@article{pmid32705984,

title = {Periodic propagating waves coordinate RhoGTPase network dynamics at the leading and trailing edges during cell migration},

author = {Alfonso Bolado-Carrancio and Oleksii S Rukhlenko and Elena Nikonova and Mikhail A Tsyganov and Anne Wheeler and Amaya Garcia-Munoz and Walter Kolch and Alex von Kriegsheim and Boris N Kholodenko},

doi = {10.7554/eLife.58165},

issn = {2050-084X},

year  = {2020},

date = {2020-07-01},

journal = {Elife},

volume = {9},

abstract = {Migrating cells need to coordinate distinct leading and trailing edge dynamics but the underlying mechanisms are unclear. Here, we combine experiments and mathematical modeling to elaborate the minimal autonomous biochemical machinery necessary and sufficient for this dynamic coordination and cell movement. RhoA activates Rac1 via DIA and inhibits Rac1 via ROCK, while Rac1 inhibits RhoA through PAK. Our data suggest that in motile, polarized cells, RhoA-ROCK interactions prevail at the rear, whereas RhoA-DIA interactions dominate at the front where Rac1/Rho oscillations drive protrusions and retractions. At the rear, high RhoA and low Rac1 activities are maintained until a wave of oscillatory GTPase activities from the cell front reaches the rear, inducing transient GTPase oscillations and RhoA activity spikes. After the rear retracts, the initial GTPase pattern resumes. Our findings show how periodic, propagating GTPase waves coordinate distinct GTPase patterns at the leading and trailing edge dynamics in moving cells.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32424125,

title = {Nanostructural Diversity of Synapses in the Mammalian Spinal Cord},

author = {Matthew J Broadhead and Calum Bonthron and Lauren Arcinas and Sumi Bez and Fei Zhu and Frances Goff and Jonathan Nylk and Kishan Dholakia and Frank Gunn-Moore and Seth G N Grant and Gareth B Miles},

doi = {10.1038/s41598-020-64874-9},

issn = {2045-2322},

year  = {2020},

date = {2020-05-01},

journal = {Sci Rep},

volume = {10},

number = {1},

pages = {8189},

abstract = {Functionally distinct synapses exhibit diverse and complex organisation at molecular and nanoscale levels. Synaptic diversity may be dependent on developmental stage, anatomical locus and the neural circuit within which synapses reside. Furthermore, astrocytes, which align with pre and post-synaptic structures to form 'tripartite synapses', can modulate neural circuits and impact on synaptic organisation. In this study, we aimed to determine which factors impact the diversity of excitatory synapses throughout the lumbar spinal cord. We used PSD95-eGFP mice, to visualise excitatory postsynaptic densities (PSDs) using high-resolution and super-resolution microscopy. We reveal a detailed and quantitative map of the features of excitatory synapses in the lumbar spinal cord, detailing synaptic diversity that is dependent on developmental stage, anatomical region and whether associated with VGLUT1 or VGLUT2 terminals. We report that PSDs are nanostructurally distinct between spinal laminae and across age groups. PSDs receiving VGLUT1 inputs also show enhanced nanostructural complexity compared with those receiving VGLUT2 inputs, suggesting pathway-specific diversity. Finally, we show that PSDs exhibit greater nanostructural complexity when part of tripartite synapses, and we provide evidence that astrocytic activation enhances PSD95 expression. Taken together, these results provide novel insights into the regulation and diversification of synapses across functionally distinct spinal regions and advance our general understanding of the 'rules' governing synaptic nanostructural organisation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32233401,

title = {Polylactic is a Sustainable, Low Absorption, Low Autofluorescence Alternative to Other Plastics for Microfluidic and Organ-on-Chip Applications},

author = {Alfredo E Ongaro and Davide Di Giuseppe and Ali Kermanizadeh and Allende Miguelez Crespo and Arianna Mencattini and Lina Ghibelli and Vanessa Mancini and Krystian L Wlodarczyk and Duncan P Hand and Eugenio Martinelli and Vicki Stone and Nicola Howarth and Vincenzo La Carrubba and Virginia Pensabene and Maïwenn Kersaudy-Kerhoas},

