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Journal articleCowman SA, James P, Wilson R, et al., 2018,
Profiling mycobacterial communities in pulmonary nontuberculous mycobacterial disease
, PLoS ONE, Vol: 13, ISSN: 1932-6203The diagnosis of pulmonary non-tuberculous mycobacterial disease (pNTM) is dependent on the isolation of NTM in culture, which is prone to overgrowth and contamination and may not capture the diversity of mycobacteria present, including rare or unidentified species. This study aimed to develop a culture independent method of detecting and identifying mycobacteria from sputum samples using partial sequencing of the hsp65 gene. DNA was extracted from sputum samples from subjects with pNTM and disease controls. Multiplexed partial sequencing of the hsp65 gene was performed using the Illumina MiSeq and custom primers. A reference database of hsp65 sequences was created for taxonomy assignment. Sequencing results were obtained from 42 subjects (31 cases, 11 controls). Mycobacterial sequences were identified in all subjects. In 90.5% of samples more than one species was found (median 5.5). The species isolated in culture was detected by sequencing in 81% of subjects and was the most abundant species in 62%. The sequencing of NTM from clinical samples reveals a far greater diversity than conventional culture and suggests NTM are present as communities rather than a single species. NTM were found to be present even in the absence of isolation in culture or clinical disease.
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Journal articleMiura M, Miyazato P, Satou Y, et al., 2018,
Epigenetic changes around the pX region and spontaneous HTLV-1 transcription are CTCF-independent [version 2; approved 2]
, Wellcome Open Research, Vol: 3, ISSN: 2398-502XBackground: The human retrovirus HTLV-1 inserts the viral complementary DNA of 9 kb into the host genome. Both plus- and minus-strands of the provirus are transcribed, respectively from the 5' and 3' long terminal repeats (LTR). Plus-strand expression is rapid and intense once activated, whereas the minus-strand is transcribed at a lower, more constant level. To identify how HTLV-1 transcription is regulated, we investigated the epigenetic modifications associated with the onset of spontaneous plus-strand expression and the potential impact of the host factor CTCF. Methods: Patient-derived peripheral blood mononuclear cells (PBMCs) and in vitro HTLV-1-infected T cell clones were examined. Cells were stained for the plus-strand-encoded viral protein Tax, and sorted into Tax + and Tax - populations. Chromatin immunoprecipitation and methylated DNA immunoprecipitation were performed to identify epigenetic modifications in the provirus. Bisulfite-treated DNA fragments from the HTLV-1 LTRs were sequenced. Single-molecule RNA-FISH was performed, targeting HTLV-1 transcripts, for the estimation of transcription kinetics. The CRISPR/Cas9 technique was applied to alter the CTCF-binding site in the provirus, to test the impact of CTCF on the epigenetic modifications. Results: Changes in the histone modifications H3K4me3, H3K9Ac and H3K27Ac were strongly correlated with plus-strand expression. DNA in the body of the provirus was largely methylated except for the pX and 3' LTR regions, regardless of Tax expression. The plus-strand promoter was hypomethylated when Tax was expressed. Removal of CTCF had no discernible impact on the viral transcription or epigenetic modifications. Conclusions: The histone modifications H3K4me3, H3K9Ac and H3K27Ac are highly dynamic in the HTLV-1 provirus: they show rapid change with the onset of Tax expression, and are reversible. The HTLV-1 provirus has an intrinsic pattern of epigenetic modifications that is independent of both the provirus inse
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Conference paperDavenport AP, Brame AL, Kuc RE, et al., 2018,
First In human study of a novel biased apelin receptor ligand, MM54, a G-alpha(i) agonist/beta-arrestin antagonist
