Genome wide identification of mutational hotspots in the apicomplexan parasite neospora caninum and the implications for virulence

© The Author(s) 2018. Neospora caninum is an apicomplexan parasite responsible for neosporosis, a disease causing hind limb paralysis in dogs and abortion in cattle, resulting in substantial economic losses to beef and dairy industries. Marked differences in pathogenicity exist between N. caninum strains suggesting that intrinsic genetic differences exist between them. These differences likely exist in genes expressed during the tachyzoite lifecycle stage which is responsible for the pathogenesis of neosporosis. An improved understanding of these genetic differences is essential to understanding N. caninum virulence, though such knowledge is scarce. Using a variant detection workflow we compared the tachyzoite transcriptomes of two N. caninum strains with different virulence properties: NC-Liverpool (virulent) and NC-Nowra (avirulent). This workflow identified 3130 SNPs and 6123 indels between the strains, and nine markers capturing 30 variants were Sanger sequenced for both strains. Sequencing of these loci was extended to an additional eight strains and subsequent phylogenetic analysis supported a genetic population structure comprised of two major clades with no geographical segregation. Sequence polymorphisms within coding regions of tachyzoiteassociated genes were concentrated on chromosomes XI and XII, with 19 distinct tachyzoite-associated SNP hotspot regions identified within coding regions of the N. caninum nuclear genome. The variants were predominantly located in loci associated with protein binding, protein-protein interactions, transcription, and translation. Furthermore, 468 nonsynonymous SNPs identified within protein-coding genes were associated with protein kinase activity, protein binding, protein phosphorylation, and proteolysis. This work may implicate these processes and the specific proteins involved as novel effectors of N. caninum tachyzoite virulence

Plant Metabolomics: An Emerging Technology for Crop Improvement

The astounding ability of plants to make smart decisions in response to environment is evident. As they have evolved a long list of complex and unique processes that involve photosynthesis, totipotency, long-distance signaling, and ability to restore structural and metabolic memory, recognition, and communication via emission of the selected class of volatiles. In recent years, use of metabolite profiling techniques in detection, unambiguous identification, quantification, and rapid analysis of the minute quantity of cellular micromolecules has increased considerably. Metabolomics is key to understand the chemical footprints during different phases of growth and development of plants. To feed the ever-increasing population with limited inputs and in a rapidly changing environment is the biggest challenges that the world agriculture faces today. To achieve the project genetic gains, the breeding strategies employing marker-assisted selection for high-yielding varieties and identifying germplasm resistant to abiotic and biotic stresses are already in vogue. Henceforth, new approaches are needed to discover and deploy agronomically important gene/s that can help crops better withstand weather extremes and growing pest prevalence worldwide. In this context, metabolic engineering technology looks viable option, with immense potential to deliver the future crops

IAPP Amyloid Aggregation and IAPP-Associated Toxicity Mitigation

Human islet amyloid polypeptide (IAPP, a.k.a. amylin) is a 37-residue peptide hormone co-synthesized and co-secreted with insulin by pancreatic β-cells for glycemic control. Extensive research indicates that the amyloid aggregation of IAPP into cross-β amyloid fibrils is a ubiquitous phenomenon as well as a major factor in the development and pathogenesis of type 2 diabetes mellitus (T2D), which is a long-term metabolic disorder compromises the quality of life of millions globally. The amyloid IAPP aggregation products, either soluble intermediate oligomers or mature fibrils, are found toxic to human cells and capable of eliciting systemic damage in T2D patients. Recent studies reveal that IAPP is able to cross the blood-brain barrier and co-aggregate with human amyloid-beta (Aβ), which is the protein associated with another amyloid neurodegenerative disorder, the Alzheimer’s disease (AD). In vitro experiments demonstrate that soluble IAPP could significantly accelerate the aggregation of Aβ, with accumulating clinical and epidemiological evidences also suggest that T2D and AD are linked together. Despite the significant differences in their pathologies, T2D is suggested as a risk factor for AD. Here, we investigate the possible mechanism of the co-aggregation of IAPP and Aβ to explore the cross-talk between these two diseases and propose that IAPP promotes Aβ aggregation by reducing the aggregation free energy barrier through its binding with Aβ. In addition, with the fact that IAPPs are stored inside β-cell granules without apparent aggregation in healthy individuals, we also study the physiological environment inside β-cell granules and its endogenous inhibition effect on IAPP aggregation. Our work demonstrates that Zn2+ coordinated molecular complex might be important to stabilize IAPP and hence the endogenous inhibition. Moreover, we study the interactions between IAPP and two different materials, the small molecule epigallocatechin gallate (EGCG) and the star-shaped polymer poly(2-hydroxyethyl acrylate) (PHEA). Our study demonstrates both EGCG and PHEA as inhibitors against amyloidogenesis, while perform in different strategies. EGCG is able to inhibit IAPP aggregation and result in minimizing the population of toxic oligomers and protofibrils, while PHEA accelerates IAPP fibrillation to circumvent accumulation of the more toxic intermediates

