Understanding the structure, toxicity and inhibition of IAPP at the nanoscale

The aggregation of peptides or proteins to form amyloid fibrils and plaques is associated with the pathologies of a range of neurological disorders and metabolic diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD) and type II diabetes mellitus (T2D). Human islet amyloid polypeptide (IAPP), specifically, contributes to glycemic control but also mediates the dysfunction of insulin-producing pancreatic β-cells in T2D. Furthermore, IAPP in circulation is capable of cross-seeding amyloid-beta and alpha-synuclein, associated with AD and PD respectively. Given no current anti-amyloid treatment is currently commercially available, fundamental understanding of amyloidosis within the complex cellular environment is required against amyloid diseases. In this thesis, conditions to slow down the rapid fibrillization of IAPP to reduce or eliminate intermediate toxic aggregates have been identified in vitro. IAPP intermediate species were characterized and a novel ‘biannular’ morphology identified, with two distinct fibrillating populations then eliciting differential cytotoxicity to mature amyloids in primary cells in vitro. The behavior of fibrillating and fibrillar amyloid species in the presence of model proteins, lipids and ultrasmall lipid membranes was investigated, with regards to their effects on amyloid fibrillization, morphology and associated cytotoxicity in vitro, progressing then towards an in-depth study into the ‘protein corona’ formation of amyloid fibrils within complex biological media. Proteomic analysis identified amyloid-enriched proteins that are known to play significant roles in mediating cellular machinery and processing, potentially leading to pathological outcomes and therapeutic targets. Finally, a biocompatible star polymer (‘PHEA’) was synthesized to mimic the anti-amyloidosis properties of natural polyphenols and effectively rescue IAPP toxicity in mouse islets. Surprisingly, the relatively rigid and amphiphilic PHEA stars induced the formation of novel amyloid morphologies through promoting fibrillization, and accelerated amyloidogenesis to eliminate toxic intermediates in a fashion reminiscent of melanin production. This thesis outlines a logical progression towards the development of effective anti-amyloid strategies: whereby through first gaining insight into amyloidosis, this knowledge can be then applied to ameliorate amyloid pathologies

Effects of Protein Corona on IAPP Amyloid Aggregation, Fibril Remodelling, and Cytotoxicity

Aggregation of islet amyloid polypeptide (IAPP), a peptide hormone co-synthesized and co-stored with insulin in pancreatic cells and also co-secreted to the circulation, is associated with beta-cell death in type-2 diabetes (T2D). In T2D patients IAPP is found aggregating in the extracellular space of the islets of Langerhans. Although the physiological environments of these intra- and extra-cellular compartments and vascular systems significantly differ, the presence of proteins is ubiquitous but the effects of protein binding on IAPP aggregation are largely unknown. Here we examined the binding of freshly-dissolved IAPP as well as pre-formed fibrils with two homologous proteins, namely cationic lysozyme (Lys) and anionic alpha-lactalbumin (aLac), both of which can be found in the circulation. Biophysical characterizations and a cell viability assay revealed distinct effects of Lys and aLac on IAPP amyloid aggregation, fibril remodelling and cytotoxicity, pointing to the role of protein “corona” in conferring the biological impact of amyloidogenic peptides. Systematic molecular dynamics simulations further provided molecular and structural details for the observed differential effects of proteins on IAPP amyloidosis. This study facilitates our understanding of the fate and transformation of IAPP in vivo, which are expected to have consequential bearings on IAPP glycemic control and T2D pathology

Effects of Protein Corona on IAPP Amyloid Aggregation, Fibril Remodelling, and Cytotoxicity

Aggregation of islet amyloid polypeptide (IAPP), a peptide hormone co-synthesized and co-stored with insulin in pancreatic cells and also co-secreted to the circulation, is associated with beta-cell death in type-2 diabetes (T2D). In T2D patients IAPP is found aggregating in the extracellular space of the islets of Langerhans. Although the physiological environments of these intra- and extra-cellular compartments and vascular systems significantly differ, the presence of proteins is ubiquitous but the effects of protein binding on IAPP aggregation are largely unknown. Here we examined the binding of freshly-dissolved IAPP as well as pre-formed fibrils with two homologous proteins, namely cationic lysozyme (Lys) and anionic alpha-lactalbumin (aLac), both of which can be found in the circulation. Biophysical characterizations and a cell viability assay revealed distinct effects of Lys and aLac on IAPP amyloid aggregation, fibril remodelling and cytotoxicity, pointing to the role of protein “corona” in conferring the biological impact of amyloidogenic peptides. Systematic molecular dynamics simulations further provided molecular and structural details for the observed differential effects of proteins on IAPP amyloidosis. This study facilitates our understanding of the fate and transformation of IAPP in vivo, which are expected to have consequential bearings on IAPP glycemic control and T2D pathology

