10,265 research outputs found

    Radial Dynamics of Pickering-stabilised Endoskeletal Antibubbles and Their Components in Pulsed Ultrasound

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    Liquids containing microscopic antibubbles may have theranostic applications in harmonic diagnostic ultrasonic imaging and in ultrasound-assisted drug delivery. Presently there are no known agents available with the acoustic properties required for use in both of these applications. The Pickering-stabilised antibubble may possess the de- sired acoustic properties to be such a theranostic agent. An antibubble is a gas bubble containing at least one incompressible core. An antibubble is inherently unstable and thus needs to be stabilised to exist for longer than a moment. One such stabilising method, involving the adsorption of nanoparticles to gasā€“liquid interfaces, is called Pickering stabilisation. A Pickering-stabilised antibubble responds to an incident sound ļ¬eld by means of radial pulsation and other, more complicated, dynamics. Despite the potential application of microscopic antibubbles in theranostics, their dynamic behaviour and the acoustic regimes in which this behaviour occurs are not known. The purpose of this research was to predict the dynamic response of Pickering- stabilised antibubbles to pulsed ultrasound, and to identify and quantify the contribution of each of the Pickering-stabilised antibubble components to that behaviour. Radial excursions of antibubbles and their components during ultrasound exposure were extracted from high-speed footage. The applied ultrasound had a centre frequency of 1 MHz and pressure amplitudes between 0.20 MPa and 1.30 MPa. Moreover, damping coeļ¬ƒcients, pulsation phases, and excursions of antibubbles and antibubble components were computed with equations describing a forced massā€“springā€“dashpot system and an adapted Rayleigh-Plesset equation. Over a range of driving pressure amplitudes, fragmentation thresholds were computed for antibubbles of varying size, core volume, shell stiļ¬€ness, and driving frequency. In addition, the feasibility of an antibubble component for the disruption of cell walls was tested. From the experimental data, it was found that antibubble contractions and expansions were symmetrical and predictable at an acoustic amplitude of 0.20 MPa, whilst the pulsations were asymmetrical and less predictable at an acoustic amplitude of 1.00 MPa. These results show that the presence of the core inside of the antibubble hampers the contraction of a collapsing antibubble and ameliorates its stability. Consequently, Pickering-stabilised antibubbles appear to be feasible candidates for ultrasonic imaging, with greater stability than the agents currently in use. Micron-sized antibubbles, much smaller than resonant size, were computed to have a pulsation phase diļ¬€erence of up to 16 th of a cycle with respect to free gas bubbles. The diļ¬€erence in oscillation phase is a result of the increased damping coefļ¬cient caused by the friction of the internal components and shell of the antibubble. This indicates that altering the damping of the shell or skeletal material of minute antibubbles can alter the degree to which the particleā€™s oscillation is in phase with the sound ļ¬eld. The shell stiļ¬€ness of Pickering-stabilised microbubbles without incompressible contents was measured to be 7.6 N māˆ’1 throughout low-amplitude sonication. Un- der high-amplitude sonication, the maximum expansions of microbubbles, measured from high-speed camera footage, were either agreeing with those computed for Pickering-stabilised microbubbles or corresponding to greater values. The diļ¬€ering oscillation amplitudes for similarly sized microbubbles is attributed to shell disruption of diļ¬€erent severity. For a 3-Ī¼m radius antibubble with a 90% core radius, subjected to a pulse of centre frequency 1 MHz, the fragmentation threshold was computed to drastically increase with shell stiļ¬€ness. At a driving frequency of 13 MHz, the fragmentation threshold was computed to correspond to a mechanical index less than 0.4, irrespective of shell stiļ¬€ness. Shell stiļ¬€ness changes the resonance frequency, and thus the fragmentation threshold of antibubbles. This means that the resonance frequency of an extremely low concentration and quantity of homogeneous agent can be determined using microscopy. At driving frequencies above 1 MHz, the fragmentation threshold was computed to correspond to a mechanical index of less than 0.5, irrespective of shell stiļ¬€ness. Antibubbles exposed to high-amplitude ultrasound were found to have an exponential fragment size distribution. This brings us closer to understanding and controlling disruption and material release for these particles. If the pressure of the regime is known, the number of antibubble fragments produced can be theoretically determined. Under low-amplitude ultrasound exposure, hydrophobic particles, a common component of antibubbles, were observed to jet through wood ļ¬bre cell walls, without causing visible internal structural damage to these cells. Hydrophobic particles can thus act as inertial cavitation nuclei which collapse asymmetrically close to solid boundaries such as wood pulp ļ¬bres. This indicates that hydrophobic particles on their own may be used for applications such as trans-dermal drug delivery. The dynamic response of Pickering-stabilised antibubbles to ultrasound has been predicted. Furthermore the respective behaviour of Pickering-stabilised antibubble components under theranostic ultrasound conditions has been identiļ¬ed. This work has led to a straightforward way to determine the elasto-mechano properties of small samples of contrast agent. Whilst possessing some theranostic properties, Pickering-stabilised antibubbles may be more suitable as replacements for current diagnostic agents. Hydrophobic particles, a current constituent of the Pickering-stabilised antibubble, may however, prove to be promising theranostic agents

