A Study on Auto-Catalysis and Product Inhibition: A Nucleophilic Aromatic Substitution Reaction Catalysed within the Cavity of an Octanuclear Coordination Cage.

The ability of an octanuclear cubic coordination cage to catalyse a nucleophilic aromatic substitution reaction on a cavity-bound guest was studied with 2,4-dinitrofluorobenzene (DNFB) as the guest/substrate. It was found that DNFB undergoes a catalysed reaction with hydroxide ions within the cavity of the cubic cage (in aqueous buffer solution, pH 8.6). The rate enhancement of kcat/kuncat was determined to be 22, with cavity binding of the guest being required for catalysis to occur. The product, 2,4-dinitrophenolate (DNP), remained bound within the cavity due to electrostatic stabilisation and exerts two apparently contradictory effects: it initially auto-catalyses the reaction when present at low concentrations, but at higher concentrations inhibits catalysis when a pair of DNP guests block the cavity. When encapsulated, the UV/Vis absorption spectrum of DNP is red-shifted when compared to the spectrum of free DNP in aqueous solution. Further investigations using other aromatic guests determined that a similar red-shift on cavity binding also occurred for 4-nitrophenolate (4NP) at pH 8.6. The red-shift was used to determine the stoichiometry of guest binding of DNP and 4NP within the cage cavity, which was confirmed by structural analysis with X-ray crystallography; and was also used to perform catalytic kinetic studies in the solution-state.

Chikungunya seroprevalence, force of infection, and prevalence of chronic disability after infection in endemic and epidemic settings: a systematic review, meta-analysis, and modelling study.

BACKGROUND: Chikungunya is an arboviral disease transmitted by Aedes aegypti and Aedes albopictus mosquitoes with a growing global burden linked to climate change and globalisation. We aimed to estimate chikungunya seroprevalence, force of infection (FOI), and prevalence of related chronic disability and hospital admissions in endemic and epidemic settings. METHODS: In this systematic review, meta-analysis, and modelling study, we searched PubMed, Ovid, and Web of Science for articles published from database inception until Sept 26, 2022, for prospective and retrospective cross-sectional studies that addressed serological chikungunya virus infection in any geographical region, age group, and population subgroup and for longitudinal prospective and retrospective cohort studies with data on chronic chikungunya or hospital admissions in people with chikungunya. We did a systematic review of studies on chikungunya seroprevalence and fitted catalytic models to each survey to estimate location-specific FOI (ie, the rate at which susceptible individuals acquire chikungunya infection). We performed a meta-analysis to estimate the proportion of symptomatic patients with laboratory-confirmed chikungunya who had chronic chikungunya or were admitted to hospital following infection. We used a random-effects model to assess the relationship between chronic sequelae and follow-up length using linear regression. The systematic review protocol is registered online on PROSPERO, CRD42022363102. FINDINGS: We identified 60 studies with data on seroprevalence and chronic chikungunya symptoms done across 76 locations in 38 countries, and classified 17 (22%) of 76 locations as endemic settings and 59 (78%) as epidemic settings. The global long-term median annual FOI was 0·007 (95% uncertainty interval [UI] 0·003-0·010) and varied from 0·0001 (0·00004-0·0002) to 0·113 (0·07-0·20). The highest estimated median seroprevalence at age 10 years was in south Asia (8·0% [95% UI 6·5-9·6]), followed by Latin America and the Caribbean (7·8% [4·9-14·6]), whereas median seroprevalence was lowest in the Middle East (1·0% [0·5-1·9]). We estimated that 51% (95% CI 45-58) of people with laboratory-confirmed symptomatic chikungunya had chronic disability after infection and 4% (3-5) were admitted to hospital following infection. INTERPRETATION: We inferred subnational heterogeneity in long-term average annual FOI and transmission dynamics and identified both endemic and epidemic settings across different countries. Brazil, Ethiopia, Malaysia, and India included both endemic and epidemic settings. Long-term average annual FOI was higher in epidemic settings than endemic settings. However, long-term cumulative incidence of chikungunya can be similar between large outbreaks in epidemic settings with a high FOI and endemic settings with a relatively low FOI. FUNDING: International Vaccine Institute.

