The YSI 2300 Analyzer Replacement Meeting Report.

This is a summary report of the most important aspects discussed during the YSI 2300 Analyzer Replacement Meeting. The aim is to provide the interested reader with an overview of the complex topic and propose solutions for the current issue. This solution should not only be adequate for the United States or Europe markets but also for all other countries. The meeting addendum presents three outcomes of the meeting.

Fast spatially-resolved T2 measurements with constant-gradient CPMG.

Speed of acquisition is paramount for the application of magnetic resonance to flow experiments through porous rocks. One popular method for imaging core floods is the spatially resolved T2 experiment which can separate fluids either by their viscosity contrast or by doping one fluid with a relaxation agent. Existing techniques for spatial-T2 may suffer from long acquisition times and eddy currents due to the pulsing of magnetic field gradients. Here, we propose a constant gradient method for 1d spatially-resolved T2 which embraces the speed of frequency encoding techniques and avoids eddy currents by the absence of any gradient ramps during the radio frequency (r.f.) pulse train. We provide the operating envelope for this kind of experiment, which is restricted due to the slice selectivity of the r.f. pulses in the presence of the magnetic field gradient. Additionally, we show that the effects of self-diffusion and the mixing of T1 and T2 contributions are manageable. As an illustration, we have applied this technique to an enhanced oil recovery experiment. The two fluid phases were tracked without any doping and with a time resolution of 40 s. In this case, the increased time resolution allowed us to observe dynamic flow phenomena such as fluid fingering and the calculation of the velocity of the fluid displacement fronts.

Under-sampling and compressed sensing of 3D spatially-resolved displacement propagators in porous media using APGSTE-RARE MRI.

A method for under-sampling and compressed sensing of 3D spatially-resolved propagators is presented and demonstrated for flow in a packed bed and a heterogeneous carbonate rock. By sampling only 12.5% of q,k-space, the experimental acquisition time was reduced by almost an order of magnitude. In particular, for both systems studied, a 3D image was acquired at 1 mm isotropic spatial resolution such that 134,400 local propagators were obtained. Data were acquired in ~1 h and ~11 h for the packed bed and rock, respectively. It is shown that spatial resolution and under-sampling using this implementation retains the quantitative nature of the propagator measurement, and differences between implementation of this measurement in two and three dimensions are identified. The potential for 3D spatially-resolved propagators to provide new insights into transport processes in porous media by characterisation of the statistical moments of the propagators is discussed.

Acquisition of spatially-resolved displacement propagators using compressed sensing APGSTE-RARE MRI.

A method is presented for accelerating the acquisition of spatially-resolved displacement propagators via under-sampling of an Alternating Pulsed Gradient Stimulated Echo - Rapid Acquisition with Relaxation Enhancement (APGSTE-RARE) data acquisition with compressed sensing image reconstruction. The method was demonstrated with respect to the acquisition of 2D spatially-resolved displacement propagators of water flowing through a packed bed of hollow cylinders. The q,k-space was under-sampled according to variable-density pseudo-random sampling patterns. The quality of compressed sensing reconstructions of spatially-resolved propagators at a range of sampling fractions was assessed using the peak signal-to-noise ratio (PSNR) as a quality metric. Propagators of good quality (PSNR 33.2 dB) were reconstructed from only 6.25% of all data points in q,k-space, resulting in a reduction in the data acquisition time from 4 h to 14 min. The spatially-resolved propagators were reconstructed using both the total variation and nuclear norm sparsifying transforms; use of total variation resulted in a slightly higher quality of the reconstructed image in most cases. To illustrate the power of this method to characterise heterogeneous flow in porous media, the method is applied to the characterisation of flow in a vuggy carbonate rock.

Application of the reference method isotope dilution gas chromatography mass spectrometry (ID/GC/MS) to establish metrological traceability for calibration and control of blood glucose test systems.

Self-monitoring of blood glucose (BG) by means of handheld BG systems is a cornerstone in diabetes therapy. The aim of this article is to describe a procedure with proven traceability for calibration and evaluation of BG systems to guarantee reliable BG measurements. Isotope dilution gas chromatography mass spectrometry (ID/GC/MS) is a method that fulfills all requirements to be used in a higher-order reference measurement procedure. However, this method is not applicable for routine measurements because of the time-consuming sample preparation. A hexokinase method with perchloric acid (PCA) sample pretreatment is used in a measurement procedure for such purposes. This method is directly linked to the ID/GC/MS method by calibration with a glucose solution that has an ID/GC/MS-determined target value. BG systems are calibrated with whole blood samples. The glucose levels in such samples are analyzed by this ID/GC/MS-linked hexokinase method to establish traceability to higher-order reference material. For method comparison, the glucose concentrations in 577 whole blood samples were measured using the PCA-hexokinase method and the ID/GC/MS method; this resulted in a mean deviation of 0.1%. The mean deviation between BG levels measured in >500 valid whole blood samples with BG systems and the ID/GC/MS was 1.1%. BG systems allow a reliable glucose measurement if a true reference measurement procedure, with a noninterrupted traceability chain using ID/GC/MS linked hexokinase method for calibration of BG systems, is implemented. Systems should be calibrated by means of a traceable and defined measurement procedure to avoid bias.

