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The clinical practice guideline for primary aldosteronism (PA) places a high value on confirmatory tests to sparing patients with false-positive results in case detection from undergoing adrenal venous sampling (AVS). However, it is unclear whether multiple types of confirmatory tests are more useful than a single type. To evaluate whether the machine-learned combination of two confirmatory tests is more useful in predicting subtypes of PA than each test alone.A retrospective cross-sectional study in referral centres.This study included 615 patients with PA randomly assigned to the training and test data sets. The participants underwent saline infusion test (SIT) and captopril challenge test (CCT) and were subtyped by AVS (unilateral, n = 99; bilateral, n = 516).The area under the curve (AUC) and clinical usefulness using decision curve analysis for the subtype prediction in the test data set.The AUCs for the combination of SIT and CCT, SIT alone and CCT alone were 0.850, 0.813 and 0.786, respectively, with no significant differences between them. The AUC for the baseline clinical characteristics alone was 0.872, whereas the AUCs for these combined with SIT, combined with CCT and combined with both SIT and CCT were 0.868, 0.854 and 0.855, respectively, with no significant improvement in AUC. The additional clinical usefulness of the second confirmatory test was unremarkable on decision curve analysis.Our data suggest that patients with positive case detection undergo one confirmatory test to determine the indication for AVS.

The position of the pelvic fins among teleost fishes has tended to shift rostrally during evolution. This positional shift seems to have led to the diversification of feeding behavior and allowed adaptation to new environments. To understand the developmental basis of this shift in pelvic fin position among teleosts, we investigated the embryonic development of the lateral plate mesoderm, which gives rise to the pelvic fins, at histological levels in the medaka Oryzias latipes (abdominal pelvic fins) and Nile tilapia Oreochromis niloticus (thoracic pelvic fins). Our histological analyses revealed that the lateral plate mesodermal cells expand not only ventrally but also rostrally to cover the yolk during embryogenesis of both medaka and Nile tilapia. In medaka, we also found that the lateral plate mesoderm completely covered the yolk prior to the initiation of the pelvic fin buds, whereas in Nile tilapia the pelvic fin buds appeared in the body wall from the lateral plate mesoderm at the thoracic level when the lateral plate mesodermal cells only covered one-third of the yolk. We discuss the relevance of such differences in the rate of the lateral plate mesoderm expansion on the yolk surface and the position of the pelvic fins.

SUMMARY Desorbed gases from an anode stimulated by electron irradiation are regarded as one of the causes of vacuum breakdown. To understand vacuum breakdown phenomena, it is important to analyze the composition and quantity of electron-stimulated desorption gases. The single-pulse electron beam irradiation method was developed for detailed analysis of desorbed gas characteristics. It is possible for this method to reduce the modification of surface conditions due to electron beam irradiation, and it makes it possible to measure the transitions of the gas desorption rate from the virgin surface state to a certain period. Using this method, we investigated the desorbed gases from an oxygen-free copper electrode. It was found that electron irradiation caused H2 gas desorption from the copper electrode and produced conditioning of the gas desorption. A large amount of H2 gas was desorbed in the initial electron beam irradiation. By analysis of the gas desorbed by irradiation with a single-pulse electron beam, it is possible to observe the change of the gas desorption rate from the virgin surface to a certain number of electron-irradiated surface states. Detailed analysis of the gases desorbed from a sample can be used to evaluate the electrode material in a vacuum.

Background and Purpose The present treatment for choroidal neovascularization (CNV) associated with age-related macular degeneration (AMD) is not sufficient. Hence, we examined the therapeutic efficacy of reducing histamine H4 receptor expression on CNV in mice. Experimental Approach H4 receptor expression was examined in CNVs from patients with AMD. In mice, laser photocoagulation was performed in the retina to induce experimental CNV (laser CNV). Protein and mRNA expression levels were determined and CNV volume measured in wild-type and Hrh4-/- mice with laser CNV. The effects of JNJ7777120, an H4 receptor antagonist, administered intravitreously, on CNV volume and pathological vessel leakage were determined in mice with laser CNV and controls. Fundus imaging, retinal histology and electroretinography were performed on eyes injected with JNJ7777120 to evaluate retinal toxicity. Key Results Human H4 receptors were only confirmed in CNV samples from AMD patients and not in the other subretinal tissues. Mouse H4 receptors were expressed in retinal pigment epithelium only after inducing laser CNV in wild-type mice, and were co-localized with the macrophage marker F4/80. Laser CNV volume was reduced in Hrh4-/- mice compared with that in wild-type mice, and JNJ7777120 suppressed laser-induced CNV volume and pathological CNV leakage in wild-type mice. Also eyes injected with JNJ7777120 did not show retinal degeneration. Conclusions and Implications H4 receptors are expressed in macrophages that accumulate around CNVs. Suppressing H4 receptor expression prevented the pathological vessel leakage without showing retinal toxicity, indicating that the H4 receptor has potential as a novel therapeutic target in AMD.