The melon Fom-1-Prv resistance gene pair: Correlated spatial expression and interaction with a viral protein.

The head-to-head oriented pair of melon resistance genes, Fom-1 and Prv, control resistance to Fusarium oxysporum races 0 and 2 and papaya ringspot virus (PRSV), respectively. They encode, via several RNA splice variants, TIR-NBS-LRR proteins, and Prv has a C-terminal extra domain with a second NBS homologous sequence. In other systems, paired R-proteins were shown to operate by "labor division," with one protein having an extra integrated domain that directly binds the pathogen's Avr factor, and the second protein executing the defense response. We report that the expression of the two genes in two pairs of near-isogenic lines was higher in the resistant isoline and inducible by F. oxysporum race 2 but not by PRSV. The intergenic DNA region separating the coding sequences of the two genes acted as a bi-directional promoter and drove GUS expression in transgenic melon roots and transgenic tobacco plants. Expression of both genes was strong in melon root tips, around the root vascular cylinder, and the phloem and xylem parenchyma of tobacco stems and petioles. The pattern of GUS expression suggests coordinated expression of the two genes. In agreement with the above model, Prv's extra domain was shown to interact with the cylindrical inclusion protein of PRSV both in yeast cells and in planta.

T6SS: A Key to Pseudomonas's Success in Biocontrol?

Bacteria from the genus Pseudomonas have been extensively studied for their capacity to act as biological control agents of disease and pests and for their ability to enhance and promote crop production in agricultural systems. While initial research primarily focused on the human pathogenic bacteria Pseudomonas aeruginosa, recent studies indicate the significance of type VI secretion (T6SS) in other Pseudomonas strains for biocontrol purposes. This system possibly plays a pivotal role in restricting the biological activity of target microorganisms and may also contribute to the bolstering of the survival capabilities of the bacteria within their applied environment. The type VI secretion system is a phage-like structure used to translocate effectors into both prokaryotic and eukaryotic target cells. T6SSs are involved in a myriad of interactions, some of which have direct implications in the success of Pseudomonas as biocontrol agents. The prevalence of T6SSs in the genomes of Pseudomonas species is notably greater than the estimated 25% occurrence rate found in Gram-negative bacteria. This observation implies that T6SS likely plays a pivotal role in the survival and fitness of Pseudomonas. This review provides a brief overview of T6SS, its role in Pseudomonas with biocontrol applications, and future avenues of research within this subject matter.

The preservation of sarin and O,O'-diisopropyl fluorophosphate inside coordination cage hosts.

The host-guest chemistry of O,O'-diisopropyl fluorophosphate (DFP), a phosphonofluoridate G-series chemical warfare agent simulant, was investigated in the presence of a number of octanuclear cubic coordination cage hosts. The aim was to demonstrate cage-catalysed hydrolysis of DFP at near neutral pH: however, two octanuclear coordination cages, HPEG (containing water-solubilising PEG groups) and HW (containing water-solubilising hydroxymethyl groups), were actually found to increase the lifetime of DFP in aqueous buffer solution (pH 8.7). Crystallographic analysis of DFP with a structurally related host cage revealed that DFP binds to windows in the cage surface, not in the internal cavity. The phosphorus-fluorine bond is directed into the cavity rather than towards the external environment, with the cage/DFP association protecting DFP from hydrolysis. Initial studies with the chemical warfare agent (CWA) sarin (GB) with HPEG cage in a buffered solution also showed a drastically reduced rate of hydrolysis for sarin when bound in the host cage. The ability of these cages to inhibit hydrolysis of these P-F bond containing organophosphorus guests, by encapsulation, may have applications in forensic sample preservation and analysis.

