Validation of Platelet Counting Accuracy With the Celltac F Automated Hematology Analyzer

Rapid and accurate analysis of platelet count plays an important role in evaluating hemorrhagic status. Therefore, we evaluated platelet counting performance of a hematology analyzer, Celltac F (MEK-8222, Nihon Kohden Corporation, Tokyo, Japan), that features easy use with low reagent consumption and high throughput while occupying minimal space in the clinical laboratory. All blood samples were anticoagulated with dipotassium ethylenediaminetetraacetic acid (EDTA-2K). The samples were stored at room temperature (18^C–22^C) and tested within 4 hours of phlebotomy. We evaluated the counting ability of the Celltac F hematology analyzer by comparing it with the platelet counts obtained by the flow cytometry method that ISLH and ICSH recommended, and also the manual visual method by Unopette (Becton Dickinson Vacutainer Systems). The ICSH/ISLH reference method is based on the fact that platelets can be stained with monoclonal antibodies to CD41 and/or CD61. The dilution ratio was optimized after the precision, coincidence events, and debris counts were confirmed by the reference method. Good correlation of platelet count between the Celltac F and the ICSH/ISLH reference method (r = 0.99, and the manual visual method (r= 0.93) were obtained. The regressions were y = 0.90 x+9.0 and y=1.11x+8.4, respectively. We conclude that the Celltac F hematology analyzer for platelet counting was well suited to the ICSH/ISLH reference method for rapidness and reliability

Inhibition of L-Arginine Metabolizing Enzymes by L-Arginine-Derived Advanced Glycation End Products

Nω-Carboxymethyl-arginine (CMA), Nω-carboxyethyl-arginine (CEA) and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) have been identified as L-arginine-derived advanced glycation end products (AGEs) formed by non-enzymatic reactions between reducing sugars such as glucose and amino groups in proteins. These AGEs are structurally analogous to endogenous inhibitors of nitric oxide synthases (NOS) including NG-monomethyl-L-arginine (L-NMMA) and asymmetric NG,NG-dimethyl-L-arginine (ADMA). Increased plasma levels of these NOS inhibitors, and thus impaired generation of NO in vivo has been associated with the pathogenesis of vascular complications such as kidney failure and atherosclerosis. For these reasons we examined whether L-arginine-derived AGEs inhibit the activities of three L-arginine metabolizing enzymes including three isoforms of NOS (endothelium, neuronal and inducible NOS), dimethylarginine dimethylaminohydrolase (DDAH) that catalyzes the hydrolytic degradation of L-NMMA and ADMA to L-citrulline, and arginase that modulates intracellular L-arginine bioavailability. We found that AGEs inhibited the in vitro activities of endothelium type NOS weakly (IC50 values of CMA, CEA and MG-H1 were 830, 3870 and 1280 µM, respectively) and were also potential endogenous inhibitors for arginase (IC50 values of CMA and CML were 1470 and 1060 µM), but were poor inhibitors for DDAH. These results suggest that the tested L-arginine- and L-lysine-derived AGEs appear not to impair NO biosynthesis directly

Auto-Luminescent Genetically-Encoded Ratiometric Indicator for Real-Time Ca2+ Imaging at the Single Cell Level

Background: Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. Methodology/Principal Findings: We applied BRET to develop an autoluminescent Ca2+ indicator, BRAC, which is composed of Ca^[2+]-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent protein's chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful visualization of Ca2+ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca^[2+]-independent signal drifts due to change in cell shape, focus shift, etc. Conclusions/Significance: The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca2+ imaging not only in single live cells but also in small living subjects

Japanese VLBI Network observations of radio-loud narrow-line Seyfert 1 galaxies

We performed phase-reference very long baseline interferometry (VLBI) observations on five radio-loud narrow-line Seyfert 1 galaxies (NLS1s) at 8.4 GHz with the Japanese VLBI Network (JVN). Each of the five targets (RXS J08066+7248, RXS J16290+4007, RXS J16333+4718, RXS J16446+2619, and B3 1702+457) in milli-Jansky levels were detected and unresolved in milli-arcsecond resolutions, i.e., with brightness temperatures higher than 10^7 K. The nonthermal processes of active galactic nuclei (AGN) activity, rather than starbursts, are predominantly responsible for the radio emissions from these NLS1s. Out of the nine known radio-loud NLS1s, including the ones chosen for this study, we found that the four most radio-loud objects exclusively have inverted spectra. This suggests a possibility that these NLS1s are radio-loud due to Doppler beaming, which can apparently enhance both the radio power and the spectral frequency.Comment: 8 pages, 2 figures, accepted for publication in PAS

