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An 11-month-old boy with productive cough was referred to our hospital. He had nasal obstruction immediately after birth, and wheezing, wet cough, and rhinorrhea were observed daily after the neonatal period. Clinical and imaging findings revealed secretory otitis media, chronic sinusitis, and bronchiectasis. Primary ciliary dyskinesia was suspected. Transmission electron microscopy of nasal cilia showed defects of the outer and inner dynein arms. Genetic examinations of the family revealed copy number variation in PIH1 domain-containing 3 (PIH1D3) in the proband and mother. This is the first report of a Japanese patient with primary ciliary dyskinesia caused by copy number variation in PIH1D3.

Angelman syndrome is a neurodevelopmental disorder characterized by intellectual disability (ID), a distinctive gait pattern, abnormal behaviors, severe impairment in language development, and characteristic facial features. Most cases are caused by the absence of a maternal contribution to the imprinted region on chromosome 15q11-q13. Here, we present the first reported case of a 3-year-old boy with an atypical phenotype of Angelman syndrome due to uniparental isodisomy with two recessive homozygous pathogenic variants: in HERC2 and AP3B2. Known phenotypes related to HERC2 and AP3B2 include ID and early infantile epileptic encephalopathy, respectively. The patient had severe global developmental delay and profound ID and showed a happy demeanor, stereotypic laughter, and hand-flapping movements, but also irritability. Craniofacial dysmorphic features, including brachycephaly, strabismus, wide ala nasi, short philtrum, wide open mouth, and slight hypopigmentation were seen. Progressive microcephaly was noted. Magnetic resonance imaging of the brain showed delayed myelination and cerebral atrophy. Trio whole exome sequencing and CGH-SNP array analysis revealed paternal uniparental isodisomy of chromosome 15 and two coexisting recessive diseases resulting from homozygous HERC2 and AP3B2 pathogenic variants. The pathogenic variant in HERC2 was inherited from his heterozygous-carrier father, and the variant in AP3B2 was de novo. We suppose that these unusual features were the combination of the effect of three concomitant disorders.

Objective To explore how cytochalasin D (CyD) affects clot initiation and to compare clotting times (CTs) of EXTEM and FIBTEM on rotational thromboelastometry in cardiac surgical patients undergoing cardiopulmonary bypass (CPB). Design Retrospective cohort study with translational in vitro coagulation experiments. Setting Single-center, tertiary, academic medical center. Participants Patients who underwent cardiac surgery with CPB between November 2015 and August 2017. Intervention None. Measurements and Main Results The study's primary measurements were CTEXTEM and CTFIBTEM before and after CPB. Additionally, the authors performed translational in vitro coagulation experiments using commercial plasma. In these experiments, the impact of CyD on in vitro thrombin generation (TG) was assessed using 10 platelet-rich plasma (PRP) samples and calibrated automated thrombogram. The impact of CyD on ROTEM-CT also was evaluated in vitro using the same 10 PRP samples. One hundred fifty-three patients had clinical CTEXTEM and CTFIBTEM measurements. CTFIBTEM was shorter than CTEXTEM before and after CPB by 6.8% (95% confidence interval [CI], 5.5-8.1) and 8.9% (95% CI, 4.7-13.0), respectively. These results correlated with in vitro experiments, where TG lag time was shortened by CyD and CTFIBTEM was shorter than CTEXTEM. Conclusion CyD shortens the onset of TG and clot formation, resulting in shorter CTFIBTEM than CTEXTEM. The authors’ data suggest that CTEXTEM and CTFIBTEM are not interchangeable. Additional clinical studies are warranted to assess if CTFIBTEM can be used to optimize the indication for plasma transfusion.

