@article{pmid29089557,

title = {High-throughput assessment of mechanical properties of stem cell derived red blood cells, toward cellular downstream processing},

author = {Ewa Guzniczak and Maryam Mohammad Zadeh and Fiona Dempsey and Melanie Jimenez and Henry Bock and Graeme Whyte and Nicholas Willoughby and Helen Bridle},

doi = {10.1038/s41598-017-14958-w},

issn = {2045-2322},

year  = {2017},

date = {2017-10-01},

journal = {Sci Rep},

volume = {7},

number = {1},

pages = {14457},

abstract = {Stem cell products, including manufactured red blood cells, require efficient sorting and purification methods to remove components potentially harmful for clinical application. However, standard approaches for cellular downstream processing rely on the use of specific and expensive labels (e.g. FACS or MACS). Techniques relying on inherent mechanical and physical properties of cells offer high-throughput scalable alternatives but knowledge of the mechanical phenotype is required. Here, we characterized for the first time deformability and size changes in CD34+ cells, and expelled nuclei, during their differentiation process into red blood cells at days 11, 14, 18 and 21, using Real-Time Deformability Cytometry (RT-DC) and Atomic Force Microscopy (AFM). We found significant differences (p < 0.0001; standardised mixed model) between the deformability of nucleated and enucleated cells, while they remain within the same size range. Expelled nuclei are smaller thus could be removed by size-based separation. An average Young's elastic modulus was measured for nucleated cells, enucleated cells and nuclei (day 14) of 1.04 ± 0.47 kPa, 0.53 ± 0.12 kPa and 7.06 ± 4.07 kPa respectively. Our identification and quantification of significant differences (p < 0.0001; ANOVA) in CD34+ cells mechanical properties throughout the differentiation process could enable development of new routes for purification of manufactured red blood cells.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28622508,

title = {SAF-A Regulates Interphase Chromosome Structure through Oligomerization with Chromatin-Associated RNAs},

author = {Ryu-Suke Nozawa and Lora Boteva and Dinesh C Soares and Catherine Naughton and Alison R Dun and Adam Buckle and Bernard Ramsahoye and Peter C Bruton and Rebecca S Saleeb and Maria Arnedo and Bill Hill and Rory R Duncan and Sutherland K Maciver and Nick Gilbert},

doi = {10.1016/j.cell.2017.05.029},

issn = {1097-4172},

year  = {2017},

date = {2017-06-01},

journal = {Cell},

volume = {169},

number = {7},

pages = {1214--1227.e18},

abstract = {Higher eukaryotic chromosomes are organized into topologically constrained functional domains; however, the molecular mechanisms required to sustain these complex interphase chromatin structures are unknown. A stable matrix underpinning nuclear organization was hypothesized, but the idea was abandoned as more dynamic models of chromatin behavior became prevalent. Here, we report that scaffold attachment factor A (SAF-A), originally identified as a structural nuclear protein, interacts with chromatin-associated RNAs (caRNAs) via its RGG domain to regulate human interphase chromatin structures in a transcription-dependent manner. Mechanistically, this is dependent on SAF-A's AAA ATPase domain, which mediates cycles of protein oligomerization with caRNAs, in response to ATP binding and hydrolysis. SAF-A oligomerization decompacts large-scale chromatin structure while SAF-A loss or monomerization promotes aberrant chromosome folding and accumulation of genome damage. Our results show that SAF-A and caRNAs form a dynamic, transcriptionally responsive chromatin mesh that organizes large-scale chromosome structures and protects the genome from instability.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28362576,

title = {Ambra1 spatially regulates Src activity and Src/FAK-mediated cancer cell invasion via trafficking networks},

author = {Christina Schoenherr and Adam Byron and Emma Sandilands and Ketevan Paliashvili and George S Baillie and Amaya Garcia-Munoz and Cristina Valacca and Francesco Cecconi and Bryan Serrels and Margaret C Frame},

