Accuracy and precision in quantitative fluorescence microscopy

The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Förster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements

Burden of disease from inadequate water, sanitation and hygiene for selected adverse health outcomes: An updated analysis with a focus on low- and middle-income countries

Background To develop updated estimates in response to new exposure and exposure-response data of the burden of diarrhoea, respiratory infections, malnutrition, schistosomiasis, malaria, soil-transmitted helminth infections and trachoma from exposure to inadequate drinking-water, sanitation and hygiene behaviours (WASH) with a focus on low- and middle-income countries. Methods For each of the analysed diseases, exposure levels with both sufficient global exposure data for 2016 and a matching exposure-response relationship were combined into population-attributable fractions. Attributable deaths and disability-adjusted life years (DALYs) were estimated for each disease and, for most of the diseases, by country, age and sex group separately for inadequate water, sanitation and hygiene behaviours and for the cluster of risk factors. Uncertainty estimates were computed on the basis of uncertainty surrounding exposure estimates and relative risks. Findings An estimated 829,000 WASH-attributable deaths and 49.8 million DALYs occurred from diarrhoeal diseases in 2016, equivalent to 60% of all diarrhoeal deaths. In children under 5 years, 297,000 WASH-attributable diarrhoea deaths occurred, representing 5.3% of all deaths in this age group. If the global disease burden from different diseases and several counterfactual exposure distributions was combined it would amount to 1.6 million deaths, representing 2.8% of all deaths, and 104.6 million DALYs in 2016. Conclusions Despite recent declines in attributable mortality, inadequate WASH remains an important determinant of global disease burden, especially among young children. These estimates contribute to global monitoring such as for the Sustainable Development Goal indicator on mortality from inadequate WASH

Gravitational Waves From Known Pulsars: Results From The Initial Detector Era

We present the results of searches for gravitational waves from a large selection of pulsars using data from the most recent science runs (S6, VSR2 and VSR4) of the initial generation of interferometric gravitational wave detectors LIGO (Laser Interferometric Gravitational-wave Observatory) and Virgo. We do not see evidence for gravitational wave emission from any of the targeted sources but produce upper limits on the emission amplitude. We highlight the results from seven young pulsars with large spin-down luminosities. We reach within a factor of five of the canonical spin-down limit for all seven of these, whilst for the Crab and Vela pulsars we further surpass their spin-down limits. We present new or updated limits for 172 other pulsars (including both young and millisecond pulsars). Now that the detectors are undergoing major upgrades, and, for completeness, we bring together all of the most up-to-date results from all pulsars searched for during the operations of the first-generation LIGO, Virgo and GEO600 detectors. This gives a total of 195 pulsars including the most recent results described in this paper.United States National Science FoundationScience and Technology Facilities Council of the United KingdomMax-Planck-SocietyState of Niedersachsen/GermanyAustralian Research CouncilInternational Science Linkages program of the Commonwealth of AustraliaCouncil of Scientific and Industrial Research of IndiaIstituto Nazionale di Fisica Nucleare of ItalySpanish Ministerio de Economia y CompetitividadConselleria d'Economia Hisenda i Innovacio of the Govern de les Illes BalearsNetherlands Organisation for Scientific ResearchPolish Ministry of Science and Higher EducationFOCUS Programme of Foundation for Polish ScienceRoyal SocietyScottish Funding CouncilScottish Universities Physics AllianceNational Aeronautics and Space AdministrationOTKA of HungaryLyon Institute of Origins (LIO)National Research Foundation of KoreaIndustry CanadaProvince of Ontario through the Ministry of Economic Development and InnovationNational Science and Engineering Research Council CanadaCarnegie TrustLeverhulme TrustDavid and Lucile Packard FoundationResearch CorporationAlfred P. Sloan FoundationAstronom

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Sensitivity of the Advanced LIGO detectors at the beginning of gravitational wave astronomy

The Laser Interferometer Gravitational Wave Observatory (LIGO) consists of two widely separated 4 km laser interferometers designed to detect gravitational waves from distant astrophysical sources in the frequency range from 10 Hz to 10 kHz. The first observation run of the Advanced LIGO detectors started in September 2015 and ended in January 2016. A strain sensitivity of better than 10−23/Hz−−−√ was achieved around 100 Hz. Understanding both the fundamental and the technical noise sources was critical for increasing the astrophysical strain sensitivity. The average distance at which coalescing binary black hole systems with individual masses of 30  M⊙ could be detected above a signal-to-noise ratio (SNR) of 8 was 1.3 Gpc, and the range for binary neutron star inspirals was about 75 Mpc. With respect to the initial detectors, the observable volume of the Universe increased by a factor 69 and 43, respectively. These improvements helped Advanced LIGO to detect the gravitational wave signal from the binary black hole coalescence, known as GW150914

Search for post-merger gravitational waves from the remnant of the binary neutron star merger GW170817

In Advanced LIGO, detection and astrophysical source parameter estimation of the binary black hole merger GW150914 requires a calibrated estimate of the gravitational-wave strain sensed by the detectors. Producing an estimate from each detector's differential arm length control loop readout signals requires applying time domain filters, which are designed from a frequency domain model of the detector's gravitational-wave response. The gravitational-wave response model is determined by the detector's opto-mechanical response and the properties of its feedback control system. The measurements used to validate the model and characterize its uncertainty are derived primarily from a dedicated photon radiation pressure actuator, with cross-checks provided by optical and radio frequency references. We describe how the gravitational-wave readout signal is calibrated into equivalent gravitational-wave-induced strain and how the statistical uncertainties and systematic errors are assessed. Detector data collected over 38 calendar days, from September 12 to October 20, 2015, contain the event GW150914 and approximately 16 of coincident data used to estimate the event false alarm probability. The calibration uncertainty is less than 10% in magnitude and 10 degrees in phase across the relevant frequency band 20 Hz to 1 kHz