Showing papers by "Randall Division of Cell and Molecular Biophysics published in 2015"
TL;DR: Recent advances in understanding of how Rho GTPases contribute to different types of migration are reviewed, comparing lamellipodium-driven versus bleb-driven migration modes and how cells migrate across the endothelium is described.
619 citations
TL;DR: Combined inhibition of DNMT activities and JAK signalling, in vitro and in vivo, results in long-term reversion of CAF-associated proinvasive activity and restoration of the wild-type fibroblast phenotype.
Abstract: Carcinoma-associated fibroblasts (CAF) mediate the onset of a proinvasive tumour microenvironment. The proinflammatory cytokine LIF reprograms fibroblasts into a proinvasive phenotype, which promotes extracellular matrix remodelling and collective invasion of cancer cells. Here we unveil that exposure to LIF initiates an epigenetic switch leading to the constitutive activation of JAK1/STAT3 signalling, which results in sustained proinvasive activity of CAF. Mechanistically, p300-histone acetyltransferase acetylates STAT3, which, in turn, upregulates and activates the DNMT3b DNA methyltransferase. DNMT3b methylates CpG sites of the SHP-1 phosphatase promoter, which abrogates SHP-1 expression, and results in constitutive phosphorylation of JAK1. Sustained JAK1/STAT3 signalling is maintained by DNA methyltransferase DNMT1. Consistently, in human lung and head and neck carcinomas, STAT3 acetylation and phosphorylation are inversely correlated with SHP-1 expression. Combined inhibition of DNMT activities and JAK signalling, in vitro and in vivo, results in long-term reversion of CAF-associated proinvasive activity and restoration of the wild-type fibroblast phenotype.
252 citations
TL;DR: Results in isoform-specific knockout mice argue for a role of ROCK1 and ROCK2 in the pathogenesis of cardiac fibrosis and cardiac hypertrophy, respectively, and pharmacological ROCK inhibition reduces the enhanced ROCK activity in patients, accompanied with a measurable improvement in medical condition.
Abstract: Rho-associated kinases ROCK1 and ROCK2 are serine/threonine kinases that are downstream targets of the small GTPases RhoA, RhoB, and RhoC. ROCKs are involved in diverse cellular activities including actin cytoskeleton organization, cell adhesion and motility, proliferation and apoptosis, remodeling of the extracellular matrix and smooth muscle cell contraction. The role of ROCK1 and ROCK2 has long been considered to be similar; however, it is now clear that they do not always have the same functions. Moreover, depending on their subcellular localization, activation, and other environmental factors, ROCK signaling can have different effects on cellular function. With respect to the heart, findings in isoform-specific knockout mice argue for a role of ROCK1 and ROCK2 in the pathogenesis of cardiac fibrosis and cardiac hypertrophy, respectively. Increased ROCK activity could play a pivotal role in processes leading to cardiovascular diseases such as hypertension, pulmonary hypertension, angina pectoris, vasospastic angina, heart failure, and stroke, and thus ROCK activity is a potential new biomarker for heart disease. Pharmacological ROCK inhibition reduces the enhanced ROCK activity in patients, accompanied with a measurable improvement in medical condition. In this review, we focus on recent findings regarding ROCK signaling in the pathogenesis of cardiovascular disease, with a special focus on differences between ROCK1 and ROCK2 function.
236 citations
TL;DR: It is shown that the switch from E- to N-cadherin during EMT is essential for acquisition of CIL behavior, which provides insight into the balance of physical forces that contributes to CIL in cells in vivo.
235 citations
TL;DR: Differences in sIgE levels and allergen specificity could not justify the clinical phenotype in all children with PA and PS children and blocking IgG4 antibodies provide an additional explanation for the absence of clinical reactivity in PS patients sensitized to major peanut allergens.
Abstract: Background Most children with detectable peanut-specific IgE (P-sIgE) are not allergic to peanut. We addressed 2 non–mutually exclusive hypotheses for the discrepancy between allergy and sensitization: (1) differences in P-sIgE levels between children with peanut allergy (PA) and peanut-sensitized but tolerant (PS) children and (2) the presence of an IgE inhibitor, such as peanut-specific IgG 4 (P-sIgG 4 ), in PS patients. Methods Two hundred twenty-eight children (108 patients with PA, 77 PS patients, and 43 nonsensitized nonallergic subjects) were studied. Levels of specific IgE and IgG 4 to peanut and its components were determined. IgE-stripped basophils or a mast cell line were used in passive sensitization activation and inhibition assays. Plasma of PS subjects and patients submitted to peanut oral immunotherapy (POIT) were depleted of IgG 4 and retested in inhibition assays. Results Basophils and mast cells sensitized with plasma from patients with PA but not PS patients showed dose-dependent activation in response to peanut. Levels of sIgE to peanut and its components could only partially explain differences in clinical reactivity between patients with PA and PS patients. P-sIgG 4 levels ( P = .023) and P-sIgG 4 /P-sIgE ( P 4 /Ara h 1–sIgE ( P = .050), Ara h 2–sIgG 4 /Ara h 2–sIgE ( P = .004), and Ara h 3–sIgG 4 /Ara h 3–sIgE ( P = .016) ratios were greater in PS children compared with those in children with PA. Peanut-induced activation was inhibited in the presence of plasma from PS children with detectable P-sIgG 4 levels and POIT but not from nonsensitized nonallergic children. Depletion of IgG 4 from plasma of children with PS (and POIT) sensitized to Ara h 1 to Ara h 3 partially restored peanut-induced mast cell activation ( P = .007). Conclusions Differences in sIgE levels and allergen specificity could not justify the clinical phenotype in all children with PA and PS children. Blocking IgG 4 antibodies provide an additional explanation for the absence of clinical reactivity in PS patients sensitized to major peanut allergens.
198 citations
TL;DR: Developing single-chain antibody fragments against tubulin to achieve super-resolution imaging of microtubules with a decreased apparent diameter and test the resolving power of these novel probes, which generate microtubule bundles with a known spacing of 50–70 nm and successfully resolve individualMicrotubules.
Abstract: Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techniques can detect specific structures at an increased resolution, but the narrow spacing between neuronal microtubules poses challenges because most existing labelling strategies increase the effective microtubule diameter by 20-40 nm and will thereby blend neighbouring microtubules into one structure. Here we develop single-chain antibody fragments (nanobodies) against tubulin to achieve super-resolution imaging of microtubules with a decreased apparent diameter. To test the resolving power of these novel probes, we generate microtubule bundles with a known spacing of 50-70 nm and successfully resolve individual microtubules. Individual bundled microtubules can also be resolved in different mammalian cells, including hippocampal neurons, allowing novel insights into fundamental mechanisms of microtubule organization in cell- and neurobiology.
173 citations
TL;DR: Redistribution and loss of AF granules are among the earliest AMD changes and could reduce fundus AF signal attributable to RPE at these locations and can enhance the interpretation of clinical fundus-AF and provide a basis for future quantitative studies.
Abstract: Purpose.
Lipofuscin (LF) and melanolipofuscin (MLF) of the retinal pigment epithelium (RPE) are the principal sources of autofluorescence (AF) signals in clinical fundus–AF imaging. Few details about the subcellular distribution of AF organelles in AMD are available. We describe the impact of aging and AMD on RPE morphology revealed by the distribution of AF LF/MLF granules and actin cytoskeleton in human tissues.
135 citations
TL;DR: A model-based Bayesian approach is presented to evaluate molecular cluster assignment proposals, generated in this study by analysis based on Ripley's K function, and takes full account of the individual localization precisions calculated for each emitter.
Abstract: This paper reports a Bayesian approach for the automatic identification of the optimal clustering proposal in the analysis of single-molecule localization-based super-resolution data. Single-molecule localization-based super-resolution microscopy techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) produce pointillist data sets of molecular coordinates. Although many algorithms exist for the identification and localization of molecules from raw image data, methods for analyzing the resulting point patterns for properties such as clustering have remained relatively under-studied. Here we present a model-based Bayesian approach to evaluate molecular cluster assignment proposals, generated in this study by analysis based on Ripley's K function. The method takes full account of the individual localization precisions calculated for each emitter. We validate the approach using simulated data, as well as experimental data on the clustering behavior of CD3ζ, a subunit of the CD3 T cell receptor complex, in resting and activated primary human T cells.
126 citations
TL;DR: The unexpected conformations adopted by functionally important Cγ2 domain loops are discussed, and potential implications for the interaction between IgG4 and FcγRs are speculated about.
Abstract: IgG4, the least represented human IgG subclass in serum, is an intriguing antibody with unique biological properties, such as the ability to undergo Fab-arm exchange and limit immune complex formation. The lack of effector functions, such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, is desirable for therapeutic purposes. IgG4 plays a protective role in allergy by acting as a blocking antibody, and inhibiting mast cell degranulation, but a deleterious role in malignant melanoma, by impeding IgG1-mediated anti-tumor immunity. These findings highlight the importance of understanding the interaction between IgG4 and Fcγ receptors. Despite a wealth of structural information for the IgG1 subclass, including complexes with Fcγ receptors, and structures for intact antibodies, high-resolution crystal structures were not reported for IgG4-Fc until recently. Here, we highlight some of the biological properties of human IgG4, and review the recent crystal structures of IgG4-Fc. We discuss the unexpected conformations adopted by functionally important Cγ2 domain loops, and speculate about potential implications for the interaction between IgG4 and FcγRs.
120 citations
TL;DR: The applicability of this novel FLIM technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand.
Abstract: We demonstrate diffraction limited multiphoton imaging in a massively parallel, fully addressable time-resolved multi-beam multiphoton microscope capable of producing fluorescence lifetime images with sub-50ps temporal resolution. This imaging platform offers a significant improvement in acquisition speed over single-beam laser scanning FLIM by a factor of 64 without compromising in either the temporal or spatial resolutions of the system. We demonstrate FLIM acquisition at 500 ms with live cells expressing green fluorescent protein. The applicability of the technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand. Furthermore, ligand-dependent association of HER2-HER3 receptor tyrosine kinases was observed on a similar timescale and involved the internalisation and accumulation or receptor heterodimers within endosomes. These data demonstrate the broad applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction.
119 citations
TL;DR: A novel mechanism by which TGF-β-induced transcription sustains actomyosin force in melanoma cells and thereby promotes melanoma progression independently of EMT is proposed.
TL;DR: It is shown that Drosophila hemocytes require a precisely orchestrated CIL response for their developmental dispersal, and the physical coupling of the flowing actin networks during CIL acts as a mechanotransducer, allowing cells to haptically sense each other and coordinate their behaviors.
TL;DR: Rab-coupling protein–mediated integrin trafficking promotes filopodia formation via RhoA-ROCK-FHOD3, generating non-lamellipodial actin spike protrusions that drive cancer cell migration in 3D extracellular matrix and in vivo.
Abstract: Invasive migration in 3D extracellular matrix (ECM) is crucial to cancer metastasis, yet little is known of the molecular mechanisms that drive reorganization of the cytoskeleton as cancer cells disseminate in vivo. 2D Rac-driven lamellipodial migration is well understood, but how these features apply to 3D migration is not clear. We find that lamellipodia-like protrusions and retrograde actin flow are indeed observed in cells moving in 3D ECM. However, Rab-coupling protein (RCP)-driven endocytic recycling of α5β1 integrin enhances invasive migration of cancer cells into fibronectin-rich 3D ECM, driven by RhoA and filopodial spike-based protrusions, not lamellipodia. Furthermore, we show that actin spike protrusions are Arp2/3-independent. Dynamic actin spike assembly in cells invading in vitro and in vivo is regulated by Formin homology-2 domain containing 3 (FHOD3), which is activated by RhoA/ROCK, establishing a novel mechanism through which the RCP–α5β1 pathway reprograms the actin cytoskeleton to promote invasive migration and local invasion in vivo.
TL;DR: It is shown that the number and position of the hydroxyl groups and the presence/absence of the galloyl moiety have significant impact on the selected catechin solvation shells and hydrogen bond capabilities, which are ultimately linked to their ability to interact with and affect the biological environment.
Abstract: Green tea catechins are polyphenols which are believed to provide health benefits; they are marketed as health supplements and are studied for their potential effects on a variety of medical conditions. However, their mechanisms of action and interaction with the environment at the molecular level are still not well-understood. Here, by means of atomistic simulations, we explore the structural properties of four green tea catechins, in the gas phase and water solution: specifically, (-)-epigallocatechin-3-gallate, which is the most abundant, (-)-epicatechin-3-gallate, (-)-epigallocatechin-3-O-(3-O-methyl)-gallate, and (-)-epigallocatechin. We characterize the free energy conformational landscapes of these catechins at ambient conditions, as a function of the torsional degrees of freedom of the pholyphenolic rings, determining the stable conformers and their connections. We show that these free energy landscapes are only subtly influenced by the interactions with the solvent and by the structural details of the polyphenolic rings. However, the number and position of the hydroxyl groups (or their sustituents) and the presence/absence of the galloyl moiety have significant impact on the selected catechin solvation shells and hydrogen bond capabilities, which are ultimately linked to their ability to interact with and affect the biological environment.
TL;DR: It is shown that distinct combinations of talin domains are required for each of three different integrin functions during Drosophila development, and that vinculin and actomyosin activity help change talin’s orientation.
TL;DR: Local allergic Rhinitis (LAR) is a consistent entity that does not evolve to allergic rhinitis with systemic atopy over time although patients have significant impairment in quality of life and increase in the severity of nasal symptoms over time.
Abstract: Local allergic rhinitis (LAR) is characterized by the presence of a nasal Th2 inflammatory response with local production of specific IgE antibodies and a positive response to a nasal allergen provocation test (NAPT) without evidence of systemic atopy. The prevalence has been shown to be up to 25% in subjects affected with rhinitis with persistence, comorbidity and evolution similar to allergic rhinitis. LAR is a consistent entity that does not evolve to allergic rhinitis with systemic atopy over time although patients have significant impairment in quality of life and increase in the severity of nasal symptoms over time. Lower airways can be also involved. The diagnosis of LAR is based mostly on demonstration of positive response to NAPT and/or local synthesis of specific IgE. Allergens involved include seasonal or perennial such as house dusts mites, pollens, animal epithelia, moulds (alternaria) and others. Basophils from peripheral blood may be activated by the involved allergens suggesting the spill over of locally synthesized specific IgE to the circulation. LAR patients will benefit from the same treatment as allergic patients using antihistamines, inhaled corticosteroids and IgE antagonists. Studies on immunotherapy are ongoing and will determine its efficacy in LAR in terms of symptoms improvement and evolution of the natural course of the disease.
TL;DR: It is proposed actin nodules are platelet podosome-related structures required for platelet–platelet interaction and their absence contributes to the bleeding diathesis of Wiskott–Aldrich syndrome.
Abstract: The actin nodule is a novel F-actin structure present in platelets during early spreading. However, only limited detail is known regarding nodule organization and function. Here we use electron microscopy, SIM and dSTORM super-resolution, and live-cell TIRF microscopy to characterize the structural organization and signalling pathways associated with nodule formation. Nodules are composed of up to four actin-rich structures linked together by actin bundles. They are enriched in the adhesion-related proteins talin and vinculin, have a central core of tyrosine phosphorylated proteins and are depleted of integrins at the plasma membrane. Nodule formation is dependent on Wiskott-Aldrich syndrome protein (WASp) and the ARP2/3 complex. WASp(-/-) mouse blood displays impaired platelet aggregate formation at arteriolar shear rates. We propose actin nodules are platelet podosome-related structures required for platelet-platelet interaction and their absence contributes to the bleeding diathesis of Wiskott-Aldrich syndrome.
TL;DR: A method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated.
Abstract: Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG1/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG4/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions.
TL;DR: The various approaches to super-resolution microscopy, the various approaches which can be successfully used in live cells, the tradeoffs in resolution, speed, and ease of implementation which one must make for each approach, and the quality of results that one might expect from each technique are discussed.
TL;DR: Appreciation of these new structural data has revised the view of IgE structure, shed light on the co‐evolution of antibodies and their receptors, and may open up new therapeutic opportunities.
Abstract: Immunoglobulin E (IgE) is well known for its role in allergic disease, the manifestations of which are mediated through its two Fc receptors, FceRI and CD23 (FceRII). IgE and its interactions with these receptors are therefore potential targets for therapeutic intervention, and exciting progress has been made in this direction. Furthermore, recent structural studies of IgE-Fc, the two receptors, and of their complexes, have revealed a remarkable degree of plasticity at the IgE-CD23 interface and an even more remarkable degree of dynamic flexibility within the IgE molecule. Indeed, there is allosteric communication between the two receptor-binding sites, which we now know are located at some distance from each other in IgE-Fc (at opposite ends of the Ce3 domain). The conformational changes associated with FceRI and CD23 binding to IgE-Fc ensure that their interactions are mutually incompatible, and it may be that this functional imperative has driven IgE to evolve such a dynamic structure. Appreciation of these new structural data has revised our view of IgE structure, shed light on the co-evolution of antibodies and their receptors, and may open up new therapeutic opportunities.
TL;DR: Removing satellite cells from a dystrophic milieu reveals that their regenerative capacity remains both intact and similar to satellite cells derived from healthy muscle, indicating that the host environment is critical for controlling satellite cell function.
TL;DR: It is argued that periconception environmental responses reflect developmental plasticity and 'decisions' made by embryos to optimise their own development, but with lasting consequences.
Abstract: Periconceptional environment may influence embryo development, ultimately affecting adult health. Here, we review the rodent model of maternal low-protein diet specifically during the preimplantation period (Emb-LPD) with normal nutrition during subsequent gestation and postnatally. This model, studied mainly in the mouse, leads to cardiovascular, metabolic and behavioural disease in adult offspring, with females more susceptible. We evaluate the sequence of events from diet administration that may lead to adult disease. Emb-LPD changes maternal serum and/or uterine fluid metabolite composition, notably with reduced insulin and branched-chain amino acids. This is sensed by blastocysts through reduced mammalian target of rapamycin complex 1 signalling. Embryos respond by permanently changing the pattern of development of their extra-embryonic lineages, trophectoderm and primitive endoderm, to enhance maternal nutrient retrieval during subsequent gestation. These compensatory changes include stimulation in proliferation, endocytosis and cellular motility, and epigenetic mechanisms underlying them are being identified. Collectively, these responses act to protect fetal growth and likely contribute to offspring competitive fitness. However, the resulting growth adversely affects long-term health because perinatal weight positively correlates with adult disease risk. We argue that periconception environmental responses reflect developmental plasticity and 'decisions' made by embryos to optimise their own development, but with lasting consequences.
TL;DR: It is proposed that loss of traction force on ligand-bound integrin-beta3 causes recruitment of Dab2/clathrin, resulting in endocytosis of integrins.
Abstract: Force is known to recruit adaptor proteins to the intracellular tails of integrin extracellular matrix receptors. Here the authors show that matrix force-dependent β3 integrin signals block endocytosis by preventing the recruitment of the clathrin adaptor Dab2.
TL;DR: A wide-field fluorescence microscopy setup demonstrating fast fluorescence lifetime data acquisition and the fastest direct TCSPC transfer from a single photon counting device to the computer to date is presented.
Abstract: We present a digital architecture for fast acquisition of time correlated single photon counting (TCSPC) events from a 32×32 complementary metal oxide semiconductor (CMOS) single photon avalanche detector (SPAD) array (Megaframe) to the computer memory. Custom firmware was written to transmit event codes from 1024-TCSPC-enabled pixels for fast transfer of TCSPC events. Our 1024-channel TCSPC system is capable of acquiring up to 0.5×10(9) TCSPC events per second with 16 histogram bins spanning a 14 ns width. Other options include 320×10(6) TCSPC events per second with 256 histogram bins spanning either a 14 or 56 ns time window. We present a wide-field fluorescence microscopy setup demonstrating fast fluorescence lifetime data acquisition. To the best of our knowledge, this is the fastest direct TCSPC transfer from a single photon counting device to the computer to date.
TL;DR: An analysis of immunoglobulin repertoire in different subclasses of antibody in five different populations of B cell, and shows how the repertoire in these different groups changes with age.
Abstract: Older people are more susceptible to infection, less responsive to vaccination and have a more inflammatory immune environment. Using spectratype analysis, we have previously shown that the B-cell repertoire of older people shows evidence of inappropriate clonal expansions in the absence of challenge, and that this loss of B-cell diversity correlates with poor health. Studies on response to vaccination, using both spectratyping and high-throughput sequencing of the repertoire, indicate that older responses to challenge are lacking in magnitude and/or delayed significantly. Also that some of the biologically significant differences may be in different classes of antibody. We have also previously shown that normal young B-cell repertoires can vary between different phenotypic subsets of B cells. In this paper, we present an analysis of immunoglobulin repertoire in different subclasses of antibody in five different populations of B cell, and show how the repertoire in these different groups changes with age. Although some age-related repertoire differences occur in naive cells, before exogenous antigen exposure, we see indications that there is a general dysregulation of the selective forces that shape memory B-cell populations in older people.
TL;DR: It is concluded that zinc promotes activation and proliferation of myogenic cells, and this activation requires phosphorylation of PI3K/Akt and ERK as part of the signaling cascade.
TL;DR: It is demonstrated that podoplanin regulates invadopodia maturation by acting upstream of the ROCK-LIMK-Cofilin pathway through the control of RhoC GTPase activity, which has a key role in the regulation ofInvadopody function in SCC cells, controlling the initial steps of cancer cell invasion.
Abstract: Invadopodia are actin-rich cell membrane projections used by invasive cells to penetrate the basement membrane. Control of invadopodia stability is critical for efficient degradation of the extracellular matrix (ECM); however, the underlying molecular mechanisms remain poorly understood. Here, we uncover a new role for podoplanin, a transmembrane glycoprotein closely associated with malignant progression of squamous cell carcinomas (SCCs), in the regulation of invadopodia-mediated matrix degradation. Podoplanin downregulation in SCC cells impairs invadopodia stability, thereby reducing the efficiency of ECM degradation. We report podoplanin as a novel component of invadopodia-associated adhesion rings, where it clusters prior to matrix degradation. Early podoplanin recruitment to invadopodia is dependent on lipid rafts, whereas ezrin/moesin proteins mediate podoplanin ring assembly. Finally, we demonstrate that podoplanin regulates invadopodia maturation by acting upstream of the ROCK-LIMK-Cofilin pathway through the control of RhoC GTPase activity. Thus, podoplanin has a key role in the regulation of invadopodia function in SCC cells, controlling the initial steps of cancer cell invasion.
TL;DR: A method of increased acquisition speed on a two-beam SIM fluorescence microscope with a lateral resolution of ~100 nm at a maximum raw data acquisition rate of 162 frames per second (fps) with a region of interest of interest free of mechanically moving components is demonstrated.
Abstract: A significant improvement in acquisition speed of structured illumination microscopy (SIM) opens a new field of applications to this already well-established super-resolution method towards 3D scanning real-time imaging of living cells. We demonstrate a method of increased acquisition speed on a two-beam SIM fluorescence microscope with a lateral resolution of ~100 nm at a maximum raw data acquisition rate of 162 frames per second (fps) with a region of interest of 16.5 × 16.5 µm2, free of mechanically moving components. We use a programmable spatial light modulator (ferroelectric LCOS) which promises precise and rapid control of the excitation pattern in the sample plane. A passive Fourier filter and a segmented azimuthally patterned polarizer are used to perform structured illumination with maximum contrast. Furthermore, the free running mode in a modern sCMOS camera helps to achieve faster data acquisition.
TL;DR: CMOS line sensors are demonstrated to be a versatile tool for time-resolved fluorescence spectroscopy by providing parallelized and flexible spectral detection of fluorescence decay.
Abstract: We present a CMOS chip 256 × 2 single photon avalanche diode (SPAD) line sensor, 2378 µm pitch, 437% fill factor, custom designed for time resolved emission spectroscopy (TRES) Integrating time-to-digital converters (TDCs) implement on-chip mono-exponential fluorescence lifetime pre-calculation allowing timing of 65k photons/pixel at 200 Hz line rate at 40 ps resolution using centre-of-mass method (CMM) Per pixel time-correlated single-photon counting (TCSPC) histograms can also be generated with 320 ps bin resolution We characterize performance in terms of dark count rate, instrument response function and lifetime uniformity for a set of fluorophores with lifetimes ranging from 4 ns to 6 ns Lastly, we present fluorescence lifetime spectra of multicolor microspheres and skin autofluorescence acquired using a custom built spectrometer In TCSPC mode, time-resolved spectra are acquired within 5 minutes whilst in CMM mode spectral lifetime signatures are acquired within 2 ms for fluorophore in cuvette and 200 ms for skin autofluorescence We demonstrate CMOS line sensors to be a versatile tool for time-resolved fluorescence spectroscopy by providing parallelized and flexible spectral detection of fluorescence decay
Guy's Hospital1, University of Cambridge2, Centre for Addiction and Mental Health3, King's College London4, Randall Division of Cell and Molecular Biophysics5, Wellcome Trust Sanger Institute6, Western General Hospital7, Newcastle University8, Guy's and St Thomas' NHS Foundation Trust9, University of Cape Town10
TL;DR: It is suggested that although rare coding variants may make a modest overall contribution to complex disease susceptibility, they can inform the understanding of the molecular pathways that contribute to pathogenesis.
Abstract: The contribution of rare coding sequence variants to genetic susceptibility in complex disorders is an important but unresolved question. Most studies thus far have investigated a limited number of genes from regions which contain common disease associated variants. Here we investigate this in inflammatory bowel disease by sequencing the exons and proximal promoters of 531 genes selected from both genome-wide association studies and pathway analysis in pooled DNA panels from 474 cases of Crohn's disease and 480 controls. 80 variants with evidence of association in the sequencing experiment or with potential functional significance were selected for follow up genotyping in 6,507 IBD cases and 3,064 population controls. The top 5 disease associated variants were genotyped in an extension panel of 3,662 IBD cases and 3,639 controls, and tested for association in a combined analysis of 10,147 IBD cases and 7,008 controls. A rare coding variant p.G454C in the BTNL2 gene within the major histocompatibility complex was significantly associated with increased risk for IBD (p = 9.65x10-10, OR = 2.3[95% CI = 1.75-3.04]), but was independent of the known common associated CD and UC variants at this locus. Rare ( T) or decreased risk (IL12B p.V298F, and NICN p.H191R) of IBD. These results provide additional insights into the involvement of the inhibition of T cell activation in the development of both sub-phenotypes of inflammatory bowel disease. We suggest that although rare coding variants may make a modest overall contribution to complex disease susceptibility, they can inform our understanding of the molecular pathways that contribute to pathogenesis.