Advancing the Protein-Catalyzed Capture Agent Technology to New Frontiers

Protein-catalyzed capture (PCC) agents are a nascent synthetic aptamer technology that was first disclosed in 2009. In addition to reviewing the different classes of peptide-based aptamers in chapter 1, this thesis records efforts to advance the PCC technology in two ways. First, in chapter 2 the development of a barcoded-rapid assay platform (B-RAP) technology enables the parallel analysis of up to fifteen PCC agents at once as well as dramatically shortening the time required to characterize the binding affinity for a pool of ligands from weeks to a couple of days. Secondly, the capture agent technology was utilized to target difficult proteins. Kirsten rat sarcoma (KRas) protein is a GTPase that acts as a light switch for several important cellular signaling pathways. Oncogenic variants of KRas are responsible for driving roughly 20-25% of all cancers, but KRas is considered “undruggable” from a small molecule targeting point of view. We report the identification of PCC ligands that bind to conserved allosteric switches on KRas and inhibit the protein’s GTPase enzymatic activity. The biomarker Plasmodium falciparum Histidine Rich Protein II (HRP2) presents an unusual challenge as it is a highly variable, unstructured and sticky protein. In chapter 3 we report on efforts to develop low nM binding capture agents against highly prevalent epitopes of HRP2, and the use of medicinal chemistry optimization to prepare structurally related variants of the lead capture agent for probing the structure-activity relationship and how it affects binding to HRP2

An Allosteric Inhibitor of KRas Identified Using a Barcoded Rapid Assay Microchip Platform

Protein catalyzed capture agents (PCCs) are synthetic antibody surrogates that can target a wide variety of biologically relevant proteins. As a step toward developing a high-throughput PCC pipeline, we report on the preparation of a barcoded rapid assay platform for the analysis of hits from PCC library screens. The platform is constructed by first surface patterning a micrometer scale barcode composed of orthogonal ssDNA strands onto a glass slide. The slide is then partitioned into microwells, each of which contains multiple copies of the full barcode. Biotinylated candidate PCCs from a click screen are assembled onto the barcode stripes using a complementary ssDNA-encoded cysteine-modified streptavidin library. This platform was employed to evaluate candidate PCC ligands identified from an epitope targeted in situ click screen against the two conserved allosteric switch regions of the Kirsten rat sarcoma (KRas) protein. A single microchip was utilized for the simultaneous evaluation of 15 PCC candidate fractions under more than a dozen different assay conditions. The platform also permitted more than a 10-fold savings in time and a more than 100-fold reduction in biological and chemical reagents relative to traditional multiwell plate assays. The best ligand was shown to exhibit an in vitro inhibition constant (IC_(50)) of ∼24 μM

Stone artefacts on the seabed at a submerged freshwater spring confirm a drowned cultural landscape in Murujuga, Western Australia

We report the discovery and identification of five ancient stone artefacts associated with a submerged freshwater spring at the underwater archaeological site WH1 in Murujuga (Dampier Archipelago), Western Australia. A limiting date applied to the site based on timing of inundation suggests it was occupied in the Late Pleistocene or Early Holocene. The site is situated well below the intertidal zone having been recorded at 14 m depth in Flying Foam Passage. This discovery highlights the high potential of these submerged springs as archaeological survey targets. We discuss results of a recent survey that expands the number of confirmed artefacts located at WH1 and the geomorphological context in a large calcareous depression associated with a freshwater source. This study demonstrates how submerged landscape research using a suite of technologies can reveal archaeological assemblages in this tropical geomorphological environment, and that adapted techniques could be applied to other tropical conditions such as mangrove coasts, large deltaic plains, or reef-building environments. There are likely thousands of drowned archaeological sites on the continental shelf of the tropics, extending from the intertidal zone to the lowest point of the culturally occupied landmass, at approximately 130 m below modern sea level

An Allosteric Inhibitor of KRas Identified Using a Barcoded Rapid Assay Microchip Platform

Protein catalyzed capture agents (PCCs) are synthetic antibody surrogates that can target a wide variety of biologically relevant proteins. As a step toward developing a high-throughput PCC pipeline, we report on the preparation of a barcoded rapid assay platform for the analysis of hits from PCC library screens. The platform is constructed by first surface patterning a micrometer scale barcode composed of orthogonal ssDNA strands onto a glass slide. The slide is then partitioned into microwells, each of which contains multiple copies of the full barcode. Biotinylated candidate PCCs from a click screen are assembled onto the barcode stripes using a complementary ssDNA-encoded cysteine-modified streptavidin library. This platform was employed to evaluate candidate PCC ligands identified from an epitope targeted in situ click screen against the two conserved allosteric switch regions of the Kirsten rat sarcoma (KRas) protein. A single microchip was utilized for the simultaneous evaluation of 15 PCC candidate fractions under more than a dozen different assay conditions. The platform also permitted more than a 10-fold savings in time and a more than 100-fold reduction in biological and chemical reagents relative to traditional multiwell plate assays. The best ligand was shown to exhibit an in vitro inhibition constant (IC_(50)) of ∼24 μM