So far, the molecular mechanisms underlying the acidic-stress responses of plants are complicated and only fragmentally understood. Here, we investigated the mechanisms responsible for acidic-stress acclimation. Previously, DNA microarray analysis identified the sll1558 gene in Synechocystis sp. PCC 6803 (hereafter called Synechocystis 6803) to be upregulated following short-term acid treatment (1 h at pH 3.0). The sll1558 gene encodes uridine diphosphate-glucose pyrophosphorylase (UDP-glucose pyrophosphorylase), which catalyzes the conversion of glucose-1-phosphate into UDP-glucose. We constructed mutant cells for this gene and analyzed their phenotype. The sll1558 gene did not completely segregate in sll1558 mutant cells; thus, Sll1558 is essential for the survival of Synechocystis 6803. Besides, the partially disrupted sll1558 mutant cells were highly sensitive to acidic stress (pH 6.0) as well as other stress conditions (high salt, high osmolality, high/low temperature, and ultraviolet-B stress); the number of sll1558 transcripts increased under these conditions. UDP-glucose is used for the synthesis of various materials, such as glycolipids. From the membrane lipid composition analysis, digalactosyldiacylglycerol decreased and phosphatidylglycerol increased in the partially disrupted sll1558 mutant cells under acidic stress. These results suggest that sll1558 is important not only for the survival of Synechocystis 6803, but also for tolerance under various stress conditions.

We report the first Japanese case of primary ciliary dyskinesia caused by DNAH9 variations. The patient, a 5-year-old girl, had repeated episodes of productive cough after contracting the common cold at the age of 1 year and 6 months. She did not have a situs abnormality or congenital heart defect. We identified two novel DNAH9 variants, NM_001372.3: c. [1298C>G];[5547_5550delTGAC], (p.[Ser433Cys];[Asp1850fs]).

Objective Primary ciliary dyskinesia (PCD) is a rare hereditary disease. Most reports of PCD in Japan are case reports, and clinical analysis has not been performed. Differences in the causative genes might affect the clinical features in different ethnic groups. The purpose of this study was to clarify the clinical features of Japanese patients with PCD. Methods We performed a retrospective chart review of PCD patients seen at Mie University Hospital and patients whose blood samples were sent to us for genetic analysis from 2011 to 2020. Data on the following items were collected and analyzed: age at first visit to the hospital, age at diagnosis of PCD, process of referral to our facility, chief complaint, situs status, PrImary CiliARy DyskinesiA Rule (PICADAR) score, nasal nitric oxide concentration, otoscopic findings, rhinoscopic findings, and paranasal computed tomography scan findings. Results Sixty-seven patients (24 male, 43 female) were diagnosed with PCD during the study period. Age at diagnosis ranged from 2 months to 69 years (median, 17 years). Respiratory symptoms (77%) were the most common complaint, followed by nasal (15%) and aural (8%) symptoms. Situs inversus was present in 17 (25%) cases. Only 2 cases had congenital cardiac anomalies. The mean PICADAR score was 7.3 (range, 3-14) points. Approximately 50% of tympanic membranes showed retraction, suggesting otitis media with effusion. The mean Lund-Mackay score was 12.8 (range, 7-17) points, suggesting that the radiographic findings were not always severe. There was no significant difference in the total Lund-Mackay score between patients with and without situs inversus (12.7 vs. 12.6, respectively). Conclusion Situs inversus was present in 25% of Japanese PCD patients, which is much lower than observed in other countries. This is a result of differences in the major disease-causing genes. The general rule that "situs inversus is observed in approximately 50% of PCD patients" cannot be applied, at least, in Japanese PCD patients.

A 19-year-old-woman experienced severe burning pain in the lower extremities with erythema and swelling. She was diagnosed with primary erythromelalgia (PE). The pain was unresponsive to medications but relieved by immersing her feet in cold water. We performed a multilevel lumbar sympathetic ganglion block (LSGB) with 5% phenol at second lumbar vertebra (L2) and third lumbar vertebra (L3), and additional fourth lumbar vertebra (L4) levels. An epidural block was intermittently combined. The pain and skin lesions dramatically improved after the procedures, and she no longer needed medications or to soak her feet in cold water. This case demonstrated that extensive LSGB may be a therapeutic option for intractable PE.

