A Fluorescence in Situ Hybridization Method To Quantify mRNA Translation by Visualizing Ribosome–mRNA Interactions in Single Cells

Single-molecule fluorescence in situ hybridization (smFISH) is a simple and widely used method to measure mRNA transcript abundance and localization in single cells. A comparable single-molecule in situ method to measure mRNA translation would enable a more complete understanding of gene regulation. Here we describe a fluorescence assay to detect ribosome interactions with mRNA (FLARIM). The method adapts smFISH to visualize and characterize translation of single molecules of mRNA in fixed cells. To visualize ribosome–mRNA interactions, we use pairs of oligonucleotide probes that bind separately to ribosomes (via rRNA) and to the mRNA of interest, and that produce strong fluorescence signals via the hybridization chain reaction (HCR) when the probes are in close proximity. FLARIM does not require genetic manipulation, is applicable to practically any endogenous mRNA transcript, and provides both spatial and temporal information. We demonstrate that FLARIM is sensitive to changes in ribosome association with mRNA upon inhibition of global translation with puromycin. We also show that FLARIM detects changes in ribosome association with an mRNA whose translation is upregulated in response to increased concentrations of iron

A simple three-dimensional-focusing, continuous-flow mixer for the study of fast protein dynamics

We present a simple, yet flexible microfluidic mixer with a demonstrated mixing time as short as 80 μs that is widely accessible because it is made of commercially available parts. To simplify the study of fast protein dynamics, we have developed an inexpensive continuous-flow microfluidic mixer, requiring no specialized equipment or techniques. The mixer uses three-dimensional, hydrodynamic focusing of a protein sample stream by a surrounding sheath solution to achieve rapid diffusional mixing between the sample and sheath. Mixing initiates the reaction of interest. Reactions can be spatially observed by fluorescence or absorbance spectroscopy. We characterized the pixel-to-time calibration and diffusional mixing experimentally. We achieved a mixing time as short as 80 μs. We studied the kinetics of horse apomyoglobin (apoMb) unfolding from the intermediate (I) state to its completely unfolded (U) state, induced by a pH jump from the initial pH of 4.5 in the sample stream to a final pH of 2.0 in the sheath solution. The reaction time was probed using the fluorescence of 1-anilinonaphthalene-8-sulfonate (1,8-ANS) bound to the folded protein. We observed unfolding of apoMb within 760 μs, without populating additional intermediate states under these conditions. We also studied the reaction kinetics of the conversion of pyruvate to lactate catalyzed by lactate dehydrogenase using the intrinsic tryptophan emission of the enzyme. We observe sub-millisecond kinetics that we attribute to Michaelis complex formation and loop domain closure. These results demonstrate the utility of the three-dimensional focusing mixer for biophysical studies of protein dynamics

Cosmology and astrophysics from relaxed galaxy clusters - IV: Robustly calibrating hydrostatic masses with weak lensing

This is the fourth in a series of papers studying the astrophysics and cosmology of massive, dynamically relaxed galaxy clusters. Here, we use measurements of weak gravitational lensing from the Weighing the Giants project to calibrate Chandra X-ray measurements of total mass that rely on the assumption of hydrostatic equilibrium. This comparison of X-ray and lensing masses provides a measurement of the combined bias of X-ray hydrostatic masses due to both astrophysical and instrumental sources. Assuming a fixed cosmology, and within a characteristic radius (r_2500) determined from the X-ray data, we measure a lensing to X-ray mass ratio of 0.96 +/- 9% (stat) +/- 9% (sys). We find no significant trends of this ratio with mass, redshift or the morphological indicators used to select the sample. In accordance with predictions from hydro simulations for the most massive, relaxed clusters, our results disfavor strong, tens-of-percent departures from hydrostatic equilibrium at these radii. In addition, we find a mean concentration of the sample measured from lensing data of c_200 = $3.0_{-1.8}^{+4.4}$. Anticipated short-term improvements in lensing systematics, and a modest expansion of the relaxed lensing sample, can easily increase the measurement precision by 30--50%, leading to similar improvements in cosmological constraints that employ X-ray hydrostatic mass estimates, such as on Omega_m from the cluster gas mass fraction.Comment: 13 pages. Submitted to MNRAS. Comments welcom

Hubble Residuals of Nearby Type Ia Supernovae Are Correlated with Host Galaxy Masses