An Autopsy Case of Carcinosarcoma of the Esophagus

A case of carcinosarcoma, a rare polypoid tumor of the esophagus is presented. The characteristic gross and microscopic features as well as a discussion of the histogenesis of the sarcomatous elements are presented by microscopic, immunohistochemical and electron micronscopic examinations. Immunohistochemically, keratin and EMA (epithelial membrane antigen) were demonstrated in the islands of squamous cell carcinoma within the sarcomatous elements and in the carcinoma in situ at the border of normal mucosa. Vimentin, desmin, actin, myoglobin, factor VIII, S-100 protein, NSE, neuraminidase were not demonstrated in both the carcinomatous and the sarcomatous elements except for a positive reactivity to a-1-antichymotrypsin in the sarcomatous elements at part. It is suggested that the sarcomatous elements are of epithelial origin based on the facts as follows : 1 transition from overlying epithelium or carcinomatous islands to sarcomatous elements existent ; 2 some small tubules were formed within the sarcomatous elements, which showed transition into the sarcomatous elements ; and 3 a part of the sarcomatous elements revealed either positive or weak reactivity to keratin and EMA. Further, weak reactivity to keratin and EMA in the more anaplastic lesion may reflect the lack of tonofilaments and desmosomes in the ultrastructural findings

A high-quality, haplotype-phased genome reconstruction reveals unexpected haplotype diversity in a pearl oyster

Homologous chromosomes in the diploid genome are thought to contain equivalent genetic information, but this common concept has not been fully verified in animal genomes with high heterozygosity. Here we report a near-complete, haplotype-phased, genome assembly of the pearl oyster, Pinctada fucata, using hi-fidelity (HiFi) long reads and chromosome conformation capture data. This assembly includes 14 pairs of long scaffolds (>38 Mb) corresponding to chromosomes (2n = 28). The accuracy of the assembly, as measured by an analysis of k-mers, is estimated to be 99.99997%. Moreover, the haplotypes contain 95.2% and 95.9%, respectively, complete and single-copy BUSCO genes, demonstrating the high quality of the assembly. Transposons comprise 53.3% of the assembly and are a major contributor to structural variations. Despite overall collinearity between haplotypes, one of the chromosomal scaffolds contains megabase-scale non-syntenic regions, which necessarily have never been detected and resolved in conventional haplotype-merged assemblies. These regions encode expanded gene families of NACHT, DZIP3/hRUL138-like HEPN, and immunoglobulin domains, multiplying the immunity gene repertoire, which we hypothesize is important for the innate immune capability of pearl oysters. The pearl oyster genome provides insight into remarkable haplotype diversity in animals

Phylogenetic comparisons reveal mosaic histories of larval and adult shell matrix protein deployment in pteriomorph bivalves

Molluscan shells are organo-mineral composites, in which the dominant calcium carbonate is intimately associated with an organic matrix comprised mainly of proteins and polysaccharides. However, whether the various shell matrix proteins (SMPs) date to the origin of hard skeletons in the Cambrian, or whether they represent later deployment through adaptive evolution, is still debated. In order to address this issue and to better understand the origins and evolution of biomineralization, phylogenetic analyses have been performed on the three SMP families, Von Willebrand factor type A (VWA) and chitin-binding domain-containing protein (VWA-CB dcp), chitobiase, and carbonic anhydrase (CA), which exist in both larval and adult shell proteomes in the bivalves, Crassostrea gigas and Pinctada fucata. In VWA-CB dcp and chitobiase, paralogs for larval and adult SMPs evolved before the divergence of these species. CA-SMPs have been taken as evidence for ancient origins of SMPs by their presumed indispensable function in biomineralization and ubiquitous distribution in molluscs. However, our results indicate gene duplications that gave rise to separate deployments as larval and adult CA-SMPs occurred independently in each lineage after their divergence, which is considerably more recent than hitherto assumed, supporting the "recent heritage and fast evolution" scenario for SMP evolution

Effects of demethylating agent 5-aza-2 '-deoxycytidine and histone deacetylase inhibitor FR901228 on maspin gene expression in oral cancer cell lines

Maspin, which belongs to the serine protease inhibitor (serpin) superfamily, has been proposed as a potent tumor suppressor that inhibits cell motility, invasion, angiogenesis, and metastasis. In the present study, we examined the effects of 5-aza-2'-deoxycytidine (5-aza-dC), a demethylating agent, and FR901228, a histone deacetylase (HDAC) inhibitor, on maspin expression in oral cancer cell tines. The expression levels of maspin mRNA were divided into two groups, which was the maspin tow-expressed and high-expressed cell lines in the 12 oral cancer cell lines. The maspin promoter contained only a few methylated CpG sites in the maspin low-expressed cell lines. Moreover, the methylation status was not altered after 5-aza-dC treatment. However, the transcription of the maspin gene was clearly increased following 5-aza-dC treatment in a number of oral cancer cell tines. These results imply that an action of 5-aza-dC is separate from induction of promoter demethylation. Treatment with FR901228 resulted in a time-dependent stimulation of the re-expression of maspin mRNA as early as 4 h after treatment in the maspin downregulated cells. The re-expression of the maspin gene may contribute to the recuperation of biological functions linked to FR901228 such as an inhibitory effect on tumor angiogenesis and cell invasion. These results indicate that maspin and its target genes may be excellent leads for future studies on the potential benefits of FR901228, a HDAC inhibitor, in cancer therapy.</p