doi = {10.7554/eLife.23172},

issn = {2050-084X},

year  = {2017},

date = {2017-03-01},

journal = {Elife},

volume = {6},

abstract = {Here, using mouse squamous cell carcinoma cells, we report a completely new function for the autophagy protein Ambra1 as the first described 'spatial rheostat' controlling the Src/FAK pathway. Ambra1 regulates the targeting of active phospho-Src away from focal adhesions into autophagic structures that cancer cells use to survive adhesion stress. Ambra1 binds to both FAK and Src in cancer cells. When FAK is present, Ambra1 is recruited to focal adhesions, promoting FAK-regulated cancer cell direction-sensing and invasion. However, when Ambra1 cannot bind to FAK, abnormally high levels of phospho-Src and phospho-FAK accumulate at focal adhesions, positively regulating adhesion and invasive migration. Spatial control of active Src requires the trafficking proteins Dynactin one and IFITM3, which we identified as Ambra1 binding partners by interaction proteomics. We conclude that Ambra1 is a core component of an intracellular trafficking network linked to tight spatial control of active Src and FAK levels, and so crucially regulates their cancer-associated biological outputs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28089515,

title = {Navigation through the Plasma Membrane Molecular Landscape Shapes Random Organelle Movement},

author = {Alison R Dun and Gabriel J Lord and Rhodri S Wilson and Deirdre M Kavanagh and Katarzyna I Cialowicz and Shuzo Sugita and Seungmee Park and Lei Yang and Annya M Smyth and Andreas Papadopulos and Colin Rickman and Rory R Duncan},

doi = {10.1016/j.cub.2016.12.002},

issn = {1879-0445},

year  = {2017},

date = {2017-02-01},

journal = {Curr Biol},

volume = {27},

number = {3},

pages = {408--414},

abstract = {Eukaryotic plasma membrane organization theory has long been controversial, in part due to a dearth of suitably high-resolution techniques to probe molecular architecture in situ and integrate information from diverse data streams [1]. Notably, clustered patterning of membrane proteins is a commonly conserved feature across diverse protein families (reviewed in [2]), including the SNAREs [3], SM proteins [4, 5], ion channels [6, 7], and receptors (e.g., [8]). Much effort has gone into analyzing the behavior of secretory organelles [9-13], and understanding the relationship between the membrane and proximal organelles [4, 5, 12, 14] is an essential goal for cell biology as broad concepts or rules may be established. Here we explore the generally accepted model that vesicles at the plasmalemma are guided by cytoskeletal tracks to specific sites on the membrane that have clustered molecular machinery for secretion [15], organized in part by the local lipid composition [16]. To increase our understanding of these fundamental processes, we integrated nanoscopy and spectroscopy of the secretory machinery with organelle tracking data in a mathematical model, iterating with knockdown cell models. We find that repeated routes followed by successive vesicles, the re-use of similar fusion sites, and the apparently distinct vesicle "pools" are all fashioned by the Brownian behavior of organelles overlaid on navigation between non-reactive secretory protein molecular depots patterned at the plasma membrane.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28089515b,

title = {Navigation through the Plasma Membrane Molecular Landscape Shapes Random Organelle Movement},

author = {Alison R Dun and Gabriel J Lord and Rhodri S Wilson and Deirdre M Kavanagh and Katarzyna I Cialowicz and Shuzo Sugita and Seungmee Park and Lei Yang and Annya M Smyth and Andreas Papadopulos and Colin Rickman and Rory R Duncan},

doi = {10.1016/j.cub.2016.12.002},

issn = {1879-0445},

year  = {2017},

date = {2017-02-01},

journal = {Curr Biol},

volume = {27},

number = {3},

pages = {408--414},

abstract = {Eukaryotic plasma membrane organization theory has long been controversial, in part due to a dearth of suitably high-resolution techniques to probe molecular architecture in situ and integrate information from diverse data streams [1]. Notably, clustered patterning of membrane proteins is a commonly conserved feature across diverse protein families (reviewed in [2]), including the SNAREs [3], SM proteins [4, 5], ion channels [6, 7], and receptors (e.g., [8]). Much effort has gone into analyzing the behavior of secretory organelles [9-13], and understanding the relationship between the membrane and proximal organelles [4, 5, 12, 14] is an essential goal for cell biology as broad concepts or rules may be established. Here we explore the generally accepted model that vesicles at the plasmalemma are guided by cytoskeletal tracks to specific sites on the membrane that have clustered molecular machinery for secretion [15], organized in part by the local lipid composition [16]. To increase our understanding of these fundamental processes, we integrated nanoscopy and spectroscopy of the secretory machinery with organelle tracking data in a mathematical model, iterating with knockdown cell models. We find that repeated routes followed by successive vesicles, the re-use of similar fusion sites, and the apparently distinct vesicle "pools" are all fashioned by the Brownian behavior of organelles overlaid on navigation between non-reactive secretory protein molecular depots patterned at the plasma membrane.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29623296,