From Sloan Digital Sky Survey u'g'r'i'z' imaging, we estimate the stellar masses of the host galaxies of 70 low redshift SN Ia (0.015 < z < 0.08) from the hosts' absolute luminosities and mass-to-light ratios. These nearby SN were discovered largely by searches targeting luminous galaxies, and we find that their host galaxies are substantially more massive than the hosts of SN discovered by the flux-limited Supernova Legacy Survey. Testing four separate light curve fitters, we detect ~2.5{\sigma} correlations of Hubble residuals with both host galaxy size and stellar mass, such that SN Ia occurring in physically larger, more massive hosts are ~10% brighter after light curve correction. The Hubble residual is the deviation of the inferred distance modulus to the SN, calculated from its apparent luminosity and light curve properties, away from the expected value at the SN redshift. Marginalizing over linear trends in Hubble residuals with light curve parameters shows that the correlations cannot be attributed to a light curve-dependent calibration error. Combining 180 higher-redshift ESSENCE, SNLS, and HigherZ SN with 30 nearby SN whose host masses are less than 10^10.8 solar masses in a cosmology fit yields 1+w=0.22 +0.152/-0.143, while a combination where the 30 nearby SN instead have host masses greater than 10^10.8 solar masses yields 1+w=-0.03 +0.217/-0.108. Progenitor metallicity, stellar population age, and dust extinction correlate with galaxy mass and may be responsible for these systematic effects. Host galaxy measurements will yield improved distances to SN Ia.Comment: 16 pages, 6 figures, published in ApJ, minor change

The LSST DESC data challenge 1: Generation and analysis of synthetic images for next-generation surveys

Data Challenge 1 (DC1) is the first synthetic data set produced by the Rubin Observatory Legacy Survey of Space and Time (LSST) Dark Energy Science Collaboration (DESC). DC1 is designed to develop and validate data reduction and analysis and to study the impact of systematic effects that will affect the LSST data set. DC1 is comprised of r-band observations of 40 deg2 to 10 yr LSST depth. We present each stage of the simulation and analysis process: (a) generation, by synthesizing sources from cosmological N-body simulations in individual sensor-visit images with different observing conditions; (b) reduction using a development version of the LSST Science Pipelines; and (c) matching to the input cosmological catalogue for validation and testing. We verify that testable LSST requirements pass within the fidelity of DC1. We establish a selection procedure that produces a sufficiently clean extragalactic sample for clustering analyses and we discuss residual sample contamination, including contributions from inefficiency in star-galaxy separation and imperfect deblending. We compute the galaxy power spectrum on the simulated field and conclude that: (i) survey properties have an impact of 50 per cent of the statistical uncertainty for the scales and models used in DC1; (ii) a selection to eliminate artefacts in the catalogues is necessary to avoid biases in the measured clustering; and (iii) the presence of bright objects has a significant impact (2-6) in the estimated power spectra at small scales (&gt; 1200), highlighting the impact of blending in studies at small angular scales in LSST

Risk Factors for African Tick-Bite Fever in Rural Central Africa

African tick-bite fever is an emerging infectious disease caused by the spotted fever group Rickettsia, Rickettsia africae, and is transmitted by ticks of the genus Amblyomma. To determine the seroprevalence of exposure to R. africae and risk factors associated with infection, we conducted a cross-sectional study of persons in seven rural villages in distinct ecological habitats of Cameroon. We examined 903 plasma samples by using an indirect immunofluorescence assay for antibodies to R. africae and analyzed demographic and occupational data collected from questionnaires. Of the 903 persons tested, 243 (26.9%) had IgG/IgM/IgA reactive with R. africae. Persons from four of the seven village sites were significantly more likely to be seropositive (P < 0.05), and lowland forest sites tended to have higher seroprevalences. These results suggest that African tick-bite fever is common in adults in rural areas of Cameroon and that ecological factors may play a role in the acquisition of R. africae infection

Near-field optical power transmission of dipole nano-antennas

Nano-antennas in functional plasmonic applications require high near-field optical power transmission. In this study, a model is developed to compute the near-field optical power transmission in the vicinity of a nano-antenna. To increase the near-field optical power transmission from a nano-antenna, a tightly focused beam of light is utilized to illuminate a metallic nano-antenna. The modeling and simulation of these structures is performed using 3-D finite element method based full-wave solutions of Maxwell’s equations. Using the optical power transmission model, the interaction of a focused beam of light with plasmonic nanoantennas is investigated. In addition, the tightly focused beam of light is passed through a band-pass filter to identify the effect of various regions of the angular spectrum to the near-field radiation of a dipole nano-antenna. An extensive parametric study is performed to quantify the effects of various parameters on the transmission efficiency of dipole nano-antennas, including length, thickness, width, and the composition of the antenna, as well as the wavelength and half-beam angle of incident light. An optimal dipole nanoantenna geometry is identified based on the parameter studies in this work. In addition, the results of this study show the interaction of the optimized dipole nano-antenna with a magnetic recording medium when it is illuminated with a focused beam of light

