Magnetic Modulation of Biochemical Synthesis in Synthetic Cells.

Synthetic cells can be constructed from diverse molecular components, without the design constraints associated with modifying 'living' biological systems. This can be exploited to generate cells with abiotic components, creating functionalities absent in biology. One example is magnetic responsiveness, the activation and modulation of encapsulated biochemical processes using a magnetic field, which is absent from existing synthetic cell designs. This is a critical oversight, as magnetic fields are uniquely bio-orthogonal, noninvasive, and highly penetrative. Here, we address this by producing artificial magneto-responsive organelles by coupling thermoresponsive membranes with hyperthermic Fe3O4 nanoparticles and embedding them in synthetic cells. Combining these systems enables synthetic cell microreactors to be built using a nested vesicle architecture, which can respond to alternating magnetic fields through in situ enzymatic catalysis. We also demonstrate the modulation of biochemical reactions by using different magnetic field strengths and the potential to tune the system using different lipid compositions. This platform could unlock a wide range of applications for synthetic cells as programmable micromachines in biomedicine and biotechnology.

Biomimetic behaviors in hydrogel artificial cells through embedded organelles.

Artificial cells are biomimetic structures formed from molecular building blocks that replicate biological processes, behaviors, and architectures. Of these building blocks, hydrogels have emerged as ideal, yet underutilized candidates to provide a gel-like chassis in which to incorporate both biological and nonbiological componentry which enables the replication of cellular functionality. Here, we demonstrate a microfluidic strategy to assemble biocompatible cell-sized hydrogel-based artificial cells with a variety of different embedded functional subcompartments, which act as engineered synthetic organelles. The organelles enable the recreation of increasingly biomimetic behaviors, including stimulus-induced motility, content release through activation of membrane-associated proteins, and enzymatic communication with surrounding bioinspired compartments. In this way, we showcase a foundational strategy for the bottom-up construction of hydrogel-based artificial cell microsystems which replicate fundamental cellular behaviors, paving the way for the construction of next-generation biotechnological devices.

Stimuli-responsive vesicles as distributed artificial organelles for bacterial activation.

Intercellular communication is a hallmark of living systems. As such, engineering artificial cells that possess this behavior has been at the heart of activities in bottom-up synthetic biology. Communication between artificial and living cells has potential to confer novel capabilities to living organisms that could be exploited in biomedicine and biotechnology. However, most current approaches rely on the exchange of chemical signals that cannot be externally controlled. Here, we report two types of remote-controlled vesicle-based artificial organelles that translate physical inputs into chemical messages that lead to bacterial activation. Upon light or temperature stimulation, artificial cell membranes are activated, releasing signaling molecules that induce protein expression in Escherichia coli. This distributed approach differs from established methods for engineering stimuli-responsive bacteria. Here, artificial cells (as opposed to bacterial cells themselves) are the design unit. Having stimuli-responsive elements compartmentalized in artificial cells has potential applications in therapeutics, tissue engineering, and bioremediation. It will underpin the design of hybrid living/nonliving systems where temporal control over population interactions can be exerted.

Magnitude of venous or capillary blood-derived SARS-CoV-2-specific T cell response determines COVID-19 immunity.

T cells specific for SARS-CoV-2 are thought to protect against infection and development of COVID-19, but direct evidence for this is lacking. Here, we associated whole-blood-based measurement of SARS-CoV-2-specific interferon-γ-positive T cell responses with positive COVID-19 diagnostic (PCR and/or lateral flow) test results up to 6 months post-blood sampling. Amongst 148 participants donating venous blood samples, SARS-CoV-2-specific T cell response magnitude is significantly greater in those who remain protected versus those who become infected (P < 0.0001); relatively low magnitude T cell response results in a 43.2% risk of infection, whereas high magnitude reduces this risk to 5.4%. These findings are recapitulated in a further 299 participants testing a scalable capillary blood-based assay that could facilitate the acquisition of population-scale T cell immunity data (14.9% and 4.4%, respectively). Hence, measurement of SARS-CoV-2-specific T cells can prognosticate infection risk and should be assessed when monitoring individual and population immunity status.

Dynamic Reconfiguration of Subcompartment Architectures in Artificial Cells.