doi = {10.1021/acs.analchem.0c00651},

issn = {1520-6882},

year  = {2020},

date = {2020-05-01},

journal = {Anal Chem},

volume = {92},

number = {9},

pages = {6693--6701},

abstract = {Organ-on-chip (OOC) devices are miniaturized devices replacing animal models in drug discovery and toxicology studies. The majority of OOC devices are made from polydimethylsiloxane (PDMS), an elastomer widely used in microfluidic prototyping, but posing a number of challenges to experimentalists, including leaching of uncured oligomers and uncontrolled absorption of small compounds. Here we assess the suitability of polylactic acid (PLA) as a replacement material to PDMS for microfluidic cell culture and OOC applications. We changed the wettability of PLA substrates and demonstrated the functionalization method to be stable over a time period of at least 9 months. We successfully cultured human cells on PLA substrates and devices, without coating. We demonstrated that PLA does not absorb small molecules, is transparent (92% transparency), and has low autofluorescence. As a proof of concept of its manufacturability, biocompatibility, and transparency, we performed a cell tracking experiment of prostate cancer cells in a PLA device for advanced cell culture.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32328637,

title = {Asymmetric assembly of centromeres epigenetically regulates stem cell fate},

author = {Anna Ada Dattoli and Ben L Carty and Antje M Kochendoerfer and Conall Morgan and Annie E Walshe and Elaine M Dunleavy},

doi = {10.1083/jcb.201910084},

issn = {1540-8140},

year  = {2020},

date = {2020-04-01},

journal = {J Cell Biol},

volume = {219},

number = {4},

abstract = {Centromeres are epigenetically defined by CENP-A-containing chromatin and are essential for cell division. Previous studies suggest asymmetric inheritance of centromeric proteins upon stem cell division; however, the mechanism and implications of selective chromosome segregation remain unexplored. We show that Drosophila female germline stem cells (GSCs) and neuroblasts assemble centromeres after replication and before segregation. Specifically, CENP-A deposition is promoted by CYCLIN A, while excessive CENP-A deposition is prevented by CYCLIN B, through the HASPIN kinase. Furthermore, chromosomes inherited by GSCs incorporate more CENP-A, making stronger kinetochores that capture more spindle microtubules and bias segregation. Importantly, symmetric incorporation of CENP-A on sister chromatids via HASPIN knockdown or overexpression of CENP-A, either alone or together with its assembly factor CAL1, drives stem cell self-renewal. Finally, continued CENP-A assembly in differentiated cells is nonessential for egg development. Our work shows that centromere assembly epigenetically drives GSC maintenance and occurs before oocyte meiosis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32067018,

title = {Chromosome compaction and chromatin stiffness enhance diffusive loop extrusion by slip-link proteins},

author = {A Bonato and C A Brackley and J Johnson and D Michieletto and D Marenduzzo},

doi = {10.1039/c9sm01875a},

issn = {1744-6848},

year  = {2020},

date = {2020-03-01},

journal = {Soft Matter},

volume = {16},

number = {9},

pages = {2406--2414},

abstract = {We use Brownian dynamics simulations to study the formation of chromatin loops through diffusive sliding of slip-link-like proteins, mimicking the behaviour of cohesin molecules. We recently proposed that diffusive sliding is sufficient to explain the extrusion of chromatin loops of hundreds of kilo-base-pairs (kbp), which may then be stabilised by interactions between cohesin and CTCF proteins. Here we show that the flexibility of the chromatin fibre strongly affects this dynamical process, and find that diffusive loop extrusion is more efficient on stiffer chromatin regions. We also show that the dynamics of loop formation are faster in confined and collapsed chromatin conformations but that this enhancement is counteracted by the increased crowding. We provide a simple theoretical argument explaining why stiffness and collapsed conformations favour diffusive extrusion. In light of the heterogeneous physical and conformational properties of eukaryotic chromatin, we suggest that our results are relevant to understand the looping and organisation of interphase chromosomes in vivo.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31727590,

title = {Three dimensional in vitro models of cancer: Bioprinting multilineage glioblastoma models},

author = {Miguel A Hermida and Jothi Dinesh Kumar and Daniela Schwarz and Keith G Laverty and Alberto Di Bartolo and Marcus Ardron and Mihails Bogomolnijs and Anne Clavreul and Paul M Brennan and Ulrich K Wiegand and Ferry Pw Melchels and Will Shu and Nicholas R Leslie},

