A simplified noninvasive method to measure airway blood flow in humans.

Our laboratory has previously developed and validated a noninvasive soluble gas uptake method to measure airway blood flow (Qaw) in humans (Onorato DJ, Demirozu MC, Breitenbücher A, Atkins ND, Chediak AD, and Wanner A. Am J Respir Crit Care Med 149: 1132-1137, 1994; Scuri M, McCaskill V, Chediak AD, Abraham WM, and Wanner A. J Appl Physiol 79: 1386-1390, 1995). The method has the disadvantage of requiring eight breath-hold maneuvers for a single Qaw measurement, a complicated data analysis, and the inhalation of a potentially explosive gas mixture containing dimethylether (DME) and O2. Because of these shortcomings, the method thus far has not been used in other laboratories. We now simplified the method by having the subjects inhale 500 ml of a 10% DME-90% N2 gas mixture to fill the anatomical dead space, followed by a 5- or 15-s breath hold, and measuring the instantaneous DME and N2 concentrations and volume at the airway opening during the subsequent exhalation. From the difference in DME concentration in phase 1 of the expired N2 wash-in curve multiplied by the phase 1 dead space volume and divided by the mean DME concentration and the solubility coefficient for DME in tissue, Qaw can be calculated by using Fick's equation. We compared the new method to the validated old method in 10 healthy subjects and found mean +/- SE Qaw values of 34.6 +/- 2.3 and 34.6 +/- 2.8 microl.min(-1).ml(-1), respectively (r = 0.93; upper and lower 95% confidence limit +2.48 and -2.47). Using the new method, the mean coefficient of variation for two consecutive measurements was 4.4% (range 0-10.4%); inhalation of 1.2 mg albuterol caused a 53 +/- 14% increase in Qaw (P = 0.02) and inhalation of 2.4 mg methoxamine caused a 32 +/- 7% decrease in Qaw (P = 0.07). We conclude that the new method provides reliable values of and detects the expected changes in Qaw with vasoactive drugs. The simplicity and improved safety of the method should improve its acceptability for the noninvasive assessment of Qaw in clinical research.     

A novel video-based method using projected light to measure trunk volumes during respiration

This paper proposes and evaluates an innovative video-based method for measuring the trunk volume during respiration, using projected light and surface reconstruction. The method consists of the following main steps: (a) to project a grid of circular light markers on the anterior and posterior human body trunk surface during breathing, (b) to register the subject's trunk surface using two pairs of pre-calibrated digital video cameras, (c) to segment the video stream and track the projected markers using pre-processing techniques, morphological operators and detection algorithms, (d) to label the corresponding markers in the video sequences registered by each pair of stereo cameras, (e) to reconstruct the 3-D coordinates of all markers, (f) to reconstruct the surfaces from the unordered cloud of points using the Power Crust method and (g) to calculate the trunk volume in function of time using the divergence theorem. The evaluation of the method was based on two experiments. (1) Comparison of the volume of a trunk model (mannequin) by immersion and using the proposed optical method. (2) Analysis of the applicability of the method for measuring a subject's trunk volume during a vital capacity respiratory manoeuvre. The results showed that the method was able to automatically measure more than 2000 projected points per image and to provide a very detailed representation of the subject's trunk. The relative accuracy of the volume measurement was estimated to be better than 3%. The analysis of the experiments revealed that signals coherent with the respiratory cycles could be identified through this method. In conclusion, the method based on light projection and surface reconstruction provides an accurate, non-invasive and useful means to calculate human trunk volumes during breathing.     

A novel video-based method using projected light to measure trunk volumes during respiration

This paper proposes and evaluates an innovative video-based method for measuring the trunk volume during respiration, using projected light and surface reconstruction. The method consists of the following main steps: (a) to project a grid of circular light markers on the anterior and posterior human body trunk surface during breathing, (b) to register the subject's trunk surface using two pairs of pre-calibrated digital video cameras, (c) to segment the video stream and track the projected markers using pre-processing techniques, morphological operators and detection algorithms, (d) to label the corresponding markers in the video sequences registered by each pair of stereo cameras, (e) to reconstruct the 3-D coordinates of all markers, (f) to reconstruct the surfaces from the unordered cloud of points using the Power Crust method and (g) to calculate the trunk volume in function of time using the divergence theorem. The evaluation of the method was based on two experiments. (1) Comparison of the volume of a trunk model (mannequin) by immersion and using the proposed optical method. (2) Analysis of the applicability of the method for measuring a subject’s trunk volume during a vital capacity respiratory manoeuvre. The results showed that the method was able to automatically measure more than 2000 projected points per image and to provide a very detailed representation of the subject's trunk. The relative accuracy of the volume measurement was estimated to be better than 3%. The analysis of the experiments revealed that signals coherent with the respiratory cycles could be identified through this method. In conclusion, the method based on light projection and surface reconstruction provides an accurate, non-invasive and useful means to calculate human trunk volumes during breathing.     