Models and metaphors: complexity theory and through-life management in the built environment

Complexity thinking may have both modelling and metaphorical applications in the through-life management of the built environment. These two distinct approaches are examined and compared. In the first instance, some of the sources of complexity in the design, construction and maintenance of the built environment are identified. The metaphorical use of complexity in management thinking and its application in the built environment are briefly examined. This is followed by an exploration of modelling techniques relevant to built environment concerns. Non-linear and complex mathematical techniques such as fuzzy logic, cellular automata and attractors, may be applicable to their analysis. Existing software tools are identified and examples of successful built environment applications of complexity modelling are given. Some issues that arise include the definition of phenomena in a mathematically usable way, the functionality of available software and the possibility of going beyond representational modelling. Further questions arising from the application of complexity thinking are discussed, including the possibilities for confusion that arise from the use of metaphor. The metaphor of a 'commentary machine' is suggested as a possible way forward and it is suggested that an appropriate linguistic analysis can in certain situations reduce perceived complexity

Calculations of collisions between cold alkaline earth atoms in a weak laser field

We calculate the light-induced collisional loss of laser-cooled and trapped magnesium atoms for detunings up to 50 atomic linewidths to the red of the ^1S_0-^1P_1 cooling transition. We evaluate loss rate coefficients due to both radiative and nonradiative state-changing mechanisms for temperatures at and below the Doppler cooling temperature. We solve the Schrodinger equation with a complex potential to represent spontaneous decay, but also give analytic models for various limits. Vibrational structure due to molecular photoassociation is present in the trap loss spectrum. Relatively broad structure due to absorption to the Mg_2 ^1Sigma_u state occurs for detunings larger than about 10 atomic linewidths. Much sharper structure, especially evident at low temperature, occurs even at smaller detunings due to of Mg_2 ^1Pi_g absorption, which is weakly allowed due to relativistic retardation corrections to the forbidden dipole transition strength. We also perform model studies for the other alkaline earth species Ca, Sr, and Ba and for Yb, and find similar qualitative behavior as for Mg.Comment: 20 pages, RevTex, 13 eps figures embedde

Presence and Persistence of Ebola or Marburg Virus in Patients and Survivors: A Rapid Systematic Review

Background: The 2013-15 Ebola outbreak was unprecedented due to sustainedtransmission within urban environments and thousands of survivors. In 2014 the World Health Organization stated that there was insufficient evidence to give definitive guidance about which body fluids are infectious and when they pose a risk to humans. We report a rapid systematic review of published evidence on the presence of filoviruses in body fluids of infected people and survivors. Methods: Scientific articles were screened for information about filovirus in human body fluids. The aim was to find primary data that suggested high likelihood of actively infectious filovirus in human body fluids (viral RNA). Eligible infections were from Marburg virus (MARV or RAVV) and Zaire, Sudan, Taï Forest and Bundibugyo species of Ebola. [1] Cause of infection had to be laboratory confirmed (in practice either tissue culture or RT-PCR tests), or evidenced by compatible clinical history with subsequent positivity for filovirus antibodies or inflammatory factors. Data were extracted and summarized narratively. Results: 6831 unique articles were found, and after screening, 33 studies were eligible. For most body fluid types there were insufficient patients to draw strong conclusions, and prevalence of positivity was highly variable. Body fluids taken >16 days after onset were usually negative. In the six studies that used both assay methods RT-PCR tests for filovirus RNA gave positive results about 4 times more often than tissue culture. Conclusions: Filovirus was reported in most types of body fluid, but not in every sample from every otherwise confirmed patient. Apart from semen, most non-blood, RT-PCR positive samples are likely to be culture negative and so possibly of low infectious risk. Nevertheless, it is not apparent how relatively infectious many body fluids are during or after illness, even when culture-positive, not least because most test results come from more severe cases. Contact with blood and blood-stained body fluids remains the major risk for disease transmission because of the known high viral loads in blood