title = {EnLightenment: High resolution smartphone microscopy as an educational and public engagement platform},

author = {Laura C Wicks and Gemma S Cairns and Jacob Melnyk and Scott Bryce and Rory R Duncan and Paul A Dalgarno},

doi = {10.12688/wellcomeopenres.12841.2},

issn = {2398-502X},

year  = {2017},

date = {2017-01-01},

journal = {Wellcome Open Res},

volume = {2},

pages = {107},

abstract = {We developed a simple, cost-effective smartphone microscopy platform for use in educational and public engagement programs. We demonstrated its effectiveness, and potential for citizen science through a national imaging initiative, . The cost effectiveness of the instrument allowed for the program to deliver over 500 microscopes to more than 100 secondary schools throughout Scotland, targeting 1000's of 12-14 year olds. Through careful, quantified, selection of a high power, low-cost objective lens, our smartphone microscope has an imaging resolution of microns, with a working distance of 3 mm. It is therefore capable of imaging single cells and sub-cellular features, and retains usability for young children. The microscopes were designed in kit form and provided an interdisciplinary educational tool. By providing full lesson plans and support material, we developed a framework to explore optical design, microscope performance, engineering challenges on construction and real-world applications in life sciences, biological imaging, marine biology, art, and technology. A national online imaging competition framed  with over 500 high quality images submitted of diverse content, spanning multiple disciplines. With examples of cellular and sub-cellular features clearly identifiable in some submissions, we show how young public can use these instruments for research-level imaging applications, and the potential of the instrument for citizen science programs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28071717,

title = {Tryptophan and Non-Tryptophan Fluorescence of the Eye Lens Proteins Provides Diagnostics of Cataract at the Molecular Level},

author = {Anna Gakamsky and Rory R Duncan and Nicola M Howarth and Baljean Dhillon and Kim K Buttenschön and Daniel J Daly and Dmitry Gakamsky},

doi = {10.1038/srep40375},

issn = {2045-2322},

year  = {2017},

date = {2017-01-01},

journal = {Sci Rep},

volume = {7},

pages = {40375},

abstract = {The chemical nature of the non-tryptophan (non-Trp) fluorescence of porcine and human eye lens proteins was identified by Mass Spectrometry (MS) and Fluorescence Steady-State and Lifetime spectroscopy as post-translational modifications (PTM) of Trp and Arg amino acid residues. Fluorescence intensity profiles measured along the optical axis of human eye lenses with age-related nuclear cataract showed increasing concentration of fluorescent PTM towards the lens centre in accord with the increased optical density in the lens nucleolus. Significant differences between fluorescence lifetimes of "free" Trp derivatives hydroxytryptophan (OH-Trp), N-formylkynurenine (NFK), kynurenine (Kyn), hydroxykynurenine (OH-Kyn) and their residues were observed. Notably, the lifetime constants of these residues in a model peptide were considerably greater than those of their "free" counterparts. Fluorescence of Trp, its derivatives and argpyrimidine (ArgP) can be excited at the red edge of the Trp absorption band which allows normalisation of the emission spectra of these PTMs to the fluorescence intensity of Trp, to determine semi-quantitatively their concentration. We show that the cumulative fraction of OH-Trp, NFK and ArgP emission dominates the total fluorescence spectrum in both emulsified post-surgical human cataract protein samples, as well as in whole lenses and that this correlates strongly with cataract grade and age.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28069661,

title = {Bimodal dynamics of granular organelles in primary renin-expressing cells revealed using TIRF microscopy},

author = {Charlotte Buckley and Alison R Dun and Audrey Peter and Christopher Bellamy and Kenneth W Gross and Rory R Duncan and John J Mullins},

