Determinants of Oral Corticosteroid Responsiveness in Wheezing Asthmatic Youth (DOORWAY): Protocol for a prospective multicentre cohort study of children with acute moderate-to-severe asthma exacerbations

Introduction: Oral corticosteroids are the cornerstone of acute asthma management in the emergency department. Recent evidence has raised doubts about the efficacy of this treatment in preschool-aged children with viral-induced wheezing and in smoking adults. The aims of the study were to: (1) document the magnitude of response to oral corticosteroids in children presenting to the emergency department with moderate or severe asthma; (2) quantify potential determinants of response to corticosteroids and (3) explore the role of gene polymorphisms associated with the responsiveness to corticosteroids. Methods and analysis: The design is a prospective cohort study of 1008 children aged 1-17 years meeting a strict definition of asthma and presenting with a clinical score of ≥4 on the validated Pediatric Respiratory Assessment Measure. All children will receive standardised severity-specific treatment with prednisone/prednisolone and cointerventions (salbutamol with/without ipratropium bromide). Determinants, namely viral aetiology, environmental tobacco smoke and single nucleotide polymorphism, will be objectively documented. The primary efficacy endpoint is the failure of emergency department (ED) management within 72 h of the ED visit. Secondary endpoints include other measures of asthma severity and time to recovery within 7 days of the index visit. The study has 80% power for detecting a risk difference of 7.5% associated with each determinant from a baseline risk of 21%, at an α of 0.05. Ethics and dissemination: Ethical approval has been obtained from all participating institutions. An impaired response to systemic steroids in certain subgroups will challenge the current standard of practice and call for the immediate search for better approaches. A potential host-environment interaction will broaden our understanding of corticosteroid responsiveness in children. Documentation of similar effectiveness of corticosteroids across determinants will provide the needed reassurance regarding current treatment recommendations. Results: Results will be disseminated at international conferences and manuscripts targeted at emergency physicians, paediatricians, geneticists and respirologists. Trial registration number: This study is registered at Clinicaltrials.gov (NCT02013076)

Long-Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation.

Developmental cell biology requires technologies in which the fate of single cells is followed over extended time periods, to monitor and understand the processes of self-renewal, differentiation, and reprogramming. A workflow is presented, in which single cells are encapsulated into droplets (Ø: 80 µm, volume: ≈270 pL) and the droplet compartment is later converted to a hydrogel bead. After on-chip de-emulsification by electrocoalescence, these 3D scaffolds are subsequently arrayed on a chip for long-term perfusion culture to facilitate continuous cell imaging over 68 h. Here, the response of murine embryonic stem cells to different growth media, 2i and N2B27, is studied, showing that the exit from pluripotency can be monitored by fluorescence time-lapse microscopy, by immunostaining and by reverse-transcription and quantitative PCR (RT-qPCR). The defined 3D environment emulates the natural context of cell growth (e.g., in tissue) and enables the study of cell development in various matrices. The large scale of cell cultivation (in 2000 beads in parallel) may reveal infrequent events that remain undetected in lower throughput or ensemble studies. This platform will help to gain qualitative and quantitative mechanistic insight into the role of external factors on cell behavior.Wellcome Trust WT108438/C/15/

Stain-Free Quantification of Chromosomes in Live Cells Using Regularized Tomographic Phase Microscopy

Refractive index imaging is a label-free technique that enables long-term monitoring of the internal structures and molecular composition in living cells with minimal perturbation. Existing tomographic methods for the refractive index imaging lack 3-D resolution and result in artifacts that prevent accurate refractive index quantification. To overcome these limitations without compromising the capability to observe a sample in its most native condition, we have developed a regularized tomographic phase microscope (RTPM) enabling accurate refractive index imaging of organelles inside intact cells. With the enhanced accuracy, we quantify the mass of chromosomes in intact living cells, and differentiate two human colon cancer lines, HT-29 and T84 cells, solely based on the non-aqueous (dry) mass of chromosomes. In addition, we demonstrate chromosomal imaging using a dual-wavelength RTPM, which shows its potential to determine the molecular composition of cellular organelles in live cells.National Institute of Biomedical Imaging and Bioengineering (U.S.) (9P41EB015871-26A1

Actomyosin and vimentin cytoskeletal networks regulate nuclear shape, mechanics and chromatin organization

This work was supported in part by a Marie Curie CIG grant (PCIG14-GA-2013-631011 CSKFingerprints) and a BBSRC grant (BB/P006108/1). MCK is supported by a PhD studentship from the Life Sciences Initiative at QMUL