    Bioactive Self-Assembled Protein Nanosheets for Stem Cell-Based Biotechnologies

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    Tissue and stem cell culture methods have been dominated by glass and plastic substrates such as Tissue culture plastic. These solid substrates, although widely used, are associated with poor scalability for adherent stem cell expansion in systems such as 3D bioreactors and the design of parallel culture systems. Therefore, investigating strategies to bypass these obstacles in stem cell expansion is essential to enable the wider translation of stem cell technologies. An alternative strategy recently proposed consists in using a liquid surface instead, such as an oil, and associated oil droplets. Indeed, emulsions can be formed using protein nanosheets to stabilise oil/water interfaces to promote the adhesion of stem cells and enable their proliferation. These nanosheets exhibit enhanced interfacial mechanics and allow the introduction of bioactive components via recombinant protein expression to promote bioactivity. Beyond the application of resulting bioemulsions for the expansion of Mesenchymal stem cells, the impact of these bioactive interfaces on the differentiation of iPSCs and the development of cerebral organoids will be presented. The Bovine serum albumin protein was recombinantly modified to attach an N-terminal Avi-Tag, this was expressed and purified from the yeast P. pastoris expression system. The Avi-tag was then biotinylated in vitro by recombinantly expressed BirA. Emulsions of a specific size were formed using the newly biotinylated Bt-BSA protein and functionalized with a cascade of components to mimic cell-cell ligands, this resulted in bioemulsions with a bioactive surface that can interact with surrounding cells. These functionalised droplets were integrated into developing cerebral organoids and their impact on phenotype was studied. The droplets were found not to deform sufficiently to allow mechanical forces to be measured, yet the many of these droplets were retained within the organoids which led to an interesting phenotype within the organoids. The developing rosettes were found to develop enlarged lumens shown by an increase in area, this phenotype did not impact the differentiation into the cerebral lineage depicted by immunohistochemistry of hallmark marker of neuronal differentiation within organoids retaining droplets. The interfacial mechanics of fibrinogen nanosheets treated with varying concentrations of thrombin was studied using interfacial shear rheology. The effect of thrombin significantly altered the interfacial mechanics with the lower concentration of thrombin significantly increasing the toughness multiple folds and decreasing the elasticity of the nanosheets. Additionally, the nanostructure of nanosheets was studied using SEM and TEM and traditional fibrin fibres were found to not form at these interfaces, but local rearrangements and retractions in the thrombin treated nanosheets were observed. Finally, these enhanced mechanical properties promoted the proliferation and expansion of Mesenchymal stem cells on quasi-2D and 3D interfaces