, Scientific Sessions of the American-Heart-Association (AHA), Publisher: American Heart Association, Pages: E75-E76, ISSN: 0009-7330Introduction: The peptide apelin acts via G proteins to cause beneficial vasodilation and potent positive inotropy to ameliorate pulmonary arterial hypertension in humans and animal models. Apelin is internalised via β-arrestin. In contrast, with loss of endogenous apelin, its receptor acts as a mechanosensor, stimulating β-arrestin to induce detrimental cardiac hypertrophy. Our aim was to characterise the action of our apelin ligand, MM54 that in cell based assays blocks β-arrestin but activates the Gαi protein pathway, in this first in human study. Method: Competition binding in human heart (n=3) used [I125] [Pyr1]apelin-13 (0.1nmol/L). β-arrestin recruitment, receptor internalization and forskolin-induced cAMP inhibition were measured in CHO-K1 cells expressing human apelin receptor. Forearm blood flow was measured in 9 volunteers using venous occlusion plethysmography at baseline and at 4 incremental doses (1, 10, 30, 100 nmol/min) of MM54, each for eight minutes. The Aellig hand vein technique was used to measure the effect of 3 incremental doses (3, 30, 300 nmol/min) of MM54 for 15 min on veins pre-constricted with noradrenaline in 6 individuals compared with 8 controls. Data are mean+SEM, n≥3. Results: MM54 had an affinity of pKi = 6.50±0.03. In β-arrestin (pKB 6.93±0.15) and receptor internalization assays (pKB 5.89±0.06) MM54 was an antagonist, but activated the G protein pathway (pD2±SEM 5.86+0.23). At the highest concentration (100 nmol/min), MM54 caused a significant absolute increase in forearm blood flow compared to control arm, representing a 76 % change from baseline (P<0.01, ANOVA with repeated measures with Dunnett’s post hoc analysis on untransformed data). In the hand vein, MM54 caused a significant concentration dependent dilatation in veins over the concentration range tested, with the highest dose causing 57% reversal (P<0.01). Conclusion: At the cellular level, the resu
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Conference paperLim W-W, Corden B, Sivakumar V, et al., 2018,
Therapeutic Targeting of Interleukin-11 with Neutralizing Antibodies Prevents Cardiac Fibrosis
, Scientific Sessions of the American-Heart-Association (AHA), Publisher: LIPPINCOTT WILLIAMS & WILKINS, Pages: E71-E72, ISSN: 0009-7330 -
Journal articleAltmann DM, 2018,
T-cell immunology of the lung: maintaining the balance between host defence and immune pathology
, IMMUNOLOGY, Vol: 156, Pages: 1-2, ISSN: 0019-2805 -
Working paperMacintyre G, Piskorz A, Ross E, et al., 2018,
frenchFISH: Poisson models for quantifying DNA copy-number from fluorescence in situ hybridisation of tissue sections
Chromosomal aberration and DNA copy number change are robust hallmarks of cancer. Imaging of spots generated using fluorescence in situ hybridisation (FISH) of locus specific probes is routinely used to detect copy number changes in tumour nuclei. However, it often does not perform well on solid tumour tissue sections, where partially represented or overlapping nuclei are common. To overcome these challenges, we have developed a computational approach called FrenchFISH, which comprises a nuclear volume correction method coupled with two types of Poisson models: either a Poisson model for improved manual spot counting without the need for control probes; or a homogenous Poisson Point Process model for automated spot counting. We benchmarked the performance of FrenchFISH against previous approaches in a controlled simulation scenario and exemplify its use in 12 ovarian cancer FFPE-tissue sections, for which we assess copy number alterations in three loci (c-Myc, hTERC and SE7). We show that FrenchFISH outperforms standard spot counting approaches and that the automated spot counting is significantly faster than manual without loss of performance. FrenchFISH is a general approach that can be used to enhance clinical diagnosis on sections of any tissue.
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Journal articleSanchez-Garrdio J, Sancho-Shimizu V, Shenoy A, 2018,
Regulated proteolysis of p62/SQSTM1 enables differential control of autophagy and nutrient sensing
, Science Signaling, Vol: 11, ISSN: 1937-9145The multidomain scaffold protein p62 (also called sequestosome-1) is involved in autophagy, antimicrobial immunity, and oncogenesis. Mutations in SQSTM1, which encodes p62, are linked to hereditary inflammatory conditions such as Paget’s disease of the bone, frontotemporal dementia (FTD), amyotrophic lateral sclerosis, and distal myopathy with rimmed vacuoles. Here, we report that p62 was proteolytically trimmed by the protease caspase-8 into a stable protein, which we called p62TRM. We found that p62TRM, but not full-length p62, was involved in nutrient sensing and homeostasis through the mechanistic target of rapamycin complex 1 (mTORC1). The kinase RIPK1 and caspase-8 controlled p62TRM production and thus promoted mTORC1 signaling. An FTD-linked p62 D329G polymorphism and a rare D329H variant could not be proteolyzed by caspase-8, and these noncleavable variants failed to activate mTORC1, thereby revealing the detrimental effect of these mutations. These findings on the role of p62TRM provide new insights into SQSTM1-linked diseases and mTORC1 signaling.
