Host plant adaptation in the polyphagous whitefly, Trialeurodes vaporariorum, is associated with transcriptional plasticity and altered sensitivity to insecticides

This is the final version. Available from BMC via the DOI in this record. Background: The glasshouse whitefly, Trialeurodes vaporariorum, is a damaging crop pest and an invasive generalist capable of feeding on a broad range of host plants. As such this species has evolved mechanisms to circumvent the wide spectrum of anti-herbivore allelochemicals produced by its host range. T. vaporariorum has also demonstrated a remarkable ability to evolve resistance to many of the synthetic insecticides used for control. Results: To gain insight into the molecular mechanisms that underpin the polyphagy of T. vaporariorum and its resistance to natural and synthetic xenobiotics, we sequenced and assembled a reference genome for this species. Curation of genes putatively involved in the detoxification of natural and synthetic xenobiotics revealed a marked reduction in specific gene families between this species and another generalist whitefly, Bemisia tabaci. Transcriptome profiling of T. vaporariorum upon transfer to a range of different host plants revealed profound differences in the transcriptional response to more or less challenging hosts. Large scale changes in gene expression (> 20% of genes) were observed during adaptation to challenging hosts with a range of genes involved in gene regulation, signalling, and detoxification differentially expressed. Remarkably, these changes in gene expression were associated with significant shifts in the tolerance of host-adapted T. vaporariorum lines to natural and synthetic insecticides. Conclusions: Our findings provide further insights into the ability of polyphagous insects to extensively reprogram gene expression during host adaptation and illustrate the potential implications of this on their sensitivity to synthetic insecticides.ER

The effects of endophytic fungi of NZ terrestrial orchids: developing methods for conservation

Nearly 40% of New Zealand (NZ) orchid species are of conservation concern, some critically endangered, largely due to habitat loss. In NZ, there are currently no propagation programs for terrestrial orchids all of which rely on symbiotic fungi to provide the nutrients required for germination, and little is known about the specific fungal species that might make this possible. To develop an understanding of the fungal interactions affecting recruitment in the field, a survey of endophytic fungal diversity from the roots of Chiloglottis valida, Microtis unifolia, Pterostylis banksii, Spiranthes novae-zelandiae and Thelymitra longifolia was carried out. The identification of fungi was assisted by obtaining sequences of the ITS rDNA gene marker. Seeds of M. unifolia, P. banksii, S. novae-zelandiae and T. longifolia were inoculated with cultured endophytes that were recovered from the roots of conspecific orchids, and their effect on seed germination evaluated. Seed viability using fluorescein diacetate was assayed on all species prior to all experiments and showed moderate to high viability scores for all species. Recovered endophytes belonged to the phyla Ascomycota, Basidiomycota, and Zygomycota. The effect of the different endophytes on seed germination was variable, with five inoculants exhibiting a positive response. Three inoculants had a consistent negative effect on seed germination. The distribution of orchid symbiotic mycorrhizae in situ was investigated at Otari-Wilton’s Bush, Wellington, NZ. Mesh seed packets containing seed of M. unifolia and T. longifolia were interred for 150 days, along transects (≤ 1 metre) that originated at adult orchids at three sites, and an additional site with no adult orchids was used as a control. No small-scale patterns were detected; however, germination rates were higher at undisturbed sites. Seed viability was considerably reduced to <2% after five months under the soil suggesting M. unifolia and T. longifolia seeds do not persist in the seed bank beyond one growing season. Sequences of ITS rDNA indicate Tulasnella calospora assists in the germination of M. unifolia at this site. Similarly, Tulasnella calospora promoted germination of the Nationally Vulnerable wetland species S. novae-zelandiae. Pelotons were isolated from the roots of S. novae-zelandiae plants from a wild population from the lower north island and cultured in Petri dishes. Germination of this orchid began after 30 days from inoculation when the pelotons are already observed inside the embryo. Chlorophyllus tissue was observed after c. 80 days of inoculation. The phylogenetic relationship of Asian-Pacific Spiranthes species with New Zealand Spiranthes was also investigated using nuclear (ITS) and chloroplast (trnL-trnF) DNA sequences. Phylogenetic analyses supported the recognition of Spiranthes novae-zelandiae ‘Motutangi’ as a distinct taxonomic unit. It was also found that the Asian-Pacific Spiranthes species are in need of taxonomic revision. Methods used and developed in this thesis study could be used to identify potential orchid symbionts and pathogens, assess suitable potential relocation sites, and propagation of NZ orchids using symbiotic fungi for restoration and conservation purposes