    Genetic analysis and molecular basis of G6PD deficiency among malaria patients in Thailand: implications for safe use of 8-aminoquinolines

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    Background: It was hypothesized that glucose-6-phosphate dehydrogenase (G6PD) deficiency confers a protective effect against malaria infection, however, safety concerns have been raised regarding haemolytic toxicity caused by radical cure with 8-aminoquinolines in G6PD-deficient individuals. Malaria elimination and control are also complicated by the high prevalence of G6PD deficiency in malaria-endemic areas. Hence, accurate identification of G6PD deficiency is required to identify those who are eligible for malaria treatment using 8-aminoquinolines. Methods: The prevalence of G6PD deficiency among 408 Thai participants diagnosed with malaria by microscopy (71), and malaria-negative controls (337), was assessed using a phenotypic test based on water-soluble tetrazolium salts. High-resolution melting (HRM) curve analysis was developed from a previous study to enable the detection of 15 common missense, synonymous and intronic G6PD mutations in Asian populations. The identified mutations were subjected to biochemical and structural characterisation to understand the molecular mechanisms underlying enzyme deficiency. Results: Based on phenotypic testing, the prevalence of G6PD deficiency (T) and intronic (c.1365-13T>C and c.486-34delT) mutations was detected with intermediate to normal enzyme activity. The double missense mutations were less catalytically active than their corresponding single missense mutations, resulting in severe enzyme deficiency. While the mutations had a minor effect on binding affinity, structural instability was a key contributor to the enzyme deficiency observed in G6PD-deficient individuals. Conclusions: With varying degrees of enzyme deficiency, G6PD genotyping can be used as a complement to phenotypic screening to identify those who are eligible for 8-aminoquinolines. The information gained from this study could be useful for management and treatment of malaria, as well as for the prevention of unanticipated reactions to certain medications and foods in the studied population

    DNMT3B PWWP mutations cause hypermethylation of heterochromatin

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    The correct establishment of DNA methylation patterns is vital for mammalian development and is achieved by the de novo DNA methyltransferases DNMT3A and DNMT3B. DNMT3B localises to H3K36me3 at actively transcribing gene bodies via its PWWP domain. It also functions at heterochromatin through an unknown recruitment mechanism. Here we find that knockout of DNMT3B causes loss of methylation predominantly at H3K9me3-marked heterochromatin and that DNMT3B PWWP domain mutations or deletion result in striking increases of methylation in H3K9me3-marked heterochromatin. Removal of the N-terminal region of DNMT3B affects its ability to methylate H3K9me3-marked regions. This region of DNMT3B directly interacts with HP1 and facilitates the bridging of DNMT3B with H3K9me3-marked nucleosomes in vitro. Our results suggest that DNMT3B is recruited to H3K9me3 marked heterochromatin in a PWWP-independent mannerthat is facilitated by the proteinā€™s N-terminal region through an interaction with a key heterochromatin protein. More generally, we suggest that DNMT3B plays a role in DNA methylation homeostasis at heterochromatin, a process which is disrupted in cancer, aging and Immunodeficiency, Centromeric Instability and Facial Anomalies (ICF) syndrome

    Cryptic bacterial pathogens of diatoms peak during senescence of a winter diatom bloom.

