scholarly journals FLIM Indicators for Quantitative Measurement of pH

2021 ◽  
Vol 6 (1) ◽  
pp. 33
Author(s):  
Tatiana R. Simonyan ◽  
Anastasia V. Mamontova ◽  
Konstantin A. Lukyanov ◽  
Alexey M. Bogdanov
Keyword(s):  
Quantitative Measure ◽  
Experimental System ◽  
Hek293 Cells ◽  
Fluorescence Excitation ◽  
Fluorescent Indicators ◽  
Ph Changes ◽  
Ph Indicators ◽  
Wide Range ◽  
Wide Ph Range ◽  
The Time Domain

Monitoring of intracellular pH changes in situ can provide valuable information about cellular metabolism and a deeper understanding of physiological processes. Most traditional fluorescent indicators are only capable of a relative assessment of changes in the studied parameter in the cell. We associate the possibility of measuring the absolute values that characterize the analyte with the detection of the indicator signal in the time domain, where its quantitative measure is the fluorescence lifetime (tau). In this project, we test promising pH-sensitive fluorophores with labile fluorescence lifetimes—EYFP-G65T and EGFP-Y145L/S205V—both as fluorescent cores for the previously described pH indicators and as independent pH indicators. Measurement of the fluorescence attenuation kinetics of four structures (EYFP-G65T, EGFP-Y145L/S205V, SypHer3s, and SypHer3s-G65T) over a wide pH range revealed areas where tau is linearly dependent on pH. The differences in the fluorescence excitation modes of these molecules makes it possible to use them in one experimental system to assess pH changes in a wide range, 4.0–9.0. We showed that under the conditions of traditional fluorescence microscopy (in the cytoplasm of HEK293 cells), the SypHer3s-G65T indicator shows a dynamic response range approximately 3 times wider than the original SypHer3s.

Controllability Ellipsoid to Evaluate the Performance of Mobile Manipulators for Manufacturing Tasks

2017 ◽  
Vol 9 (6) ◽  
Author(s):  
Stephen L. Canfield ◽  
Reabetswe M. Nkhumise
Keyword(s):  
Time Domain ◽  
Controller Design ◽  
Control Design ◽  
Quantitative Measure ◽  
Space Representation ◽  
System Response ◽  
Geometric Representation ◽  
Mobile Manipulators ◽  
Control Parameters ◽  
The Time Domain

This paper develops an approach to evaluate a state-space controller design for mobile manipulators using a geometric representation of the system response in tool space. The method evaluates the robot system dynamics with a control scheme and the resulting response is called the controllability ellipsoid (CE), a tool space representation of the system’s motion response given a unit input. The CE can be compared with a corresponding geometric representation of the required motion task (called the motion polyhedron) and evaluated using a quantitative measure of the degree to which the task is satisfied. The traditional control design approach views the system response in the time domain. Alternatively, the proposed CE views the system response in the domain of the input variables. In order to complete the task, the CE must fully contain the motion polyhedron. The optimal robot arrangement would minimize the total area of the CE while fully containing the motion polyhedron. This is comparable to minimizing the power requirements of robot design when applying a uniform scale to all inputs. It will be shown that changing the control parameters changes the eccentricity and orientation of the CE, implying a preferred set of control parameters to minimize the design motor power. When viewed in the time domain, the control parameters can be selected to achieve desired stability and time response. When coupled with existing control design methods, the CE approach can yield robot designs that are stable, responsive, and minimize the input power requirements.

scholarly journals Fluorescence lifetime imaging of pH along the secretory pathway

2021 ◽  
Author(s):  
Peter Linders ◽  
Martin ter Beest ◽  
Geert van den Bogaart
Keyword(s):  
Temporal Resolution ◽  
Fluorescence Lifetime ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Excitation Wavelength ◽  
Fluorescence Lifetime Imaging ◽  
Ph Homeostasis ◽  
Ph Changes ◽  
Lifetime Imaging ◽  
Wide Range

Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles cause a wide range of diseases, including disorders of glycosylation and lysosomal storage diseases. Ratiometric imaging of the pH-sensitive mutant of green fluorescent protein (GFP), pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution unsuitable to follow the rapid transit of cargo between organelles. We therefore applied fluorescence lifetime imaging microscopy (FLIM) to measure intraorganellar pH with just a single excitation wavelength. We first validated this method by confirming the pH in multiple compartments along the secretory pathway. Then, we analyze the dynamic pH changes within cells treated with Brefeldin A, a COPI coat inhibitor. Finally, we followed the pH changes of newly-synthesized molecules of the inflammatory cytokine tumor necrosis factor (TNF)-α while it was in transit from the endoplasmic reticulum via the Golgi to the plasma membrane. The toolbox we present here can be applied to measure intracellular pH with high spatial and temporal resolution, and can be used to assess organellar pH in disease models.