RAD and the demographic history of a hybrid zone - new insights into the evolution of hybrid sterility

\gls{rad} is a molecular method involving restriction digestion and high throughput DNA sequencing. It promises the systematic subsampling of the genome and highly repeatable scoring of genetic variation in hundreds of individuals at current sequencing costs. However, it comes with its own problems. De novo assembly of \gls{rad} sequence data usually creates many putative reference tags that are only found in one or a few individuals leaving only relatively few markers for population genomic analyses. In the first chapter, I investigate three potential reasons for this outcome -- incomplete digestion, genomic religation and insufficient DNA template amount -- by looking at the occurrence of restriction enzyme recognition sequences within the resultant sequencing data of two different types of \gls{rad} libraries. Analysis of sequence clusters as well as the proportion of concordantly mapping read pairs against a \textit{Locusta} reference sequence suggest that incomplete digestion has affected one of the restriction enzymes used and thereby the number of loci that could be sequenced at sufficient depth across individuals. The other restriction enzyme is found to be much less affected by incomplete digestion and instead random religation of restriction fragments indicates an inefficient adapter ligation step that also leads to low read depths across individuals. Finally, qPCR and read mapping against four newly reconstructed \gls{pe} contig pair reference sequences suggests that low amount of starting DNA and/or high loss of DNA during the library preparation are a major cause for the locus drop-out observed in the de novo assembled read data. In the second part of this thesis, I use \gls{rad} sequence data to make inferences about several aspects of the demographic history of two grasshopper subspecies that form a hybrid zone in the Pyrenees between France and Spain. Sequence data was generated from 36 individuals sampled at the two opposite ends of a hybrid zone that is characterised by hybrid male sterility. I use a state-of-the-art de novo assembly strategy that utilises the shotgun-type \gls{pe} reads from standard \gls{rad} to distinguish alleles from paralogs. I then conduct several population genomic analyses with the programme \texttt{ANGSD} that incorporates uncertainty in genotypes by using genotype likelihoods instead of called genotypes. Results based on more than 1 million filtered sites confirm the high genetic differentiation of the two subspecies found in pre-genomic studies and a surprisingly high genetic diversity in the subspecies that is thought to be derived from a very distant glacial refuge. Further, demographic modelling with the programme $\delta a \delta i$ reveals a robust signal of low but significant gene flow during the divergence of the two subspecies ($Nm \simeq 0.471$, until about 25 \gls{kya}). Allowing for gene flow roughly doubles the divergence time estimate from about 0.5 to 1.1 \gls{mya}. The divergence time estimate without allowing for gene flow is highly consistent with previous estimates from a mitochondrial sequence marker. A history of divergence with gene flow also indicates that alleles causing \glspl{DMI} are unlikely to have risen in frequency by genetic drift alone. The gene flow is clearly asymmetric between the two subspecies in line with many previous studies of the hybrid zone that indicated asymmetric introgression in the same direction. There is no signal of recent (postglacial) gene flow in the data set. However, this may well be due to a lack of power. Further analysis of this data set promises to yield more insights, e.g. loci potentially under divergent selection between the two subspecies

What carcinoembryonic antigen level should trigger further investigation during colorectal cancer follow-up? A systematic review and secondary analysis of a randomised controlled trial