Profiling the serum protein corona of fibrillar human islet amyloid polypeptide

Amyloids may be regarded as native nanomaterials that form in the presence of complex protein mixtures. By drawing an analogy with the physicochemical properties of nanoparticles in biological fluids, we hypothesized that amyloids should form a protein corona in vivo that would imbue the underlying amyloid with a modified biological identity. To explore this hypothesis we characterized the protein corona of human islet amyloid polypeptide (IAPP) fibrils in FBS using two complementary methodologies developed herein; quartz crystal microbalance and ‘centrifugal capture’, coupled with nano-liquid chromatography tandem mass spectroscopy. Clear evidence for a significant protein corona was obtained. No trends were identified for amyloid corona proteins based on their physicochemical properties, while strong binding with IAPP fibrils occurred for linear proteins or multi-domain proteins with structural plasticity. Proteomic analysis identified amyloid-enriched proteins that are known to play significant roles in mediating cellular machinery and processing, potentially leading to pathological outcomes and therapeutic targets

Star polymers reduce IAPP toxicity via accelerated amyloid aggregation

Protein aggregation into amyloid fibrils is a ubiquitous phenomenon across the spectrum of neurodegenerative disorders and type 2 diabetes. A common strategy against amyloidogenesis is to minimize the populations of toxic oligomers and protofibrils by inhibiting protein aggregation with small molecules or nanoparticles. However, melanin synthesis in nature is realized by accelerated protein fibrillation to circumvent accumulation of toxic intermediates. Accordingly, we designed and demonstrated the use of star-shaped poly(2-hydroxyethyl acrylate) (PHEA) nanostructures for promoting the aggregation while ameliorating the toxicity of human islet amyloid polypeptide (IAPP), the peptide involved in glycemic control and the pathology of type 2 diabetes. The binding of PHEA elevated the beta-sheet content in IAPP aggregates while rendered a new morphology of ‘stelliform’ amyloids originating from the polymers. Atomistic molecular dynamics simulations revealed that the PHEA arms served as rod-like scaffolds for IAPP binding and subsequently accelerated IAPP aggregation by increased local peptide concentration. The tertiary structure of the star nanoparticles was found to be essential to drive the specific interactions required to impel the accelerated IAPP aggregation. This study shed new light on the structure-toxicity relationship of IAPP and points to the potential of exploiting star polymers as a new class of therapeutic agents against amyloidogenesis

Testing a global standard for quantifying species recovery and assessing conservation impact.

Recognizing the imperative to evaluate species recovery and conservation impact, in 2012 the International Union for Conservation of Nature (IUCN) called for development of a "Green List of Species" (now the IUCN Green Status of Species). A draft Green Status framework for assessing species' progress toward recovery, published in 2018, proposed 2 separate but interlinked components: a standardized method (i.e., measurement against benchmarks of species' viability, functionality, and preimpact distribution) to determine current species recovery status (herein species recovery score) and application of that method to estimate past and potential future impacts of conservation based on 4 metrics (conservation legacy, conservation dependence, conservation gain, and recovery potential). We tested the framework with 181 species representing diverse taxa, life histories, biomes, and IUCN Red List categories (extinction risk). Based on the observed distribution of species' recovery scores, we propose the following species recovery categories: fully recovered, slightly depleted, moderately depleted, largely depleted, critically depleted, extinct in the wild, and indeterminate. Fifty-nine percent of tested species were considered largely or critically depleted. Although there was a negative relationship between extinction risk and species recovery score, variation was considerable. Some species in lower risk categories were assessed as farther from recovery than those at higher risk. This emphasizes that species recovery is conceptually different from extinction risk and reinforces the utility of the IUCN Green Status of Species to more fully understand species conservation status. Although extinction risk did not predict conservation legacy, conservation dependence, or conservation gain, it was positively correlated with recovery potential. Only 1.7% of tested species were categorized as zero across all 4 of these conservation impact metrics, indicating that conservation has, or will, play a role in improving or maintaining species status for the vast majority of these species. Based on our results, we devised an updated assessment framework that introduces the option of using a dynamic baseline to assess future impacts of conservation over the short term to avoid misleading results which were generated in a small number of cases, and redefines short term as 10 years to better align with conservation planning. These changes are reflected in the IUCN Green Status of Species Standard

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030