A novel flow cytometry-based technique to measure adult neurogenesis in the brain

The stimulation of neurogenesis is an exciting novel therapeutic option for diseases of the central nervous system, ranging from depression to neurodegeneration. One major bottleneck in screening approaches for neurogenesis-inducing compounds is the very demanding in vivo quantification of newborn neurons based on stereological techniques. To effectively develop compounds in this area, novel fast and reliable techniques for quantification of in vivo neurogenesis are needed. In this study, we introduce a flow cytometry-based method for quantifying newly generated neurons in the brain based on the counting of cell nuclei from dissected brain regions. Important steps involve density sedimentation of the cell nuclei, and staining for the proliferation marker bromodeoxy uridine and nuclear cell type markers such as NeuN. We demonstrate the ability of the technique to detect increased neurogenesis in the hippocampus of animals which underwent physical exercise and received fluoxetine treatment.     

A novel method to measure cryoprotectant permeation into intact articular cartilage

Successful cryopreservation of articular cartilage (AC) could improve clinical results of osteochondral allografting and provide a useful treatment alternative for large cartilage defects. However, successful cartilage cryopreservation is limited by the time required for cryoprotective agent (CPA) permeation into the matrix and high CPA toxicity. This study describes a novel, practical method to examine the time-dependent permeation of CPAs [dimethyl sulfoxide (DMSO) and propylene glycol (PG)] into intact porcine AC. Dowels of porcine AC (10 mm diameter) were immersed in solutions containing high concentrations of each CPA for different times (0, 15, 30, 60 min, 3, 6, and 24 h) at three temperatures (4, 22, and 37 degrees C), with and without cartilage attachment to bone. The cartilage was isolated and the amount of cryoprotective agent within the matrix was determined. The results demonstrated a sharp rise in the CPA concentration within 15-30 min exposure to DMSO and PG. The concentration plateaued between 3 and 6 h of exposure at a concentration approximately 88-99% of the external concentration (6.8 M). This observation was temperature-dependent with slower permeation at lower temperatures. This study demonstrated the effectiveness of a novel technique to measure CPA permeation into intact AC, and describes permeation kinetics of two common CPAs into intact porcine AC.     

A novel,FFT-based one-dimensional blood flow solution method for arterial network

In the present work, we propose an FFT-based method for solving blood flow equations in an arterial network with variable properties and geometrical changes. An essential advantage of this approach is in correctly accounting for the vessel skin friction through the use of Womersley solution. To incorporate nonlinear effects, a novel approximation method is proposed to enable calculation of nonlinear corrections. Unlike similar methods available in the literature, the set of algebraic equations required for every harmonic is constructed automatically. The result is a generalized, robust and fast method to accurately capture the increasing pulse wave velocity downstream as well as steepening of the pulse front. The proposed method is shown to be appropriate for incorporating correct convection and diffusion coefficients. We show that the proposed method is fast and accurate and it can be an effective tool for 1D modelling of blood flow in human arterial networks.

     

Density indicator method to measure pulmonary blood flows

The injection of plasma, saline, or erythrocyte (RBC) concentrate into the pulmonary circulation produces a change in the gravimetric density of the blood outflow similar to the dilution curve of dye. We used an improved density-measuring system to assess the flow of these density indicators through the lung in vivo and in vitro perfused dog lobe. From the in vitro density-dilution curves of plasma and RBC concentrate we calculated the pulmonary flow rate and found it to be 1.04 +/- 0.02 (SD) times the measured one. The outflow-dilution curves of gravimetric density were not as broad as those of optical density following in vivo injection of plasma bolus containing indocyanine green, and the gravimetric measurements dipped to base line, whereas the optical measurement did not. The density-dilution curves of isotonic saline injection are similar to that of plasma. Following injection of RBC concentrates with the dye, density changes in the pulmonary outflow lag behind the emergence of the dye. This was presumably related to RBC aggregation in the concentrates. In reference to the injected plasma, no loss in the density indicators for saline and RBC injection was observed. Based on these results and the similarity of the density indicators to the blood, we conclude that the plasma and isotonic saline are good density indicators to be used for the determination of pulmonary blood flows.     