Transcriptome analysis reveals novel patterning and pigmentation genes underlying Heliconius butterfly wing pattern variation

BACKGROUND: Heliconius butterfly wing pattern diversity offers a unique opportunity to investigate how natural genetic variation can drive the evolution of complex adaptive phenotypes. Positional cloning and candidate gene studies have identified a handful of regulatory and pigmentation genes implicated in Heliconius wing pattern variation, but little is known about the greater developmental networks within which these genes interact to pattern a wing. Here we took a large-scale transcriptomic approach to identify the network of genes involved in Heliconius wing pattern development and variation. This included applying over 140 transcriptome microarrays to assay gene expression in dissected wing pattern elements across a range of developmental stages and wing pattern morphs of Heliconius erato. RESULTS: We identified a number of putative early prepattern genes with color-pattern related expression domains. We also identified 51 genes differentially expressed in association with natural color pattern variation. Of these, the previously identified color pattern “switch gene” optix was recovered as the first transcript to show color-specific differential expression. Most differentially expressed genes were transcribed late in pupal development and have roles in cuticle formation or pigment synthesis. These include previously undescribed transporter genes associated with ommochrome pigmentation. Furthermore, we observed upregulation of melanin-repressing genes such as ebony and Dat1 in non-melanic patterns. CONCLUSIONS: This study identifies many new genes implicated in butterfly wing pattern development and provides a glimpse into the number and types of genes affected by variation in genes that drive color pattern evolution

Genomic Instability in Severe Congenital Neutropenia, a Leukemia Predisposition Syndrome

Severe congenital neutropenia (SCN) is a rare blood disorder characterized by abnormally low levels of circulating neutrophils. Mutations in multiple genes like neutrophil elastase gene (ELANE) and granulocyte colony stimulating factor receptor (CSF3R) may cause SCN. The treatment of choice for SCN is the administration of granulocyte-colony stimulating factor (G-CSF) which elevates the neutrophil count and hence improves the survival and quality of life. Long term survivorship on G-CSF is however linked to development of MDS (myelodysplastic syndrome)/AML (acute myeloid leukemia). About 70% of MDS/AML patients acquire nonsense mutations affecting the cytoplasmic domain of CSF3R. In this project, we hypothesized that this coding region of CSF3R constitutes a hotspot, vulnerable to mutations resulting from excessive oxidative stress or endoplasmic reticulum (ER) stress. We used the murine Ba/F3 cell line to study the effect of induced oxidative or ER stress on the mutation rate in our hypothesized hotspot of the exogenous human CSF3R, the corresponding region in the endogenous Csf3r, and a leukemia-associated gene Runx1. Ba/F3 cells transduced with the cDNA for partial C-terminal of CSF3R fused in-frame with a Green Fluorescent Protein (GFP) tag was subjected to cellular stress inducing mutagen treatment for a prolonged period of time (30 days). The amplicon based targeted deep sequencing data for days 15 and 30 samples show that although there was increased mutagenesis observed in all genes, there were more mutations in the GFP region as compared to the GC-rich partial CSF3R region. Our findings also indicate that there is no correlation between the stress-inducing chemical treatments and mutagenesis in Ba/F3 cells. Thus, we conclude that there are other mechanisms to acquired mutations of CSF3R that help drive the evolution of SCN to MDS/AML. To test this hypothesis, further experiments using unique barcoding system are in progress to characterize the clonal competition between different mutant CSF3R and ELANE expressing cell lines. This study will shed further light on the selection advantage that is provided to cells because cooperativity between mutations in different genes