Artificial cells are minimal structures constructed from biomolecular building blocks designed to mimic cellular processes, behaviors, and architectures. One near-ubiquitous feature of cellular life is the spatial organization of internal content. We know from biology that organization of content (including in membrane-bound organelles) is linked to cellular functions and that this feature is dynamic: the presence, location, and degree of compartmentalization changes over time. Vesicle-based artificial cells, however, are not currently able to mimic this fundamental cellular property. Here, we describe an artificial cell design strategy that addresses this technological bottleneck. We create a series of artificial cell architectures which possess multicompartment assemblies localized either on the inner or on the outer surface of the artificial cell membrane. Exploiting liquid-liquid phase separation, we can also engineer spatially segregated regions of condensed subcompartments attached to the cell surface, aligning with coexisting membrane domains. These structures can sense changes in environmental conditions and respond by reversibly transitioning from condensed multicompartment layers on the membrane surface to a dispersed state in the cell lumen, mimicking the dynamic compartmentalization found in biological cells. Likewise, we engineer exosome-like subcompartments that can be released to the environment. We can achieve this by using two types of triggers: chemical (addition of salts) and mechanical (by pulling membrane tethers using optical traps). These approaches allow us to control the compartmentalization state of artificial cells on population and single-cell levels.

Standardisation of allergen products: 4. Validation of a candidate European Pharmacopoeia standard method for quantification of major grass pollen allergen Phl p 5.

BACKGROUND: The aim of the BSP090 project is the establishment of European Pharmacopoeia Chemical Reference Substances (CRSs) in combination with corresponding standard ELISA methods for quantification of major allergens in allergen products. Here, we present data of a Phl p 5-specific sandwich ELISA that proved suitable for the quantification of Phl p 5, one of the major Timothy grass (Phleum pratense) pollen allergens. METHODS: A Phl p 5-specific ELISA system was assessed with respect to accuracy, precision, inter-assay (within laboratory) and inter-laboratory variations, in a ring trial including 14 laboratories in Europe and the USA. Model samples containing recombinant Phl p 5a CRS as well as native grass pollen extracts were analysed. Each participant was instructed to perform at least one preliminary assay to familiarise with the protocol, followed by three independent assays. RESULTS: The candidate standard ELISA proved suitable to quantify recombinant and native Phl p 5 with satisfactory precision (93% of results within ±30% acceptance range). Inter-assay variation (max. GCV 24%) and especially inter-laboratory variation (max. GCV 13%) showed conclusive results. When assessing accuracy by means of recovery of recombinant spikes from a grass pollen extract matrix, similarly satisfactory spike recovery results were observed for the two spikes with higher concentrations (all within ±30% acceptance range), whereas recovery of the lowest concentration spike was slightly poorer with mean results of six laboratories exceeding acceptance range. CONCLUSIONS: Based on the collaborative study results, the assessed Phl p 5-specific immunoassay is appropriate to be proposed as European Pharmacopoeia standard method.

Hydrogels as functional components in artificial cell systems.

Recent years have seen substantial efforts aimed at constructing artificial cells from various molecular components with the aim of mimicking the processes, behaviours and architectures found in biological systems. Artificial cell development ultimately aims to produce model constructs that progress our understanding of biology, as well as forming the basis for functional bio-inspired devices that can be used in fields such as therapeutic delivery, biosensing, cell therapy and bioremediation. Typically, artificial cells rely on a bilayer membrane chassis and have fluid aqueous interiors to mimic biological cells. However, a desire to more accurately replicate the gel-like properties of intracellular and extracellular biological environments has driven increasing efforts to build cell mimics based on hydrogels. This has enabled researchers to exploit some of the unique functional properties of hydrogels that have seen them deployed in fields such as tissue engineering, biomaterials and drug delivery. In this Review, we explore how hydrogels can be leveraged in the context of artificial cell development. We also discuss how hydrogels can potentially be incorporated within the next generation of artificial cells to engineer improved biological mimics and functional microsystems.

Activating mechanosensitive channels embedded in droplet interface bilayers using membrane asymmetry.

Droplet microcompartments linked by lipid bilayers show great promise in the construction of synthetic minimal tissues. Central to controlling the flow of information in these systems are membrane proteins, which can gate in response to specific stimuli in order to control the molecular flux between membrane separated compartments. This has been demonstrated with droplet interface bilayers (DIBs) using several different membrane proteins combined with electrical, mechanical, and/or chemical activators. Here we report the activation of the bacterial mechanosensitive channel of large conductance (MscL) in a dioleoylphosphatidylcholine:dioleoylphosphatidylglycerol DIB by controlling membrane asymmetry. We show using electrical measurements that the incorporation of lysophosphatidylcholine (LPC) into one of the bilayer leaflets triggers MscL gating in a concentration-dependent manner, with partial and full activation observed at 10 and 15 mol% LPC respectively. Our findings could inspire the design of new minimal tissues where flux pathways are dynamically defined by lipid composition.

