Smart Nanomaterials from Repeat Proteins and Amyloid Fibrils

Protein-based materials are an important area of research for various reasons. Natural protein materials such as spider silk have mechanical properties which compare favourably to artificial or inorganic materials, and in addition are biodegradable and can be produced from easily available feedstocks. It is also possible to produce materials that incorporate the functionality of a natural protein, such as ligand-binding or catalysis of reactions, thus allowing this functionality to be used in the solid rather than solution phase. Two particularly interesting components for protein-based materials are amyloid fibrils and tandem repeat proteins. Amyloid fibrils are exceptionally strong, tough, highly-ordered structures that self-assemble from a wide range of simple building blocks. Meanwhile, tandem repeat proteins are a class of proteins that act as scaffolds to mediate protein-protein interactions and are known to act as elastic springs. Unlike globular proteins, tandem repeat proteins can be designed to bind specific ligands, and their ligand-binding properties and stability can be tuned separately. This work details the synthesis and characterisation of repeat protein and amyloid fibril components for a “smart” hydrogel, the production of these gels, and their characterisation using a microfluidic method that I developed. Although amyloid fibrils have previously been decorated with functional proteins, hitherto, this has usually been done by assembling the fibrils from already-functionalised components. This approach limits the functionality to species that can survive the harsh conditions of amyloid aggregation and do not disturb fibril assembly. Therefore, a method was developed to produce amyloid fibrils that displayed an alkyne functionality on their surface to allow functional proteins or other species to be attached after assembly. This involved the design and synthesis (using solid-phase peptide chemistry) of a peptide based on the previously known TTR105-115 peptide (derived from the amyloidogenic Transthyretin protein). These fibrils were characterised by AFM and TEM and it was then shown that the assembled fibrils could be functionalised using an azide-alkyne “click” reaction. The reaction was shown to work with a variety of ligands including proteins, which were found to retain their structure and function after crosslinking to the fibril. The fibrils with ligands attached were characterised by a variety of methods including LCMS (liquid chromatography-mass spectrometry) and super-resolution optical microscopy. Next, repeat proteins were produced recombinantly containing non-natural azido amino acids at their termini. Incorporation of non-natural amino acids was carried out using a number of different methods including amber codon suppression and methionine replacement. Micron-sized hydrogels were then formed from microfluidic-generated droplets by covalently crosslinking the alkyne-functionalised fibrils with the azide-functionalised repeat proteins. The initial experiments to show proof of principle were carried out with consensus-designed repeat proteins, but repeat proteins based on natural sequences were also used to make hydrogels that could later be tested for potential uptake of peptides known to bind these proteins. These hydrogels could potentially be used for drug delivery or other applications in which a chemical response to a mechanical stimulus is desired. The mechanical properties of the hydrogels were measured using novel microfluidic devices, which were designed and fabricated using standard PDMS-based soft lithographyPhD research was funded through the EPSRC nanoDTC, EP/G037221/

Nanoscale click-reactive scaffolds from peptide self-assembly.

Background Due to their natural tendency to self-assemble, proteins and peptides are important components for organic nanotechnology. One particular class of peptides of recent interest is those that form amyloid fibrils, as this self-assembly results in extremely strong, stable quasi-one-dimensional structures which can be used to organise a wide range of cargo species including proteins and oligonucleotides. However, as the amyloid state is accessible to a large number of proteins via misfolding, assembly of peptides already conjugated to proteins is limited to certain cargo species. Therefore, a general method is needed to conjugate proteins and other molecules to amyloid fibrils after the fibrils have self-assembled. Results Here we have designed an amyloidogenic peptide based on the TTR105-115 fragment of transthyretin to form fibrils that display an alkyne functionality, important for bioorthogonal chemical reactions, on their surface. The fibrils were formed and reacted both with an azide-containing amino acid and with an azide-functionalised dye by the Huisgen azidoalkyne cycloaddition, one of the class of “click” reactions. Mass spectrometry and total internal reflection fluorescence optical microscopy were used to show that peptides incorporated into the fibrils reacted with the azide while maintaining the structure of the fibril. These click-functionalised amyloid fibrils have a variety of potential uses in materials and as scaffolds for bionanotechnology. Discussion Although previous studies have produced peptides that can both form amyloid fibrils and undergo “click”-type reactions, this is the first example of amyloid fibrils that can undergo such a reaction after they have been formed. Our approach has the advantage that self-assembly takes place before click functionalization rather than pre-functionalised building blocks self-assembling. Therefore, the molecules used to functionalise the fibril do not themselves have to be exposed to harsh, amyloid-forming conditions. This means that a wider range of proteins can be used as ligands in this process. For instance, the fibrils can be functionalised with a green fluorescent protein that retains its fluorescence after it is attached to the fibrils, whereas this protein loses its fluorescence if it is exposed to the conditions used for aggregation

Do non-philosophers think epistemic consequentialism is counterintuitive?