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    This is the final version. Available from Wiley via the DOI in this record.Data availability: Sequence data of bacterial strains isolated during this study are deposited in NCBI GenBank with accession nos.: OR776937ā€“ OR776933.Diatoms are globally abundant microalgae that form extensive blooms in aquatic ecosystems. Certain bacteria behave antagonistically towards diatoms, killing or inhibiting their growth. Despite their crucial implications to diatom bloom and population health, knowledge of diatom antagonists in the environment is fundamentally lacking. We report systematic characterisation of the diversity and seasonal dynamics of bacterial antagonists of diatoms via plaque assay sampling in the Western English Channel, where diatoms frequently bloom. Unexpectedly, peaks in detection did not occur during characteristic spring diatom blooms, but coincided with a winter bloom of Coscinodiscus, suggesting that these bacteria likely influence distinct diatom host populations. We isolated multiple bacterial antagonists, spanning 4 classes and 10 bacterial orders. Notably, a diatom attaching Roseobacter Ponticoccus alexandrii was isolated multiple times, indicative of a persistent environmental presence. Moreover, many isolates had no prior reports of antagonistic activity towards diatoms. We verified diatom growth inhibitory effects of eight isolates. In all cases tested, these effects were activated by pre-exposure to diatom organic matter. Discovery of widespread 'cryptic' antagonistic activity indicates that bacterial pathogenicity towards diatoms is more prevalent than previously recognised. Finally, examination of the global biogeography of WEC antagonists revealed co-occurrence patterns with diatom host populations in marine waters globally.Natural Environment Research CouncilNatural Environment Research CouncilNatural Environment Research Counci

    Elucidating How MLH1 Loss Regulates a Metabolic Phenotype in Endometrial Cancer

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    Endometrial cancer is the fourth most common cancer in women and the most common gynaecological malignancy in the developed world. No new systemic treatments for endometrial cancer have been developed in recent years and its incidence is expected to double over the next decade. As such, there is a need to better understand key molecular pathways that are altered in the disease and could be targeted by novel treatments. The DNA MMR pathway is lost in approximately 30% of endometrial cancers. A small proportion of these are caused by germline mutations in one of the four MMR genes, however, the majority result from the epigenetic silencing of MLH1. Recently, our lab has shown that MLH1-deficient cells demonstrate a mitochondrial phenotype characterised by reduced OXPHOS, reduced mtDNA copy number and Complex I inhibition. OXPHOS-deficient cells must adapt their metabolism to compensate for energy defects and the inability to efficiently use the tricarboxylic acid cycle to generate energy. We hypothesise that this altered metabolism is driving tumourigenesis by increasing the tumour cells' metastatic potential. In this PhD we aimed to further investigate the influence MLH1 loss has on cellular metabolism using MLH1 positive and negative paired endometrial cell lines. Ultimately, we aim to understand whether altered metabolism in MLH1-deficient endometrial cancer may be therapeutically targeted