International audience; In the forward end-cap of the Belle II spectrometer, an innovative proximity focusing Ring Imaging Cherenkov counter with a multilayer focusing aerogel radiator has been installed. The detector is designed to be operated in a magentic field of 1.5 T, and consists of a double layer aerogel radiator, an expansion volume and a photon detector. In total 420 Hamamatsu hybrid avalanche photo sensors with 144 channels each are used to read out single Cherenkov photons with high efficiency. We expect the device to provide better than 4s separation of pions from kaons in the full kinmatic region of the experiment, from 0.5 GeV/c to 4 GeV/c. The detector components have been successfully produced and installed in the spectrometer. After the commissioning phase in 2018, the detector is now included in the Belle II data taking, and is expected to contribute substantially to the performance of the spectrometer in looking for rare decays of B and D mesons, and of tau leptons. In this contribution we review the detector design and present results of the first detector performance studies.

Background Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by functional impairment of cilia throughout the body. The involvement of copy number variation (CNV) in the development of PCD is largely unknown. Methods We examined 93 Japanese patients with clinically suspected PCD from 84 unrelated families. CNV was examined either by exome sequencing of a PCD gene panel or by whole‐exome sequencing (WES). The identified alterations were validated by PCR and Sanger sequencing. Nasal ciliary ultrastructure was examined by electron microscopy. Results Analysis of CNV by the panel or WES revealed a biallelic deletion in the dynein regulatory complex subunit 1 (DRC1) gene in 21 patients, which accounted for 49% of the PCD patients in whom a disease‐causing gene was found. Sanger sequencing of the PCR product revealed a 27,748‐bp biallelic deletion including exons 1–4 of DRC1 with identical breakpoints in all 21 patients. The ciliary ultrastructure of the patients with this CNV showed axonemal disorganization and the loss or gain of central microtubules. Conclusion The deletion of DRC1 is the major cause of PCD in Japan and this alteration can cause various ciliary ultrastructural abnormalities., Analysis of copy number variation revealed a biallelic deletion in the dynein regulatory complex subunit 1 (DRC1) gene in 21 patients, which accounted for 49% of the primary ciliary dyskinesia patients in whom a disease‐causing gene was found. Sanger sequencing of the PCR product revealed a 27,748‐bp biallelic deletion including exons 1–4 of DRC1 with identical breakpoints in all 21 patients. The ciliary ultrastructure of the patients with this copy number variation showed axonemal disorganization and the loss or gain of central microtubules.

Background The main cause of unsuccessful peripheral radial artery catheterization using traditional palpation is imprecisely locating the arterial center. The authors evaluated factors causing disparities between the arterial centers determined by palpation versus ultrasound. The authors applied them to create and test a novel catheterization training program. Methods The arterial central axis was determined by ultrasound and palpation in 350 adults. Potential independent predictors of disparity included sex, body mass index, pulse pressure, transverse arterial diameter, subcutaneous arterial depth, chronic hypertension, and experience as an anesthesiologist (less than 3 vs. greater than or equal to 3 yr). Using the results, the authors developed a radial artery catheterization training program. It was tested by enrolling 20 first-year interns, randomized to a training or control group. The time to successful insertion was the primary outcome measure. The success rate and time required for catheterization by palpation were evaluated in 100 adult patients per group. Results Independent predictors of central axis disparity were pulse pressure, subcutaneous radial artery depth, years of experience, and chronic hypertension. Training improved the catheterization time (training group 56 ± 2 s vs. control group 109 ± 2 s; difference –53 ± 3 s; 95% CI, –70 to –36 s; P < 0.0001) and total success rate (training group 83 of 100 attempts, 83%; 95% CI, 75 to 90 vs. control group 57 of 100, 57%; 95% CI, 47 to 66; odds ratio, 3.7; 95% CI, 2.7 to 5.1). Conclusions Misjudging the central axis position of the radial artery is common with a weak pulse and/or deep artery. The authors’ program, which focused on both these issues, shortened the time for palpation-guided catheterization and improved success.

Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by structural and/or functional impairment of cilia throughout the whole body. Early diagnosis of PCD is important for the prevention of long‑term sequelae, however early diagnosis is a challenge due to the phenotypic heterogeneity of PCD. In the current study, the patient with PCD was diagnosed at nine years old following several efforts to control intractable airway symptoms. The patient experienced a chronic productive cough beginning in early childhood and had multiple episodes of pneumonia and otitis media with effusion and sinusitis. No situs inversus or other heterotaxias were reported. Serial chest X‑rays exhibited persistent atelectasis and bronchiectasis in the right middle lobe. When the patient was nine years old, electron microscopy of his cilia and genetic analysis were conducted. Electron microscopy of a biopsy specimen from the nasal mucosa indicated loss of the outer dynein arms. Whole‑exome analysis of the genome demonstrated the presence of compound heterozygous mutations in DNAH5: NM_001369.2:c.5983C>T, p.Arg1995X in exon 36 and NM_001369.2:c.9101delG, p.Gly3034ValfsX22 in exon 54; neither of which have been previously reported in the literature in a Japanese patient. Notably, this case is, to the best of our knowledge, the first reported case of PCD caused by the DNAH5 mutation in a Japanese patient.

BACKGROUND Incomplete reversal with a recommended 5-g dose of idarucizumab has been reported in patients with excessively high dabigatran concentrations. A timely detection of reversal failure after idarucizumab using whole blood (WB) coagulation testing is clinically useful. The aims of this study were to determine residual dabigatran activity after idarucizumab on thrombin generation (TG) using in vitro supratherapeutic dabigatran models and to compare 4 WB point-of-care tests (activated partial thromboplastin time [aPTT], prothrombin time [PT], and 2 thromboelastometry tests) with the TG results. METHODS Blood samples from 12 healthy volunteers were spiked in vitro with 0-5000 ng/mL of dabigatran. Dabigatran reversal was evaluated by adding 1000 μg/mL of idarucizumab (Praxbind) to dabigatran-spiked samples, which reflect the administration of 5-g idarucizumab to a 70-kg patient. Residual dabigatran activity was assessed using the calibrated automated TG (Thrombinoscope) in platelet-poor plasma samples. The TG results were compared with WB aPTT (DRIHEMATO APTT-S) and PT (DRIHEMATO PT-S) using CG02N analyzer, thromboelastometry (ROTEM) triggered by ellagic acid (INTEM) and tissue factor (EXTEM). RESULTS At a therapeutic concentration of dabigatran (200 ng/mL), the lag time was prolonged, and peak TG was decreased. The effects of dabigatran on TG were increased up to 1000 ng/mL, and TG was obliterated at higher supratherapeutic dabigatran levels (P < .001 versus control, respectively). TG was fully restored with idarucizumab when dabigatran was ≤2000 ng/mL, but residual anticoagulant activity was observed at higher dabigatran levels. Dabigatran prolonged WB aPTT and PT concentration dependently, and residual prolongations were observed when idarucizumab was added to 3000 or 5000 ng/mL of dabigatran (P < .001 versus control, respectively). In contrast, both INTEM and EXTEM clotting times were reversed toward reference ranges at all dabigatran concentrations when idarucizumab was added. CONCLUSIONS Our data indicate that the recommended dose of idarucizumab may not restore TG completely with excessively elevated concentrations of dabigatran. All WB measurements with aPTT, PT, and thromboelastometry predicted supratherapeutic dabigatran concentrations, whereas those tests varied in sensitivity to residual anticoagulant activity after reversal. WB aPTT corresponded well with plasma TG changes among those measurements, but the use of thromboelastometry may overestimate the effect of idarucizumab. Caution should be exercised before extrapolating in vitro point-of-care data to the clinical monitoring of dabigatran reversal.