Direct epistemic consequentialism is the idea that X is epistemically permissible iff X maximizes epistemic value. It has received lots of attention in recent years and is widely accepted by philosophers to have counterintuitive implications. There are various reasons one might suspect that the relevant intuitions will not be widely shared among non-philosophers. This paper presents an initial empirical study of ordinary intuitions. The results of two experiments demonstrate that the counterintuitiveness of epistemic consequentialism is more than a philosophers' worry---the folk seem to agree

Deformable and Robust Core–Shell Protein Microcapsules Templated by Liquid–Liquid Phase‐Separated Microdroplets

Abstract: Microcapsules are a key class of microscale materials with applications in areas ranging from personal care to biomedicine, and with increasing potential to act as extracellular matrix (ECM) models of hollow organs, tissues, or biomolecular condensates. Such capsules are conventionally generated from non‐ECM materials including synthetic polymers. Here, robust microcapsules with controllable shell thickness from physically‐ and enzymatically‐crosslinked gelatin are fabricated, and a core–shell architecture is achieved by exploiting a liquid–liquid phase‐separated aqueous system in a one‐step microfluidic process. Microfluidic mechanical testing reveals that the mechanical robustness of thicker‐shell capsules could be controlled through modulation of the shell thickness. Furthermore, the microcapsules demonstrate environmentally‐responsive deformation, including buckling driven by osmosis and external mechanical forces. A sequential release of cargo species is obtained through the degradation of the capsules. Stability measurements show the capsules are stable at 37 °C for more than 2 weeks. Finally, through gel–sol transition, microgels function as precursors for the formation of all‐aqueous liquid–liquid phase‐separated systems that are two‐phase or multiphase. These smart capsules that can undergo phase transition are promising models of hollow biostructures, microscale drug carriers, and building blocks or compartments for active soft materials and robots

International Consensus Statement on Rhinology and Allergy: Rhinosinusitis

Background: The 5 years since the publication of the first International Consensus Statement on Allergy and Rhinology: Rhinosinusitis (ICAR‐RS) has witnessed foundational progress in our understanding and treatment of rhinologic disease. These advances are reflected within the more than 40 new topics covered within the ICAR‐RS‐2021 as well as updates to the original 140 topics. This executive summary consolidates the evidence‐based findings of the document. Methods: ICAR‐RS presents over 180 topics in the forms of evidence‐based reviews with recommendations (EBRRs), evidence‐based reviews, and literature reviews. The highest grade structured recommendations of the EBRR sections are summarized in this executive summary. Results: ICAR‐RS‐2021 covers 22 topics regarding the medical management of RS, which are grade A/B and are presented in the executive summary. Additionally, 4 topics regarding the surgical management of RS are grade A/B and are presented in the executive summary. Finally, a comprehensive evidence‐based management algorithm is provided. Conclusion: This ICAR‐RS‐2021 executive summary provides a compilation of the evidence‐based recommendations for medical and surgical treatment of the most common forms of RS