doi = {10.1016/j.jbior.2019.100658},

issn = {2212-4934},

year  = {2020},

date = {2020-01-01},

journal = {Adv Biol Regul},

volume = {75},

pages = {100658},

abstract = {Three dimensional (3D) bioprinting of multiple cell types within optimised extracellular matrices has the potential to more closely model the 3D environment of human physiology and disease than current alternatives. In this study, we used a multi-nozzle extrusion bioprinter to establish models of glioblastoma made up of cancer and stromal cells printed within matrices comprised of alginate modified with RGDS cell adhesion peptides, hyaluronic acid and collagen-1. Methods were developed using U87MG glioblastoma cells and MM6 monocyte/macrophages, whilst more disease relevant constructs contained glioblastoma stem cells (GSCs), co-printed with glioma associated stromal cells (GASCs) and microglia. Printing parameters were optimised to promote cell-cell interaction, avoiding the 'caging in' of cells due to overly dense cross-linking. Such printing had a negligible effect on cell viability, and cells retained robust metabolic activity and proliferation. Alginate gels allowed the rapid recovery of printed cell protein and RNA, and fluorescent reporters provided analysis of protein kinase activation at the single cell level within printed constructs. GSCs showed more resistance to chemotherapeutic drugs in 3D printed tumour constructs compared to 2D monolayer cultures, reflecting the clinical situation. In summary, a novel 3D bioprinting strategy is developed which allows control over the spatial organisation of tumour constructs for pre-clinical drug sensitivity testing and studies of the tumour microenvironment.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31494034,

title = {Decreased Enhancer-Promoter Proximity Accompanying Enhancer Activation},

author = {Nezha S Benabdallah and Iain Williamson and Robert S Illingworth and Lauren Kane and Shelagh Boyle and Dipta Sengupta and Graeme R Grimes and Pierre Therizols and Wendy A Bickmore},

doi = {10.1016/j.molcel.2019.07.038},

issn = {1097-4164},

year  = {2019},

date = {2019-11-01},

journal = {Mol Cell},

volume = {76},

number = {3},

pages = {473--484.e7},

abstract = {Enhancers can regulate the promoters of their target genes over very large genomic distances. It is widely assumed that mechanisms of enhancer action involve the reorganization of three-dimensional chromatin architecture, but this is poorly understood. The predominant model involves physical enhancer-promoter interaction by looping out the intervening chromatin. However, studying the enhancer-driven activation of the Sonic hedgehog gene (Shh), we have identified a change in chromosome conformation that is incompatible with this simple looping model. Using super-resolution 3D-FISH and chromosome conformation capture, we observe a decreased spatial proximity between Shh and its enhancers during the differentiation of embryonic stem cells to neural progenitors. We show that this can be recapitulated by synthetic enhancer activation, is impeded by chromatin-bound proteins located between the enhancer and the promoter, and appears to involve the catalytic activity of poly (ADP-ribose) polymerase. Our data suggest that models of enhancer-promoter communication need to encompass chromatin conformations other than looping.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31645626,

title = {Altered dendritic spine function and integration in a mouse model of fragile X syndrome},

author = {Sam A Booker and Aleksander P F Domanski and Owen R Dando and Adam D Jackson and John T R Isaac and Giles E Hardingham and David J A Wyllie and Peter C Kind},

doi = {10.1038/s41467-019-11891-6},

issn = {2041-1723},

year  = {2019},

date = {2019-10-01},

journal = {Nat Commun},

volume = {10},

number = {1},

pages = {4813},

abstract = {Cellular and circuit hyperexcitability are core features of fragile X syndrome and related autism spectrum disorder models. However, the cellular and synaptic bases of this hyperexcitability have proved elusive. We report in a mouse model of fragile X syndrome, glutamate uncaging onto individual dendritic spines yields stronger single-spine excitation than wild-type, with more silent spines. Furthermore, fewer spines are required to trigger an action potential with near-simultaneous uncaging at multiple spines. This is, in part, from increased dendritic gain due to increased intrinsic excitability, resulting from reduced hyperpolarization-activated currents, and increased NMDA receptor signaling. Using super-resolution microscopy we detect no change in dendritic spine morphology, indicating no structure-function relationship at this age. However, ultrastructural analysis shows a 3-fold increase in multiply-innervated spines, accounting for the increased single-spine glutamate currents. Thus, loss of FMRP causes abnormal synaptogenesis, leading to large numbers of poly-synaptic spines despite normal spine morphology, thus explaining the synaptic perturbations underlying circuit hyperexcitability.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30962624,