Membrane vesicles released from bacteria contribute to cell–cell communication by carrying various cargos such as proteins, nucleic acids and signaling molecules. Cellulolytic bacteria have been isolated from many environments, yet the function of membrane vesicles for cellulolytic ability has been rarely described. Here, we show that a Gram-positive cellulolytic bacterium Clostridium thermocellum released membrane vesicles, each approximately 50–300 nm in diameter, into the broth. The observations with immunoelectron microscopy also revealed that cellulosomes, which are carbohydrate-active enzyme complexes that give C. thermocellum high cellulolytic activity, localized on the surface of the membrane vesicles. The membrane vesicles collected by ultracentrifugation maintained the cellulolytic activity. Supplementation with the biosurfactant surfactin or sonication treatment disrupted the membrane vesicles in the exoproteome of C. thermocellum and significantly decreased the degradation activity of the exoproteome for microcrystalline cellulose. However, these did not affect the degradation activity for soluble carboxymethyl cellulose. These results suggest a novel function of membrane vesicles: C. thermocellum releases cellulolytic enzymes on the surface of membrane vesicles to enhance the cellulolytic activity of C. thermocellum for crystalline cellulose.

BACKGROUND Recombinant activated factor VII (rFVIIa) concentrate reduces allogeneic blood transfusions, but it may increase thromboembolic complications in complex cardiac surgery. The mixture of activated factor VII (FVIIa) and factor X (FX) (FVIIa/FX) (FVIIa:FX = 1:10) is a novel bypassing agent for hemophilia patients. We hypothesized that the combination of FX and FVIIa could improve thrombin generation (TG) in acquired multifactorial coagulation defects such as seen in cardiac surgery and conducted in vitro evaluation of FVIIa/FX in parallel with other coagulation factor concentrates using in vitro and in vivo diluted plasma samples. METHODS Plasma samples were collected from 9 healthy volunteers and 12 cardiac surgical patients. We measured TG (Thrombinoscope) using in vitro 50% dilution plasma and in vivo dilution plasma after cardiopulmonary bypass, in parallel with thromboelastometry (ROTEM) and standard coagulation assays. In vitro additions of FVIIa/FX (0.35, 0.7, and 1.4 μg/mL, based on the FVIIa level), rFVIIa (1.4, 2.8, and 6.4 μg/mL), prothrombin complex concentrate (0.3 international unit), and 20% plasma replacement were evaluated. RESULTS In diluted plasma, the addition of either FVIIa/FX or rFVIIa shortened the lag time and increased the peak TG, but the effect in lag time of FVIIa/FX at 0.35 μg/mL was more extensive than rFVIIa at 6.4 μg/mL. Prothrombin complex concentrate increased peak TG by increasing the prothrombin level but failed to shorten the lag time. No improvement in any of the TG variables was observed after 20% volume replacement with plasma. The addition of factor concentrates normalized prothrombin time/international normalized ratio but not with plasma replacement. In cardiac patients, similar patterns were observed on TG in post-cardiopulmonary bypass samples. FVIIa/FX shortened clotting time (CT) in a concentration-dependent manner on CT on thromboelastometry. Plasma replacement did not improve CT, but a combination of plasma and FVIIa/FX (0.35 μg/mL) more effectively shortened CT than FVIIa/FX alone. CONCLUSIONS The combination of FVIIa and FX improved TG more efficiently than rFVIIa alone or plasma in dilutional coagulopathy models. The required FVIIa dose in FVIIa/FX was considerably lower than those reported during bypassing therapy in hemophilia patients (1.4-2.8 μg/mL). The combination of plasma could restore coagulation more efficiently compared to FVIIa/FX alone. Lesser FVIIa requirement to exert procoagulant activity may be favorable in terms of reducing systemic thromboembolic complications.