Dimerization between G protein-coupled receptors (GPCRs) is a clearly established phenomenon. However, limited information is currently available on the interface essential for this process. Based on structural comparisons and sequence homology between rhodopsin and A1 adenosine receptor (A1R), we initially hypothesized that four residues in transmembrane (TM) 4 and TM5 are involved in A1R homodimerization. Accordingly, these residues were substituted with Ala by site-directed mutagenesis. Interestingly, the mutant protein displayed no significant decrease in homodimer formation compared with wild-type A1R, as evident from coimmunoprecipitation and BRET2 analyses (improved bioluminescence resonance energy transfer system offered by Perkin-Elmer Life Sciences), but lost ligand binding activity almost completely. Further studies disclosed that this effect was derived from the mutation of one particular residue, Trp132, which is highly conserved among many GPCRs. Confocal immunofluorescence and cell-surface biotinylation studies revealed that the mutant receptors localized normally at transfected cell membranes, signifying that loss of ligand binding was not because of defective cellular trafficking. Molecular modeling of the A1R-ligand complex disclosed that Trp132 interacted with several residues located in TM3 and TM5 that stabilized agonist binding. Thus, loss of interactions of Trp with these residues may, in turn, disrupt binding to agonists. Our study provides strong evidence of the essential role of the highly conserved Trp132 in TM4 of adenosine receptors.

We have obtained two types of thermostable mutant lactate oxidase - one that exhibited an E-to-G point mutation at position 160 (E160G) through error-prone PCR-based random mutagenesis, and another that exhibited an E-to-G mutation at position 160 and a V-to-I mutation at position 198 (E160G/V198I) through DNA shuffling-based random mutagenesis - both of which we have previously reported. Our molecular modeling of lactate oxidase suggests that the substitution of G for E at position 160 reduces the electrostatic repulsion between the negative charges of E160 and E130 in the (beta/alpha)8 barrel structure, but a thermal-inactivation experiment on the five kinds of single-mutant lactate oxidase at position 160 (E160A, E160Q, E160H, E160R, and E160K) showed that the side-chain volume of the amino acid at position 160 mainly contributes to the thermostability of lactate oxidase. We also produced V198I single-mutant lactate oxidase through site-directed mutagenesis, and analysed the thermostability of wild-type, V198I, E160G, and E160G/V198I lactate oxidase enzymes. The half-life of E160G/V198I lactate oxidase at 70 degrees C was about three times longer than that of E160G lactate oxidase, and was about 20 times longer than that of wild-type lactate oxidase. In contrast, the thermostability of the V198I lactate oxidase was almost identical to that of wild-type lactate oxidase. This indicates that the V198I mutation alone does not affect lactate oxidase thermostability, but does affect it when combined with the E160G mutation.

Many biologically active heterodimeric proteins of snake venom consist of two C-type lectin-like subunits. One of these proteins, habu IX/X-bp, is a Gla domain-binding protein whose subunits both bind to a Ca2+ ion, with a total of two Ca2+-binding sites. The molecular modeling and Ca2+-binding analysis of echis IX/X-bp revealed that it lacks one of two Ca2+-binding sites, though the folding of this subunit is conserved. It is concluded that heterodimeric C-type lectin-like proteins function independent of Ca2+ and have essentially a similar folding to habu IX/X-bp.

The fast multipole method proposed by Greengard and Rokhlin (GR) is applied to large biomacromolecular systems. In this method, the system is divided into a hierarchy of cells, and electric field exerted on a particle is decomposed into two parts. The first part is a rapidly varying field due to nearby cells, so that it needs rigorous pairwise calculations. The second part is a slowly varying local field due to distant cells; hence, it allows rapid calculations through a multipole expansion technique. In this work, two additional possibilities for improving the performance are numerically examined. The first is an improvement of the convergence of the expansion by increasing the number of nearby cells, without including higher-order multipole moments. The second is an acceleration of the calculations by the particle–particle and particle–mesh/multipole expansion (PPPM/MPE) method, which uses fast Fourier transform instead of the hierarchy. For this purpose, the PPPM/MPE method originally developed by the authors for a periodic system is extended to a nonperiodic isolated system. The advantages and disadvantages of the GR and PPPM/MPE methods are discussed for both periodic and isolated systems. It is numerically shown that these methods with reasonable costs can reduce the error in potential felt by each particle to 0.1–1 kcal/mol, much smaller than the 30-kcal/mol error involved in conventional simple truncations. © 1994 by John Wiley & Sons, Inc.

To make improved treatments of electrostatic interactions in biomacromolecular simulations, two possibilities are considered. The first is the famous particle–particle and particle–mesh (PPPM) method developed by Hockney and Eastwood, and the second is a new one developed here in their spirit but by the use of the multipole expansion technique suggested by Ladd. It is then numerically found that the new PPPM method gives more accurate results for a two-particle system at small separation of particles. Preliminary numerical examination of the various computational methods for a single configuration of a model BPTI–water system containing about 24,000 particles indicates that both of the PPPM methods give far more accurate values with reasonable computational cost than do the conventional truncation methods. It is concluded the two PPPM methods are nearly comparable in overall performance for the many-particle systems, although the first method has the drawback that the accuracy in the total electrostatic energy is not high for configurations of charged particles randomly generated. © 1993 John Wiley & Sons, Inc.

We have developed a 20” desktop autostereoscopic projection display using a Curved-Directional-Reflection (CDR) screen. A liquid crystal on silicon (LCOS) and light emitting diode (LED) light sources have allowed the realization of compact 65 mm width projectors that are able to be put side by side at the interocular distance. The efficient optical system of an LED array as area-light-sources combined with an ultra high gain (