title = {Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software},

author = {Daniel Sage and Thanh-An Pham and Hazen Babcock and Tomas Lukes and Thomas Pengo and Jerry Chao and Ramraj Velmurugan and Alex Herbert and Anurag Agrawal and Silvia Colabrese and Ann Wheeler and Anna Archetti and Bernd Rieger and Raimund Ober and Guy M Hagen and Jean-Baptiste Sibarita and Jonas Ries and Ricardo Henriques and Michael Unser and Seamus Holden},

doi = {10.1038/s41592-019-0364-4},

issn = {1548-7105},

year  = {2019},

date = {2019-05-01},

journal = {Nat Methods},

volume = {16},

number = {5},

pages = {387--395},

abstract = {With the widespread uptake of two-dimensional (2D) and three-dimensional (3D) single-molecule localization microscopy (SMLM), a large set of different data analysis packages have been developed to generate super-resolution images. In a large community effort, we designed a competition to extensively characterize and rank the performance of 2D and 3D SMLM software packages. We generated realistic simulated datasets for popular imaging modalities-2D, astigmatic 3D, biplane 3D and double-helix 3D-and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D SMLM software and provides a holistic view of how the latest 2D and 3D SMLM packages perform in realistic conditions. This resource allows researchers to identify optimal analytical software for their experiments, allows 3D SMLM software developers to benchmark new software against the current state of the art, and provides insight into the current limits of the field.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30655294,

title = {A VPS33A-binding motif on syntaxin 17 controls autophagy completion in mammalian cells},

author = {Rebecca S Saleeb and Deirdre M Kavanagh and Alison R Dun and Paul A Dalgarno and Rory R Duncan},

doi = {10.1074/jbc.RA118.005947},

issn = {1083-351X},

year  = {2019},

date = {2019-03-01},

journal = {J Biol Chem},

volume = {294},

number = {11},

pages = {4188--4201},

abstract = {Autophagy is an intracellular degradation pathway that transports cytoplasmic material to the lysosome for hydrolysis. It is completed by SNARE-mediated fusion of the autophagosome and endolysosome membranes. This process must be carefully regulated to maintain the organization of the membrane system and prevent mistargeted degradation. As yet, models of autophagosomal fusion have not been verified within a cellular context because of difficulties with assessing protein interactions  Here, we used high-resolution fluorescence lifetime imaging (FLIM)-FRET of HeLa cells to identify protein interactions within the spatiotemporal framework of the cell. We show that autophagosomal syntaxin 17 (Stx17) heterotrimerizes with synaptosome-associated protein 29 (SNAP29) and vesicle-associated membrane protein 7 (VAMP7) , highlighting a functional role for VAMP7 in autophagosome clearance that has previously been sidelined in favor of a role for VAMP8. Additionally, we identified multimodal regulation of SNARE assembly by the Sec1/Munc18 (SM) protein VPS33A, mirroring other syntaxin-SM interactions and therefore suggesting a unified model of SM regulation. Contrary to current theoretical models, we found that the Stx17 N-peptide appears to interact in a positionally conserved, but mechanistically divergent manner with VPS33A, providing a late "go, no-go" step for autophagic fusion via a phosphoserine master-switch. Our findings suggest that Stx17 fusion competency is regulated by a phosphosite in its N-peptide, representing a previously unknown regulatory step in mammalian autophagy.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30692205,

title = {Nuclear pore density controls heterochromatin reorganization during senescence},

author = {Charlene Boumendil and Priya Hari and Karl C F Olsen and Juan Carlos Acosta and Wendy A Bickmore},

doi = {10.1101/gad.321117.118},

issn = {1549-5477},

year  = {2019},

date = {2019-02-01},

journal = {Genes Dev},

volume = {33},

number = {3-4},

pages = {144--149},

abstract = {During oncogene-induced senescence (OIS), heterochromatin is lost from the nuclear periphery and forms internal senescence-associated heterochromatin foci (SAHFs). We show that an increased nuclear pore density during OIS is responsible for SAHF formation. In particular, the nucleoporin TPR is necessary for both formation and maintenance of SAHFs. Loss of SAHFs does not affect cell cycle arrest but abrogates the senescence-associated secretory phenotype-a program of inflammatory cytokine gene activation. Our results uncover a previously unknown role of nuclear pores in heterochromatin reorganization in mammalian nuclei and demonstrate the importance of heterochromatin organization for a specific gene activation program.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30636036,