Rituximab or Cyclosporine in the Treatment of Membranous Nephropathy

Background: B-cell anomalies play a role in the pathogenesis of membranous nephropathy. B-cell depletion with rituximab may therefore be noninferior to treatment with cyclosporine for inducing and maintaining a complete or partial remission of proteinuria in patients with this condition. Methods: We randomly assigned patients who had membranous nephropathy, proteinuria of at least 5 g per 24 hours, and a quantified creatinine clearance of at least 40 ml per minute per 1.73 m(2) of body-surface area and had been receiving angiotensin-system blockade for at least 3 months to receive intravenous rituximab (two infusions, 1000 mg each, administered 14 days apart; repeated at 6 months in case of partial response) or oral cyclosporine (starting at a dose of 3.5 mg per kilogram of body weight per day for 12 months). Patients were followed for 24 months. The primary outcome was a composite of complete or partial remission of proteinuria at 24 months. Laboratory variables and safety were also assessed. Results: A total of 130 patients underwent randomization. At 12 months, 39 of 65 patients (60%) in the rituximab group and 34 of 65 (52%) in the cyclosporine group had a complete or partial remission (risk difference, 8 percentage points; 95% confidence interval [CI], -9 to 25; P=0.004 for noninferiority). At 24 months, 39 patients (60%) in the rituximab group and 13 (20%) in the cyclosporine group had a complete or partial remission (risk difference, 40 percentage points; 95% CI, 25 to 55; P<0.001 for both noninferiority and superiority). Among patients in remission who tested positive for anti-phospholipase A(2) receptor (PLA2R) antibodies, the decline in autoantibodies to anti-PLA2R was faster and of greater magnitude and duration in the rituximab group than in the cyclosporine group. Serious adverse events occurred in 11 patients (17%) in the rituximab group and in 20 (31%) in the cyclosporine group (P=0.06). Conclusions: Rituximab was noninferior to cyclosporine in inducing complete or partial remission of proteinuria at 12 months and was superior in maintaining proteinuria remission up to 24 months. (Funded by Genentech and the Fulk Family Foundation; MENTOR ClinicalTrials.gov number, .) In a randomized, controlled trial involving patients with membranous nephropathy, rituximab was noninferior to cyclosporine in inducing complete or partial remission of proteinuria at 12 months and was superior in maintaining proteinuria remission for up to 24 months.Genentech; Fulk Family Foundation6 month embargo; published July 4, 2019This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Characterization of functional methylomes by next-generation capture sequencing identifies novel disease-associated variants.

Most genome-wide methylation studies (EWAS) of multifactorial disease traits use targeted arrays or enrichment methodologies preferentially covering CpG-dense regions, to characterize sufficiently large samples. To overcome this limitation, we present here a new customizable, cost-effective approach, methylC-capture sequencing (MCC-Seq), for sequencing functional methylomes, while simultaneously providing genetic variation information. To illustrate MCC-Seq, we use whole-genome bisulfite sequencing on adipose tissue (AT) samples and public databases to design AT-specific panels. We establish its efficiency for high-density interrogation of methylome variability by systematic comparisons with other approaches and demonstrate its applicability by identifying novel methylation variation within enhancers strongly correlated to plasma triglyceride and HDL-cholesterol, including at CD36. Our more comprehensive AT panel assesses tissue methylation and genotypes in parallel at ∼4 and ∼3 M sites, respectively. Our study demonstrates that MCC-Seq provides comparable accuracy to alternative approaches but enables more efficient cataloguing of functional and disease-relevant epigenetic and genetic variants for large-scale EWAS.This work was supported by a Canadian Institute of Health Research (CIHR) team grant awarded to E.G., A.T., M.C.V. and M.L. (TEC-128093) and the CIHR funded Epigeneome Mapping Centre at McGill University (EP1-120608) awarded to T.P. and M.L., and the Swedish Research Council, Knut and Alice Wallenberg Foundation and the Torsten Söderberg Foundation awarded to L.R. F.A. holds studentship from The Research Institute of the McGill University Health Center (MUHC). F.G. is a recipient of a research fellowship award from the Heart and Stroke Foundation of Canada. A.T. is the director of a Research Chair in Bariatric and Metabolic Surgery. M.C.V. is the recipient of the Canada Research Chair in Genomics Applied to Nutrition and Health (Tier 1). E.G. and T.P. are recipients of a Canada Research Chair Tier 2 award. The MuTHER Study was funded by a programme grant from the Wellcome Trust (081917/Z/07/Z) and core funding for the Wellcome Trust Centre for Human Genetics (090532). TwinsUK was funded by the Wellcome Trust; European Community's Seventh Framework Programme (FP7/2007-2013). The study also receives support from the National Institute for Health Research (NIHR)-funded BioResource, Clinical Research Facility and Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust in partnership with King's College London. T.D.S. is a holder of an ERC Advanced Principal Investigator award. SNP genotyping was performed by The Wellcome Trust Sanger Institute and National Eye Institute via NIH/CIDR. Finally, we thank the NIH Roadmap Epigenomics Consortium and the Mapping Centers (http://nihroadmap.nih.gov/epigenomics/) for the production of publicly available reference epigenomes. Specifically, we thank the mapping centre at MGH/BROAD for generation of human adipose reference epigenomes used in this study.This is the final version. It was first published by NPG at http://www.nature.com/ncomms/2015/150529/ncomms8211/full/ncomms8211.html#abstrac