Lichen Conservation in Eastern North America: Population Genomics, Climate Change, and Translocations

Conservation biology is a scientific discipline that draws on methods from diverse fields to address specific conservation concerns and inform conservation actions. This field is overwhelmingly focused on charismatic animals and vascular plants, often ignoring other diverse and ecologically important groups. This trend is slowly changing in some ways; for example, increasing number of fungal species are being added to the IUCN Red-List. However, a strong taxonomic bias still exists. Here I contribute four research chapters to further the conservation of lichens, one group of frequently overlooked organisms. I address specific conservation concerns in eastern North America using modern methods. The results of these studies provide insight into lichen conservation in each situation, implications for the broader ecosystems within the study regions, and advancement of methods for the study of lichen conservation and biology. The first research chapter (Chapter 2) is a population genomics study based on whole genome shotgun sequencing of Cetradonia linearis, an endangered, lichenized fungus. These data were used to 1) assemble and annotate a reference genome, 2) characterize the mating system, 3) test for isolation by distance (IBD) and isolation by environment (IBE), and 4) investigate the biogeographic history of the species. Approximately 70% of the genome (19.5 Mb) was assembled. Using this assembly, only a single mating type was located, suggesting the species could be unisexual. There was strong evidence for both low rates of recombination and for Isolation by Distance, but no evidence for Isolation by Environment. The hypothesis that C. linearis had a larger range during the last glacial maximum, especially in the southern portion of its current extent, was supported by Hindcast species distribution models and the spatial distribution of genetic diversity. Given the findings here, it is recommended that C. linearis remain protected by the U.S. Endangered Species Act and listed as Vulnerable on the International Union for the Conservation of Nature Red-List. The third chapter is an estimation of the impacts of climate change on high-elevation, endemic lichens in the southern Appalachians, a global diversity hotspot for many groups, including lichens. Extensive field surveys in the high elevations of the region were carried out to accurately document the current distributions of eight narrowly endemic species. These data were compared with herbarium records, and species distribution modeling was used to predict how much climatically suitable area will remain within, and north of, the current range of the target species at multiple time points and climate change scenarios. Fieldwork showed that target species ranged from extremely rare to locally abundant and models predicted average losses of suitable area within the current distribution of species ranging from 93.8 to 99.7%. The results indicate that climate change poses a significant threat to high-elevation lichens, and illustrates the application of current modeling techniques for rare, montane species. In the fourth chapter, a dataset of \u3e13,000 occurrence records for lichens in the Mid-Atlantic Coastal Plain (MACP) of eastern North America was used to model distributions of 193 species. The resulting models were used to quantify the amount of each species’ distribution that is occupied by unsuitable land use types, along with the potential area that will be lost to sea-level rise (SLR). These analyses showed that species have likely already lost an average of 32% of their distributional area to development and agriculture, and are predicted to lose an average of 12.4 and 33.7% of their distributional area with one foot (~0.3 m) and six feet (~1.8 m) of SLR, respectively. Functional and taxonomic groups were compared to identify specific effects of SLR. Species reproducing with symbiotic propagules were found to have significantly larger distributions than species that reproduce sexually with fungal spores alone, and the sexually reproducing species were predicted to lose greater distributional area to SLR. Cladonia species occupy significantly less area in the MACP than Parmotrema species and were predicted to lose more of their distributions to SLR. Patterns of total species diversity showed that the area with the highest diversity is the Dare Peninsula in North Carolina, which was also predicted to lose the most land area to SLR. The workflow established here is flexible and applicable to estimating SLR impacts worldwide and can provide essential insights for local conservation planning. The fifth chapter describes the results of three experiments conducted to test new and established methods for lichen transplantation. First, small fragments of Graphis sterlingiana, Hypotrachyna virginica, and Lepraria lanata were placed on medical gauze attached to each of the species’ most common substrate to test the feasibility of transplanting narrowly endemic species. Second, burlap, cheesecloth, medical gauze, and a plastic air filter were directly compared for their use as artificial transplant substrates with Lepraria finkii as the test lichen. Third, transplants of Usnea angulata were established to test its amenability to transplantation via hanging fragments on monofilament. The first two experiments were established on Roan Mountain, North Carolina and the third experiment at Highlands Biological Station, North Carolina. In the first two experiments medical gauze did not withstand local weather conditions and nearly all pieces fell from the trees within 6 months. The plastic air filter and burlap performed best as artificial substrates for transplants, with a 100% and 80% success rate, respectively. Cheesecloth remained attached to the trees, but only 20% of lichen fragments remained attached to the substrate after one year. In the third experiment U. angulata grew 3.5 ± 1.4 cm in 5 months, exceeding previously reported growth rates for this species. These results advance methods for conservation-focused lichen transplants, and expand established methods to a new region and new species