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Journal articleLoh AYY, Burgess CH, Tanase DA, et al., 2018,
Electric single-molecule hybridization detector for short DNA fragments
, Analytical Chemistry, Vol: 90, Pages: 14063-14071, ISSN: 0003-2700By combining DNA nanotechnology and high-bandwidth single-molecule detection in nanopipets, we demonstrate an electric, label-free hybridization sensor for short DNA sequences (<100 nucleotides). Such short fragments are known to occur as circulating cell-free DNA in various bodily fluids, such as blood plasma and saliva, and have been identified as disease markers for cancer and infectious diseases. To this end, we use as a model system an 88-mer target from the RV1910c gene in Mycobacterium tuberculosis, which is associated with antibiotic (isoniazid) resistance in TB. Upon binding to short probes attached to long carrier DNA, we show that resistive-pulse sensing in nanopipets is capable of identifying rather subtle structural differences, such as the hybridization state of the probes, in a statistically robust manner. With significant potential toward multiplexing and high-throughput analysis, our study points toward a new, single-molecule DNA-assay technology that is fast, easy to use, and compatible with point-of-care environments.
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Journal articleTrantidou T, Friddin M, Gan KB, et al., 2018,
Mask-free laser lithography for rapid and low-cost microfluidic device fabrication
, Analytical Chemistry, Vol: 90, Pages: 13915-13921, ISSN: 0003-2700Microfluidics has become recognized as a powerful platform technology associated with a constantly increasing array of applications across the life sciences. This surge of interest over recent years has led to an increased demand for microfluidic chips, resulting in more time being spent in the cleanroom fabricating devices using soft lithography—a slow and expensive process that requires extensive materials, training and significant engineering resources. This bottleneck limits platform complexity as a byproduct of lengthy delays between device iterations and affects the time spent developing the final application. To address this problem, we report a new, rapid, and economical approach to microfluidic device fabrication using dry resist films to laminate laser cut sheets of acrylic. We term our method laser lithography and show that our technique can be used to engineer 200 μm width channels for assembling droplet generators capable of generating monodisperse water droplets in oil and micromixers designed to sustain chemical reactions. Our devices offer high transparency, negligible device to device variation, and low X-ray background scattering, demonstrating their suitability for real-time X-ray-based characterization applications. Our approach also requires minimal materials and apparatus, is cleanroom free, and at a cost of around $1.00 per chip could significantly democratize device fabrication, thereby increasing the interdisciplinary accessibility of microfluidics.
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Journal articleScalfari A, Muraro PA, 2018,
Monoclonal antibody therapy and long-term outcomes in multiple sclerosis - The challenge of treatment optimisation
, European Neurological Review, Vol: 13, Pages: 78-85, ISSN: 1758-3837The therapeutic landscape of multiple sclerosis (MS) has been transformed by the advent of several new monoclonal antibody (MAb) therapies that can potentially lead to full stabilisation of detectable disease activity. Natalizumab, alemtuzumab and ocrelizumab are currently licensed MAbs for the treatment of MS. Daclizumab was licensed for the treatment of MS, although it has been recently withdrawn from the market by the manufacturer. Most patients are initially managed with first-line treatments, and, if disease breakthrough occurs, are escalated to a stronger compound, yet the available evidence indicates an early window of therapeutic opportunity for MAbs to exert most of their efficacy. It is important to balance the superior efficacy of MAbs compared with injectable treatments against more serious side effects, although these are well recognised and can be monitored where indicated and treated. In particular, the risk of progressive multifocal leucoencephalopathy with natalizumab can be managed by screening potential patients for the John Cunningham virus. The MAbs also have the benefit of convenience to patients compared with daily or weekly treatments since they are given via less frequent administration. The cost of these treatments, compared with other therapies, may be an important issue in many countries where healthcare budgets are under pressure. The complex decision of choosing the best treatment for an individual should be made jointly between the doctor and the patient after careful consideration of the many factors to be weighed.
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