Primary ciliary dyskinesia (PCD) is a genetic disease inherited in an autosomal recessive manner. The prevalence of PCD is estimated to be 1 in 20,000 live births. Congenital abnormality of the primary cilia results in situs inversus in 50% of patients. Decreased function of motile cilia causes chronic rhinosinusitis, otitis media with effusion, bronchiectasis and infertility. Cases with situs inversus are considered to show "Kartagener's syndrome", and diagnosis is not difficult. However, in cases without situs inversus, the diagnosis is much more troublesome. PCD without situs inversus is thus probably underdiagnosed. Prolonged chronic cough represents an important symptom that is seen in most patients. The diagnosis of PCD requires the presence of the characteristic clinical phenotypes and either: (1) specific ciliary ultrastructural defects identified by transmission electron microscopy in biopsy samples of respiratory epithelium; or (2) identification of mutation in one of the genes known to be associated with PCD. Nasal nitric oxide concentration is extremely low in PCD, and this could be useful for screening of the disease. At present, no fundamental therapies are available for PCD. Diagnosis in the early stages is important to prevent progression of bronchiectasis and deterioration of lung function by guidance for daily life, immunization, cessation of smoking and prompt therapy at the time of respiratory tract infection. Since PCD is inherited in an autosomal-recessive manner, genetic counseling is necessary after definite diagnosis.

Objectives Platelet defect mechanisms after cardiopulmonary bypass remain unclear. Our hypothesis microRNA expressions in circulating platelets significantly change between pre and post cardiopulmonary bypass, and consequent messenger RNA and protein expression level alterations cause postcardiopulmonary bypass platelet defect. Design Single-center prospective observational study. Setting Operating room of Kyoto Prefectural University of Medicine. Patients Twenty-five adult patients scheduled for elective cardiac surgeries under cardiopulmonary bypass. Interventions None. Measurements and main results In the initial phase, changes in microRNA expression between pre and post cardiopulmonary bypass underwent next generation sequencing analysis (10 patients). Based on the results, we focused on changes in mir-10b and mir-96, which regulate glycoprotein 1b and vesicle-associated membrane protein 8, respectively, and followed them until messenger RNA and protein syntheses (15 patients) using quantitative polymerase chain reaction and Western blotting. Seven microRNAs including mir-10b and mir-96 exhibited significant differences in the initial phase. In the subsequent phase, mir-10b-5p and mir-96-5p overexpressions were confirmed, and glycoprotein 1b and vesicle-associated membrane protein 8 messenger RNA levels were significantly decreased after cardiopulmonary bypass: fold differences (95% CI): mir-10b-5p: 1.35 (1.05-2.85), p value equals to 0.01; mir-96-5p: 1.59 (1.06-2.13), p value equals to 0.03; glycoprotein 1b messenger RNA: 0.46 (0.32-0.60), p value of less than 0.001; and vesicle-associated membrane protein messenger RNA: 0.70 (0.56-0.84), p value of less than 0.001. Glycoprotein 1b and vesicle-associated membrane protein 8 were also significantly decreased after cardiopulmonary bypass: glycoprotein 1b: 82.6% (71.3-93.8%), p value equals to 0.005; vesicle-associated membrane protein 8: 79.0% (70.7-82.3%), p value of less than 0.001. Conclusions Expressions of several microRNAs in circulating platelets significantly changed between pre and post cardiopulmonary bypass. Overexpressions of mir-10b and mir-96 decreased glycoprotein 1b and vesicle-associated membrane protein 8 messenger RNA as well as protein, possibly causing platelet defect after cardiopulmonary bypass.