scholarly journals A toolbox of Stable Integration Vectors (SIV) in the fission yeast Schizosaccharomyces pombe

2019 ◽  
Author(s):  
Aleksandar Vještica ◽  
Magdalena Marek ◽  
Pedro N’kosi ◽  
Laura Merlini ◽  
Gaowen Liu ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Model Organism ◽  
Experimental System ◽  
Single Copy ◽  
Cell Physiology ◽  
Large Set ◽  
Stable Integration ◽  
Wide Range ◽  
Fluorescent Tags

AbstractSchizosaccharomyces pombe is a widely used model organism that resembles higher eukaryotes in many aspects of cell physiology. Its popularity as an experimental system partially stems from the ease of genetic manipulations, where the innate homology-targeted repair is exploited to precisely edit the genome. While vectors to incorporate exogenous sequences into the chromosomes are available, most are poorly characterized. Here we show that commonly used fission yeast vectors, which upon integration produce repetitive genomic regions, yield unstable genomic loci. We overcome this problem by designing a new series of Stable Integration Vectors (SIV) that target four different prototrophy genes. SIV produce non-repetitive, stable genomic loci and integrate predominantly as single copy. Additionally, we develop a set of complementary auxotrophic alleles that preclude false-positive integration events. We expand the vector series to include antibiotic resistance markers, promoters, fluorescent tags and terminators, and build a highly modular toolbox to introduce heterologous sequences. Finally, as proof of concept, we generate a large set of ready-to-use, fluorescent probes to mark organelles and cellular processes with a wide range of applications in fission yeast research.

scholarly journals DIRECTED EVOLUTION OF METABOLIC PATHWAYS IN MICROBIAL POPULATIONS. I. MODIFICATION OF THE ACID PHOSPHATASE pH OPTIMUM IN S. CEREVISIAE

Genetics ◽  
1972 ◽  
Vol 70 (1) ◽  
pp. 59-73 ◽  
Author(s):  
J C Francis ◽  
P E Hansche
Keyword(s):  
Acid Phosphatase ◽  
Metabolic Pathways ◽  
Experimental System ◽  
Microbial Populations ◽  
Kinetic Properties ◽  
Mutational Event ◽  
Ph Optimum ◽  
Functional Changes ◽  
Evolution Of Metabolic Pathways ◽  
Wide Range

ABSTRACT An experimental system for directing the evolution of enzymes and metabolic pathways in microbial populations is proposed and an initial test of its power is provided.—The test involved an attempt to genetically enhance certain functional properties of the enzyme acid phosphatase in S. cerevisiae by constructing an environment in which the functional changes desired would be "adaptive". Naturally occurring mutations in a population of 109 cells were automatically and continuously screened, over 1,000 generations, for their effect on the efficiency (Km) and activity of acid phosphatase at pH 6, and for their effect on the efficiency of orthophosphate metabolism.—The first adaptation observed, M1, was due to a single mutational event that effected a 30% increase in the efficiency of orthophosphate metabolism. The second, M2, effected an adaptive shift in the pH optimum of acid phosphatase and an increase in its activity over a wide range of pH values (an increment of 60% at pH 6). M2 was shown to result from a single mutational event in the region of the acid phosphatase structural gene. The third, M3, effected cell clumping, an adaptation to the culture apparatus that had no effect on phosphate metabolism.—The power of this system for directing the evolution of enzymes and of metabolic pathways is discussed in terms of the kinetic properties of the experimental system and in terms of the results obtained.

scholarly journals Plasticity in Escherichia coli cell wall metabolism promotes fitness and mediates intrinsic antibiotic resistance across environmental conditions

2018 ◽  
Author(s):  
Elizabeth A Mueller ◽  
Petra Anne Levin
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Growth Parameter ◽  
Culture Conditions ◽  
Cell Wall Integrity ◽  
Bacterial Cells ◽  
Intrinsic Resistance ◽  
Wide Range ◽  
Peptidoglycan Cell Wall ◽  
Wide Ph Range

ABSTRACTAlthough the peptidoglycan cell wall is an essential structural and morphological feature of most bacterial cells, the extracytoplasmic enzymes involved in its synthesis are frequently dispensable under standard culture conditions. By modulating a single growth parameter—extracellular pH—we discovered a subset of these so-called “redundant” enzymes in Escherichia coli are required for maximal fitness across pH environments. Among these pH specialists are the class A penicillin binding proteins PBP1 a and PBP1 b; defects in these enzymes attenuate growth in alkaline and acidic conditions, respectively. Genetic, biochemical, and cytological studies demonstrate that synthase activity is required for cell wall integrity across a wide pH range, and differential activity across pH environments significantly alters intrinsic resistance to cell wall active antibiotics. Together, our findings reveal previously thought to be redundant enzymes are instead specialized for distinct environmental niches, thereby ensuring robust growth and cell wall integrity in a wide range of conditions.

