Exponential gradient maker using a disposable syringe

With a simple modification, any disposable syringe can become a reliable and easy to use exponential gradient maker. The modification consists of two notches, made with a razor blade, in the borders of the rubber sealing tip of the plunger. A clamp in the tube connected to the syringe allows control over solution flow. With the clamp prohibiting drainage, the body of the syringe is filled with the desired volume of starting solution I. A magnetic stir bar, small enough to spin inside the syringe is included. The notched plunger is introduced until no air space remains. This forms the fixed volume, closed mixing chamber, while the rest of the volume of the syringe forms the open chamber. The two chambers are connected through the notches in the plunger. The ending solution II is poured after the introduction of the plunger. Opening the clamp allows solution I in the closed chamber to flow out, and the solution II in the open chamber flows through the notches and mixes with solution I. This exponential gradient maker can be reused many times, but the low cost of the components makes it potentially disposable. This feature is especially useful when using toxic chemicals, or when pouring polyacrylamide gradient gels, since the apparatus may be disposed of after contamination or eventual polymerization.     

Design and calibration of unicapillary pneumotachographs

Giannella-Neto, A., C. Bellido, R. B. Barbosa, and M. F. Vidal Melo. Design and calibration of unicapillarypneumotachographs. J. Appl. Physiol.84(1): 335-343, 1998.This study presents a method for design andcalibration of unicapillary pneumotachographs for small-animalexperiments. The design, based on Poiseuille's law, defines a set ofinternal radius and length values that allows for laminar flow,measurable pressure differences, and minimal interference withanimal's respiratory mechanics and gas exchange. A third-orderpolynomial calibration (Pol) of the pressure-flow relationship wasemployed and compared with linear calibration (Lin). Tests were donefor conditions of ambient pressure (Pam) and positive pressure (Ppos)ventilation at different flow ranges. A physical model designed tomatch normal and low compliance in rats was used. At normal compliance,Pol provided lower errors than Lin for mixed (1-12 ml/s), mean(4-10 ml/s), and high (8-12 ml/s) flow rate calibrations forboth Pam and Ppos inspiratory tests (P < 0.001 for all conditions)and expiratory tests (P < 0.001 forall conditions). At low compliance, they differed significantly with8.6 ± 4.1% underestimation when Lin at Pam was used in Ppos tests.Ppos calibration, preferably in combination with Pol, should be used inthis case to minimize errors (Pol = 0.8 ± 0.5%, Lin = 6.5 ± 4.0%, P < 0.0005). Nonlinearcalibration may be useful for improvement of flow and volumemeasurements in small animals during both Pam and Ppos ventilation.

     

Optimized 3D coordinate reconstruction from paired stereographs using a calibrated phantom

A refinement to a previously described three-dimensional reconstruction algorithm based on point identification in calibrated non-orthogonal radiograms (stereo-pairs) is described. The modification involves a computation of the focal point magnitude of the point in three dimensions, analogous to focusing in two dimensions, as well as the most likely location of the target point in 3-space; the focal point magnitude may be thought of as the precision of the point identification. Multiple observer studies of the same stereopair can be used to estimate three-dimensional reconstruction accuracy by providing an average location and a mean distance from average. Both measures are useful parameters for initial selection of bone landmark references and for error propagation studies.     

Cyclamen: time, sea and speciation biogeography using a temporally calibrated phylogeny

Aim The Mediterranean region is a species‐rich area with a complex geographical history. Geographical barriers have been removed and restored due to sea level changes and local climatic change. Such barriers have been proposed as a plausible mechanism driving the high levels of speciation and endemism in the Mediterranean basin. This raises the fundamental question: is allopatric isolation the mechanism by which speciation occurs? This study explores the potential driving influence of palaeo‐geographical events on the speciation of Cyclamen (Myrsinaceae), a group with most species endemic to the Mediterranean region. Cyclamen species have been shown experimentally to have few genetic barriers to hybridization. Location The Mediterranean region, including northern Africa, extending eastwards to the Black Sea coast. Methods A generic level molecular phylogeny of Myrsinaceae and Primulaceae is constructed, using Bayesian approximation, to produce a secondary age estimate for the stem lineage of Cyclamen. This estimate is used to calibrate temporally an infrageneric phylogeny of Cyclamen, built with nrDNA ITS, cpDNA trnL‐F and cpDNA rps16 sequences. A biogeographical analysis of Cyclamen is performed using dispersal–vicariance analysis. Results The emergence of the Cyclamen stem lineage is estimated at 30.1–29.2 Ma, and the crown divergence at 12.9–12.2 Ma. The average age of Cyclamen species is 3.7 Myr. Every pair of sister species have mutually exclusive, allopatric distributions relative to each other. This pattern appears typical of divergence events throughout the evolutionary history of the genus. Main conclusions Geographical barriers, such as the varying levels of the Mediterranean Sea, are the most plausible explanation for speciation events throughout the phylogenetic history of Cyclamen. The genus demonstrates distributional patterns congruent with the temporally reticulate palaeogeography of the Mediterranean region.     