Lempäälän matkailun tila matkailuyrittäjien näkökulmasta

Opinnäytetyön tarkoituksena oli selvittää Lempäälän kunnan tämän hetkistä matkailun tilaa paikallisten matkailuyrittäjien näkökulmasta. Tämän opinnäytetyön toimeksiantajana toimi Lempäälän Kehitys Oy, joka vastaa Lempäälän kunnan elinkeinotoimen palveluista ja kehittämisestä. Tavoite oli saada mahdollisimman moni lempääläläinen matkailuyrittäjä mukaan tutkimukseen, jotta kaikki kehitysehdotukset ja ideat saataisiin eteenpäin toimeksiantajalle. Toimeksiantajan toiveen mukaisesti selvitettiin kunnan matkailun tilaa kyselylomakkeen avulla. Tutkimuksen kysely toteutettiin haastattelemalla paikallisia yrittäjiä heidän yrityksissään tai vaihtoehtoisesti sähköpostitse. Haastattelumuotona käytettiin strukturoitua haastattelua eli lomakehaastattelua sillä vastausprosentin pelättiin jäävän alhaiseksi. Aineistoina käytettiin haastattelujen tuloksia, Lempäälän tietopaketti 2014 -esitettä sekä matkailuyrityksen omia nettisivuja. Tutkimuksen tuloksia analysoitiin vetovoimateorian ja segmentoinnin teorian avulla. Tutkimuksen perusteella Lempäälän matkailun tila on kokonaisuudessaan hyvä sillä paikallisilla yrittäjillä on vireää yritystoimintaa. Monet yrittäjistä ovat kiinnostuneita yhteistyöstä muiden yrittäjien kanssa mikä parantaisi koko Lempäälän matkailua. Lempäälässä vierailee paljon lapsiperheitä ja pariskuntia, jotka nauttivat rauhallisuudesta ja luonnosta. Lempäälän kunnalla on matkailupotentiaalia luontomatkailukohteena, jota pitäisi tulevaisuudessa osata hyödyntää ja markkinoida oikein.The purpose of this thesis was to find out the current situation in tourism in Lempäälä from the point of view tourism entrepreneurs. The thesis was commissioned by Lempäälän Kehitys Oy, which is responsible for the industrial development and services in the municipality of Lempäälä. The aim of this thesis was to make as many tourism entrepreneurs as possible to participate in this survey, so that all the ideas and improvement suggestions would reach the commissioning company. Lempäälän Kehitys wanted the present state of tourism to be investigated by means of an inquiry. The survey was carried out by interviewing local entrepreneurs either face to face or via e-mail. The type of the interview was half structured, that is a form interview, since the percent of the answers was scared to be low. The material of the study consisted of the results of the interviews, the 2014 brochure on Lempäälä and entrepreneur`s homepages. The results of the survey were analyzed in relation to the theories of attraction and segmentation. On the basis of the survey, the situation in tourism in Lempäälä is good, because many local entrepreneurs have active business. Also many entrepreneurs are also interested in cooperation with others which improves the entire tourism in the municipality of Lempäälä. The main visitors in Lempäälä are families and couples, who enjoy peacefulness and nature. The municipality of Lempäälä has a lot of tourism potential as a nature attraction, which should be utilized and marketed in the right way in the future

MOESM1 of Nanoscale click-reactive scaffolds from peptide self-assembly

Additional file 1. Synthesis of o-propargyl serine, image processing details and fluorescence polarization result

Monitoring Cell Spheroid Formation And Culture In Porous Membrane Microwells By Electrical Impedance

We have constructed a reusable flow cell in which the aggregation of cells in low-attachment microwells to form spheroids can be monitored in real-time using electrical impedance. Culture in dynamic flow conditions allows media to be refreshed without disturbing the cells. The use of transparent ITO electrodes for sensing, and the methods used for fabrication of porous membrane microwells, allow the spheroids to be observed using a microscope. Electrodes for addressable measurement of individual microwells have also been developed

The role of ENPP1/PC-1 in osteoinduction by calcium phosphate ceramics

In the past decade, calcium phosphate (CaP) ceramics have emerged as alternatives to autologous bone grafts for the treatment of large, critical-sized bone defects. In order to be effective in the regeneration of such defects, ceramics must show osteoinductive behaviour, defined as the ability to induce de novo heterotopic bone formation. While a set of osteoinductive CaP ceramics has been developed, the exact processes underlying osteoinduction, and the role of the physical and chemical properties of the ceramics, remain largely unknown. Previous studies have focused on the role of the transcriptome to shed light on the mechanism of osteoinduction at the mRNA level. To complement these studies, a proteomic analysis was performed to study the behaviour of hMSCs on osteoinductive and non-osteoinductive CaPs. The results of this analysis suggest that plasma cell glycoprotein 1 (PC-1), encoded by the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene, plays a key role in the process of osteoinduction by CaP ceramics. Validation experiments have confirmed that indeed, the mRNA expression of ENPP1 and the production of PC-1 are higher on osteoinductive than on non-osteoinductive CaP ceramics, a trend that was also observed for other osteogenic markers such as bone morphogenetic protein 2 (BMP2) and osteopontin (OPN), but not for alkaline phosphatase (ALP). Our results also showed that the expression of PC-1 is restricted to those cells which are in direct contact with the CaP ceramic surface, plausibly due to the localised depletion of calcium and inorganic phosphate ions from the supersaturated cell culture medium as CaP crystallises on the ceramic surface. Replicating the surface of the osteoinductive ceramic in polystyrene resulted in a significant decrease in ENPP1 expression, suggesting that surface structural properties alone are not sufficient to induce ENPP1 expression. Finally, knocking down ENPP1 expression in hMSCs resulted in increased BMP2 expression, both at the mRNA and protein level, suggesting that ENPP1 is a negative regulator of BMP-2 signalling. Taken together, this study shows, for the first time, that ENPP1/PC-1 plays an important role in CaP-induced osteogenic differentiation of hMSCs and thus possibly osteoinduction by CaP ceramics. Furthermore, we have identified a crucial role for the interfacial (chemical) events occurring on the CaP ceramic surface in the process of osteoinduction. This knowledge can contribute to the development of new bone graft substitutes, with improved osteoinductive potential.</p