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

Geotechnical Appraisal of the Thar Open Cut Mining Project

This paper is concerned with a slope stability appraisal of the proposed open cut mining operations in the Thar lignite field in Sindh, Pakistan. The Thar coalfield covers an area of approximately 9 000 km2 and is estimated to contain 193 billion tonnes of lignite resources. The design of safe high wall slopes is necessary to ensure mine safety and overall economical viability of the mining operations. In the Thar lignite field, the presence of three main aquifers induces pore pressure in the rock mass surrounding the lignite seams and makes high wall slopes potentially unsafe. It is, therefore, necessary to dewater the rock mass before commencing mining excavations. A proposed mine dewatering scheme to facilitate rock mass dewatering surrounding the mining excavations and a description of the slope stability analysis of the high wall using the software “SLIDE” version 5 is outlined. Three computer models with slope angles of 28o, 29o and 30o, incorporating a plane failure mode, were analyzed to investigate the stability of pit slopes. The generalized stratigraphy of borehole RE-25 has been used for the development of the computer models. The main conclusions of this study are that the slope angle of 28o is quite acceptable for a Stability Factor (SF) ≤ 1.3 whereas the excavated slopes with slope angles ≥ 29o are not safe against the plane failure for SF\u3e1.3. This assessment was followed by a slope stability analysis incorporating circular failure modes. Five models incorporating various slope angles ranging from 23o to 27o and one model incorporating combined slope angles of 23o in dune sand and 26o in the rest of the strata were developed and analysed. The main conclusions from this study are that the dune sand layer (having a thickness of 48 m) is acceptable for a SF of 1.3 at slope angle ≤ 23o, while the rest of the strata is acceptable for SF=1.3 at slope angles ≤ 26o. The overburden to lignite extraction ratio for this slope design has been calculated as 3:1 or 3 m3 of overburden over 1 t of lignite

Effect of Ca2+ on the Steady-State and Time-Resolved Emission Properties of the Genetically Encoded Fluorescent Sensor CatchER

We previously designed a calcium sensor CatchER (a GFP-based Calcium sensor for detecting high concentrations in the high calcium concentration environment such as ER) with a capability for monitoring calcium ion responses in various types of cells. Calcium binding to CatchER induces the ratiometric changes in the absorption spectra, as well as an increase in fluorescence emission at 510 nm upon excitation at both 395 and 488 nm. Here, we have applied the combination of the steady-state and time-resolved optical methods and Hydrogen/Deuterium isotope exchange to understand the origin of such calcium-induced optical property changes of CatchER. We first demonstrated that calcium binding results in a 44% mean fluorescence lifetime increase of the indirectly excited anionic chromophore. Thus, CatchER is the first protein-based calcium indicator with the single fluorescent moiety to show the direct correlation between the lifetime and calcium binding. Calcium exhibits a strong inhibition on the excited-state proton transfer nonadiabatic geminate recombination in protic (vs deuteric) medium. Analysis of CatchER crystal structures and the MD simulations reveal the proton transfer mechanism in which the disrupted proton migration path in CatchER is rescued by calcium binding. Our finding provides important insights for a strategy to design calcium sensors and suggests that CatchER could be a useful probe for FLIM imaging of calcium in situ

Optical photometric GTC/OSIRIS observations of the young massive association Cygnus OB2

In order to fully understand the gravitational collapse of molecular clouds, the star formation process and the evolution of circumstellar disks, these phenomena must be studied in different Galactic environments with a range of stellar contents and positions in the Galaxy. The young massive association Cygnus OB2, in the Cygnus-X region, is an unique target to study how star formation and the evolution of circumstellar disks proceed in the presence of a large number of massive stars. We present a catalog obtained with recent optical observations in r,i,z filters with OSIRIS, mounted on the $10.4\,m$ GTC telescope, which is the deepest optical catalog of Cyg OB2 to date. The catalog consist of 64157 sources down to M=0.15 solar masses at the adopted distance and age of Cyg OB2. A total of 38300 sources have good photometry in all three bands. We combined the optical catalog with existing X-ray data of this region, in order to define the cluster locus in the optical diagrams. The cluster locus in the r-i vs. i-z diagram is compatible with an extinction of the optically selected cluster members in the 2.64<AV<5.57 range. We derive an extinction map of the region, finding a median value of AV=4.33 in the center of the association, decreasing toward the north-west. In the color-magnitude diagrams, the shape of the distribution of main sequence stars is compatible with the presence of an obscuring cloud in the foreground at about 850+/-25 pc from the Sun.Comment: Accepted for publication ApJS 201

Observation of interference between two distinct autoionizing states in dissociative photoionization of H 2

Dissociative photoionization (DPI) of randomly oriented H2 molecules has been studied using linearly polarized synchrotron radiation at selected photon energies of 31, 33, and 35 eV. Large amplitude oscillations in the photoelectron asymmetry parameter β, as a function of electron energy, have been observed. The phase of these β oscillations are in excellent agreement with the results of recent close coupling calculations [Fernández and Martín, New J. Phys. 11, 043020 (2009)]. We show that the oscillations are the signature of interferences between the 1Q1 Σu+1 and 1Q2 Πu1 doubly excited states decaying at different internuclear distances. The oscillations thus provide information about the classical paths followed by the nuclei. The presence of such oscillations is predicted to be a general phenomenon in DPIWe acknowledge NSERC, CLS, and UoW for their support. We thank Mare Nostrum BSC and CCC-UAM for allocation of computer time. Work partially supported by the MICINN Projects No. FIS2010-15127, No. ACI2008-0777, and No. CSD 2007-00010, the European MC-ITN CORINF, and the COST Action CM070

The Massive Star Forming Region Cygnus OB2. I. Chandra catalogue of association members