Shunt effect of gas compression inside pneumotachographs during forced oscillations

Determination of the frequency response of pneumotachographs is needed whenever they are used to measure high-frequency flows, such as in the forced oscillation method. When screen and capillary pneumotachographs are calibrated using an adiabatic compression in a closed box as a reference impedance, they can be adequately described by a series of inertial-resistive elements. However, this type of reference impedance strongly differs from the actual respiratory impedance (ZL). We studied the frequency response of pneumotachographs up to 250 Hz in reference to the impedance of a compressible gas oscillating in a long tube, taken as a more generalizable model of actual ZL. We found that, with this device, the series resistance-inertance models fail to describe the frequency response of the pneumotachograph. However, when compressible effects in the pneumotachograph are taken into account by adding to the resistive models a compliance (Cpn) corresponding to the compression in half of the inner volume of the pneumotachograph, the agreement with experiments becomes satisfactory. Gas compression-related phenomena were demonstrated to be negligible only when the parameter omega Cpn magnitude of ZL is much smaller than 1 (omega pulsation). Results obtained in normal humans have shown that such a correction is required above 100 Hz. Similar correction at lower frequency might also be necessary in cases of large respiratory impedance (e.g., babies, subjects with pathological lungs, and intubated subjects).     

Properties of cell death models calibrated and compared using Bayesian approaches

Using models to simulate and analyze biological networks requires principled approaches to parameter estimation and model discrimination. We use Bayesian and Monte Carlo methods to recover the full probability distributions of free parameters (initial protein concentrations and rate constants) for mass‐action models of receptor‐mediated cell death. The width of the individual parameter distributions is largely determined by non‐identifiability but covariation among parameters, even those that are poorly determined, encodes essential information. Knowledge of joint parameter distributions makes it possible to compute the uncertainty of model‐based predictions whereas ignoring it (e.g., by treating parameters as a simple list of values and variances) yields nonsensical predictions. Computing the Bayes factor from joint distributions yields the odds ratio (～20‐fold) for competing ‘direct’ and ‘indirect’ apoptosis models having different numbers of parameters. Our results illustrate how Bayesian approaches to model calibration and discrimination combined with single‐cell data represent a generally useful and rigorous approach to discriminate between competing hypotheses in the face of parametric and topological uncertainty.     

Calibration of stopped-flow spectrophotometers using a two-step disulfide exchange reaction

A coupled two-step reaction of Ellman's reagent (5,5′-dithiobis(2-nitrobenzoic acid)) with excess thioglycerol produces a progress curve composed of two superimposed exponentials. The ratio of the two pseudo-first-order rate constants equals 22.5 and does not vary appreciably with ehanges of either pH value or temperature. Because the ratio of the two amplitudes is defined by the ratio of the rate constants, the reaction can be used to estimate both the apparent zero time of mixing (or the dead time) and the detector linearity of a stopped-flow instrument with a single mixing experiment. The reaction is used as a standard of performance for both absorbance and fluorescence measurements with a stopped-flow spectrophotometer.     

Slow viscous flow in a syringe

The slow viscous flow in a syringe is modeled by the quasi-steady axisymmetric Stokes equation with a point sink for the needle hole. The governing equations are approximated using nonstandard finite difference formulas optimized for the boundary conditions, and solved numerically using a SOR technique. Streamlines and pressure profiles are computed for a variety of syringe configurations.