doi = {10.1152/ajprenal.00384.2016},

issn = {1522-1466},

year  = {2017},

date = {2017-01-01},

journal = {Am J Physiol Renal Physiol},

volume = {312},

number = {1},

pages = {F200--F209},

abstract = {Renin is the initiator and rate-limiting factor in the renin-angiotensin blood pressure regulation system. Although renin is not exclusively produced in the kidney, in nonmurine species the synthesis and secretion of the active circulatory enzyme is confined almost exclusively to the dense core granules of juxtaglomerular (JG) cells, where prorenin is processed and stored for release via a regulated pathway. Despite its importance, the structural organization and regulation of granules within these cells is not well understood, in part due to the difficulty in culturing primary JG cells in vitro and the lack of appropriate cell lines. We have streamlined the isolation and culture of primary renin-expressing cells suitable for high-speed, high-resolution live imaging using a Percoll gradient-based procedure to purify cells from RenGFP transgenic mice. Fibronectin-coated glass coverslips proved optimal for the adhesion of renin-expressing cells and facilitated live cell imaging at the plasma membrane of primary renin cells using total internal reflection fluorescence microscopy (TIRFM). To obtain quantitative data on intracellular function, we stained mixed granule and lysosome populations with Lysotracker Red and stimulated cells using 100 nM isoproterenol. Analysis of membrane-proximal acidic granular organelle dynamics and behavior within renin-expressing cells revealed the existence of two populations of granular organelles with distinct functional responses following isoproterenol stimulation. The application of high-resolution techniques for imaging JG and other specialized kidney cells provides new opportunities for investigating renal cell biology.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27876857,

title = {Smart-aggregation imaging for single molecule localisation with SPAD cameras},

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

doi = {10.1038/srep37349},

issn = {2045-2322},

year  = {2016},

date = {2016-11-01},

journal = {Sci Rep},

volume = {6},

pages = {37349},

abstract = {Single molecule localisation microscopy (SMLM) has become an essential part of the super-resolution toolbox for probing cellular structure and function. The rapid evolution of these techniques has outstripped detector development and faster, more sensitive cameras are required to further improve localisation certainty. Single-photon avalanche photodiode (SPAD) array cameras offer single-photon sensitivity, very high frame rates and zero readout noise, making them a potentially ideal detector for ultra-fast imaging and SMLM experiments. However, performance traditionally falls behind that of emCCD and sCMOS devices due to lower photon detection efficiency. Here we demonstrate, both experimentally and through simulations, that the sensitivity of a binary SPAD camera in SMLM experiments can be improved significantly by aggregating only frames containing signal, and that this leads to smaller datasets and competitive performance with that of existing detectors. The simulations also indicate that with predicted future advances in SPAD camera technology, SPAD devices will outperform existing scientific cameras when capturing fast temporal dynamics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27852806,

title = {SBE6: a novel long-range enhancer involved in driving sonic hedgehog expression in neural progenitor cells},

author = {Nezha S Benabdallah and Philippe Gautier and Betul Hekimoglu-Balkan and Laura A Lettice and Shipra Bhatia and Wendy A Bickmore},

doi = {10.1098/rsob.160197},

issn = {2046-2441},

year  = {2016},

date = {2016-11-01},

journal = {Open Biol},

volume = {6},

number = {11},

abstract = {The expression of genes with key roles in development is under very tight spatial and temporal control, mediated by enhancers. A classic example of this is the sonic hedgehog gene (Shh), which plays a pivotal role in the proliferation, differentiation and survival of neural progenitor cells both in vivo and in vitro. Shh expression in the brain is tightly controlled by several known enhancers that have been identified through genetic, genomic and functional assays. Using chromatin profiling during the differentiation of embryonic stem cells to neural progenitor cells, here we report the identification of a novel long-range enhancer for Shh-Shh-brain-enhancer-6 (SBE6)-that is located 100 kb upstream of Shh and that is required for the proper induction of Shh expression during this differentiation programme. This element is capable of driving expression in the vertebrate brain. Our study illustrates how a chromatin-focused approach, coupled to in vivo testing, can be used to identify new cell-type specific cis-regulatory elements, and points to yet further complexity in the control of Shh expression during embryonic brain development.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27737961,

title = {Condensin II mutation causes T-cell lymphoma through tissue-specific genome instability},

author = {Jessica Woodward and Gillian C Taylor and Dinesh C Soares and Shelagh Boyle and Daoud Sie and David Read and Keerthi Chathoth and Milica Vukovic and Nuria Tarrats and David Jamieson and Kirsteen J Campbell and Karen Blyth and Juan Carlos Acosta and Bauke Ylstra and Mark J Arends and Kamil R Kranc and Andrew P Jackson and Wendy A Bickmore and Andrew J Wood},

