The Role of Sexual Recombination in the Evolution of the Protozoan Parasite, Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan parasite with a haploid genome. Toxoplasma has the capacity to infect any warm-blooded animal and infects between 30-80% of humans and 47% of wildlife surveyed globally. Although this parasite possesses an exceedingly flexible replication strategy, with both a prevalent asexual replication cycle in any intermediate host, and a fecund sexual cycle capable of producing in excess of 100 million infectious oocysts exclusively in its definitive felid host; the extent to which each replication method is utilized to transmit this generalist parasite in nature has long been under debate. Sexual recombination in Toxoplasma is an advantageous trait that allows the parasite to admix population genetic diversity to generate new biological potential, such as the ability to invade new hosts, evade host immunity, or cause outbreaks. However, despite its fecund sexual cycle, Toxoplasma has a genetically limited population structure that is dominated by just a few clonal lines that circulate predominantly in North America and Europe. Two theories exist to explain this clonal population structure, the first suggests that the clonal lines are highly adapted for oral transmission among intermediate hosts and are expanding exclusively asexually within this population of hosts, the second states that the clonal lines are particularly fit clones that are being expanded in cats by uniparental self-mating due to Toxoplasma’s lack of mating types and its ability to undergo unisexual expansion via single clone infection in the definitive host. In support of the latter theory, fungal pathogens have been shown previously to cryptically expand certain clones when sexual recombination occurs between closely related strains. The recombinant progeny produced are typically indistinguishable from each other due to the use of limited, low-resolution markers that fail to capture the genetic differences between the two mating clones. During the tenure of this thesis, studies have been published that chart Toxoplasma global population genetic diversity, but no study has examined intra-typic (i.e., within a clonal lineage) population genetics at whole genome resolution. Work shown here interrogates the most prevalent clonal lineage characterized worldwide, Type II, and the most prevalent clonal lineage in North America, Type X. Work here aims to determine at whole genome resolution the intratypic genetic relationship within two previously reported clonal lineages: Type II and Type X (also referred to as HG12), and whether strains within these two clonal groups isolated across different geographies and animal host species, are evolving via sexual recombination or genetic drift. Strains studied in this thesis were examined by limited sequencing at established Toxoplasma genotyping markers, as well as by whole genome sequencing, which has only recently come into use to interrogate genetic diversity and determine the extent to which recombination is occurring within the genome of this parasite. Type II strains were identified to have undergone limited mitotic drift within the lineage, and the polymorphism detected was generally correlated with the region of the strains’ geographic isolation. However, distinct haploblocks of Type II sequences bearing the hallmarks of different geographic regions were identified in a subset of Type II isolates, and these admixtures suggested that intra-typic recombination (unisexual mating) was occurring between Type II strains of distinct geographies. Proof of unisexual mating between two highly homologous, but independent clones, was identified phylogenetically by an incongruence between nuclear and maternally inherited organellar genomes among these Type II strains. Importantly, genotyping using just ten low resolution PCR-RFLP markers generally predicted that the strain belonged to the Type II lineage, because relatively few admixtures between Type II and other genetic backgrounds were identified after increasing the resolution to WGS. Although at first glance this appears to support a model whereby Type II strains bypass their sexual cycle, and are expanding largely asexually, closer examination of the haploblocks of geographically shared polymorphism identified a number of Type II strains that possessed different admixtures of these Type II, geographically derived haploblocks. This observation was parsimonious with recombination among cladespecific genotypes and supports expansion by unisexual mating. The other clonotype supported by low-resolution PCR-RFLP analyses, and interrogated here, Type X, is the most prevalent genotype infecting wildlife in North America. This thesis performed an unprecedented, longitudinal study of isolates collected over a 7-year period from a single host species, Southern sea otters, in a localized geographic region. In contrast to the Type II lineage, outcrossing was especially pronounced among the Type X strains examined. In fact, Type X was determined to be a clade of sexually-related progeny from at least one sexual cross between a Type II ancestral strain and a previously unknown strain of mosaic ancestry, referred to as g/d. When infected in mice, Type X strains displayed a range of virulence phenotypes from highly virulent to avirulent, like a Type II strain. Because these natural isolates resembled recombinant progeny, the Type X strains were utilized as if they were true F1 progeny from a sexual cross and a QTL was performed to identify genes conferring mouse virulence. The analysis identified a novel virulence gene, ROP33. Evidence from whole genome sequencing demonstrated that while Type X strains resembled closely related progeny, mitotic drift had occurred within these strains prior to their infection in sea otters. However, the observed mitotic drift, in combination with the clear and limited number of crossover break points in Type X, supports a model in which only a limited number of crosses between Type II and a strain of distinct ancestry occurred to generate the Type X strains, but that the Type II strain that crossed with the g/d parent was divergent. Two mouse virulent Type X strains were engineered to be drug resistant and crossed with a cat-competent Type II ME49 strain to perform a forward genetic screen to identify the murine virulence loci. Rather than selecting progeny that were double-drug resistant, progeny from these crosses were individually isolated prior to drug selection and then tested to establish whether they were recombinant. Progeny isolated in this unbiased manner allowed for a more accurate determination of the degree to which clones derived from meiosis undergo outcrossing versus self-mating. In four independent genetic crosses, the relative recombination rate was low (approximately 1-3%), rather than the expected 25% rate indicating that in all instances, self-mating was favored. Taken together, the evidence from the Type II and Type X population genetic analyses, as well as from several independent sexual crosses between these two clonal lineages, established that sexual outcrossing is more prevalent than previously envisaged, but that the majority of clones derived from sexual replication closely resemble a single parental type that was amplified by self-mating. And in geographies with predominantly clonal population genetic structures, it is clear that unisexual mating between closely-related strains will be underestimated and not considered as a major contributor to maintaining the clonal population genetic structure. As a consequence, whole genome typing is required to resolve differences between strains within a clonal group, which are preferentially admixing with each other