Engineering motile aqueous phase-separated droplets via liposome stabilisation.

There are increasing efforts to engineer functional compartments that mimic cellular behaviours from the bottom-up. One behaviour that is receiving particular attention is motility, due to its biotechnological potential and ubiquity in living systems. Many existing platforms make use of the Marangoni effect to achieve motion in water/oil (w/o) droplet systems. However, most of these systems are unsuitable for biological applications due to biocompatibility issues caused by the presence of oil phases. Here we report a biocompatible all aqueous (w/w) PEG/dextran Pickering-like emulsion system consisting of liposome-stabilised cell-sized droplets, where the stability can be easily tuned by adjusting liposome composition and concentration. We demonstrate that the compartments are capable of negative chemotaxis: these droplets can respond to a PEG/dextran polymer gradient through directional motion down to the gradient. The biocompatibility, motility and partitioning abilities of this droplet system offers new directions to pursue research in motion-related biological processes.

Comparison of house dust mite sensitization profiles in allergic adults from Canada, Europe, South Africa and USA.

BACKGROUND: Sensitization to house dust mite (HDM) is a leading cause of allergic rhinitis and asthma. Despite more than 30 HDM-derived allergens having been identified to date, specific therapeutic approaches do not yet take into account the local sensitization profiles of patients. This study aimed to identify patterns of HDM sensitization in HDM-allergic adults living in distinct geographic areas, to inform the development of targeted diagnostic and therapeutic tools. METHODS: Serum samples from 685 HDM-allergic subjects from Canada, Europe, South Africa, and the USA were tested for levels of IgE specific for 17 micro-arrayed HDM allergens by ImmunoCAP Immuno Solid-phase Allergen Chip (ISAC) technology. RESULTS: The results confirmed significant geographical variability in sensitization patterns and levels of IgE. In all areas, the major sensitizers were the group 1 and group 2 allergens and Der p 23. Der p 23 was a frequent sensitizer: 64% of the subjects had IgE specific for Der p 23, and 2.3% were monosensitized to it. In South Africa, Der p 23 was the dominant HDM allergen (86% prevalence) and Der p 7 achieved major allergen status (56%). IgE sensitization to HDM was influenced by asthmatic status, levels of allergen exposure, age, race-ethnicity and smoking status, but not by BMI. CONCLUSION: Sensitization profiles to HDM allergens differ considerably among distinct geographic areas, with Der p 7 and Der p 23 being major sensitizers in South Africa. Such heterogeneity should be taken into account in the diagnosis and treatment of HDM-allergic patients.

Bos d 11 in baked milk poses a risk for adverse reactions in milk-allergic patients.

BACKGROUND: Patients are commonly challenged with foods containing baked milk, for example muffins, yet little is known about the specific allergen content of muffins used in milk challenges or of the effect that baking has on allergenicity. OBJECTIVE: Our objective was to compare the levels of major milk allergens in uncooked and baked muffins using monoclonal immunoassays and IgE antibody binding before and after baking. METHODS: Uncooked and baked muffins were prepared using recipes from Mount Sinai and Imperial College. Allergen levels were compared by ELISA for Bos d 5 (ß-lactoglobulin) and Bos d 11 (ß-casein). IgE reactivity was assessed using sera from milk-sensitized donors in direct binding and inhibition ELISA. RESULTS: Bos d 5 was reduced from 680 µg/g in uncooked muffin mix to 0.17 µg/g in baked muffins, representing a >99% decrease after baking. Conversely, Bos d 11 levels in baked muffin remained high and only decreased by 30% from a mean of 4249 µg/g in uncooked muffin mix to 2961 µg/g when baked (~181 mg Bos d 11 per muffin). Baked muffins retained ~70% of the IgE binding to uncooked muffin mix. Baked muffin extract inhibited IgE binding to uncooked muffin mix by up to 80%, demonstrating retention of in vitro IgE reactivity. CONCLUSIONS AND CLINICAL RELEVANCE: High levels of Bos d 11 in baked muffins pose a risk for adverse reactions, especially in patients who have high anti-casein IgE antibodies.

Immune Remodeling of the Extracellular Matrix Drives Loss of Cancer Stem Cells and Tumor Rejection.