Objective To evaluate otologic features of primary ciliary dyskinesia (PCD), especially eardrum features, audiometric findings, and clinical course. Study design Retrospective patient review. Setting Tertiary referral center. Patients Fifteen patients (mean age, 16.9 years [range, 1-32 yr]; 8 males and 7 females) diagnosed with PCD at our university hospital in the last 12 years. Intervention Diagnostic. Main outcome measures Electron microscopy of nasal cilia, gene mutation analysis, endoscopy of 30 eardrums, pure-tone audiometry, and tympanometry. Results All 15 patients showed ciliary ultrastructural abnormalities on electron microscopy and/or biallelic mutations in genes associated with ciliary function or structure. All 30 eardrums examined showed certain abnormalities. Fourteen patients had otitis media with effusion or its sequelae. The remaining patient had chronic otitis media. Pure-tone audiometry revealed the mean air conduction thresholds to be 25.0 and 26.4 dB in the right and left ears, respectively. In the ears with better hearing and worse hearing, the mean air conduction thresholds were 22.3 and 29.0 dB respectively. Conclusion Otologic disease among patients with PCD essentially comprised otitis media with effusion, and the patients' eardrums showed a variety of findings. Knowledge of these otologic features may lead to the early detection of PCD.

Point-of-care (POC) devices allow for prothrombin time/international normalized ratio (PT/INR) testing in whole blood (WB) and timely administration of plasma or prothrombin complex concentrate during cardiopulmonary bypass surgery. This study evaluated the sensitivities of a new POC PT test, a dry-hematology method with heparin neutralization technology (DRIHEMATO PT-S [DRI PT-S]; AT Corporation, Kanagawa, Japan), and compared it with other POC tests currently available.Prospective, observational study.University hospital, single center.Healthy volunteers and warfarin-treated and cardiac surgical patients.In WB samples obtained from 6 healthy volunteers, PT-INR results of DRI PT-S were not affected by an in vitro addition of heparin6.0 U/mL. In warfarin-treated samples (n = 88, PT/INR 0.98-3.87), PT-INR with DRI PT-S showed acceptable correlation with the laboratory method (rThis study demonstrated that PT/INR can be accurately assessed using the dry-hematology method in WB under therapeutic heparin levels. Currently available other POC PT/INR tests are affected by heparin, and thus they are not recommended for coagulation monitoring during cardiopulmonary bypass.

Using recently-developed Ag stress-migration bonding (SMB), a light-emitting diode (LED) die can be bonded on a glass substrate above 200 °C in air without pressure, where both the die and substrate surfaces are coated with a few-microns-thick Ag layer. We investigate this emerging die-attach method and reveal the detailed bonding processes. The thermal residual stress in the Ag coating layer causes hillock formation on both the surfaces, and the extruded Ag grains grow to fill the gap between the contacted surfaces. A sound contact over the large bonded area can be achieved without applying any pressure. However, the LED dies exposed to a high working temperature may increase the risk of void formation in the bonding interface structure, leading to a crack or interface failure due to the bond strength degradation. In this study, we insert a barrier layer of a high melting-point metal like Pt or Ru under the Ag bond layer to prevent void diffusion from the Ag layer. The high bond strength achieved by Ag SMB is then unaffected by the heat exposure tests even at 440 °C for 2 h, assuring sufficiently long lifetime for the LED die-attach. Our pressureless Ag SMB method thus achieves excellent thermal stability, in addition to the ideal reflectivity and electric/heat conductivities of Ag, perfectly suitable for LED applications, as well as various other power devices of high-temperature wide-bandgap power semiconductors like GaN and SiC.