doi = {10.1101/gad.284562.116},

issn = {1549-5477},

year  = {2016},

date = {2016-10-01},

journal = {Genes Dev},

volume = {30},

number = {19},

pages = {2173--2186},

abstract = {Chromosomal instability is a hallmark of cancer, but mitotic regulators are rarely mutated in tumors. Mutations in the condensin complexes, which restructure chromosomes to facilitate segregation during mitosis, are significantly enriched in cancer genomes, but experimental evidence implicating condensin dysfunction in tumorigenesis is lacking. We report that mice inheriting missense mutations in a condensin II subunit (Caph2) develop T-cell lymphoma. Before tumors develop, we found that the same Caph2 mutation impairs ploidy maintenance to a different extent in different hematopoietic cell types, with ploidy most severely perturbed at the CD4CD8 T-cell stage from which tumors initiate. Premalignant CD4CD8 T cells show persistent catenations during chromosome segregation, triggering DNA damage in diploid daughter cells and elevated ploidy. Genome sequencing revealed that Caph2 single-mutant tumors are near diploid but carry deletions spanning tumor suppressor genes, whereas P53 inactivation allowed Caph2 mutant cells with whole-chromosome gains and structural rearrangements to form highly aggressive disease. Together, our data challenge the view that mitotic chromosome formation is an invariant process during development and provide evidence that defective mitotic chromosome structure can promote tumorigenesis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27402708,

title = {Shh and ZRS enhancer colocalisation is specific to the zone of polarising activity},

author = {Iain Williamson and Laura A Lettice and Robert E Hill and Wendy A Bickmore},

doi = {10.1242/dev.139188},

issn = {1477-9129},

year  = {2016},

date = {2016-08-01},

journal = {Development},

volume = {143},

number = {16},

pages = {2994--3001},

abstract = {Limb-specific Shh expression is regulated by the (∼1 Mb distant) ZRS enhancer. In the mouse, limb bud-restricted spatiotemporal Shh expression occurs from ∼E10 to E11.5 at the distal posterior margin and is essential for correct autopod formation. Here, we have analysed the higher-order chromatin conformation of Shh in expressing and non-expressing tissues, both by fluorescence in situ hybridisation (FISH) and by chromosome conformation capture (5C). Conventional and super-resolution light microscopy identified significantly elevated frequencies of Shh/ZRS colocalisation only in the Shh-expressing regions of the limb bud, in a conformation consistent with enhancer-promoter loop formation. However, in all tissues and at all developmental stages analysed, Shh-ZRS spatial distances were still consistently shorter than those to a neural enhancer located between Shh and ZRS in the genome. 5C identified a topologically associating domain (TAD) over the Shh/ZRS genomic region and enriched interactions between Shh and ZRS throughout E11.5 embryos. Shh/ZRS colocalisation, therefore, correlates with the spatiotemporal domain of limb bud-specific Shh expression, but close Shh and ZRS proximity in the nucleus occurs regardless of whether the gene or enhancer is active. We suggest that this constrained chromatin configuration optimises the opportunity for the active enhancer to locate and instigate the expression of Shh.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26571506,

title = {Estimation of Fluorescence Lifetimes Via Rotational Invariance Techniques},

author = {Hongqi Yu and Rebecca Saleeb and Paul Dalgarno and David Day-Uei Li},

doi = {10.1109/TBME.2015.2491364},

issn = {1558-2531},

year  = {2016},

date = {2016-06-01},

journal = {IEEE Trans Biomed Eng},

volume = {63},

number = {6},

pages = {1292--1300},

abstract = {Estimation of signal parameters via rotational invariance techniques is a classical algorithm widely used in array signal processing for direction-of-arrival estimation of emitters. Inspired by this method, a new signal model and new fluorescence lifetime estimation via rotational invariance techniques (FLERIT) were developed for multiexponential fluorescence lifetime imaging (FLIM) experiments. The FLERIT only requires a few time bins of a histogram generated by a time-correlated single-photon counting FLIM system, greatly reducing the data throughput from the imager to the signal processing units. As a noniterative method, the FLERIT does not require initial conditions, prior information nor model selection that are usually required by widely used traditional fitting methods, including nonlinear least square methods or maximum-likelihood methods. Moreover, its simplicity means it is suitable for implementations in embedded systems for real-time applications. FLERIT was tested on synthesized and experimental fluorescent cell data showing the potentials to be widely applied in FLIM data analysis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27108798,