The nature of the tumor microenvironment (TME) influences the ability of tumor-specific T cells to control tumor growth. In this study, we performed an unbiased comparison of the TME of regulatory T-cell (Treg)-replete and Treg-depleted carcinogen-induced tumors, including Treg-depleted responding (regressing) and non-responding (growing) tumors. This analysis revealed an inverse relationship between extracellular matrix (ECM) and T-cell infiltrates where responding tumors were T-cell rich and ECM poor, whereas the converse was observed in non-responder tumors. For this reason, we hypothesized that the ECM acted as a barrier to successful T-cell infiltration and tumor rejection. However, further experiments revealed that this was not the case but instead showed that an effective T-cell response dramatically altered the density of ECM in the TME. Along with loss of ECM and high numbers of infiltrating T cells, responder tumors were distinguished by the development of lymphatic and blood vessel networks with specialized immune function. ECM-rich tumors exhibited a stem cell-like gene expression profile and superior tumor-initiating capacity, whereas such features were absent in responder tumors. Overall, these findings define an extended role for an effective immune response, not just in direct killing of tumor cells but in widescale remodeling of the TME to favor loss of ECM, elimination of cancer stem cells, and propagation of adaptive immunity.

Are current analytical methods suitable to verify VITAL® 2.0/3.0 allergen reference doses for EU allergens in foods?

Food allergy affects up to 6% of Europeans. Allergen identification is important for the risk assessment and management of the inadvertent presence of allergens in foods. The VITAL® initiative for voluntary incidental trace allergen labeling suggests protein reference doses, based on clinical reactivity in food challenge studies, at or below which voluntary labelling is unnecessary. Here, we investigated if current analytical methodology could verify the published VITAL® 2.0 doses, that were available during this analysis, in serving sizes between 5 and 500 g. Available data on published and commercial ELISA, PCR and mass spectrometry methods, especially for the detection of peanuts, soy, hazelnut, wheat, cow's milk and hen's egg were reviewed in detail. Limit of detection, quantitative capability, matrix compatibility, and specificity were assessed. Implications by the recently published VITAL® 3.0 doses were also considered. We conclude that available analytical methods are capable of reasonably robust detection of peanut, soy, hazelnut and wheat allergens for levels at or below the VITAL® 2.0 and also 3.0 doses, with some methods even capable of achieving this in a large 500 g serving size. Cow's milk and hen's egg are more problematic, largely due to matrix/processing incompatibility. An unmet need remains for harmonized reporting units, available reference materials, and method ring-trials to enable validation and the provision of comparable measurement results.

Membrane protein mediated bilayer communication in networks of droplet interface bilayers.

Droplet interface bilayers (DIBs) are model membranes formed between lipid monolayer-encased water droplets in oil. Compared to conventional methods, one of the most unique properties of DIBs is that they can be connected together to generate multi-layered 'tissue-like' networks, however introducing communication pathways between these compartments typically relies on water-soluble pores that are unable to gate. Here, we show that network connectivity can instead be achieved using a water-insoluble membrane protein by successfully reconstituting a chemically activatable mutant of the mechanosensitive channel MscL into a network of DIBs. Moreover, we also show how the small molecule activator can diffuse through an open channel and across the neighbouring droplet to activate MscL present in an adjacent bilayer. This demonstration of membrane protein mediated bilayer communication could prove key toward developing the next generation of responsive bilayer networks capable of defining information flow inside a minimal tissue.

Size dependency of gold nanoparticles interacting with model membranes.

The rapid development of nanotechnology has led to an increase in the number and variety of engineered nanomaterials in the environment. Gold nanoparticles (AuNPs) are an example of a commonly studied nanomaterial whose highly tailorable properties have generated significant interest through a wide range of research fields. In the present work, we characterise the AuNP-lipid membrane interaction by coupling qualitative data with quantitative measurements of the enthalpy change of interaction. We investigate the interactions between citrate-stabilised AuNPs ranging from 5 to 60 nm in diameter and large unilamellar vesicles acting as a model membrane system. Our results reveal the existence of two critical AuNP diameters which determine their fate when in contact with a lipid membrane. The results provide new insights into the size dependent interaction between AuNPs and lipid bilayers which is of direct relevance to nanotoxicology and to the design of NP vectors.

Building a synthetic mechanosensitive signaling pathway in compartmentalized artificial cells.