Conformations of NhaA, the Na+/H+ exchanger from Escherichia coli, in the pH-activated and ion-translocating states.

NhaA, the main sodium-proton exchanger in the inner membrane of Escherichia coli, regulates the cytosolic concentrations of H+ and Na+. It is inactive at acidic pH, becomes active between pH 6 and pH 7, and reaches maximum activity at pH 8. By cryo-electron microscopy of two-dimensional crystals grown at pH 4 and incubated at higher pH, we identified two sequential conformational changes in the protein in response to pH or substrate ions. The first change is induced by a rise in pH from 6 to 7 and marks the transition from the inactive state to the pH-activated state. pH activation, which precedes the ion-induced conformational change, is accompanied by an overall expansion of the NhaA monomer and a local ordering of the N-terminus. The second conformational change is induced by the substrate ions Na+ and Li+ at pH above 7 and involves a 7-A displacement of helix IVp. This movement would cause a charge imbalance at the ion-binding site that may trigger the release of the substrate ion and open a periplasmic exit channel.

Conformations of NhaA, the Na/H exchanger from Escherichia coli, in the pH-activated and ion-translocating states.

NhaA, the main sodium-proton exchanger in the inner membrane of Escherichia coli, regulates the cytosolic concentrations of H and Na. It is inactive at acidic pH, becomes active between pH 6 and pH 7, and reaches maximum activity at pH 8. By cryo-electron microscopy of two-dimensional crystals grown at pH 4 and incubated at higher pH, we identified two sequential conformational changes in the protein in response to pH or substrate ions. The first change is induced by a rise in pH from 6 to 7 and marks the transition from the inactive state to the pH-activated state. pH activation, which precedes the ion-induced conformational change, is accompanied by an overall expansion of the NhaA monomer and a local ordering of the N-terminus. The second conformational change is induced by the substrate ions Na and Li at pH above 7 and involves a 7-A displacement of helix IVp. This movement would cause a charge imbalance at the ion-binding site that may trigger the release of the substrate ion and open a periplasmic exit channel.

Examining the dynamic energy landscape of an antiporter upon inhibitor binding.

Previously, we applied single-molecule force spectroscopy to detect and locate interactions within the functional Na(+)/H(+) antiporter NhaA from Escherichia coli. It was observed that the binding of the inhibitor 2-aminoperimidine established interactions different from those introduced by the binding of the native ligand. To understand the inhibitory mechanism of the inhibitor, we applied single-molecule dynamic force spectroscopy to reconstruct the energy landscape of NhaA. Dynamic force spectroscopy revealed that the energy landscape of the antiporter remained mainly unchanged except for the energy barrier of the functionally important transmembrane alpha-helix IX. Inhibitor binding set this domain into a newly formed deep and narrow energy minimum that kinetically stabilized alpha-helix IX and reduced its conformational entropy. The entropy reduction of alpha-helix IX is thought to inhibit its functionally important structural flexibility, while the deeper energy barrier shifted the population of active antiporters towards inhibited antiporters.

Structure and function of prokaryotic glutamate transporters from Escherichia coli and Pyrococcus horikoshii.

The glutamate transporters GltP(Ec) from Escherichia coli and GltP(Ph) from Pyrococcus horikoshii were overexpressed in E. coli and purified to homogeneity with a yield of 1-2 mg/L of culture. Single-particle analysis and electron microscopy indicate that GltP(Ph) is a trimer in detergent solution. Electron microscopy of negatively stained GltP(Ph) two-dimensional crystals shows that the transporter is a trimer also in the membrane. Gel filtration of GltP(Ec) indicates a reversible equilibrium of two oligomeric states in detergent solution that we identified as a trimer and hexamer by blue-native gel electrophoresis and cross-linking. The purified transporters were fully active upon reconstitution into liposomes, as demonstrated by the uptake of radioactively labeled L-aspartate or L-glutamate. L-aspartate/L-glutamate transport of GltP(Ec) involves the cotransport of protons and depends only on pH, whereas GltP(Ph) catalyzes L-glutamate transport with a cotransport of H+ or Na+. L-glutamate induces a fast transient current in GltP(Ph) proteoliposomes coupled to a solid supported membrane (SSM). We show that the electric signal depends on the concentration of Na+ or H+ outside the proteoliposomes and that GltP(Ph) does not require K+ inside the proteoliposomes. In addition, the electrical currents are inhibited by TBOA and HIP-B. The half-saturation concentration for activation of GltP(Ph) glutamate transport (K0.5(glut)) is 194 microM.