title = {A Restricted Repertoire of De Novo Mutations in ITPR1 Cause Gillespie Syndrome with Evidence for Dominant-Negative Effect},

author = {Meriel McEntagart and Kathleen A Williamson and Jacqueline K Rainger and Ann Wheeler and Anne Seawright and Elfride De Baere and Hannah Verdin and L Therese Bergendahl and Alan Quigley and Joe Rainger and Abhijit Dixit and Ajoy Sarkar and Eduardo López Laso and Rocio Sanchez-Carpintero and Jesus Barrio and Pierre Bitoun and Trine Prescott and Ruth Riise and Shane McKee and Jackie Cook and Lisa McKie and Berten Ceulemans and Françoise Meire and I Karen Temple and Fabienne Prieur and Jonathan Williams and Penny Clouston and Andrea H Németh and Siddharth Banka and Hemant Bengani and Mark Handley and Elisabeth Freyer and Allyson Ross and  and Veronica van Heyningen and Joseph A Marsh and Frances Elmslie and David R FitzPatrick},

doi = {10.1016/j.ajhg.2016.03.018},

issn = {1537-6605},

year  = {2016},

date = {2016-05-01},

journal = {Am J Hum Genet},

volume = {98},

number = {5},

pages = {981--992},

abstract = {Gillespie syndrome (GS) is characterized by bilateral iris hypoplasia, congenital hypotonia, non-progressive ataxia, and progressive cerebellar atrophy. Trio-based exome sequencing identified de novo mutations in ITPR1 in three unrelated individuals with GS recruited to the Deciphering Developmental Disorders study. Whole-exome or targeted sequence analysis identified plausible disease-causing ITPR1 mutations in 10/10 additional GS-affected individuals. These ultra-rare protein-altering variants affected only three residues in ITPR1: Glu2094 missense (one de novo, one co-segregating), Gly2539 missense (five de novo, one inheritance uncertain), and Lys2596 in-frame deletion (four de novo). No clinical or radiological differences were evident between individuals with different mutations. ITPR1 encodes an inositol 1,4,5-triphosphate-responsive calcium channel. The homo-tetrameric structure has been solved by cryoelectron microscopy. Using estimations of the degree of structural change induced by known recessive- and dominant-negative mutations in other disease-associated multimeric channels, we developed a generalizable computational approach to indicate the likely mutational mechanism. This analysis supports a dominant-negative mechanism for GS variants in ITPR1. In GS-derived lymphoblastoid cell lines (LCLs), the proportion of ITPR1-positive cells using immunofluorescence was significantly higher in mutant than control LCLs, consistent with an abnormality of nuclear calcium signaling feedback control. Super-resolution imaging supports the existence of an ITPR1-lined nucleoplasmic reticulum. Mice with Itpr1 heterozygous null mutations showed no major iris defects. Purkinje cells of the cerebellum appear to be the most sensitive to impaired ITPR1 function in humans. Iris hypoplasia is likely to result from either complete loss of ITPR1 activity or structure-specific disruption of multimeric interactions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27293801,

title = {Automated single particle detection and tracking for large microscopy datasets},

author = {Rhodri S Wilson and Lei Yang and Alison Dun and Annya M Smyth and Rory R Duncan and Colin Rickman and Weiping Lu},

doi = {10.1098/rsos.160225},

issn = {2054-5703},

year  = {2016},

date = {2016-05-01},

journal = {R Soc Open Sci},

volume = {3},

number = {5},

pages = {160225},

abstract = {Recent advances in optical microscopy have enabled the acquisition of very large datasets from living cells with unprecedented spatial and temporal resolutions. Our ability to process these datasets now plays an essential role in order to understand many biological processes. In this paper, we present an automated particle detection algorithm capable of operating in low signal-to-noise fluorescence microscopy environments and handling large datasets. When combined with our particle linking framework, it can provide hitherto intractable quantitative measurements describing the dynamics of large cohorts of cellular components from organelles to single molecules. We begin with validating the performance of our method on synthetic image data, and then extend the validation to include experiment images with ground truth. Finally, we apply the algorithm to two single-particle-tracking photo-activated localization microscopy biological datasets, acquired from living primary cells with very high temporal rates. Our analysis of the dynamics of very large cohorts of 10 000 s of membrane-associated protein molecules show that they behave as if caged in nanodomains. We show that the robustness and efficiency of our method provides a tool for the examination of single-molecule behaviour with unprecedented spatial detail and high acquisition rates.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27109929,