    Spatiotemporal Control of Chemical Reaction Networks using Droplet Microfluidics

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    A number of cellular organisms, such as yeast, bacteria and slime moulds, exhibit dynamic behaviour, in particular switching and rhythms that are controlled by feedback mechanisms in enzyme-catalysed reactions. The mechanisms of these processes are well understood, and recently there has been a focus on generating similar reactions in synthetic biocatalytic systems to establish bioinspired analogues for applications in materials and medicine. In this context, compartmentalisation of biochemical reactions within synthetic cell models such as micelles, vesicles, and W/O/W-based double emulsions is attracting growing attention for applications in the field of therapeutics. In this respect, it is necessary to adopt easier-to-use stimuli-responsive (react to pH, temperature or light) biochemical reactions, to apply artificial cell models to the biomedical context, and regulate artificial cell communication in a spatiotemporal controlled way. As a first step, it is crucial to control the output of a chemical reaction that maybe exploited for applications in the field of programmable materials and biomedicine. Droplet emulsion and synthetic vesicle systems have been widely employed as bioinspired micro- or nanoreactors for production of materials such as hydrogels and ceramic particles. They also provide test platform for biomimetic cell like behaviour. To address this, we have developed and fine-tuned a platform with synthetic bottom-up chemistry that has enabled us to systematically and thoroughly investigate the effects of entrapment on a feedback-driven enzymatic reaction. As a result of this process, we have revealed a system that is more intricate than originally thought. Firstly, taking advantage from pressure driven droplet microfluidics, we developed a system of enzyme-encapsulated (urea-urease) double emulsion (W/O/W) droplets to obtain a localised pH pulse, with a controllable induction time to program material properties. The urease-catalysed hydrolysis of urea (urea-urea reaction), has a feedback through the production of the base (NH3). This leads to a change from an acidic to a basic pH after an induction time (Tind), resulting in an environment with auto-changing pH conditions. Reaction was initiated by addition of urea and a pulse in base (ammonia) was observed in the droplets after a time lag of the order of minutes. The pH-time profile can be manipulated by the diffusion timescale of urea and ammonia through the oil layer, resulting in localised pH changes not accessible in bulk solutions. Secondly, we performed a computational investigation of the nonlinear reaction chemistry (urea-urease) within the designed platform of the W/O/W-based reactor. A radially distributed reaction diffusion model is presented for a layered sphere mimicking a double emulsion. Here we have combined the experiments with simulations (shell-core model) to demonstrate the influence of urea transport triggered by the shell, the core and the external solution surrounding the cell model (Āµ-reactor) on the induction time/period (Tind) of urea-urease reaction. Third, inspired from natural cellular systems (e.g. bacterial quorum sensing), we focus on the use of urea-urease reaction confined to double emulsions to investigate chemical communications. We observed a system that resulted in a system of microreactors acting as individual units with distinct induction periods (Tind) for the first time. We show that in contrast to other systems, the release of ammonia can accelerate the reaction in all the droplets but there is no evident synchronisation of activity characterised by a wide distribution of induction times across the population of micro-reactors. However, the investigation of behaviour of population/group of Āµ-reactors as a function of substrate urea concentration and the density of Āµ-reactors highlights the possibility of transitions to collective behaviours. Finally, we aimed to use the double emulsion template for potential biomedical and therapeutic applications using the autocatalytic urea-urease reaction. We used the platform to produce thiol-acrylate gels in the form of double emulsion loaded gel films and spherical microcapsules for potential drug delivery applications. In addition, we employed the encapsulated double emulsion platform of the enzyme urease to study the inhibition of the enzyme itself; which is important in the development of anti-microbials for ureolytic bacteria. By building this platform, we have not only learned how to control the kinetic output of the reaction (urea-urease), but have also demonstrated its potential in future applications

    Spider venom neurotoxin based bioinsecticides: A novel bioactive for the control of the Asian citrus psyllid Diaphorina citri (Hemiptera)

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    The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a key vector of the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas) associated with huanglongbing (HLB), the most serious and currently incurable disease of citrus worldwide. Here we report the first investigation into the potential use of a spider venom-derived recombinant neurotoxin, Ļ‰/Īŗ-HxTx-Hv1h (hereafter HxTx-Hv1h) when delivered alone or when fused to snowdrop lectin (Galanthus nivalis agglutinin; GNA) to control D. citri. Proteins, including GNA alone, were purified from fermented transformed yeast Pichia pastoris cultures. Recombinant HxTx-Hv1h, HxTx-Hv1h/GNA and GNA were all orally toxic to D. citri, with Day 5 median lethal concentrations (LC 50) derived from dose-response artificial diet assays of 27, 20 and 52 Ī¼M, respectively. Western analysis of whole insect protein extracts confirmed that psyllid mortality was attributable to protein ingestion and that the fusion protein was stable to cleavage by D. citri proteases. When applied topically (either via droplet or spray) HxTx-Hv1h/GNA was the most effective of the proteins causing >70 % mortality 5 days post treatment, some 2 to 3-fold higher levels of mortality as compared to the toxin alone. By contrast, no significant mortality or phenotypic effects were observed for bumble bees (Bombus terrestris L.) fed on the recombinant proteins in acute toxicity assays. This suggests that HxTx-Hv1h/GNA has potential as a novel bioinsecticide for the management of D. citri offering both enhanced target specificity as compared to chemical pesticides and compatibility with integrated pest management (IPM) strategies
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