Statistical characteristics of finger-tapping data in Huntington’s disease

Measuring the rate of finger tapping is a technique commonly used as an indicator of impairment in degenerative neurological conditions, such as Huntington’s disease. The information it provides can be greatly enhanced by analysing not simply the overall tapping rate, but also the statistical characteristics of the individual times between each successive response. Recent technological improvements in the recording equipment allow the responses to be analysed extremely quickly, and permit modification of the task in the interest of greater clinical specificity. Here we illustrate its use with some pilot data from a group of manifest HD patients and age-matched controls. Even in this small cohort, differences in the responses are apparent that appear to relate to the severity of the disease as measured by conventional behavioural tests

Discrimination of biofilm samples using pattern recognition techniques

Biofilms are complex aggregates formed by microorganisms such as bacteria, fungi and algae, which grow at the interfaces between water and natural or artificial materials. They are actively involved in processes of sorption and desorption of metal ions in water and reflect the environmental conditions in the recent past. Therefore, biofilms can be used as bioindicators of water quality. The goal of this study was to determine whether the biofilms, developed in different aquatic systems, could be successfully discriminated using data on their elemental compositions. Biofilms were grown on natural or polycarbonate materials in flowing water, standing water and seawater bodies. Using an unsupervised technique such as principal component analysis (PCA) and several supervised methods like classification and regression trees (CART), discriminant partial least squares regression (DPLS) and uninformative variable elimination–DPLS (UVE-DPLS), we could confirm the uniqueness of sea biofilms and make a distinction between flowing water and standing water biofilms. The CART, DPLS and UVE-DPLS discriminant models were validated with an independent test set selected either by the Kennard and Stone method or the duplex algorithm. The best model was obtained from CART with 100% correct classification rate for the test set designed by the Kennard and Stone algorithm. With CART, one variable describing the Mg content in the biofilm water phase was found to be important for the discrimination of flowing water and standing water biofilms

A rapid and non-invasive method for authenticating the origin of pistachio samples by NIR spectroscopy and chemometrics