Background Following primary surgical and adjuvant treatment for colorectal cancer, many patients are routinely followed up with blood carcinoembryonic antigen (CEA) testing. Objective To determine how the CEA test result should be interpreted to inform the decision to undertake further investigation to detect treatable recurrences. Design Two studies were conducted: (1) a Cochrane review of existing studies describing the diagnostic accuracy of blood CEA testing for detecting colorectal recurrence; and (2) a secondary analysis of data from the two arms of the FACS (Follow-up After Colorectal Surgery) trial in which CEA testing was carried out. Setting and participants The secondary analysis was based on data from 582 patients recruited into the FACS trial between 2003 and 2009 from 39 NHS hospitals in England with access to high-volume services offering surgical treatment of metastatic recurrence and followed up for 5 years. CEA testing was undertaken in general practice. Results In the systematic review we identified 52 studies for meta-analysis, including in aggregate 9717 participants (median study sample size 139, interquartile range 72–247). Pooled sensitivity at the most commonly recommended threshold in national guidelines of 5 µg/l was 71% [95% confidence interval (CI) 64% to 76%] and specificity was 88% (95% CI 84% to 92%). In the secondary analysis of FACS data, the diagnostic accuracy of a single CEA test was less than was suggested by the review [area under the receiver operating characteristic curve (AUC) 0.74, 95% CI 0.68 to 0.80]. At the commonly recommended threshold of 5 µg/l, sensitivity was estimated as 50.0% (95% CI 40.1% to 59.9%) and lead time as about 3 months. About four in 10 patients without a recurrence will have at least one false alarm and six out of 10 tests will be false alarms (some patients will have multiple false alarms, particularly smokers). Making decisions to further investigate based on the trend in serial CEA measurements is better (AUC for positive trend 0.85, 95% CI 0.78 to 0.91), but to maintain approximately 70% sensitivity with 90% specificity it is necessary to increase the frequency of testing in year 1 and to apply a reducing threshold for investigation as measurements accrue. Limitations The reference standards were imperfect and the main analysis was subject to work-up bias and had limited statistical precision and no external validation. Conclusions The results suggest that (1) CEA testing should not be used alone as a triage test; (2) in year 1, testing frequency should be increased (to monthly for 3 months and then every 2 months); (3) the threshold for investigating a single test result should be raised to 10 µg/l; (4) after the second CEA test, decisions to investigate further should be made on the basis of the trend in CEA levels; (5) the optimal threshold for investigating the CEA trend falls over time; and (6) continuing smokers should not be monitored with CEA testing. Further research is needed to explore the operational feasibility of monitoring the trend in CEA levels and to externally validate the proposed thresholds for further investigation. Study registration This study is registered as PROSPERO CRD42015019327 and Current Controlled Trials ISRCTN93652154. Funding The main FACS trial and this substudy were funded by the National Institute for Health Research Health Technology Assessment programme