Abstract 434: A Novel 5-Step Model for the Mechanism of ABCA1-Mediated Nascent HDL Biogenesis

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Shuhui Wang ◽  
Gregory Brubaker ◽  
Kailash Gulshan ◽  
Jonathan D Smith
Keyword(s):  
Plasma Membrane ◽  
Membrane Lipids ◽  
Sodium Taurocholate ◽  
Hek293 Cells ◽  
Membrane Remodeling ◽  
Fluorescent Indicators ◽  
Step Model ◽  
Partial Unfolding ◽  
Cell Membrane Protein ◽  
Partially Unfolded

Objective— Lipid-poor apoA-I acts as an acceptor for cell cholesterol and phospholipids via the cell membrane protein ABCA1, generating nascent HDL. However, the mechanism of this process is not understood at the molecular level. Methods and Results— We propose a novel five-step model of nascent HDL biogenesis: ABCA1 remodeling of the plasma membrane lipids exposing phosphatidylserine and apoA-I binding to ABCA1 are the first two independent steps; third, ABCA1 facilitates apoA-I partial unfolding; forth, partially unfolded apoA-I inserts into the modified plasma membrane resulting in apoA-I lipidation; and fifth, nascent HDL is released from the cell. We created fluorescent apoA-I indicators that can monitor apoA-I unfolding and lipidation states. In cell free assays of reconstituted HDL (rHDL) generation from apoAI and DMPC liposomes, the fluorescent indicators demonstrated apoA-I unfolding and lipidation concurrent with rHDL formation. Next, HEK293 cells were stably transfected with different ABCA1 vectors encoding wild type (WT) and W590S and C1477R Tangier disease mutation isoforms. WT ABCA1 mediated cholesterol efflux to apoA-I (requires all steps) and sodium taurocholate (NaTC, requires only the membrane remodeling step,). Although neither mutant could efflux cholesterol efficiently to apoA-I, they were blocked at different steps. The W590S mutant bound apoAI but could not efflux cholesterol to NaTC, thus it was blocked at the membrane remodeling step. However, the C1477R mutant could not bind apoAI but could efflux cholesterol to NaTC, thus its activity was blocked at the apoAI binding step. When the lipidation indicator apoA-I was incubated with stably transfected HEK cells, we observed cell associated lipidated apoA-I in cells expressing WT ABCA1, but mostly unlipidated apoA-I was associated with the cells expressing W590S ABCA1. Conclusion— Our results support a novel five-step model for nascent HDL biogenesis: 1, 2) ABCA1 remodeling of the plasma membrane and apoA-I binding to ABCA1, which facilitate 3) apoA-I partial unfolding and 4) and lipidation by the remodeled membrane, followed by 5) the release of nascent HDL.

Electromagnetic modeling of 3-D sources over 2-D inhomogeneities in the time domain

Geophysics ◽  
1992 ◽  
Vol 57 (8) ◽  
pp. 994-1003 ◽  
Author(s):  
Michael Leppin
Keyword(s):  
Differential Equations ◽  
Diffusion Equation ◽  
Time Domain ◽  
Magnetic Induction ◽  
Host Rock ◽  
Vertical Component ◽  
Wavenumber Domain ◽  
Transient Electromagnetic ◽  
Wide Range ◽  
The Time Domain

A numerical method is presented by which the transient electromagnetic response of a two‐dimensional (2-D) conductor, embedded in a conductive host rock and excited by a rectangular current loop, can be modeled. This 2.5-D modeling problem has been formulated in the time domain in terms of a vector diffusion equation for the scattered magnetic induction, which is Fourier transformed into the spatial wavenumber domain in the strike direction of the conductor. To confine the region of solution of the diffusion equation to the conductive earth, boundary values for the components of the magnetic induction on the ground surface have been calculated by means of an integral transform of the vertical component of the magnetic induction at the air‐earth interface. The system of parabolic differential equations for the three magnetic components has been integrated for 9 to 15 discrete spatial wavenumbers ranging from [Formula: see text] to [Formula: see text] using an implicit homogeneous finite‐difference scheme. The discretization of the differential equations on a grid representing a cross‐section of the conductive earth results in a large, sparse system of linear equations, which is solved by the successive overrelaxation method. The three‐dimensional (3-D) response has been computed by an inverse Fourier transformation of the cubic spline interpolated scattered magnetic induction in the wavenumber domain using a digital filtering technique. To test the algorithm, responses have been computed for a two‐layered half‐space and a vertical prism embedded in a conductive host rock. These examples were then compared with results obtained analytically or numerically using frequency‐domain finite‐element and time‐domain integral equation methods. The new numerical procedure gives satisfactory results for a wide range of 2-D conductivity distributions with conductivity ratios exceeding 1:100, provided the grid is sufficiently refined at the corners of the conductivity anomalies.