title = {SWAP70 undergoes dynamic conformational regulation at the leading edge of migrating cells},

author = {Nisha Kriplani and Rory R Duncan and Nicholas R Leslie},

doi = {10.1002/1873-3468.13326},

issn = {1873-3468},

year  = {2019},

date = {2019-02-01},

journal = {FEBS Lett},

volume = {593},

number = {4},

pages = {395--405},

abstract = {Rearrangements of the actin cytoskeleton are regulated in part by dynamic localised activation and inactivation of Rho family small GTPases. SWAP70 binds to and activates the small GTPase RAC1 as well as binding to filamentous actin and PIP . We have developed an encoded biosensor, which uses Forster resonance energy transfer to reveal conformational changes in SWAP70 in live cells. SWAP70 adopts a distinct conformation at the plasma membrane, which in migrating glioma cells is enriched at the leading edge but does not always associate with its PIP -dependent translocation to the membrane. This supports a role for SWAP70 in positive feedback activation of RAC1 at sites of filamentous actin, PIP and active RAC1.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30212615,

title = {A Catch-and-Release Approach to Selective Modification of Accessible Tyrosine Residues},

author = {Christopher Allan and Miroslav Kosar and Christina V Burr and C Logan Mackay and Rory R Duncan and Alison N Hulme},

doi = {10.1002/cbic.201800532},

issn = {1439-7633},

year  = {2018},

date = {2018-12-01},

journal = {Chembiochem},

volume = {19},

number = {23},

pages = {2443--2447},

abstract = {The tyrosine side chain is amphiphilic leading to significant variations in the surface exposure of tyrosine residues in the folded structure of a native sequence protein. This variability can be exploited to give residue-selective functionalization of a protein substrate by using a highly reactive diazonium group tethered to an agarose-based resin. This novel catch-and-release approach to protein modification has been demonstrated for proteins with accessible tyrosine residues, which are compared with a control group of proteins in which there are no accessible tyrosine residues. MS analysis of the modified proteins showed that functionalization was highly selective, but reactivity was further attenuated by the electrostatic environment of any individual residue. Automated screening of PDB structures allows identification of potential candidates for selective modification by comparison with the accessibility of the tyrosine residue in a benchmark peptide (GYG).},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30049712,

title = {Mitochondrial inner membrane permeabilisation enables mtDNA release during apoptosis},

author = {Joel S Riley and Giovanni Quarato and Catherine Cloix and Jonathan Lopez and Jim O'Prey and Matthew Pearson and James Chapman and Hiromi Sesaki and Leo M Carlin and João F Passos and Ann P Wheeler and Andrew Oberst and Kevin M Ryan and Stephen Wg Tait},

doi = {10.15252/embj.201899238},

issn = {1460-2075},

year  = {2018},

date = {2018-09-01},

journal = {EMBO J},

volume = {37},

number = {17},

abstract = {During apoptosis, pro-apoptotic BAX and BAK are activated, causing mitochondrial outer membrane permeabilisation (MOMP), caspase activation and cell death. However, even in the absence of caspase activity, cells usually die following MOMP Such caspase-independent cell death is accompanied by inflammation that requires mitochondrial DNA (mtDNA) activation of cGAS-STING signalling. Because the mitochondrial inner membrane is thought to remain intact during apoptosis, we sought to address how matrix mtDNA could activate the cytosolic cGAS-STING signalling pathway. Using super-resolution imaging, we show that mtDNA is efficiently released from mitochondria following MOMP In a temporal manner, we find that following MOMP, BAX/BAK-mediated mitochondrial outer membrane pores gradually widen. This allows extrusion of the mitochondrial inner membrane into the cytosol whereupon it permeablises allowing mtDNA release. Our data demonstrate that mitochondrial inner membrane permeabilisation (MIMP) can occur during cell death following BAX/BAK-dependent MOMP Importantly, by enabling the cytosolic release of mtDNA, inner membrane permeabilisation underpins the immunogenic effects of caspase-independent cell death.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30190480,

title = {DISC1 regulates N-methyl-D-aspartate receptor dynamics: abnormalities induced by a Disc1 mutation modelling a translocation linked to major mental illness},