Correction: Disentangling contributions to guest binding inside a coordination cage host: analysis of a set of isomeric guests with differing polarities.

Correction for 'Disentangling contributions to guest binding inside a coordination cage host: analysis of a set of isomeric guests with differing polarities' by Cristina Mozaceanu et al., Dalton Trans., 2022, 51, 15263-15272, https://doi.org/10.1039/D2DT02623F.

Direct digital sensing of protein biomarkers in solution.

The detection of proteins is of central importance to biomolecular analysis and diagnostics. Typical immunosensing assays rely on surface-capture of target molecules, but this constraint can limit specificity, sensitivity, and the ability to obtain information beyond simple concentration measurements. Here we present a surface-free, single-molecule microfluidic sensing platform for direct digital protein biomarker detection in solution, termed digital immunosensor assay (DigitISA). DigitISA is based on microchip electrophoretic separation combined with single-molecule detection and enables absolute number/concentration quantification of proteins in a single, solution-phase step. Applying DigitISA to a range of targets including amyloid aggregates, exosomes, and biomolecular condensates, we demonstrate that the assay provides information beyond stoichiometric interactions, and enables characterization of immunochemistry, binding affinity, and protein biomarker abundance. Taken together, our results suggest a experimental paradigm for the sensing of protein biomarkers, which enables analyses of targets that are challenging to address using conventional immunosensing approaches.

Disentangling contributions to guest binding inside a coordination cage host: analysis of a set of isomeric guests with differing polarities.

Binding of a set of three isomeric guests (1,2-, 1,3- and 1,4-dicyanobenzene, abbreviated DCB) inside an octanuclear cubic coordination cage host H (bearing different external substitutents according to solvent used) has been studied in water/dmso (98 : 2) and CD2Cl2. These guests have essentially identical molecular surfaces, volumes and external functional groups to interact with the cage interior surface; but they differ in polarity with dipole moments of ca. 7, 4 and 0 Debye respectively. In CD2Cl2 guest binding is weak but we observe a clear correlation of binding free energy with guest polarity, with 1,4-DCB showing no detectable binding by NMR spectroscopy but 1,2-DCB having -ΔG = 9 kJ mol-1. In water (containing 2% dmso to solubilise the guests) we see the same trend but all binding free energies are much higher due to an additional hydrophobic contribution to binding, with -ΔG varying from 16 kJ mol-1 for 1,4-DCB to 22 kJ mol-1 for 1,4-DCB: again we see an increase associated with guest polarity but the increase in -ΔG per Debye of dipole moment is around half what we observe in CD2Cl2 which we ascribe to the fact the more polar guests will be better solvated in the aqueous solvent. A van't Hoff analysis by variable-temperature NMR showed that the improvement in guest binding in water/dmso is entropy-driven, which suggests that the key factor is not direct electrostatic interactions between a polar guest and the cage surface, but the variation in guest desolvation across the series, with the more polar (and hence more highly solvated) guests having a greater favourable entropy change on desolvation.

Inside or outside the box? Effect of substrate location on coordination-cage based catalysis.