Infekce bartonel u plchů velkých a jejich blech

Bartonellosis is a group of emerging infectious diseases caused by facultatively intraerythrocytic bacteria transmitted between vertebrate hosts by arthropod vectors. Although only some Bartonella spp. are traditionally associated with human diseases, it was proposed that virtually any species can have zoonotic potential. The highest diversity of species was described from rodents acting as reservoirs with variable prevalence reaching up to 90%. Fleas associated with mammalian hosts play an important role in the Bartonella sp. lifecycle and are described to act not only as competent vectors but also as reservoirs for several species. The role of edible dormice (Glis glis), which often interacts with humans, and their fleas in the bartonellae transmission and lifecycle has so far been unknown; therefore, the prevalence and diversity of Bartonella spp. in edible dormice in the Czech Republic were studied. Complementary PCR analysis of four loci (gltA, rpoB, ftsZ, ITS), MALDI- TOF, and cultivation was performed to identify Bartonella sp. infections. The prevalence reaching 90% was found in dormice and their fleas. Altogether, four species were found and several genotypes of B. grahamii-like and yet undescribed Bartonella sp. were cultivated, and their morphology and growth requirements were...Zástupci rodu Bartonella jsou fakultativně intraerytrocytární bakterie, které jsou mezi obratlovčími hostiteli přenášené krevsajícími členovci. Bartonely mohou způsobovat lidská onemocnění, bartonelózy, které se řadí do skupiny tzv. emerging infectious diseases. Předpokládá se, že všechny druhy tohoto rodu mohou mít zoonotický potenciál. Nejvyšší diverzita bartonel byla popsána u hlodavců, přičemž detekována prevalence dosahuje až 90 %. Role plcha velkého (Glis glis) a s ním asociovaných blech v životním cyklu bartonel dosud není známá. Plši mohou sloužit jako hostitelé zoonotických patogenů, navíc epidemiologicky významní vzhledem k přítomnosti v lidských obydlích a jejich okolí. Blechy mohou sloužit nejen jako kompetentní přenašeči bartonel, ale i jako rezervoároví hostitelé. Cílem této práce je přispět k poznání prevalence a diverzity bartonel u plchů velkých v České republice. K identifikaci bartonelových infekcí byly použity metody PCR (gltA, rpoB, ftsZ, ITS), MALDI- TOF a kultivace. Detekovaná prevalence u plchů a jejich blech dosahovala až 90 %. Celkem byly detekovány čtyři druhy bartonel a dva (v několika genotypech) byly i úspěšně kultivovány (B. grahamii- like a dosud nepopsaný druh Bartonella sp.). Kultury byly použity k charakterizaci morfologie a optimálních růstových podmínek....Department of ParasitologyKatedra parazitologieFaculty of SciencePřírodovědecká fakult

Comparison Of iNfliximab and ciclosporin in STeroid Resistant Ulcerative Colitis: pragmatic randomised Trial and economic evaluation (CONSTRUCT)

Background: The efficacy of infliximab and ciclosporin in treating severe ulcerative colitis (UC) is proven, but there has been no comparative evaluation of effectiveness. Objective: To compare the clinical effectiveness and cost-effectiveness of infliximab and ciclosporin in treating steroid-resistant acute severe UC. Method: Between May 2010 and February 2013 we recruited 270 participants from 52 hospitals in England, Scotland and Wales to an open-label parallel-group, pragmatic randomised trial. Consented patients admitted with severe colitis completed baseline quality-of-life questionnaires before receiving intravenous hydrocortisone. If they failed to respond within about 5 days, and met other inclusion criteria, we invited them to participate and used a web-based adaptive randomisation algorithm to allocate them in equal proportions between 5 mg/kg of intravenous infliximab at 0, 2 and 6 weeks or 2 mg/kg/day of intravenous ciclosporin for 7 days followed by 5.5 mg/kg/day of oral ciclosporin until 12 weeks from randomisation. Further treatment was at the discretion of physicians responsible for clinical management. The primary outcome was quality-adjusted survival (QAS): the area under the curve (AUC) of scores derived from Crohn’s and Ulcerative Colitis Questionnaires completed by participants at 3 and 6 months, and then 6-monthly over 1–3 years, more frequently after surgery. Secondary outcomes collected simultaneously included European Quality of Life-5 Dimensions (EQ-5D) scores and NHS resource use to estimate cost-effectiveness. Blinding was possible only for data analysts. We interviewed 20 trial participants and 23 participating professionals. Funded data collection finished in March 2014. Most participants consented to complete annual questionnaires and for us to analyse their routinely collected health data over 10 years. Results: The 135 participants in each group were well matched at baseline. In 121 participants analysed in each group, we found no significant difference between infliximab and ciclosporin in QAS [mean difference in AUC/day 0.0297 favouring ciclosporin, 95% confidence interval (CI) –0.0088 to 0.0682; p = 0.129]; EQ-5D scores (quality-adjusted life-year mean difference 0.021 favouring ciclosporin, 95% CI –0.032 to 0.096; p = 0.350); Short Form questionnaire-6 Dimensions scores (mean difference 0.0051 favouring ciclosporin, 95% CI –0.0250 to 0.0353; p = 0.737). There was no statistically significant difference in colectomy rates [odds ratio (OR) 1.350 favouring infliximab, 95% CI 0.832 to 2.188; p = 0.223]; numbers of serious adverse reactions (event ratio = 0.938 favouring ciclosporin, 95% CI 0.590 to 1.493; p = 0.788); participants with serious adverse reactions (OR 0.660 favouring ciclosporin, 95% CI 0.282 to 1.546; p = 0.338); numbers of serious adverse events (event ratio 1.075 favouring infliximab, 95% CI 0.603 to 1.917; p = 0.807); participants with serious adverse events (OR 0.999 favouring infliximab, 95% CI 0.473 to 2.114; p = 0.998); deaths (all three who died received infliximab; p = 0.247) or concomitant use of immunosuppressants. The lower cost of ciclosporin led to lower total NHS costs (mean difference –£5632, 95% CI –£8305 to –£2773; p < 0.001). Interviews highlighted the debilitating effect of UC; participants were more positive about infliximab than ciclosporin. Professionals reported advantages and disadvantages with both drugs, but nurses disliked the intravenous ciclosporin. Conclusions: Total cost to the NHS was considerably higher for infliximab than ciclosporin. Nevertheless, there was no significant difference between the two drugs in clinical effectiveness, colectomy rates, incidence of SAEs or reactions, or mortality, when measured 1–3 years post treatment. To assess long-term outcome participants will be followed up for 10 years post randomisation, using questionnaires and routinely collected data. Further studies will be needed to evaluate the efficacy and effectiveness of new anti-tumour necrosis factor drugs and formulations of ciclosporin.322 page(s