In this study, near-infrared spectroscopy coupled to chemometrics is used to build an analytical protocol to authenticate the origin of pistachio nuts (Pistacia vera L.), a high value-added food product. In particular, 483 samples from six different origins (Sicily, India, Iran, Syria, Turkey and U.S.A.) were analyzed by NIR spectroscopy. Spectra were recorded on half seeds cut longitudinally in reflectance mode. Spectral data were then processed by chemometrics to build classification models by SIMCA and PLS-DA. The discriminant approach resulted in classification accuracies higher than 90% for most of the classes. On the other hand, SIMCA built class-models with high sensitivity and specificities, the only exception being the two categories Turkey and Iran, whose heterogeneity resulted in a poorer specificity (anyway higher than 80%). In particular, the results obtained for the samples coming from Bronte (Sicily), the only PDO pistachio production in Europe 95.5% non-error rate in PLS-DA, 90% sensitivity and 97% specificity in SIMCA, as evaluated on the external test set are very promising from the viewpoint of the authentication of this product. In general, the results show that the coupling of NIR spectroscopy to chemometric classification techniques can be a valuable tool for tracing the origin of pistachio nuts, providing a reliable authentication in a rapid, relatively cheap and non-invasive way.Vitale, R.; Bevilacqua, M.; Bucci, R.; Magrì, A.; Magri, A.; Marini, F. (2013). A rapid and non-invasive method for authenticating the origin of pistachio samples by NIR spectroscopy and chemometrics. Chemometrics and Intelligent Laboratory Systems. 121:90-99. doi:10.1016/j.chemolab.2012.11.019S909912

A survey of psychosocial adaptation in long-term survivors of pediatric liver transplants

We assessed 41 children and adolescents who had received liver transplants at least 4 years ago, for social, behavioral, and emotional adaptation; physical function; and family stress. We compared their level of adaptive functioning to published data from chronically ill and medically well children. On many measures, transplant recipients had equivalent levels of function to the comparison groups. However, 6- to 11-year-old patients showed mild social and scholastic deficits. Patients' parents report less negative impact of the illness on the family than do parents of other chronically ill children. A listing of medication side effects and the degree to which they are problematic was obtained.published_or_final_versio

Evenness mediates the global relationship between forest productivity and richness

1. Biodiversity is an important component of natural ecosystems, with higher species richness often correlating with an increase in ecosystem productivity. Yet, this relationship varies substantially across environments, typically becoming less pronounced at high levels of species richness. However, species richness alone cannot reflect all important properties of a community, including community evenness, which may mediate the relationship between biodiversity and productivity. If the evenness of a community correlates negatively with richness across forests globally, then a greater number of species may not always increase overall diversity and productivity of the system. Theoretical work and local empirical studies have shown that the effect of evenness on ecosystem functioning may be especially strong at high richness levels, yet the consistency of this remains untested at a global scale. 2. Here, we used a dataset of forests from across the globe, which includes composition, biomass accumulation and net primary productivity, to explore whether productivity correlates with community evenness and richness in a way that evenness appears to buffer the effect of richness. Specifically, we evaluated whether low levels of evenness in speciose communities correlate with the attenuation of the richness–productivity relationship. 3. We found that tree species richness and evenness are negatively correlated across forests globally, with highly speciose forests typically comprising a few dominant and many rare species. Furthermore, we found that the correlation between diversity and productivity changes with evenness: at low richness, uneven communities are more productive, while at high richness, even communities are more productive. 4. Synthesis. Collectively, these results demonstrate that evenness is an integral component of the relationship between biodiversity and productivity, and that the attenuating effect of richness on forest productivity might be partly explained by low evenness in speciose communities. Productivity generally increases with species richness, until reduced evenness limits the overall increases in community diversity. Our research suggests that evenness is a fundamental component of biodiversity–ecosystem function relationships, and is of critical importance for guiding conservation and sustainable ecosystem management decisions

Perspectives from the NanoSafety Modelling Cluster on the validation criteria for (Q)SAR models used in nanotechnology

Nanotechnology and the production of nanomaterials have been expanding rapidly in recent years. Since many types of engineered nanoparticles are suspected to be toxic to living organisms and to have a negative impact on the environment, the process of designing new nanoparticles and their applications must be accompanied by a thorough exposure risk analysis. (Quantitative) Structure-Activity Relationship ([Q]SAR) modelling creates promising options among the available methods for the risk assessment. These in silico models can be used to predict a variety of properties, including the toxicity of newly designed nanoparticles. However, (Q)SAR models must be appropriately validated to ensure the clarity, consistency and reliability of predictions. This paper is a joint initiative from recently completed European research projects focused on developing (Q)SAR methodology for nanomaterials. The aim was to interpret and expand the guidance for the well-known “OECD Principles for the Validation, for Regulatory Purposes, of (Q)SAR Models”, with reference to nano-(Q)SAR, and present our opinions on the criteria to be fulfilled for models developed for nanoparticles

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation