Publications
102 results found
Darling C, Davis SPX, Kumar S, et al., 2023, Single-shot optical projection tomography for high-speed volumetric imaging of dynamic biological samples, JOURNAL OF BIOPHOTONICS, Vol: 16, ISSN: 1864-063X
Van de Pette M, Dimond A, Galvao AM, et al., 2022, Epigenetic changes induced by in utero dietary challenge result in phenotypic variability in successive generations of mice, Nature Communications, Vol: 13, ISSN: 2041-1723
Transmission of epigenetic information between generations occurs in nematodes, flies and plants, mediated by specialised small RNA pathways, modified histones and DNA methylation. Similar processes in mammals can also affect phenotype through intergenerational or trans-generational mechanisms. Here we generate a luciferase knock-in reporter mouse for the imprinted Dlk1 locus to visualise and track epigenetic fidelity across generations. Exposure to high-fat diet in pregnancy provokes sustained re-expression of the normally silent maternal Dlk1 in offspring (loss of imprinting) and increased DNA methylation at the somatic differentially methylated region (sDMR). In the next generation heterogeneous Dlk1 mis-expression is seen exclusively among animals born to F1-exposed females. Oocytes from these females show altered gene and microRNA expression without changes in DNA methylation, and correct imprinting is restored in subsequent generations. Our results illustrate how diet impacts the foetal epigenome, disturbing canonical and non-canonical imprinting mechanisms to modulate the properties of successive generations of offspring.
Fox MFJ, Bland S, Mangles SPD, et al., 2022, Expectations of how student views on experimental physics develop during an undergraduate degree, Physics Education Research Conference (PERC), Publisher: AMER ASSOC PHYSICS TEACHERS, Pages: 182-187, ISSN: 2377-2379
Guglielmi L, Heliot C, Kumar S, et al., 2021, Smad4 controls signaling robustness and morphogenesis by differentially contributing to the Nodal and BMP pathways, NATURE COMMUNICATIONS, Vol: 12
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- Citations: 7
Darling C, Davis SPX, Kumar S, et al., 2021, Single-Shot Volumetric Imaging Using Optical Projection Tomography, ISSN: 1605-7422
We present a single-shot volumetric imaging method, utilising optical projection tomography. We record projections simultaneously, implementing compressive sensing and machine learning to record up to 70 (camera limited) 1x1x1.9mm volumes/second.
Darling C, Davis SPX, Kumar S, et al., 2021, Single-Shot Volumetric Imaging Using Optical Projection Tomography, European Conferences on Biomedical Optics - Advances in Microscopic Imaging III, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
Georgiadou E, Haythorne E, Dickerson MT, et al., 2020, The pore-forming subunit MCU of the mitochondrial Ca2+ uniporter is required for normal glucose-stimulated insulin secretion in vitro and in vivo in mice, Diabetologia, Vol: 63, Pages: 1368-1381, ISSN: 0012-186X
Aims/hypothesisMitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+ uptake into pancreatic beta cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial Ca2+ importer (MCU) complex is thought to represent the main route for Ca2+ transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo.MethodsHere, we inactivated the pore-forming subunit of the MCU, encoded by Mcu, selectively in mouse beta cells using Ins1Cre-mediated recombination. Whole or dissociated pancreatic islets were isolated and used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca2+ concentration and ATP production in response to increasing glucose concentrations. Electrophysiological recordings were also performed on whole islets. Serum and blood samples were collected to examine oral and i.p. glucose tolerance.ResultsGlucose-stimulated mitochondrial Ca2+ accumulation (p< 0.05), ATP production (p< 0.05) and insulin secretion (p< 0.01) were strongly inhibited in beta cell-specific Mcu-null (βMcu-KO) animals, in vitro, as compared with wild-type (WT) mice. Interestingly, cytosolic Ca2+ concentrations increased (p< 0.001), whereas mitochondrial membrane depolarisation improved in βMcu-KO animals. βMcu-KO mice displayed impaired in vivo insulin secretion at 5 min (p< 0.001) but not 15 min post-i.p. injection of glucose, whilst the opposite phenomenon was observed following an oral gavage at 5 min. Unexpectedly, glucose tolerance was improved (p< 0.05) in young βMcu-KO (<12 weeks), but not in older animals vs WT mice.Conclusions/interpretationMCU is crucial for mitochondrial Ca2+ uptake in pancreatic beta cells and is required for normal GSIS. The apparent compensatory mechanisms that maintain glucose tolerance in βMcu-KO mice remain
Davis SPX, Kumar S, Alexandrov Y, et al., 2019, Convolutional neural networks for reconstruction of undersampled optical projection tomography data applied to in vivo imaging of zebrafish., Journal of Biophotonics, Vol: 12, ISSN: 1864-063X
Optical projection tomography (OPT) is a 3D mesoscopic imaging modality that can utilize absorption or fluorescence contrast. 3D images can be rapidly reconstructed from tomographic data sets sampled with sufficient numbers of projection angles using the Radon transform, as is typically implemented with optically cleared samples of the mm-to-cm scale. For in vivo imaging, considerations of phototoxicity and the need to maintain animals under anesthesia typically preclude the acquisition of OPT data at a sufficient number of angles to avoid artifacts in the reconstructed images. For sparse samples, this can be addressed with iterative algorithms to reconstruct 3D images from undersampled OPT data, but the data processing times present a significant challenge for studies imaging multiple animals. We show here that convolutional neural networks (CNN) can be used in place of iterative algorithms to remove artifacts - reducing processing time for an undersampled in vivo zebrafish dataset from 77 to 15 minutes. We also show that using CNN produces reconstructions of equivalent quality to CS with 40% fewer projections. We further show that diverse training data classes, for example ex vivo mouse tissue data, can be used for CNN-based reconstructions of OPT data of other species including live zebrafish.
Chen L, Li G, Tang L, et al., 2019, Hyperspectral scanning laser optical tomography, Journal of Biophotonics, Vol: 12, ISSN: 1864-063X
In order to study physical relationships within tissue volumes or even organism-level systems, the spatial distribution of multiple fluorescent markers needs to be resolved efficiently in three dimensions. Here, rather than acquiring discrete spectral images sequentially using multiple emission filters, a hyperspectral scanning laser optical tomography system is developed to obtain hyperspectral volumetric data sets with 2-nm spectral resolution of optically transparent mesoscopic (millimeter-centimeter) specimens. This is achieved by acquiring a series of point-scanning hyperspectral extended depth of field images at different angles and subsequently tomographically reconstructing the 3D intensity distribution for each wavelength. This technique is demonstrated to provide robust measurements via the comparison of spectral and intensity profiles of fluorescent bead phantoms. Due to its enhanced spectral resolving ability, this technique is also demonstrated to resolve largely overlapping fluorophores, as demonstrated by the 3D fluorescence hyperspectral reconstruction of a dual-labeled mouse thymus gland sample and the ability to distinguish tumorous and normal tissues of an unlabeled mouse intestine sample.
French P, Davis S, Wisniewski L, et al., 2018, Slice-illuminated optical projection tomography, Optics Letters, Vol: 43, Pages: 5555-5558, ISSN: 0146-9592
To improve the imaging performance of optical projection tomography (OPT) in live samples, we have explored a parallelized implementation of semi-confocal line illumination and detection to discriminate against scattered photons. Slice-illuminated OPT (sl-OPT) improves reconstruction quality in scattering samples by reducing interpixel crosstalk at the cost of increased acquisition time. For in vivo imaging, this can be ameliorated through the use of compressed sensing on angularly undersampled OPT data sets. Here, we demonstrate sl-OPT applied to 3D imaging of bead phantoms and live adult zebrafish.
Wisniewski L, Davis S, Lockwood N, et al., 2018, A role for p130Cas in venous sprouting and lymphangiogenesis in the zebrafish, 5th Congress of the ESC-Council-on-Basic-Cardiovascular-Science on Frontiers in Cardio Vascular Biology, Publisher: OXFORD UNIV PRESS, Pages: S90-S90, ISSN: 0008-6363
Rutter GA, Haythorne EA, Georgiadou E, et al., 2018, Pancreatic beta cell-selective deletion of the mitochondrial calcium uniporter (MCU) impairs glucose-stimulated insulin secretion <i>in vitro</i> but not <i>in vivo</i>, Publisher: WILEY, Pages: 42-42, ISSN: 0742-3071
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Chen L, Li G, Li Y, et al., 2017, UbasM: An effective balanced optical clearing method for intact biomedical imaging, Scientific Reports, Vol: 7, ISSN: 2045-2322
Optical clearing methods can facilitate deep optical imaging in biological tissue by reducing light scattering and this has enabled accurate three-dimensional signal visualization and quantification of complex biological structures. Unfortunately, existing optical clearing approaches present a compromise between maximizing clearing capability, the preservation of fluorescent protein emission and membrane integrity and the speed of sample processing – with the latter typically requiring weeks for cm scale tissue samples. To address this challenge, we present a new, convenient, aqueous optical clearing agent, termed UbasM: Urea-Based Amino-Sugar Mixture, that rapidly renders fixed tissue samples highly transparent and reliably preserves emission from fluorescent proteins and lipophilic dyes in membrane integrity preserved tissues. UbasM is simple, inexpensive, reproducible and compatible with all labeling methods that we have encountered. It can enable convenient, volumetric imaging of tissue up to the scale of whole adult mouse organs and should be useful for a wide range of light microscopy and tomography techniques applied to biomedical research, especially the study on organism-level systems biology at multiple levels.
Watson, Andrews N, Davis S, et al., 2017, OPTiM: optical projection tomography integrated microscope using open-source hardware and software, PLOS One, Vol: 12, ISSN: 1932-6203
We describe the implementation of an OPT plate to perform optical projection tomography (OPT) on a commercial wide-field inverted microscope, using our open-source hardware and software. The OPT plate includes a tilt adjustment for alignment and a stepper motor for sample rotation as required by standard projection tomography. Depending on magnification requirements, three methods of performing OPT are detailed using this adaptor plate: a conventional direct OPT method requiring only the addition of a limiting aperture behind the objective lens; an external optical-relay method allowing conventional OPT to be performed at magnifications >4x; a remote focal scanning and region-of-interest method for improved spatial resolution OPT (up to ~1.6 μm). All three methods use the microscope’s existing incoherent light source (i.e. arc-lamp) and all of its inherent functionality is maintained for day-to-day use. OPT acquisitions are performed on in vivo zebrafish embryos to demonstrate the implementations’ viability.
Andrews N, Davis S, Hay C, et al., 2017, Functional imaging of live Zebrafish using fluorescence lifetime optical projection tomography, Conference on Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XV, Publisher: Society of Photo-Optical Instrumentation Engineers (SPIE), ISSN: 0277-786X
Van de Pette M, Abbas A, Feytout A, et al., 2017, Visualizing changes in Cdkn1c expression links early life adversity to imprint mis-regulation in adults, Cell Reports, Vol: 31, Pages: 1090-1099, ISSN: 2211-1247
Imprinted genes are regulated according to parental origin and can influence embryonic growth and metabolism and confer disease susceptibility.Here we designed sensitive allele-specific reporters to non-invasively monitor imprinted Cdkn1cexpression in mice and showed that expression was modulated by environmental factors encounteredin utero.Acute exposure to chromatin modifyingdrugs resulted in de-repression of paternally inherited (silent) Cdkn1calleles in embryos that was temporary and resolved after birth.In contrast, deprivation of maternal dietary proteinin uteroprovoked permanent de-repression of imprinted Cdkn1cexpression that was sustained into adulthood and occurred through a folate-dependent mechanism of DNA methylation loss.Given the function of imprinted genes in regulating behavior and metabolic processes in adults, these results establish imprinting deregulation as a credible mechanism linking early life adversity to later-life outcomes.Furthermore,Cdkn1c-luciferasemice offer non-invasivetools to identify factors that disrupt epigenetic processes and strategies to limit their long-term impact.
French PMW, Görlitz F, Kelly D, et al., 2017, Open source high content analysis utilizing automated fluorescence lifetime imaging microscopy, Jove-Journal of Visualized Experiments, Vol: 119, ISSN: 1940-087X
We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe herethe functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in μ Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.
Chandaria VV, McGinty J, Nowlan NC, 2016, Characterising the effects of in vitro mechanical stimulation on morphogenesis of developing limb explants, Journal of Biomechanics, Vol: 49, Pages: 3635-3642, ISSN: 1873-2380
Mechanical forces due to fetal movements play an important role in joint shape morphogenesis, and abnormalities of the joints relating to abnormal fetal movements can have long-term health implications. While mechanical stimulation during development has been shown to be important for joint shape, the relationship between the quantity of mechanical stimulation and the growth and shape change of developing cartilage has not been quantified. In this study, we culture embryonic chick limb explants in vitro in order to reveal how the magnitude of applied movement affects key aspects of the developing joint shape. We hypothesise that joint shape is affected by movement magnitude in a dose-dependent manner, and that a movement regime most representative of physiological fetal movements will promote characteristics of normal shape development. Chick hindlimbs harvested at seven days of incubation were cultured for six days, under either static conditions or one of three different dynamic movement regimes, then assessed for joint shape, cell survival and proliferation. We demonstrate that a physiological magnitude of movement in vitro promotes the most normal progression of joint morphogenesis, and that either under-stimulation or over-stimulation has detrimental effects. Providing insight into the optimal level of mechanical stimulation for cartilage growth and morphogenesis is pertinent to gaining a greater understanding of the etiology of conditions such as developmental dysplasia of the hip, and is also valuable for cartilage tissue engineering.
Rutter GA, Semplici F, Mondragon A, et al., 2016, Cell type-specific deletion in mice reveals roles for PAS kinase in insulin and glucagon production, Diabetologia, Vol: 59, Pages: 1938-1947, ISSN: 1432-0428
Background and Aims. Per-Arnt-Sim domain containing kinase (PASK) is a nutrient regulated protein kinase previously implicated in the control of insulin gene expression and glucagon secretion. Here, we explore the roles of the kinase in the control of islet hormone release by generating mice deleted selectively for the Pask gene in pancreatic beta or alpha cells. Methods. Floxed alleles of Pask were produced by homologous recombination and animals bred with mice bearing beta (Ins1Cre, PaskBKO), or alpha (PPG-Cre; PaskAKO) cell selective Cre recombinase alleles. Glucose homeostasis and hormone secretion in vivo and in vitro, gene expression, and islet cell mass, were measured using standard techniques.Results. Ins1Cre-based recombination led to efficient beta cell targeted deletion of Pask. Beta cell mass was reduced by 36.5% (p<0.05) compared to controls in PaskBKO mice, as well as in global null Pask mice (38%, p<0.05). PaskBKO mice displayed normal body weight and fasting glycemia, but slightly impaired glucose tolerance, and beta cell proliferation, after maintenance on a high fat diet. Whilst glucose tolerance was unaffected in PaskAKO mice, glucose infusion rates were increased, and glucagon secretion tended to be lower, during hypoglycemic clamps. Though alpha cell mass was increased (21.9%, p<0.05), glucagon release at low glucose was impaired (p<0.05) in PaskAKO islets. Conclusions. The present findings demonstrate cell autonomous roles for PASK in the control of pancreatic endocrine hormone secretion. Differencesbetween the glycemic phenotype of global versus cell type specific null mice suggest important roles for tissue interactions in the control of glycemia by the kinase.
Kumar S, Lockward N, Ramel M-C, et al., 2016, Quantitative in vivo optical tomography of cancer progression & vasculature development in adult zebrafish, Oncotarget, Vol: 7, Pages: 43939-43948, ISSN: 1949-2553
We describe a novel approach to study tumour progression and vasculature development in vivo via global 3-D fluorescence imaging of live non-pigmented adult zebrafish utilising angularly multiplexed optical projection tomography with compressive sensing (CS-OPT). This “mesoscopic” imaging method bridges a gap between established ~μm resolution 3-D fluorescence microscopy techniques and ~mm-resolved whole body planar imaging and diffuse tomography. Implementing angular multiplexing with CS-OPT, we demonstrate the in vivo global imaging of an inducible fluorescently labelled genetic model of liver cancer in adult non-pigmented zebrafish that also present fluorescently labelled vasculature. In this disease model, addition of a chemical inducer (doxycycline) drives expression of eGFP tagged oncogenic K-RASV12 in the liver of immune competent animals. We show that our novel in vivo global imaging methodology enables non-invasive quantitative imaging of the development of tumour and vasculature throughout the progression of the disease, which we have validated against established methods of pathology including immunohistochemistry. We have also demonstrated its potential for longitudinal imaging through a study of vascular development in the same zebrafish from early embryo to adulthood. We believe that this instrument, together with its associated analysis and data management tools, constitute a new platform for in vivo cancer studies and drug discovery in zebrafish disease models.
Andrews N, Ramel M-C, Kumar S, et al., 2016, Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time, Journal of Biophotonics, Vol: 9, Pages: 414-424, ISSN: 1864-0648
Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3-D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region.
Watson TJ, Andrews N, Harry E, et al., 2016, Remote focal scanning and sub-volume optical projection tomography
We present a platform for sub-volume optical projection tomography utilising an electrically tunable lens and tracking technology. Applied to 3D fluorescent bead phantoms and zebrafish embryos, we demonstrate an improvement in resolution and light collection efficiency with respect to conventional optical projection tomography.
Andrews N, Ramel MC, Kumar S, et al., 2016, Fluorescence lifetime optical projection tomography and FRET applied to visualizing apoptosis in live zebrafish larvae
We present the application of FLIM-OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed cleavable FRET biosensor for Caspase-3 as an indicator of gamma radiation induced apoptosis.
Kumar S, Lockwood N, Ramel MC, et al., 2016, In vivo multiplexed OPT and FLIM OPT of an adult zebrafish cancer disease model
We report angular multiplexed OPT and FLIM OPT applied to in vivo imaging of cancer and FRET biosensors in adult zebrafish. Multiple-spectral 3-D datasets of entire adult zebrafish can be acquired in 3 minutes.
McGinty J, French P, Frankel P, 2016, Novel 3D imaging platform tracks cancer progression in vivo, Biochemist, Vol: 38, Pages: 12-15, ISSN: 0954-982X
Optical imaging underpins biomedical research in many respects and recent decades have seen spectacular advances, particularly in fluorescence imaging where genetic engineering approaches to labelling have been combined with new light sources, detectors and data analysis techniques to provide capabilities like super-resolution beyond the diffraction limit, exquisite spectroscopic contrast for molecular readouts and high-speed image capture for in vivo and high-throughput applications. However, the main impact of such advanced instrumentation and data analysis has been to provide unprecedented quantitative 2D and 3D information concerning samples compatible with microscopy where volumes of less than 1 mm3 are typically imaged in a single 'acquisition'. The ability to view and measure cellular processes and signalling pathways in live cells has been a significant advance for biomedical research and drug discovery. However, for conventional microscope-based assays and experiments, the samples typically comprise thin layers of cells that are not experiencing the same signals that they would in a 3D tissue context and any findings may not directly translate to live organisms. It is desirable to study disease processes in live intact organisms that can provide appropriate physiological complexity. For cancer studies, recent research from our group shows that optical tomography can be used to directly monitor in vivo changes in tumour growth and vascular development in a zebrafish cancer model over time. This technique not only improves the value of the collected data, but if used on a wider scale should result in a reduction in the number of animals used in biomedical research.
Correia T, Lockwood N, Kumar S, et al., 2015, Accelerated optical projection tomography applied to in vivo imaging of zebrafish, PLOS One, Vol: 10, ISSN: 1932-6203
Optical projection tomography (OPT) provides a non-invasive 3-D imaging modality that can be applied to longitudinal studies of live disease models, including in zebrafish. Current limitations include the requirement of a minimum number of angular projections for reconstruction of reasonable OPT images using filtered back projection (FBP), which is typically several hundred, leading to acquisition times of several minutes. It is highly desirable to decrease the number of required angular projections to decrease both the total acquisition time and the light dose to the sample. This is particularly important to enable longitudinal studies, which involve measurements of the same fish at different time points. In this work, we demonstrate that the use of an iterative algorithm to reconstruct sparsely sampled OPT data sets can provide useful 3-D images with 50 or fewer projections, thereby significantly decreasing the minimum acquisition time and light dose while maintaining image quality. A transgenic zebrafish embryo with fluorescent labelling of the vasculature was imaged to acquire densely sampled (800 projections) and under-sampled data sets of transmitted and fluorescence projection images. The under-sampled OPT data sets were reconstructed using an iterative total variation-based image reconstruction algorithm and compared against FBP reconstructions of the densely sampled data sets. To illustrate the potential for quantitative analysis following rapid OPT data acquisition, a Hessian-based method was applied to automatically segment the reconstructed images to select the vasculature network. Results showed that 3-D images of the zebrafish embryo and its vasculature of sufficient visual quality for quantitative analysis can be reconstructed using the iterative algorithm from only 32 projections—achieving up to 28 times improvement in imaging speed and leading to total acquisition times of a few seconds.
McGinty J, Chen L, Kumar S, et al., 2015, Techniques to improve the spatial and temporal resolution in optical projection tomography: Remote focal scanning and time-lapse cell tracking
Optical projection tomography is a 3-D imaging approach applicable to transparent samples and model organisms like zebrafish embryos. We present methods to improve the spatial resolution and realize 3-D cell tracking in OPT.
Chen L, Alexandrov Y, Kumar S, et al., 2015, Mesoscopic <i>in vivo</i> 3-D tracking of sparse cell populations using angular multiplexed optical projection tomography, BIOMEDICAL OPTICS EXPRESS, Vol: 6, Pages: 1253-1261, ISSN: 2156-7085
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Mitchell RK, Mondragon A, Chen L, et al., 2015, Selective disruption of <i>Tcf7l2</i> in the pancreatic β cell impairs secretory function and lowers β cell mass, HUMAN MOLECULAR GENETICS, Vol: 24, Pages: 1390-1399, ISSN: 0964-6906
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Sayers S, Kantor C, Pullen TJ, et al., 2015, Preserved insulin secretion despite impaired glucose signalling after pancreatic beta cell selective deletion of the tumour suppressor LKB1, Publisher: WILEY-BLACKWELL, Pages: 13-13, ISSN: 0742-3071
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