Privacy protection in location based services

This thesis takes a multidisciplinary approach to understanding the characteristics of Location Based Services (LBS) and the protection of location information in these transactions. This thesis reviews the state of the art and theoretical approaches in Regulations, Geographic Information Science, and Computer Science. Motivated by the importance of location privacy in the current age of mobile devices, this thesis argues that failure to ensure privacy protection under this context is a violation to human rights and poses a detriment to the freedom of users as individuals. Since location information has unique characteristics, existing methods for protecting other type of information are not suitable for geographical transactions. This thesis demonstrates methods that safeguard location information in location based services and that enable geospatial analysis. Through a taxonomy, the characteristics of LBS and privacy techniques are examined and contrasted. Moreover, mechanisms for privacy protection in LBS are presented and the resulting data is tested with different geospatial analysis tools to verify the possibility of conducting these analyses even with protected location information. By discussing the results and conclusions of these studies, this thesis provides an agenda for the understanding of obfuscated geospatial data usability and the feasibility to implement the proposed mechanisms in privacy concerning LBS, as well as for releasing crowdsourced geographic information to third-parties

Quantitative investigation of protein-RNA interactions and regulation by phosphorylation

Phosphorylierung modulieren. Obwohl heute bereits Tausende von Phosphorylierungsstellen annotiert sind, sind entsprechende funktionelle Informationen begrenzt. Dies ist zum Teil darauf zurückzuführen, dass es keine Hochdurchsatzmethoden zur Erforschung der Funktion einer Phosphorylierungsstelle gibt. Um dieser Herausforderung zu begegnen, habe ich eine auf Shotgun-Proteomik basierende Strategie zur Messung der RNA-Bindungsaktivität von RBPs und ihren phosphorylierten Proteoformen entwickelt, die 'quantitative RNA-Interactome Capture (qRIC)' genannt wird. QRIC quantifiziert die Pull-Down-Effizienz von RBPs, die mit Oligo(dT)-Magnetbeads isoliert werden. Diese Effizienz korreliert mit der Anzahl der RNA-Bindungsstellen und der Spezifität der Motivbindung, und spiegelt so die RNA-Bindung in vivo wieder. In einer Gegenüberstellung der Pull-Down-Effizienz verschiedener Proteoformen in unbehandelten Zellen, habe ich qRIC als unvoreingenommenes Screening von regulatorischen Phosphorylierungsstellen in RBPs eingesetzt. Für jede einzelne Phosphorylierungsstelle wurde ein Delta-Effizienzwert berechnet, der den Einfluss auf die RNA-Bindung in vivo reflektiert. Die Effizienzunterschiede spiegelten das erwartete Verhalten von RBPs während der Phasentrennung von membranlosen Organellen und die Ladungsabstoßung zwischen Phosphorylierungsstellen und Nukleotiden bei physiologischem pH-Wert wider. Mithilfe des Delta-Effizienzwertes identifizierte ich mehrere bereits bekannte regulatorische Phosphorylierungsstellen in SF3B1, UPF1 und ELAVL1, sowie neue, bisher unbekannte und möglicherweise regulatorische Phosphorylierungsstellen in SERBP1, LARP1 und RBM20. Phosphomimetische Mutationsvarianten dieser Phosphorylierungsstellen wurden analysiert, um den molekularen Einfluss auf die Regulation der RBP-Funktion zu untersuchen. Es konnte gezeigt werden, dass die Phosphorylierung bestimmter Stellen im Spleißregulator RBM20 dessen nukleo-zytoplasmatische Lokalisierung, die Assoziation mit zytosolischen RNA-Granula und die Spleißfunktion beeinflusst. Diese Erkenntnisse könnten sich beispielsweise auf die Entwicklung neuer Behandlungsmethoden für Patienten mit dysfunktionalen RBM20-Mutationen auswirken, die zu dilatativer Kardiomyopathie führen. QRIC kann als Hochdurchsatzverfahren dazu beitragen, unser Wissen über die Regulierung von Protein-RNA-Interaktionen durch Phosphorylierung