author = {Elise L V Malavasi and Kyriakos D Economides and Ellen Grünewald and Paraskevi Makedonopoulou and Philippe Gautier and Shaun Mackie and Laura C Murphy and Hannah Murdoch and Darragh Crummie and Fumiaki Ogawa and Daniel L McCartney and Shane T O'Sullivan and Karen Burr and Helen S Torrance and Jonathan Phillips and Marion Bonneau and Susan M Anderson and Paul Perry and Matthew Pearson and Costas Constantinides and Hazel Davidson-Smith and Mostafa Kabiri and Barbara Duff and Mandy Johnstone and H Greg Polites and Stephen M Lawrie and Douglas H Blackwood and Colin A Semple and Kathryn L Evans and Michel Didier and Siddharthan Chandran and Andrew M McIntosh and David J Price and Miles D Houslay and David J Porteous and J Kirsty Millar},

doi = {10.1038/s41398-018-0228-1},

issn = {2158-3188},

year  = {2018},

date = {2018-09-01},

journal = {Transl Psychiatry},

volume = {8},

number = {1},

pages = {184},

abstract = {The neuromodulatory gene DISC1 is disrupted by a t(1;11) translocation that is highly penetrant for schizophrenia and affective disorders, but how this translocation affects DISC1 function is incompletely understood. N-methyl-D-aspartate receptors (NMDAR) play a central role in synaptic plasticity and cognition, and are implicated in the pathophysiology of schizophrenia through genetic and functional studies. We show that the NMDAR subunit GluN2B complexes with DISC1-associated trafficking factor TRAK1, while DISC1 interacts with the GluN1 subunit and regulates dendritic NMDAR motility in cultured mouse neurons. Moreover, in the first mutant mouse that models DISC1 disruption by the translocation, the pool of NMDAR transport vesicles and surface/synaptic NMDAR expression are increased. Since NMDAR cell surface/synaptic expression is tightly regulated to ensure correct function, these changes in the mutant mouse are likely to affect NMDAR signalling and synaptic plasticity. Consistent with these observations, RNASeq analysis of the translocation carrier-derived human neurons indicates abnormalities of excitatory synapses and vesicle dynamics. RNASeq analysis of the human neurons also identifies many differentially expressed genes previously highlighted as putative schizophrenia and/or depression risk factors through large-scale genome-wide association and copy number variant studies, indicating that the translocation triggers common disease pathways that are shared with unrelated psychiatric patients. Altogether, our findings suggest that translocation-induced disease mechanisms are likely to be relevant to mental illness in general, and that such disease mechanisms include altered NMDAR dynamics and excitatory synapse function. This could contribute to the cognitive disorders displayed by translocation carriers.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30867863,

title = {Impact of poloxamer 188 (Pluronic F-68) additive on cell mechanical properties, quantification by real-time deformability cytometry},

author = {Ewa Guzniczak and Melanie Jimenez and Matthew Irwin and Oliver Otto and Nicholas Willoughby and Helen Bridle},

doi = {10.1063/1.5040316},

issn = {1932-1058},

year  = {2018},

date = {2018-07-01},

journal = {Biomicrofluidics},

volume = {12},

number = {4},

pages = {044118},

abstract = {Advances in cellular therapies have led to the development of new approaches for cell product purification and formulation, e.g., utilizing cell endogenous properties such as size and deformability as a basis for separation from potentially harmful undesirable by-products. However, commonly used additives such as Pluronic F-68 and other poloxamer macromolecules can change the mechanical properties of cells and consequently alter their processing. In this paper, we quantified the short-term effect of Pluronic F-68 on the mechanotype of three different cell types (Jurkat cells, red blood cells, and human embryonic kidney cells) using real-time deformability cytometry. The impact of the additive concentration was assessed in terms of cell size and deformability. We observed that cells respond progressively to the presence of Pluronic F-68 within first 3 h of incubation and become significantly stiffer (p-value < 0.001) in comparison to a serum-free control and a control containing serum. We also observed that the short-term response manifested as cell stiffening is true (p-value < 0.001) for the concentration reaching 1% (w/v) of the poloxamer additive in tested buffers. Additionally, using flow cytometry, we assessed that changes in cell deformability triggered by addition of Pluronic F-68 are not accompanied by size or viability alterations.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29401768,

title = {Cylindrical microlensing for enhanced collection efficiency of small pixel SPAD arrays in single-molecule localisation microscopy},

author = {Istvan Gyongy and Amy Davies and Benjamin Gallinet and Neale A W Dutton and Rory R Duncan and Colin Rickman and Robert K Henderson and Paul A Dalgarno},