title = {PSD95 nanoclusters are postsynaptic building blocks in hippocampus circuits},

author = {Matthew J Broadhead and Mathew H Horrocks and Fei Zhu and Leila Muresan and Ruth Benavides-Piccione and Javier DeFelipe and David Fricker and Maksym V Kopanitsa and Rory R Duncan and David Klenerman and Noboru H Komiyama and Steven F Lee and Seth G N Grant},

doi = {10.1038/srep24626},

issn = {2045-2322},

year  = {2016},

date = {2016-04-01},

journal = {Sci Rep},

volume = {6},

pages = {24626},

abstract = {The molecular features of synapses in the hippocampus underpin current models of learning and cognition. Although synapse ultra-structural diversity has been described in the canonical hippocampal circuitry, our knowledge of sub-synaptic organisation of synaptic molecules remains largely unknown. To address this, mice were engineered to express Post Synaptic Density 95 protein (PSD95) fused to either eGFP or mEos2 and imaged with two orthogonal super-resolution methods: gated stimulated emission depletion (g-STED) microscopy and photoactivated localisation microscopy (PALM). Large-scale analysis of ~100,000 synapses in 7 hippocampal sub-regions revealed they comprised discrete PSD95 nanoclusters that were spatially organised into single and multi-nanocluster PSDs. Synapses in different sub-regions, cell-types and locations along the dendritic tree of CA1 pyramidal neurons, showed diversity characterised by the number of nanoclusters per synapse. Multi-nanocluster synapses were frequently found in the CA3 and dentate gyrus sub-regions, corresponding to large thorny excrescence synapses. Although the structure of individual nanoclusters remained relatively conserved across all sub-regions, PSD95 packing into nanoclusters also varied between sub-regions determined from nanocluster fluorescence intensity. These data identify PSD95 nanoclusters as a basic structural unit, or building block, of excitatory synapses and their number characterizes synapse size and structural diversity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26822455,

title = {Translation Microscopy (TRAM) for super-resolution imaging},

author = {Zhen Qiu and Rhodri S Wilson and Yuewei Liu and Alison R Dun and Rebecca S Saleeb and Dongsheng Liu and Colin Rickman and Margaret Frame and Rory R Duncan and Weiping Lu},

doi = {10.1038/srep19993},

issn = {2045-2322},

year  = {2016},

date = {2016-01-01},

journal = {Sci Rep},

volume = {6},

pages = {19993},

abstract = {Super-resolution microscopy is transforming our understanding of biology but accessibility is limited by its technical complexity, high costs and the requirement for bespoke sample preparation. We present a novel, simple and multi-color super-resolution microscopy technique, called translation microscopy (TRAM), in which a super-resolution image is restored from multiple diffraction-limited resolution observations using a conventional microscope whilst translating the sample in the image plane. TRAM can be implemented using any microscope, delivering up to 7-fold resolution improvement. We compare TRAM with other super-resolution imaging modalities, including gated stimulated emission deletion (gSTED) microscopy and atomic force microscopy (AFM). We further developed novel 'ground-truth' DNA origami nano-structures to characterize TRAM, as well as applying it to a multi-color dye-stained cellular sample to demonstrate its fidelity, ease of use and utility for cell biology.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{@ARTICLE{7348657,

title = {Marker-Less Stage Drift Correction in Super-Resolution Microscopy Using the Single-Cluster PHD Filter},

author = {Schlangen, Isabel and Franco, José and Houssineau, Jérémie and Pitkeathly, William T. E. and Clark, Daniel and Smal, Ihor and Rickman, Colin},