In this work we compare and contrast the hydrolysis of two different aromatic esters using an octanuclear cubic Co8 coordination cage host as the catalyst. Diacetyl fluorescein (DAF) is too large to bind inside the cage cavity, but in aqueous solution it interacts with the exterior surface of the cage via a hydrophobic interaction with K = 1.5(2) × 104 M-1. This is sufficient to bring it into close proximity to the layer of hydroxide ions which also surrounds the 16+ cage surface even at modest pH values, accelerating the hydrolysis of DAF to fluorescein with kcat/kuncat (the rate acceleration for that fraction of DAF in contact with the cage surface in the equilibrium) ≈50. This is far smaller than many known examples of catalysis inside a cage cavity, but at the exterior surface it is potentially general with no cavity-imposed size/shape limitations for guest binding. In contrast 4-nitrophenyl acetate (4NPA) binds inside the cage cavity with K = 3.5(3) × 103 M-1 and as such is surrounded in solution by the hydroxide ions which accumulate around the cage surface. However its hydrolysis is actually inhibited: either because of a geometrically unfavourable geometry of the bound substrate which makes it inaccessible to surface-bound hydroxide, or because the necessary volume expansion/geometry change associated with formation of a tetrahedral intermediate cannot be accommodated inside the cavity. Any 4NPA that is free in solution as part of the equilibrium undergoes catalysed hydrolysis at the cage exterior surface in the same way as DAF, but the effect is limited by the low affinity of 4NPA for the exterior surface. We conclude that exterior-surface catalysis can be effective and potentially general; and that cavity-binding of guests can result in negative, rather than positive, catalysis.

Imaging protein aggregates in the serum and cerebrospinal fluid in Parkinson's disease.

Aggregation of α-synuclein plays a key role in the development of Parkinson's disease. Soluble aggregates are present not only within human brain but also the CSF and blood. Characterizing the aggregates present in these biofluids may provide insights into disease mechanisms and also have potential for aiding diagnosis. We used two optical single-molecule imaging methods called aptamer DNA-PAINT and single-aggregate confocal fluorescence, together with high-resolution atomic force microscopy for specific detection and characterization of individual aggregates with intermolecular ß-sheet structure, present in the CSF and serum of 15 early stage Parkinson's disease patients compared to 10 healthy age-matched controls. We found aggregates ranging in size from 20 nm to 200 nm, in both CSF and serum. There was a difference in aggregate size distribution between Parkinson's disease and control groups with a significantly increased number of larger aggregates (longer than 150 nm) in the serum of patients with Parkinson's disease. To determine the chemical composition of the aggregates, we performed aptamer DNA-PAINT on serum following α-synuclein and amyloid-ß immunodepletion in an independent cohort of 11 patients with early stage Parkinson's disease and 10 control subjects. ß-Sheet aggregates in the serum of Parkinson's disease patients were found to consist of, on average, 50% α-synuclein and 50% amyloid-ß in contrast to 30% α-synuclein and 70% amyloid-ß in control serum [the differences in the proportion of these aggregates were statistically significant between diseased and control groups (P = 1.7 × 10-5 for each species)]. The ratio of the number of ß-sheet α-synuclein aggregates to ß-sheet amyloid-ß aggregates in serum extracted using our super-resolution method discriminated Parkinson's disease cases from controls with an accuracy of 98.2% (AUC = 98.2%, P = 4.3 × 10-5). Our data suggest that studying the protein aggregates present in serum can provide information about the disruption of protein homeostasis occurring in Parkinson's disease and warrants further investigation as a potential biomarker of disease.

Interaction of anions with the surface of a coordination cage in aqueous solution probed by their effect on a cage-catalysed Kemp elimination.

An octanuclear M8L12 coordination cage catalyses the Kemp elimination reaction of 5-nitro-1,2-benzisoxazole (NBI) with hydroxide to give 2-cyano-4-nitrophenolate (CNP) as the product. In contrast to the previously-reported very efficient catalysis of the Kemp elimination reaction of unsubstituted benzisoxazole, which involves the substrate binding inside the cage cavity, the catalysed reaction of NBI with hydroxide is slower and occurs at the external surface of the cage, even though NBI can bind inside the cage cavity. The rate of the catalysed reaction is sensitive to the presence of added anions, which bind to the 16+ cage surface, displacing the hydroxide ions from around the cage which are essential reaction partners in the Kemp elimination. Thus we can observe different binding affinities of anions to the surface of the cationic cage in aqueous solution by the extent to which they displace hydroxide and thereby inhibit the catalysed Kemp elimination and slow down the appearance of CNP. For anions with a -1 charge the observed affinity order for binding to the cage surface is consistent with their ease of desolvation and their ordering in the Hofmeister series. With anions that are significantly basic (fluoride, hydrogen carbonate, carboxylates) the accumulation of the anion around the cage surface accelerates the Kemp elimination compared to the background reaction with hydroxide, which we ascribe to the ability of these anions to participate directly in the Kemp elimination. This work provides valuable mechanistic insights into the role of the cage in co-locating the substrate and the anionic reaction partners in a cage-catalysed reaction.

