate of awareness (62.3%) about this parasite amongst farmers and that specific knowledge about its epidemiology and potential clinical impact was lacking. Only 2.5% of farmers used pyrantel treatment of calves at the recommended age and dose rate. Results of the farmer survey (n=326) for F.gigantica showed smallholders had very limited knowledge about Fasciolosis in large ruminants despite 20.6% reporting having seen leaf shaped parasites in livers of slaughtered cattle or buffalo in the past. None of the interviewed farmers treated larger ruminants to control liver fluke. Analyses of large ruminant production data found annual calf morbidity and mortality rates of 42.6% (CI 0.38-0.47) and 37.3% (CI 0.33-0.42) respectively; and adult morbidity and mortality rates of 7.4% (CI 0.06-0.09) and 2.8% (CI 0.003-0.05) respectively. Further, results showed low reproductive performance of 0.6 and 0.4 calf per year for cattle and buffalo respectively with first calving ages of 36 months reported for both species. Two separate field treatment trials were conducted. For T.vitulorum calves were treated when they were 90% reduction of faecal egg counts in adult cattle and buffalo four, eight and twelve weeks post treatment compared to untreated animals. In addition there was a trend of increased weight gain in treated buffalo compared to the untreated control group indicating that treatment of Fasciolosis may result in heavier buffalo. Financial analysis using partial budgeting and data from our surveys showed that there was a large net benefit of USD 3.69-14.86 per calf for treatment with pyrantel (12.5 mg/kg) once only between 14-21 days of age compared to no treatment. It was concluded that both T.vitulorum and F.gigantica are endemic in northern Laos and contribute to substantial production losses in this area. Smallholder farmers still keep large ruminants mostly as an asset and there is also a large knowledge gap amongst smallholder producers about internal parasites, their health and production effects and effective control methods. This knowledge gap and the lack of commercial driver contribute to the deficit of widespread adoption of parasite control methods by smallholder farmers despite their availability and known effectiveness. These results suggest, especially for T.vitulorum that if recommended control methods were widely adopted, large ruminant production output from smallholder farming systems could be increased through reduced calf morbidity and mortality rates