Simultaneous Analysis of Multiple Mycobacterium tuberculosis Knockdown Mutants In Vitro and In Vivo

Mycobacterium tuberculosis (Mtb) represents one of the most persistent bacterial threats to human health and new drugs are needed to limit its impact. Conditional knockdown mutants can help validate new drug targets, but the analysis of individual mutants is laborious and time consuming. Here, we describe quantitative DNA tags (qTags) and their use to simultaneously analyze conditional Mtb knockdown mutants that allowed silencing the glyoxylate and methylcitrate cycles (via depletion of isocitrate lyase, ICL), the serine protease Rv3671c, and the core subunits of the mycobacterial proteasome, PrcB and PrcA. The impact of gene silencing in multi-strain cultures was determined by measuring the relative abundance of mutant-specific qTags with real-time PCR. This achieved accurate quantification over a broad range of qTag abundances and depletion of ICL, Rv3671c, or PrcBA resulted in the expected impairment of growth of Mtb with butyrate as the primary carbon source, survival during oxidative stress, acid stress and starvation. The impact of depleting ICL, Rv3671c, or PrcBA in multi-strain mouse infections was analyzed with two approaches. We first measured the relative abundance of mutant-specific qTags in total chromosomal DNA isolated from bacteria that were recovered from infected lungs on agar plates. We then developed a two-step amplification procedure, which allowed us to measure the abundances of individual mutants directly in infected lung tissue. Both strategies confirmed that inactivation of Rv3671c and PrcBA severely reduced persistence of Mtb in mice. The multi-strain infections furthermore suggested that silencing ICL not only prevented growth of Mtb during acute infections but also prevented survival of Mtb during chronic infections. Analyses of the ICL knockdown mutant in single-strain infections confirmed this and demonstrated that silencing of ICL during chronic infections impaired persistence of Mtb to the extent that the pathogen was cleared from the lungs of most mice

Bone fragility and decline in stem cells in prematurely aging DNA repair deficient trichothiodystrophy mice

Trichothiodystrophy (TTD) is a rare, autosomal recessive nucleotide excision repair (NER) disorder caused by mutations in components of the dual functional NER/basal transcription factor TFIIH. TTD mice, carrying a patient-based point mutation in the Xpd gene, strikingly resemble many features of the human syndrome and exhibit signs of premature aging. To examine to which extent TTD mice resemble the normal process of aging, we thoroughly investigated the bone phenotype. Here, we show that female TTD mice exhibit accelerated bone aging from 39 weeks onwards as well as lack of periosteal apposition leading to reduced bone strength. Before 39 weeks have passed, bones of wild-type and TTD mice are identical excluding a developmental defect. Albeit that bone formation is decreased, osteoblasts in TTD mice retain bone-forming capacity as in vivo PTH treatment leads to increased cortical thickness. In vitro bone marrow cell cultures showed that TTD osteoprogenitors retain the capacity to differentiate into osteoblasts. However, after 13 weeks of age TTD females show decreased bone nodule formation. No increase in bone resorption or the number of osteoclasts was detected. In conclusion, TTD mice show premature bone aging, which is preceded by a decrease in mesenchymal stem cells/osteoprogenitors and a change in systemic factors, identifying DNA damage and repair as key determinants for bone fragility by influencing osteogenesis and bone metabolism

Apoptosis- and necrosis-induced changes in light attenuation measured by optical coherence tomography

Optical coherence tomography (OCT) was used to determine optical properties of pelleted human fibroblasts in which necrosis or apoptosis had been induced. We analysed the OCT data, including both the scattering properties of the medium and the axial point spread function of the OCT system. The optical attenuation coefficient in necrotic cells decreased from 2.2 ± 0.3 mm−1 to 1.3 ± 0.6 mm−1, whereas, in the apoptotic cells, an increase to 6.4 ± 1.7 mm−1 was observed. The results from cultured cells, as presented in this study, indicate the ability of OCT to detect and differentiate between viable, apoptotic, and necrotic cells, based on their attenuation coefficient. This functional supplement to high-resolution OCT imaging can be of great clinical benefit, enabling on-line monitoring of tissues, e.g. for feedback in cancer treatment

The Deflection of Charged Particles in an Electric Dodecapole Field

The properties of an ion-optical device consisting of twelve long parallel rods placed equidistantly on a circle are briefly reviewed. The use of this device in its dodecapole configuration to deflect ions in a plane perpendicular to the axis of the dodecapole is discussed. Results of trajectory computations made with the SIMION ion-optics computer program demonstrate that the device works remarkably well in separating the constituents of a multiply-charged ion beam. The use of this device to merge an ion beam with an optical beam is proposed