Orthogonal binding and displacement of different guest types using a coordination cage host with cavity-based and surface-based binding sites.

The octanuclear Co(ii) cubic coordination cage system H (or HW if it bears external water-solubilising substituents) has two types of binding site for guests. These are (i) the partially-enclosed central cavity where neutral hydrophobic organic species can bind, and (ii) the six 'portals' in the centres of each of the faces of the cubic cage where anions bind via formation of a network of CH⋯X hydrogen bonds between the anion and CH units on the positively-charged cage surface, as demonstrated by a set of crystal structures. The near-orthogonality of these guest binding modes provides the basis for an unusual dual-probe fluorescence displacement assay in which either a cavity-bound fluorophore (4-methyl-7-amino-coumarin, MAC; λ em = 440 nm), or a surface-bound anionic fluorophore (fluorescein, FLU; λ em = 515 nm), is displaced and has its emission 'switched on' according to whether the analyte under investigation is cavity-binding, surface binding, or a combination of both. A completely orthogonal system is demonstrated based using a Hw/MAC/FLU combination: addition of the anionic analyte ascorbate displaced solely FLU from the cage surface, increasing the 515 nm (green) emission component, whereas addition of a neutral hydrophobic guest such as cyclooctanone displaced solely MAC from the cage central cavity, increasing the 440 nm (blue) emission component. Addition of chloride results in some release of both components, and an intermediate colour change, as chloride is a rare example of a guest that shows both surface-binding and cavity-binding behaviour. Thus we have a colourimetric response based on differing contributions from blue and green emission components in which the specific colour change signals the binding mode of the analyte. Addition of a fixed red emission component from the complex [Ru(bipy)3]2+ (Ru) provides a baseline colour shift of the overall colour of the luminescence closer to neutral, meaning that different types of guest binding result in different colour changes which are easily distinguishable by eye.

On-Farm Evaluations of Anaerobic Soil Disinfestation and Grafting for Management of a Widespread Soilborne Disease Complex in Protected Culture Tomato Production.

Tomato production in Ohio protected culture systems is hindered by a soilborne disease complex consisting of corky root rot (Pyrenochaeta lycopersici), black dot root rot (Colletotrichum coccodes), Verticillium wilt (Verticillium dahliae), and root-knot (Meloidogyne hapla and M. incognita). In a survey of 71 high tunnels, C. coccodes was detected in 90% of high tunnels, and P. lycopersici (46%), V. dahliae (48%), and Meloidogyne spp. (45%) were found in nearly half of high tunnels. Anaerobic soil disinfestation (ASD) with wheat bran (20.2 Mg/ha) plus molasses (10.1 Mg/ha) and grafting onto 'Maxifort' or 'Estamino' rootstocks were evaluated in high tunnels on five farms. In post-ASD bioassays of trial soils, root and taproot rot severity were significantly reduced after ASD, and root-knot galling was also reduced by ASD. Soilborne pathogenic fungi were isolated less frequently from bioassay plants grown in ASD-treated soils than control soils. Similar results were observed in tomato plants grown in high tunnels. Root rot was significantly reduced by ASD in nearly all trials. Corky root rot severity was highest in nongrafted plants grown in nontreated soils, and the lowest levels of corky root rot were observed in 'Maxifort'-grafted plants. Black dot root rot severity was higher or equivalent in grafted plants compared with nongrafted plants. Root-knot severity was lower in plants grown in ASD-treated soils in high tunnels compared with plants grown in control soils, but grafting did not significantly decrease root-knot severity. However, soil treatment did not significantly affect yield, and grafting led to inconsistent impacts on yield.