doi = {10.1364/OE.26.002280},

issn = {1094-4087},

year  = {2018},

date = {2018-02-01},

journal = {Opt Express},

volume = {26},

number = {3},

pages = {2280--2291},

abstract = {Single-photon avalanche photodiode (SPAD) image sensors offer time-gated photon counting, at high binary frame rates of >100 kFPS and with no readout noise. This makes them well-suited to a range of scientific applications, including microscopy, sensing and quantum optics. However, due to the complex electronics required, the fill factor tends to be significantly lower (< 10%) than that of EMCCD and sCMOS cameras (>90%), whilst the pixel size is typically larger, impacting the sensitivity and practicalities of the SPAD devices. This paper presents the first characterisation of a cylindrical-shaped microlens array applied to a small, 8 micron, pixel SPAD imager. The enhanced fill factor, ≈50% for collimated light, is the highest reported value amongst SPAD sensors with comparable resolution and pixel pitch. We demonstrate the impact of the increased sensitivity in single-molecule localisation microscopy, obtaining a resolution of below 40nm, the best reported figure for a SPAD sensor.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29146773,

title = {Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy},

author = {Katharina N Richter and Natalia H Revelo and Katharina J Seitz and Martin S Helm and Deblina Sarkar and Rebecca S Saleeb and Elisa D'Este and Jessica Eberle and Eva Wagner and Christian Vogl and Diana F Lazaro and Frank Richter and Javier Coy-Vergara and Giovanna Coceano and Edward S Boyden and Rory R Duncan and Stefan W Hell and Marcel A Lauterbach and Stephan E Lehnart and Tobias Moser and Tiago F Outeiro and Peter Rehling and Blanche Schwappach and Ilaria Testa and Bolek Zapiec and Silvio O Rizzoli},

doi = {10.15252/embj.201695709},

issn = {1460-2075},

year  = {2018},

date = {2018-01-01},

journal = {EMBO J},

volume = {37},

number = {1},

pages = {139--159},

abstract = {Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {A 256×256 , 100-kfps, 61% Fill-Factor SPAD Image Sensor for Time-Resolved Microscopy Applications},

author = {Gyongy, Istvan and Calder, Neil and Davies, Amy and Dutton, Neale A. W. and Duncan, Rory R. and Rickman, Colin and Dalgarno, Paul and Henderson, Robert K},

doi = {10.1109/TED.2017.2779790},

year  = {2017},

date = {2017-12-21},

urldate = {2017-12-21},

journal = {IEEE Transactions on Electron Devices},

volume = {65},

issue = {2},

pages = {547-554},

abstract = {A 256 × 256 single-photon avalanche diode image sensor operating at 100kfps with fill factor of 61% and pixel pitch of 16 μm is reported. An all-nMOS 7T pixel allows gated operation down to 4 ns and ~600-ps fall time with on-chip delay generation. The sensor operates with 0.996 temporal aperture ratio in rolling shutter. Gating and cooling allow the suppression of dark noise, which, in combination with the high fill factor, enables competitive low-light performance with electron multiplying charge-coupled devices while offering time-resolved imaging modes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29234100,

title = {A Bi-fluorescence complementation system to detect associations between the Endoplasmic reticulum and mitochondria},

author = {Mark Harmon and Philip Larkman and Giles Hardingham and Mandy Jackson and Paul Skehel},

doi = {10.1038/s41598-017-17278-1},

issn = {2045-2322},

year  = {2017},

date = {2017-12-01},

journal = {Sci Rep},

volume = {7},

number = {1},

pages = {17467},

abstract = {Close contacts between the endoplasmic reticulum membrane and the mitochondrial outer membrane facilitate efficient transfer of lipids between the organelles and coordinate Ca signalling and stress responses. Changes to this coupling is associated with a number of metabolic disorders and neurodegenerative diseases including Alzheimer's, Parkinson's and motor neuron disease. The distance between the two membranes at regions of close apposition is below the resolution of conventional light microscopy, which makes analysis of these interactions challenging. Here we describe a new bifluorescence complementation (BiFC) method that labels a subset of ER-mitochondrial associations in fixed and living cells. The total number of ER-mitochondria associations detected by this approach increases in response to tunicamycin-induced ER stress, serum deprivation or reduced levels of mitofusin 2 (MFN2). This method will facilitate the analysis of dynamic interactions between the ER and mitochondrial membranes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}