zu erweitern.Post-transcriptional regulation of gene expression is fundamental in health and disease. RNA-binding proteins (RBPs) directly bind and govern the fate of RNAs in cells. At the same time, cell signaling cascades control RBP functions by modulating their physicochemical properties through post-translational modifications, like phosphorylation. Although thousands of phosphorylation sites have been annotated, functional information is limited. This, in part, is due to the lack of high-throughput methods that measure function. To tackle this challenge I developed a shotgun proteomics-based strategy for measuring the RNA-binding activity of RBPs and their phosphorylated proteoforms, named quantitative RNA-interactome capture (qRIC). In qRIC, pull-down efficiency of RBPs isolation with oligo(dT) magnetic beads is quantified in cells at steady state and correlates with the number of RNA-binding sites and motif binding specificity, reflecting a link to RNA-binding in vivo. By contrasting pull-down efficiency of different proteoforms in the cells, I applied qRIC as an unbiased screening of regulatory phosphorylation sites in RBPs affecting pull-down efficiency. A delta efficiency score was calculated for each individual phosphorylation site to denote its influence on RNA-binding in vivo. Efficiency differences globally reflected the expected behavior of RBPs during phase separation of membraneless organelles and charge repulsion between phosphorylation sites and nucleotides in physiological pH. Using the delta efficiency score, I identified several previously known regulatory phosphorylation sites in SF3B1, UPF1 and ELAVL1, plus novel candidate regulatory sites in SERBP1, LARP1 and RBM20. Phosphomimetic mutant variants of these sites were analysed to investigate the molecular mechanism of regulation. Importantly, I show that phosphorylation of candidate sites in the splicing regulator RBM20 affects its nucleo-cytoplasmic localization, association with cytosolic RNA granules, and splicing function. These findings could have implications for the development of novel treatments based on kinase activity for patients with dysfunctional RBM20 mutations leading to congenital dilated cardiomyopathy. I anticipate that qRIC, as a high throughput approach, will help to expand our knowledge about the regulation of protein-RNA interactions and their regulation by phosphorylation

Assessment of spatio-temporal patterns of black spruce bud phenology across Quebec based on MODIS-NDVI time series and field observations

Satellite remote sensing is a widely accessible tool to investigate the spatiotemporal variations in the bud phenology of evergreen species, which show limited seasonal changes in canopy greenness. However, there is a need for precise and compatible data to compare remote sensing time series with field observations. In this study, fortnightly MODIS-NDVI was fitted using double-logistic functions and calibrated using ordinal logit models with the sequential phases of bud phenology collected during 2015, 2017 and 2018 in a black spruce stand. Bud break and bud set were spatialized for the period 2009–2018 across 5000 stands in Quebec, Canada. The first phase of bud break and the last phase of bud set were observed in the field in mid-May and at the beginning of September, when NDVI was 80.5% and 92.2% of its maximum amplitude, respectively. The NDVI rate of change was estimated at 0.07 in spring and 0.04 in autumn. When spatialized on the black spruce stands, bud break was detected earlier in the southwestern regions (April–May), and later in the northeastern regions (mid to end of June). No clear trend was observed for bud set, with different patterns being detected among the years. Overall, the process bud break and bud set lasted 51 and 87 days, respectively. Our results demonstrate the potential of satellite remote sensing for providing reliable timings of bud phenological events using calibrated NDVI time series on wide regions that are remote or with limited access