Digital Sensing and Molecular Computation by an Enzyme-Free DNA Circuit.

DNA circuits form the basis of programmable molecular systems capable of signal transduction and algorithmic computation. Some classes of molecular programs, such as catalyzed hairpin assembly, enable isothermal, enzyme-free signal amplification. However, current detection limits in DNA amplification circuits are modest, as sensitivity is inhibited by signal leakage resulting from noncatalyzed background reactions inherent to the noncovalent system. Here, we overcome this challenge by optimizing a catalyzed hairpin assembly for single-molecule sensing in a digital droplet assay. Furthermore, we demonstrate digital reporting of DNA computation at the single-molecule level by employing ddCHA as a signal transducer for simple DNA logic gates. By facilitating signal transduction of molecular computation at pM concentration, our approach can improve processing density by a factor of 104 relative to conventional DNA logic gates. More broadly, we believe that digital molecular computing will broaden the scope and efficacy of isothermal amplification circuits within DNA computing, biosensing, and signal amplification in general.

One Guest or Two? A Crystallographic and Solution Study of Guest Binding in a Cubic Coordination Cage.

Invited for the cover of this issue is the group of Michael D. Ward at the University of Warwick. The image depicts structures of the host cage containing one guest or two guests. Read the full text of the article at 10.1002/chem.201905499.

A family of diastereomeric dodecanuclear coordination cages based on inversion of chirality of individual triangular cyclic helicate faces.

The dodecanuclear coordination cage [Cd12(Lnaph)12(Lmes)4](BF4)24 consists of a set of four triangular, trinuclear helical panels {Cd3(µ-Lnaph)3}6+ (based on ditopic bridging ligands Lnaph), which are connected by four tritopic ligands Lmes. The result is that the four triangular helical panels and the four Lmes-capped triangular faces of the cuboctahedral core form two alternating subsets of the eight triangular faces of the cuboctahedron. Crystallographic investigations revealed that the triangular helicate faces can have 'clockwise' (C) or 'anticlockwise' (A) helicity, and that the helicity of each face can vary independently of the others as they are mechanically separated. This generates a set of three diastereoisomers in which all four cyclic helicate faces in the cuboctahedron have the same chirality (AAAA/CCCC enantiomers with T symmetry; AAAC/CCCA enantiomers with C 3 symmetry; and achiral AACC with S 4 symmetry). This mirrors the known behaviour of many simpler M4L6 tetrahedral cages which can likewise exist as T, C 3 or S 4 isomers according to the sense of tris-chelate chirality around each individual metal centre: but here it is translated onto a much larger scale by the four chiral units being entire trinuclear helicate faces rather than single metal centres. 1H NMR spectroscopy confirms the presence of the three diastereoisomers with their different molecular symmetries in a ratio slightly different from what is expected on purely statistical grounds; and 1H NMR measurements on a non-equilibrium sample (enriched by manual crystal-picking before preparing the solution) showed that the distribution does not change over several weeks in solution, indicating the kinetic inertness of the cage assemblies.

One Guest or Two? A Crystallographic and Solution Study of Guest Binding in a Cubic Coordination Cage.

A crystallographic investigation of a series of host-guest complexes in which small-molecule organic guests occupy the central cavity of an approximately cubic M8 L12 coordination cage has revealed some unexpected behaviour. Whilst some guests form 1:1 H⋅G complexes as we have seen before, an extensive family of bicyclic guests-including some substituted coumarins and various saturated analogues-form 1:2 H⋅G2 complexes in the solid state, despite the fact that solution titrations are consistent with 1:1 complex formation, and the combined volume of the pair of guests significantly exceeds the Rebek 55±9 % packing for optimal guest binding, with packing coefficients of up to 87 %. Re-examination of solution titration data for guest binding in two cases showed that, although conventional fluorescence titrations are consistent with 1:1 binding model, alternative forms of analysis-Job plot and an NMR titration-at higher concentrations do provide evidence for 1:2 H⋅G2 complex formation. The observation of guests binding in pairs in some cases opens new possibilities for altered reactivity of bound guests, and also highlights the recently articulated difficulties associated with determining stoichiometry of supramolecular complexes in solution.

