Proteomic and transcriptomic analysis of the Protozoan Parasite Neospora caninum

Neospora caninum is an economically significant parasitic protozoan causing the disease neosporosis in cattle and dogs. Although a close relative of the zoonotic apicomplexan Toxoplasma gondii, the two organisms exhibit differing host ranges and infection dynamics. T. gondii is a model organism that has been much studied, and a great deal is known about the genes and proteins involved when it invades a host cell. This thesis explores protein expression in the proliferative and invasive tachyzoite stage of N. caninum, in particular the expression of proteins pertaining to the apical complex of organelles; those responsible for entry and establishment within a host cell. Almost 20 % of the predicted proteome has been identified by this analysis to be expressed in the tachyzoite stage, with approximately 50 % of the predicted repertoire of apical proteins being detected. The discovery of differences between these two parasites’ highly syntenic genomes could lead to a better understanding of the process by which T. gondii is able to cause disease in humans, while N. caninum has not been observed to do so. One finding of the recent genome sequencing and annotation project in N. caninum was that a key T. gondii virulence determinant, rhoptry gene 18 (ROP18) was pseudogenised in N. caninum. This finding was investigated further in this thesis to demonstrate that the pseudogenisation of ROP18 was conserved across a range of N. caninum isolates and that in vitro, N. caninum was not able to subvert the murine interferon-gamma (IFN-γ) immune response using ROP18 in the way that virulent T. gondii tachyzoites do. The tissue-dwelling Coccidia have a multi-stage life cycle which includes a latent tissue cyst-encapsulated stage called the bradyzoite. Tachyzoites convert to this more quiescent form when induced by cellular stress, and are able to remain as such for long periods, even years. At times of weakened host immunity, bradyzoites can recrudesce to produce an active infection, which can cross the placenta in a pregnant animal to infect the foetus. This a major route by which N. caninum infection is maintained within cattle herds, therefore the biology of stage conversion from tachyzoite to bradyzoite and vice-versa is of interest to researchers. An RNA-Seq analysis of cultured tachyzoites and bradyzoites identified a marked reduction in rhoptry gene expression, and differing expression profiles of other invasion-related genes from the micronemes and dense granules. Overall, these data identify proteins released from the apical organelles in N. caninum and give an insight into the different repertoires expressed by the tachyzoite and bradyzoite life stages. Furthermore, a comparison between N. caninum and T. gondii predicted apical proteomes indicates that although most genes are shared in a one-to-one orthologous relationship between the two organisms, there are a small number of differences which may turn out to be important to the biology of the parasite, as in the case of ROP18

Localisation de Visages par Boosting de Classificateurs Lineaires

Dans ce rapport, une methode pour la localisation de visages est presentee. La localisation de visages est une des composantes importantes dans l analyse et la comprehension de visages. Les methodes existantes pour localiser des visages peuvent etre subdivisees en methodes image-based et methodes feature-based. Le programme implemente utilise une methode image-based. Pour entrainer les classificateurs, nous avons utilise un algorithme de boosting (AdaBoost) qui permet d avoir une bonne performance de detection. AdaBoost est un algorithme d apprentissage agressif qui construit un classificateur fort a partir d un ensemble de classificateurs faibles. Comme classificateurs faibles, des classificateurs lineaires ont ete utilises. Chaque classificateur lineaire utilise un descripteur, base sur des masques visuels qui sont une variante d ondelette de Haar module par une Gaussienne. Les classificateurs ont ete entraines et testes sur la base de donnees classique BANCA. Pour la detection des objets caracteristiques, une technique de fenetre glissante a taille variable a ete employee. Pour trouver a partir de toutes les detections retournees par les classificateurs les positions les plus probables des objets caracteristiques, trois methodes d arbitrage ont ete implementees et testees sur la base de donnees classique BANCA

A large-scale proteogenomics study of apicomplexan pathogens-Toxoplasma gondii and Neospora caninum

Proteomics data can supplement genome annotation efforts, for example being used to confirm gene models or correct gene annotation errors. Here, we present a large‐scale proteogenomics study of two important apicomplexan pathogens: Toxoplasma gondii and Neospora caninum. We queried proteomics data against a panel of official and alternate gene models generated directly from RNASeq data, using several newly generated and some previously published MS datasets for this meta‐analysis. We identified a total of 201 996 and 39 953 peptide‐spectrum matches for T. gondii and N. caninum, respectively, at a 1% peptide FDR threshold. This equated to the identification of 30 494 distinct peptide sequences and 2921 proteins (matches to official gene models) for T. gondii, and 8911 peptides/1273 proteins for N. caninum following stringent protein‐level thresholding. We have also identified 289 and 140 loci for T. gondii and N. caninum, respectively, which mapped to RNA‐Seq‐derived gene models used in our analysis and apparently absent from the official annotation (release 10 from EuPathDB) of these species. We present several examples in our study where the RNA‐Seq evidence can help in correction of the current gene model and can help in discovery of potential new genes

Modeling the Emissions of Heavy-Duty Diesel Vehicles on Interstate 89/189 and US Route 7 in the Burlington Area [from cover]

This study compares the modeled exhaust emissions for heavy-duty diesel vehicles of various weights traveling two different bi-directional routes through the Burlington, Vermont, area. The two routes are I- 89/I-189, between the junction at US-7 and Exit 16 in Colchester, and US-7, between the same two locations. Currently, heavy vehicles exceeding 80,000 pounds are prohibited from traveling on the analyzed section of interstate highway without permit due to federal regulations, and instead travel through the Burlington area along the designated US-7 truck route. For the analysis, real-world velocity, acceleration, and roadway grade data are used to model the pollutant emissions using the Comprehensive Modal Emissions Model to ascertain the differences in emissions, if any, between the interstate and US-7 routes. Results indicated that overall emissions inventories and per-mile emission rate were lower by up to about 50% for the I-89/I-189 routes compared to the US-7 routes. However, emissions of NOx were very similar between the counterpart routes, and in one case, the southbound interstate\u2019s NOx emissions were greater than US-7, by approximately 2-6% depending on vehicle weight. Lastly, there would be an overall savings in diesel fuel use up to approximately one gallon per truck traveling on I-89/I-189 compared to US-7

Intelligent Trac Signals: Extending the Range of Self-Organization in the BML Model

The two-dimensional tra\ufb03c model of Biham, Middleton and Levine (BML) is a simple cellular automaton that exhibits a wide range of complex behavior. It consists of both northbound and eastbound cars traveling on a rectangular array of cells, each cell equipped with a tra\ufb03c signal. The tra\ufb03c signals switch synchronously from allowing northbound \ufb02ow to eastbound \ufb02ow. By gating individual tra\ufb03c signals, i.e. allowing individual tra\ufb03c signals to break from synchrony in predetermined, deterministic scenarios based on the local state of tra\ufb03c, the range for which the system self-organizes into a state of unimpeded \ufb02ow is extended. On a 100x100 cell array, this additional intelligence enables accommodation of 200 cars more than the original BML model, without any reduction in average velocity

Transient RUNX1 Expression during Early Mesendodermal Differentiation of hESCs Promotes Epithelial to Mesenchymal Transition through TGFB2 Signaling

The transition of human embryonic stem cells (hESCs) from pluripotency to lineage commitment is not fully understood, and a role for phenotypic transcription factors in the initial stages of hESC differentiation remains to be explored. From a screen of candidate factors, we found that RUNX1 is selectively and transiently upregulated early in hESC differentiation to mesendodermal lineages. Transcriptome profiling and functional analyses upon RUNX1 depletion established a role for RUNX1 in promoting cell motility. In parallel, we discovered a loss of repression for several epithelial genes, indicating that loss of RUNX1 impaired an epithelial to mesenchymal transition during differentiation. Cell biological and biochemical approaches revealed that RUNX1 depletion specifically compromised TGFB2 signaling. Both the decrease in motility and deregulated epithelial marker expression upon RUNX1 depletion were rescued by reintroduction of TGFB2, but not TGFB1. These findings identify roles for RUNX1-TGFB2 signaling in early events of mesendodermal lineage commitment

Management of Autoimmune Encephalitis in a 7-Year-Old Child With CTLA-4 Haploinsufficiency and AMPA Receptor Antibodies:A Case Report

OBJECTIVES: We report on the therapeutic management of early-onset severe neurologic symptoms in cytotoxic T lymphocyte antigen-4 haploinsufficiency (CTLA-4h) and the presence of antibodies to the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) as an important finding. METHODS: This is a case report from a Dutch academic hospital. Repeated clinical examinations, repeated brain MRI and extended diagnostics on serum and CSF were performed. We used the CARE checklist. RESULTS: A 7-year-old boy was diagnosed with CTLA-4h based on family screening. On diagnosis, he had mild chronic diarrhea and autism spectrum disorder, but no abnormalities in extensive laboratory screening. Six months later, he presented with sudden-onset autoimmune encephalitis. Repeated brain MRI revealed no abnormalities, but immunohistochemistry analysis on serum and CSF showed the presence of AMPAR antibodies. Treatment was initially focused on immunomodulation and targeted CTLA-4 replacement therapy. Because of the persistent fluctuating cerebellar and neuropsychiatric symptoms and the potential clinical significance of the AMPAR antibodies, treatment was intensified with repetition of first-line immunomodulation and rituximab. This combined therapy resulted in sustained clinical improvement and served as a bridge to curative hematopoietic stem cell transplantation. DISCUSSION: This case illustrates the rare early onset of autoimmune encephalitis and presence of AMPAR antibodies in CTLA-4h. Targeted CTLA-4 replacement therapy resulted in a partial response. However, awaiting its optimal therapeutic effect, refractory CNS symptoms required intensification of immunomodulation. The identification of AMPAR antibodies guided our treatment decisions. CLASSIFICATION OF EVIDENCE: This provides Class IV evidence. It is a single observational study without controls.</p

Management of Autoimmune Encephalitis in a 7-Year-Old Child With CTLA-4 Haploinsufficiency and AMPA Receptor Antibodies:A Case Report

OBJECTIVES: We report on the therapeutic management of early-onset severe neurologic symptoms in cytotoxic T lymphocyte antigen-4 haploinsufficiency (CTLA-4h) and the presence of antibodies to the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) as an important finding. METHODS: This is a case report from a Dutch academic hospital. Repeated clinical examinations, repeated brain MRI and extended diagnostics on serum and CSF were performed. We used the CARE checklist. RESULTS: A 7-year-old boy was diagnosed with CTLA-4h based on family screening. On diagnosis, he had mild chronic diarrhea and autism spectrum disorder, but no abnormalities in extensive laboratory screening. Six months later, he presented with sudden-onset autoimmune encephalitis. Repeated brain MRI revealed no abnormalities, but immunohistochemistry analysis on serum and CSF showed the presence of AMPAR antibodies. Treatment was initially focused on immunomodulation and targeted CTLA-4 replacement therapy. Because of the persistent fluctuating cerebellar and neuropsychiatric symptoms and the potential clinical significance of the AMPAR antibodies, treatment was intensified with repetition of first-line immunomodulation and rituximab. This combined therapy resulted in sustained clinical improvement and served as a bridge to curative hematopoietic stem cell transplantation. DISCUSSION: This case illustrates the rare early onset of autoimmune encephalitis and presence of AMPAR antibodies in CTLA-4h. Targeted CTLA-4 replacement therapy resulted in a partial response. However, awaiting its optimal therapeutic effect, refractory CNS symptoms required intensification of immunomodulation. The identification of AMPAR antibodies guided our treatment decisions. CLASSIFICATION OF EVIDENCE: This provides Class IV evidence. It is a single observational study without controls.</p

Comprehensive evaluation of Toxoplasma gondii VEG and Neospora caninum LIV genomes with tachyzoite stage transcriptome and proteome defines novel transcript features.

Toxoplasma gondii is an important protozoan parasite that infects all warm-blooded animals and causes opportunistic infections in immuno-compromised humans. Its closest relative, Neospora caninum, is an important veterinary pathogen that causes spontaneous abortion in livestock. Comparative genomics of these two closely related coccidians has been of particular interest to identify genes that contribute to varied host cell specificity and disease. Here, we describe a manual evaluation of these genomes based on strand-specific RNA sequencing and shotgun proteomics from the invasive tachyzoite stages of these two parasites. We have corrected predicted structures of over one third of the previously annotated gene models and have annotated untranslated regions (UTRs) in over half of the predicted protein-coding genes. We observe distinctly long UTRs in both the organisms, almost four times longer than other model eukaryotes. We have also identified a putative set of cis-natural antisense transcripts (cis-NATs) and long intergenic non-coding RNAs (lincRNAs). We have significantly improved the annotation quality in these genomes that would serve as a manually curated dataset for Toxoplasma and Neospora research communities

Raising AWaRe-ness of Antimicrobial Stewardship Challenges in Pediatric Emergency Care: Results from the PERFORM Study Assessing Consistency and Appropriateness of Antibiotic Prescribing Across Europe

Background Optimization of antimicrobial stewardship is key to tackling antimicrobial resistance, which is exacerbated by overprescription of antibiotics in pediatric emergency departments (EDs). We described patterns of empiric antibiotic use in European EDs and characterized appropriateness and consistency of prescribing. Methods Between August 2016 and December 2019, febrile children attending EDs in 9 European countries with suspected infection were recruited into the PERFORM (Personalised Risk Assessment in Febrile Illness to Optimise Real-Life Management) study. Empiric systemic antibiotic use was determined in view of assigned final “bacterial” or “viral” phenotype. Antibiotics were classified according to the World Health Organization (WHO) AWaRe classification. Results Of 2130 febrile episodes (excluding children with nonbacterial/nonviral phenotypes), 1549 (72.7%) were assigned a bacterial and 581 (27.3%) a viral phenotype. A total of 1318 of 1549 episodes (85.1%) with a bacterial and 269 of 581 (46.3%) with a viral phenotype received empiric systemic antibiotics (in the first 2 days of admission). Of those, the majority (87.8% in the bacterial and 87.0% in the viral group) received parenteral antibiotics. The top 3 antibiotics prescribed were third-generation cephalosporins, penicillins, and penicillin/β-lactamase inhibitor combinations. Of those treated with empiric systemic antibiotics in the viral group, 216 of 269 (80.3%) received ≥1 antibiotic in the “Watch” category. Conclusions Differentiating bacterial from viral etiology in febrile illness on initial ED presentation remains challenging, resulting in a substantial overprescription of antibiotics. A significant proportion of patients with a viral phenotype received systemic antibiotics, predominantly classified as WHO Watch. Rapid and accurate point-of-care tests in the ED differentiating between bacterial and viral etiology could significantly improve antimicrobial stewardship