We present a catalogue of 1696 X-ray sources detected in the massive star forming region (SFR) Cygnus OB2 and extracted from two archival Chandra observations of the center of the region. A deep source extraction routine, exploiting the low background rates of Chandra observations was employed to maximize the number of sources extracted. Observations at other wavelengths were used to identify low count-rate sources and remove likely spurious sources. Monte Carlo simulations were also used to assess the authenticity of these sources. We used a Bayesian technique to identify optical or near-IR counterparts for 1501 (89%) of our sources, using deep observations from the INT Photometric H-alpha Survey, the Two Micron All Sky Survey, and the UKIRT Infrared Deep Sky Survey-Galactic plane Survey. 755 (45%) of these objects have six-band r', H-alpha, i', J, H, and K optical and near-IR photometry. From an analysis of the Poisson false-source probabilities for each source we estimate that our X-ray catalogue includes < 1% of false sources, and an even lower fraction when only sources with optical or near-IR associations are considered. A Monte Carlo simulation of the Bayesian matching scheme allows this method to be compared to more simplified matching techniques and enables the various sources of error to be quantified. The catalogue of 1696 objects presented here includes X-ray broad band fluxes, model fits, and optical and near-IR photometry in what is one of the largest X-ray catalogue of a single SFR to date. The high number of stellar X-ray sources detected from relatively shallow observations confirms the status and importance of Cygnus OB2 as one of our Galaxy's most massive SFRs.Comment: Accepted for publication in ApJS. 39 pages, 5 figures, 5 tables (full tables available in the published version or on request to the author

OB Stars & Stellar Bowshocks in Cygnus-X: A Novel Laboratory Estimating Stellar Mass Loss Rates

We use mid-IR images from the Spitzer Cygnus~X Legacy Survey to search for stellar bowshocks, a signature of early type "runaway" stars with high space velocities. We identify ten arc-shaped nebulae containing centrally located stars as candidate bowshocks. New spectroscopic observations of five stars show that all are late O to early B dwarfs. Our morphologically selected sample of bowshock candidates encompasses diverse physical phenomena. Three of the stars appear to be pre-main-sequence objects on the basis of rising SEDs in the mid-IR, and their nebulae may be photon-dominated regions (PDRs). Four objects have ambiguous classification. These may be partial dust shells or bubbles. We conclude that three of the objects are probable bowshocks, based on their morphological similarity to analytic prescriptions. Their nebular morphologies reveal no systematic pattern of orientations that might indicate either a population of stars ejected from or large-scale hydrodynamic outflows from Cyg OB2. The fraction of runaways among OB stars near Cyg OB2 identified either by radial velocity or bowshock techniques is ~0.5%, much smaller than the 8% estimated among field OB stars. We also obtained a heliocentric radial velocity for the previously known bowshock star, BD+43\degr3654, of -66.2+/-9.4 km/s, solidifying its runaway status and implying a space velocity of 77+/-10 km/s. We use the principles of momentum-driven bowshocks to arrive at a novel method for estimating stellar mass loss rates. Derived mass loss rates range between 10^-7 and few x10^-6 solar masses/yr for the three O5V -- ~B2V stars identified as generating bowshocks. These values are at the upper range of, but broadly consistent with, estimates from other methods. (Abridged)Comment: 49 pages, 19 figures; Accepted for publication in ApJ; full-resolution color figure version available at http://physics.uwyo.edu/~chip/Papers/CygXBowshocks; comments invite

An R- and I-Band Photometric Variability Survey of the Cygnus OB2 Association

We present a catalog of photometrically variable stars discovered within two 21'.3 X 21'.3 fields centered on the Cygnus OB2 association. There have hitherto been no deep optical variability studies of Cyg OB2 despite it being replete with early-type massive stars, perhaps due to the high and variable extinction (up to A_V ~ 20) that permeates much of the region. Here we provide results of the first variability study with this combination of spatial coverage (~ 0.5 deg) and photometric depth (R ~ 21 mag). We find 121 stars to be variable in both R- and I-band, 116 of them newly discovered. Of the 121 variables, we identify 27 eclipsing binaries (EBs) and eclipsing binary candidates, 20 potential Herbig Ae/Be stars, and 52 pulsating variables. Confirming both the status and the cluster membership of the Herbig Ae/Be stars would address the uncertainty regarding the age and star formation history of Cyg OB2. We match our catalog to known variables and binaries in the region, 2MASS near-IR (NIR) data, and Chandra X-ray observations to find counterparts to new variables in other wavelengths.Comment: 34 pages, 12 figures, submitted to Ap

Sex‐specific alterations in whole body energetics and voluntary activity in heterozygous R163C malignant hyperthermia‐susceptible mice

Malignant hyperthermia (MH) is characterized by induction of skeletal muscle hyperthermia in response to a dysregulated increase in myoplasmic calcium. Although altered energetics play a central role in MH, MH‐susceptible humans and mouse models are often described as having no phenotype until exposure to a triggering agent. The purpose of this study was to determine the influence of the R163C ryanodine receptor 1 mutation, a common MH mutation in humans, on energy expenditure, and voluntary wheel running in mice. Energy expenditure was measured by indirect respiration calorimetry in wild‐type (WT) and heterozygous R163C (HET) mice over a range of ambient temperatures. Energy expenditure adjusted for body weight or lean mass was increased (P < .05) in male, but not female, HET mice housed at 22°C or when housed at 28°C with a running wheel. In female mice, voluntary wheel running was decreased (P < .05) in the HET vs WT animals when analyzed across ambient temperatures. The thermoneutral zone was also widened in both male and female HET mice. The results of the study show that the R163C mutations alters energetics even at temperatures that do not typically induce MH