Reactogenicity, safety and antibody response, after one and two doses of mRNA-1273 in seronegative and seropositive healthcare workers

SCOPUS: le.jinfo:eu-repo/semantics/publishe

Variable precision rough set theory decision support system: With an application to bank rating prediction

This dissertation considers, the Variable Precision Rough Sets (VPRS) model, and its development within a comprehensive software package (decision support system), incorporating methods of re sampling and classifier aggregation. The concept of /-reduct aggregation is introduced, as a novel approach to classifier aggregation within the VPRS framework. The software is applied to the credit rating prediction problem, in particularly, a full exposition of the prediction and classification of Fitch's Individual Bank Strength Ratings (FIBRs), to a number of banks from around the world is presented. The ethos of the developed software was to rely heavily on a simple 'point and click' interface, designed to make a VPRS analysis accessible to an analyst, who is not necessarily an expert in the field of VPRS or decision rule based systems. The development of the software has also benefited from consultations with managers from one of Europe's leading hedge funds, who gave valuable insight, advice and recommendations on what they considered as pertinent issues with regards to data mining, and what they would like to see from a modern data mining system. The elements within the developed software reflect each stage of the knowledge discovery process, namely, pre-processing, feature selection, data mining, interpretation and evaluation. The developed software encompasses three software packages, a pre-processing package incorporating some of the latest pre-processing and feature selection methods a VPRS data mining package, based on a novel "vein graph" interface, which presents the analyst with selectable /-reducts over the domain of / and a third more advanced VPRS data mining package, which essentially automates the vein graph interface for incorporation into a re-sampling environment, and also implements the introduced aggregated /-reduct, developed to optimise and stabilise the predictive accuracy of a set of decision rules induced from the aggregated /-reduct

Odyssée au fil des interfaces: de la physico-chimie des macromolécules à l'enveloppe bactérienne, plate-forme interactive du micro-organisme avec son micro-environnement

This find is registered at Portable Antiquities of the Netherlands with number PAN-0001909