doi = {10.1109/JSTSP.2015.2506402},

year  = {2015},

date = {2015-12-07},

urldate = {2015-12-07},

journal = {IEEE Journal of Selected Topics in Signal Processing},

volume = {10},

number = {1},

pages = {193-202},

abstract = {Fluorescence microscopy is a technique which allows the imaging of cellular and intracellular dynamics through the activation of fluorescent molecules attached to them. It is a very important technique because it can be used to analyze the behavior of intracellular processes in vivo in contrast to methods like electron microscopy. There are several challenges related to the extraction of meaningful information from images acquired from optical microscopes due to the low contrast between objects and background and the fact that point-like objects are observed as blurred spots due to the diffraction limit of the optical system. Another consideration is that for the study of intracellular dynamics, multiple particles must be tracked at the same time, which is a challenging task due to problems such as the presence of false positives and missed detections in the acquired data. Additionally, the objective of the microscope is not completely static with respect to the cover slip due to mechanical vibrations or thermal expansions which introduces bias in the measurements. In this paper, a Bayesian approach is used to simultaneously track the locations of objects with different motion behaviors and the stage drift using image data obtained from fluorescence microscopy experiments. Namely, detections are extracted from the acquired frames using image processing techniques, and then these detections are used to accurately estimate the particle positions and simultaneously correct the drift introduced by the motion of the sample stage. A single cluster Probability Hypothesis Density (PHD) filter with object classification is used for the estimation of the multiple target state assuming different motion behaviors. The detection and tracking methods are tested and their performance is evaluated on both simulated and real data.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26689257,

title = {Three-dimensional bioprinting of complex cell laden alginate hydrogel structures},

author = {Atabak Ghanizadeh Tabriz and Miguel A Hermida and Nicholas R Leslie and Wenmiao Shu},

doi = {10.1088/1758-5090/7/4/045012},

issn = {1758-5090},

year  = {2015},

date = {2015-12-01},

journal = {Biofabrication},

volume = {7},

number = {4},

pages = {045012},

abstract = {Different bioprinting techniques have been used to produce cell-laden alginate hydrogel structures, however these approaches have been limited to 2D or simple three-dimension (3D) structures. In this study, a new extrusion based bioprinting technique was developed to produce more complex alginate hydrogel structures. This was achieved by dividing the alginate hydrogel cross-linking process into three stages: primary calcium ion cross-linking for printability of the gel, secondary calcium cross-linking for rigidity of the alginate hydrogel immediately after printing and tertiary barium ion cross-linking for long-term stability of the alginate hydrogel in culture medium. Simple 3D structures including tubes were first printed to ensure the feasibility of the bioprinting technique and then complex 3D structures such as branched vascular structures were successfully printed. The static stiffness of the alginate hydrogel after printing was 20.18 ± 1.62 KPa which was rigid enough to sustain the integrity of the complex 3D alginate hydrogel structure during the printing. The addition of 60 mM barium chloride was found to significantly extend the stability of the cross-linked alginate hydrogel from 3 d to beyond 11 d without compromising the cellular viability. The results based on cell bioprinting suggested that viability of U87-MG cells was 93 ± 0.9% immediately after bioprinting and cell viability maintained above 88% ± 4.3% in the alginate hydrogel over the period of 11 d.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26655907,

title = {ADF and Cofilin1 Control Actin Stress Fibers, Nuclear Integrity, and Cell Survival},

author = {Georgios Kanellos and Jing Zhou and Hitesh Patel and Rachel A Ridgway and David Huels and Christine B Gurniak and Emma Sandilands and Neil O Carragher and Owen J Sansom and Walter Witke and Valerie G Brunton and Margaret C Frame},

doi = {10.1016/j.celrep.2015.10.056},

issn = {2211-1247},

year  = {2015},

date = {2015-12-01},

journal = {Cell Rep},

volume = {13},

number = {9},

pages = {1949--1964},

abstract = {Genetic co-depletion of the actin-severing proteins ADF and CFL1 triggers catastrophic loss of adult homeostasis in multiple tissues. There is impaired cell-cell adhesion in skin keratinocytes with dysregulation of E-cadherin, hyperproliferation of differentiated cells, and ultimately apoptosis. Mechanistically, the primary consequence of depleting both ADF and CFL1 is uncontrolled accumulation of contractile actin stress fibers associated with enlarged focal adhesions at the plasma membrane, as well as reduced rates of membrane protrusions. This generates increased intracellular acto-myosin tension that promotes nuclear deformation and physical disruption of the nuclear lamina via the LINC complex that normally connects regulated actin filaments to the nuclear envelope. We therefore describe a pathway involving the actin-severing proteins ADF and CFL1 in regulating the dynamic turnover of contractile actin stress fibers, and this is vital to prevent the nucleus from being damaged by actin contractility, in turn preserving cell survival and tissue homeostasis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}