Coordination-Cage-Catalysed Hydrolysis of Organophosphates: Cavity- or Surface-Based?

The hydrophobic central cavity of a water-soluble M8 L12 cubic coordination cage can accommodate a range of phospho-diester and phospho-triester guests such as the insecticide "dichlorvos" (2,2-dichlorovinyl dimethyl phosphate) and the chemical warfare agent analogue di(isopropyl) chlorophosphate. The accumulation of hydroxide ions around the cationic cage surface due to ion-pairing in solution generates a high local pH around the cage, resulting in catalysed hydrolysis of the phospho-triester guests. A series of control experiments unexpectedly demonstrates that-in marked contrast to previous cases-it is not necessary for the phospho-triester substrates to be bound inside the cavity for catalysed hydrolysis to occur. This suggests that catalysis can occur on the exterior surface of the cage as well as the interior surface, with the exterior-binding catalysis pathway dominating here because of the small binding constants for these phospho-triester substrates in the cage cavity. These observations suggest that cationic but hydrophobic surfaces could act as quite general catalysts in water by bringing substrates into contact with the surface (via the hydrophobic effect) where there is also a high local concentration of anions (due to ion pairing/electrostatic effects).

Soluble aggregates present in cerebrospinal fluid change in size and mechanism of toxicity during Alzheimer's disease progression.

Soluble aggregates of amyloid-ß (Aß) have been associated with neuronal and synaptic loss in Alzheimer's disease (AD). However, despite significant recent progress, the mechanisms by which these aggregated species contribute to disease progression are not fully determined. As the analysis of human cerebrospinal fluid (CSF) provides an accessible window into the molecular changes associated with the disease progression, we characterised soluble aggregates present in CSF samples from individuals with AD, mild cognitive impairment (MCI) and healthy controls using a range of sensitive biophysical methods. We used super-resolution imaging and atomic force microscopy to characterise the size and structure of the aggregates present in CSF and correlate this with their ability to permeabilise lipid membranes and induce an inflammatory response. We found that these aggregates are extremely heterogeneous and exist in a range of sizes, varying both structurally and in their mechanisms of toxicity during the disease progression. A higher proportion of small aggregates of Aß that can cause membrane permeabilization are found in MCI CSF; in established AD, a higher proportion of the aggregates were larger and more prone to elicit a pro-inflammatory response in glial cells, while there was no detectable change in aggregate concentration. These results show that large aggregates, some longer than 100 nm, are present in the CSF of AD patients and suggest that different neurotoxic mechanisms are prevalent at different stages of AD.

Fabrication and Characterization of Reconstituted Silk Microgels for the Storage and Release of Small Molecules.

Silk fibroin is a natural protein obtained from the Bombyx mori silkworm. In addition to being the key structural component in silkworm cocoons, it also has the propensity to self-assemble in vitro into hierarchical structures with desirable properties such as high levels of mechanical strength and robustness. Furthermore, it is an appealing biopolymer due to its biocompatability, low immunogenicity, and lack of toxicity, making it a prime candidate for biomedical material applications. Here, it is demonstrated that nanofibrils formed by reconstituted silk fibroin can be engineered into supramolecular microgels using a soft lithography-based microfluidic approach. Building on these results, a potential application for these protein microgels to encapsulate and release small molecules in a controlled manner is illustrated. Taken together, these results suggest that the tailored self-assembly of biocompatible and biodegradable silk nanofibrils can be used to generate functional micromaterials for a range of potential applications in the biomedical and pharmaceutical fields.