A novel method to measure hairiness in bees and other insect pollinators

Hairiness is a salient trait of insect pollinators that has been linked to thermoregulation, pollen uptake and transportation, and pollination success. Despite its potential importance in pollination ecology, hairiness is rarely included in pollinator trait analyses. This is likely due to the lack of standardized and efficient methods to measure hairiness. We describe a novel methodology that uses a stereomicroscope equipped with a live measurement module software to quantitatively measure two components of hairiness: hair density and hair length. We took measures of the two hairiness components in 109 insect pollinator species (including 52 bee species). We analyzed the relationship between hair density and length and between these two components and body size. We combined hair density and length measures to calculate a hairiness index and tested whether hairiness differed between major pollinator groups and bee genera. Body size was strongly and positively correlated to hair length and weakly and negatively correlated to hair density. The correlation between the two hairiness components was weak and negative. According to our hairiness index, butterflies and moths were the hairiest pollinator group, followed by bees, hoverflies, beetles, and other flies. Among bees, bumblebees (Bombus) and mason bees (Osmia) were the hairiest taxa, followed by digger bees (Anthophorinae), sand bees (Andrena), and sweat bees (Halictini). Our methodology provides an effective and standardized measure of the two components of hairiness (hair density and length), thus allowing for a meaningful interpretation of hairiness. We provide a detailed protocol of our methodology, which we hope will contribute to improve our understanding of pollination effectiveness, thermal biology, and responses to climate change in insects.     

A novel magnetic resonance-based method to measure gene expression in living cells

In unicellular and multicellular eukaryotes, elaborate gene regulatory mechanisms facilitate a broad range of biological processes from cell division to morphological differentiation. In order to fully understand the gene regulatory networks involved in these biological processes, the spatial and temporal patterns of expression of many thousands of genes will need to be determined in real time in living organisms. Currently available techniques are not sufficient to achieve this goal; however, novel methods based on magnetic resonance (MR) imaging may be particularly useful for sensitive detection of gene expression in opaque tissues. This report describes a novel reporter gene system that monitors gene expression dynamically and quantitatively, in yeast cells, by measuring the accumulation of inorganic polyphosphate (polyP) using MR spectroscopy (MRS) or MR spectroscopic imaging (MRI). Because this system is completely non-invasive and does not require exogenous substrates, it is a powerful tool for studying gene expression in multicellular organisms, as well.     

Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA-binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.     

A novel experimental method for evaluation of effect of nitrites on blood deoxygenation kinetics]

A new method for studying the oxygen-binding properties of blood is proposed, which is based on the analysis of transient processes on oxygen electrodes. The method was used to study the effect of sodium nitrite on the kinetics of dissociation of oxyhemoglobin in rat blood. It was shown that the rate of oxygen transport decreases at nitrite concentrations 1-5 mg/100 g body weight.     

A theory of blood flow in skeletal muscle

A theoretical analysis of blood flow in the microcirculation of skeletal muscle is provided. The flow in the microvessels of this organ is quasi steady and has a very low Reynolds number. The blood is non-Newtonian and the blood vessels are distensible with viscoelastic properties. A formulation of the problem is provided using a viscoelastic model for the vessel wall which was recently derived from measurements in the rat spinotrapezius muscle (Skalak and Schmid-Sch?nbein, 1986b). Closed form solutions are derived for several physiologically important cases, such as perfusion at steady state, transient and oscillatory flows. The results show that resting skeletal muscle has, over a wide range of perfusion pressures an almost linear pressure-flow curve. At low flow it exhibits nonlinearities. Vessel distensibility and the non-Newtonian properties of blood both have a strong influence on the shape of the pressure-flow curve. During oscillatory flow the muscle exhibits hysteresis. The theoretical results are in qualitative agreement with experimental observations.     

Validity of a novel method to measure vertical oscillation during running using a depth camera

Recent advancements in low-cost depth cameras may provide a clinically accessible alternative to conventional three-dimensional (3D) multi-camera motion capture systems for gait analysis. However, there remains a lack of information on the validity of clinically relevant running gait parameters such as vertical oscillation (VO). The purpose of this study was to assess the validity of measures of VO during running gait using raw depth data, in comparison to a 3D multi-camera motion capture system. Sixteen healthy adults ran on a treadmill at a standard speed of 2.7 m/s. The VO of their running gait was simultaneously collected from raw depth data (Microsoft Kinect v2) and 3D marker data (Vicon multi-camera motion capture system). The agreement between the VO measures obtained from the two systems was assessed using a Bland-Altman plot with 95% limits of agreement (LOA), a Pearson’s correlation coefficient (r), and a Lin’s concordance correlation coefficient (r_c). The depth data from the Kinect v2 demonstrated excellent results across all measures of validity (r = 0.97; r_c = 0.97; 95% LOA = −8.0 mm – 8.7 mm), with an average absolute error and percent error of 3.7 (2.1) mm and 4.0 (2.0)%, respectively. The findings of this study have demonstrated the ability of a low cost depth camera and a novel tracking method to accurately measure VO in running gait.     

A novel cell immunoassay to measure survival of motor neurons protein in blood cells

Background  

The motor neuron degenerative disease spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality and is caused by mutations in the survival of motor neurons (SMN) gene that reduce the expression levels of the SMN protein. A major goal of current therapeutic approaches is to increase SMN levels in SMA patients. The purpose of this study was to develop a reliable assay to measure SMN protein levels from peripheral blood samples.