To date, reconstitution of one of the fundamental methods of cell communication, the signaling pathway, has been unaddressed in the bottom-up construction of artificial cells (ACs). Such developments are needed to increase the functionality and biomimicry of ACs, accelerating their translation and application in biotechnology. Here, we report the construction of a de novo synthetic signaling pathway in microscale nested vesicles. Vesicle-cell models respond to external calcium signals through activation of an intracellular interaction between phospholipase A2 and a mechanosensitive channel present in the internal membranes, triggering content mixing between compartments and controlling cell fluorescence. Emulsion-based approaches to AC construction are therefore shown to be ideal for the quick design and testing of new signaling networks and can readily include synthetic molecules difficult to introduce to biological cells. This work represents a foundation for the engineering of multicompartment-spanning designer pathways that can be utilized to control downstream events inside an AC, leading to the assembly of micromachines capable of sensing and responding to changes in their local environment.

Engineering thermoresponsive phase separated vesicles formed via emulsion phase transfer as a content-release platform.

Giant unilamellar vesicles (GUVs) are a well-established tool for the study of membrane biophysics and are increasingly used as artificial cell models and functional units in biotechnology. This trend is driven by the development of emulsion-based generation methods such as Emulsion Phase Transfer (EPT), which facilitates the encapsulation of almost any water-soluble compounds (including biomolecules) regardless of size or charge, is compatible with droplet microfluidics, and allows GUVs with asymmetric bilayers to be assembled. However, the ability to control the composition of membranes formed via EPT remains an open question; this is key as composition gives rise to an array of biophysical phenomena which can be used to add functionality to membranes. Here, we evaluate the use of GUVs constructed via this method as a platform for phase behaviour studies and take advantage of composition-dependent features to engineer thermally-responsive GUVs. For the first time, we generate ternary GUVs (DOPC/DPPC/cholesterol) using EPT, and by compensating for the lower cholesterol incorporation efficiencies, show that these possess the full range of phase behaviour displayed by electroformed GUVs. As a demonstration of the fine control afforded by this approach, we demonstrate release of dye and peptide cargo when ternary GUVs are heated through the immiscibility transition temperature, and show that release temperature can be tuned by changing vesicle composition. We show that GUVs can be individually addressed and release triggered using a laser beam. Our findings validate EPT as a suitable method for generating phase separated vesicles and provide a valuable proof-of-concept for engineering content release functionality into individually addressable vesicles, which could have a host of applications in the development of smart synthetic biosystems.

Light-triggered enzymatic reactions in nested vesicle reactors.

Cell-sized vesicles have tremendous potential both as miniaturised pL reaction vessels and in bottom-up synthetic biology as chassis for artificial cells. In both these areas the introduction of light-responsive modules affords increased functionality, for example, to initiate enzymatic reactions in the vesicle interior with spatiotemporal control. Here we report a system composed of nested vesicles where the inner compartments act as phototransducers, responding to ultraviolet irradiation through diacetylene polymerisation-induced pore formation to initiate enzymatic reactions. The controlled release and hydrolysis of a fluorogenic ß-galactosidase substrate in the external compartment is demonstrated, where the rate of reaction can be modulated by varying ultraviolet exposure time. Such cell-like nested microreactor structures could be utilised in fields from biocatalysis through to drug delivery.

Treg Depletion Licenses T Cell-Driven HEV Neogenesis and Promotes Tumor Destruction.

T-cell infiltration into tumors represents a critical bottleneck for immune-mediated control of cancer. We previously showed that this bottleneck can be overcome by depleting immunosuppressive Foxp3+ regulatory T cells (Tregs), a process that can increase frequencies of tumor-infiltrating lymphocytes through promoting the development of specialized portals for lymphocyte entry, namely high endothelial venules (HEVs). In this paper, we used a carcinogen-induced tumor model that allows for coevolution of the tumor microenvironment and the immune response to demonstrate that Treg depletion not only results in widespread disruption to HEV networks in lymph nodes (LNs) but also activates CD8+ T cells, which then drive intratumoral HEV development. Formation of these vessels contrasts with ontogenic HEV development in LNs in that the process is dependent on the TNF receptor and independent of lymphotoxin ß receptor-mediated signaling. These intratumoral HEVs do not express the chemokine CCL21, revealing a previously undescribed intratumoral blood vessel phenotype. We propose a model where Treg depletion enables a self-amplifying loop of T-cell activation, which promotes HEV development, T-cell infiltration, and ultimately, tumor destruction. The findings point to a need to test for HEV development as part of ongoing clinical studies in patients with cancer. Cancer Immunol Res; 5(11); 1005-15. ©2017 AACR.