Dynamic Light Scattering and Zeta Potential Studies of Ceria Nanoparticles

2018 ◽  
Vol 278 ◽  
pp. 112-120 ◽  
Author(s):  
Ishaq Yusuf Habib ◽  
N.T.R.N. Kumara ◽  
Chee Ming Lim ◽  
Abdul Hanif Mahadi
Keyword(s):  
Average Particle Size ◽  
Nanoparticle Synthesis ◽  
Average Particle ◽  
Wide Range ◽  
Isoelectric Points ◽  
Wide Ph Range ◽  
Electrolyte Mixture ◽  
Oxide Nanoparticle ◽  
Ph Range ◽  
Adsorption Desorption

A Cerium (IV) oxide nanoparticle (nanoceria) is widely used in different applications such as biomedicine and catalysis due to its unique structural, morphological and catalytic properties. In this report, the dispersion of nanoceria in both aqueous and non-aqueous (methanol and ethanol) media were studied. Adsorption-desorption processes were observed upon addition of different classes of surfactants such as citric acid (CA), cetrimonium bromide (CTAB) and diethanolamine (DEA). Stable dispersions were obtained in both aqueous, non-aqueous and electrolyte assisted media with the overall mechanism being hydrolysis, dissolution and adsorption. XRD, FE-SEM, FTIR and DLS have been used in the present study to characterize the nanoceria and to quantitatively analyze their average particle size distributions in a unique electrolyte mixture of (0.1 M NaOH/ 65% HNO3:H2O, 1:1 v/v) which has not been reported previously. The surface charge study was carried out across a wide pH range between 1.4 – 9.6 and the isoelectric points (IEP) with respect to 15 ml H2O and 50 ml H2O dispersed phases occurred at a pH of about 6.5 and 6.7 respectively. The present study could be useful in a wide range of applications including nanoparticle synthesis, stabilization, and adsorption of toxic materials, biomedical and pharmaceutical.

Development and Test of a Crash Experimental System of the Level Ejection Type Automobile Bumper

2011 ◽  
Vol 486 ◽  
pp. 274-278
Author(s):  
Rui Yang ◽  
Jia Qian Li
Keyword(s):  
Experimental System ◽  
Test Method ◽  
Passive Safety ◽  
Crash Test ◽  
Successful Development ◽  
Experiment System ◽  
Wide Range ◽  
Experimental Process ◽  
Side Crash ◽  
Structure And Design

Based on the analysis of automobile bumper crash, and combined with the features of the bumper absorbing energy, a rig for bumper crash test was developed. The rig has advanced features of small size, wide-range of usage and arbitrary movement. The frontal and side crash can be tested according to different models and materials, and, hence, to provide a viable test method to study the general crash performance and energy absorption situation of the bumper, and reliable test data for investigation of automotive passive safety. In this paper, the structure and design of the rig is presented fist, followed by the working principles of the experiment system and theoretical analysis of the experimental process. An experiment was conducted which proved the successful development of the experimental system.

Experimental Study on Single-Phase Gas Flow in Microtubes

2011 ◽  
Vol 133 (11) ◽  
Author(s):  
T. T. Zhang ◽  
L. Jia ◽  
C. W. Li ◽  
L. X. Yang ◽  
Y. Jaluria
Keyword(s):  
Heat Transfer ◽  
Gas Flow ◽  
Single Phase ◽  
Slip Flow ◽  
Experimental System ◽  
Flow Region ◽  
Flow And Heat Transfer ◽  
Wide Range ◽  
Physical Insight ◽  
Insight Into

An experimental system for single-phase gas flow in microtubes has been developed. The effects of viscous heating and compressibility on the flow and temperature field were studied for a wide range of governing parameters. Also, an analytical/numerical model of the flow was developed. Numerical results for the flow and heat transfer in the slip flow region were found to agree quite well with the experimental data, lending support to the model. The study provides greater physical insight into and understanding the effects of viscous dissipation and compressibility in microtube flow and the associated heat transfer. In addition, the combined experimental and numerical simulation approaches of the process can be used for control and optimization of systems based on microtube heat transfer.

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