Pharmacogenetic meta-analysis of genome-wide association studies of LDL cholesterol response to statins

Statins effectively lower LDL cholesterol levels in large studies and the observed interindividual response variability may be partially explained by genetic variation. Here we perform a pharmacogenetic meta-analysis of genome-wide association studies (GWAS) in studies addressing the LDL cholesterol response to statins, including up to 18,596 statin-treated subjects. We validate the most promising signals in a further 22,318 statin recipients and identify two loci, SORT1/CELSR2/PSRC1 and SLCO1B1, not previously identified in GWAS. Moreover, we confirm the previously described associations with APOE and LPA. Our findings advance the understanding of the pharmacogenetic architecture of statin response

Insights into hominid evolution from the gorilla genome sequence.

Gorillas are humans' closest living relatives after chimpanzees, and are of comparable importance for the study of human origins and evolution. Here we present the assembly and analysis of a genome sequence for the western lowland gorilla, and compare the whole genomes of all extant great ape genera. We propose a synthesis of genetic and fossil evidence consistent with placing the human-chimpanzee and human-chimpanzee-gorilla speciation events at approximately 6 and 10 million years ago. In 30% of the genome, gorilla is closer to human or chimpanzee than the latter are to each other; this is rarer around coding genes, indicating pervasive selection throughout great ape evolution, and has functional consequences in gene expression. A comparison of protein coding genes reveals approximately 500 genes showing accelerated evolution on each of the gorilla, human and chimpanzee lineages, and evidence for parallel acceleration, particularly of genes involved in hearing. We also compare the western and eastern gorilla species, estimating an average sequence divergence time 1.75 million years ago, but with evidence for more recent genetic exchange and a population bottleneck in the eastern species. The use of the genome sequence in these and future analyses will promote a deeper understanding of great ape biology and evolution

COVID-19 trajectories among 57 million adults in England: a cohort study using electronic health records

BACKGROUND: Updatable estimates of COVID-19 onset, progression, and trajectories underpin pandemic mitigation efforts. To identify and characterise disease trajectories, we aimed to define and validate ten COVID-19 phenotypes from nationwide linked electronic health records (EHR) using an extensible framework. METHODS: In this cohort study, we used eight linked National Health Service (NHS) datasets for people in England alive on Jan 23, 2020. Data on COVID-19 testing, vaccination, primary and secondary care records, and death registrations were collected until Nov 30, 2021. We defined ten COVID-19 phenotypes reflecting clinically relevant stages of disease severity and encompassing five categories: positive SARS-CoV-2 test, primary care diagnosis, hospital admission, ventilation modality (four phenotypes), and death (three phenotypes). We constructed patient trajectories illustrating transition frequency and duration between phenotypes. Analyses were stratified by pandemic waves and vaccination status. FINDINGS: Among 57 032 174 individuals included in the cohort, 13 990 423 COVID-19 events were identified in 7 244 925 individuals, equating to an infection rate of 12·7% during the study period. Of 7 244 925 individuals, 460 737 (6·4%) were admitted to hospital and 158 020 (2·2%) died. Of 460 737 individuals who were admitted to hospital, 48 847 (10·6%) were admitted to the intensive care unit (ICU), 69 090 (15·0%) received non-invasive ventilation, and 25 928 (5·6%) received invasive ventilation. Among 384 135 patients who were admitted to hospital but did not require ventilation, mortality was higher in wave 1 (23 485 [30·4%] of 77 202 patients) than wave 2 (44 220 [23·1%] of 191 528 patients), but remained unchanged for patients admitted to the ICU. Mortality was highest among patients who received ventilatory support outside of the ICU in wave 1 (2569 [50·7%] of 5063 patients). 15 486 (9·8%) of 158 020 COVID-19-related deaths occurred within 28 days of the first COVID-19 event without a COVID-19 diagnoses on the death certificate. 10 884 (6·9%) of 158 020 deaths were identified exclusively from mortality data with no previous COVID-19 phenotype recorded. We observed longer patient trajectories in wave 2 than wave 1. INTERPRETATION: Our analyses illustrate the wide spectrum of disease trajectories as shown by differences in incidence, survival, and clinical pathways. We have provided a modular analytical framework that can be used to monitor the impact of the pandemic and generate evidence of clinical and policy relevance using multiple EHR sources. FUNDING: British Heart Foundation Data Science Centre, led by Health Data Research UK