Living organisms must defend themselves against various environmental stresses. Extracellular polysaccharide-producing cells exhibit enhanced tolerance toward adverse environmental stress. In Synechocystis sp. PCC6803 (Synechocystis), lipopolysaccharide (LPS) may play a role in this protection. To examine the relationship between stress tolerance of Synechocystis and LPS, we focused on Slr2019 because Slr2019 is homologous to MsbA in Escherichia coli, which is related to LPS synthesis. First, to obtain a defective mutant of LPS, we constructed the slr2019 insertion mutant (slr2019) strain. Sodium deoxycholate-polyacrylamide gel electrophoresis indicated that slr2019 strain did not synthesize normal LPS. Second, to clarify the participation of LPS in acid tolerance, wild type (WT) and slr2019 strain were grown under acid stress; slr2019 strain growth was significantly weaker than WT growth. Third, to examine influences on stress tolerance, slr2019 strain was grown under various stresses. Under salinity and temperature stress, slr2019 strain grew significantly slower than WT. To confirm cell morphology, cell shape and envelope of slr2019 strain were observed by transmission electron microscopy; slr2019 cells contained more electron-transparent bodies than WT cells. Finally, to confirm whether electron-transparent bodies are poly-3-hydroxybutyrate (PHB), slr2019 strain was stained with Nile Blue A, a PHB detector, and observed by fluorescence microscopy. The PHB granule content ratio of WT and slr2019 strain grown at BG-11 pH 8.0 was each 7.18 and 8.41 %. At pH 6.0, the PHB granule content ratio of WT and slr2019 strain was 2.99 and 2.60 %. However, the PHB granule content ratio of WT and slr2019 strain grown at BG-11N-reduced was 10.82 and 0.56 %. Because slr2019 strain significantly decreased PHB under BG-11N-reduced compared with WT, LPS synthesis may be related to PHB under particular conditions. These results indicated that Slr2019 is necessary for Synechocystis survival in various stresses.

Background Anucleate platelets can undergo apoptosis in response to various stimuli, as do nucleated cells. Cardiopulmonary bypass (CPB) causes platelet dysfunction and can also activate platelet apoptotic pathways. We therefore evaluated time-dependent changes in blood platelet Bax (a pro-apoptotic molecule) levels and platelet dysfunction after cardiac surgery. Methods We assessed blood samples obtained from subjects having on-pump or off-pump coronary artery bypass graft surgery (n=20 each). We also evaluated the in vitro effects of platelet Bax increase in eight healthy volunteers. Results Thrombin-induced platelet calcium mobilisation and platelet-surface glycoprotein Ib (GPIb) expression were lowest at weaning from CPB and did not recover on postoperative day one. On-pump surgery increased platelet expression of Bax, especially the oligomerised form, along with translocation of Bax from the cytosol to mitochondria and platelet-surface tumour necrosis factor-alpha (TNF-α)–converting enzyme (TACE) expression. In contrast, mitochondrial cytochrome c expression was reduced. While similar in direction, the magnitude of the observed changes was smaller in patients having off-pump surgery. In vitro, a cell-permeable Bax peptide increased platelet Bax expression to the same extent seen during bypass and produced similar platelet changes. These apoptotic-like changes were largely reversed by Bcl-xL pre-administration, and were completely reversed by combined application of inhibitors that stabilise outer mitochondrial membrane permeability and TACE. Conclusions CPB increases platelet Bax expression, which contributes to reduced platelet-surface GPIb expression and thrombin-induced platelet calcium changes. These changes in platelet apoptotic signalling might contribute to platelet dysfunction after CPB. Clinical trial registration UMIN Clinical Trials Registry (number UMIN000006033).

BackgroundChildren with cyanotic heart disease develop secondary erythrocytosis and thrombocytopenia via unknown mechanisms. Mature erythrocyte microRNAs may reflect clinical pathologies and cell differentiation processes pre-enucleation. This study evaluated erythrocyte microRNAs in children with cyanotic heart disease.MethodsErythrocyte microRNAs from children with cyanotic and acyanotic heart disease and without cardiac disease were quantified with Ion PGM System (n=10 per group). Differential expression was confirmed by quantitative PCR (qPCR; n=20 per group).ResultsMir-486-3p, mir-486-5p, and mir-155-5p increased in patients with cyanotic heart disease compared with those without heart disease: fold differences (95% confidence interval): mir-486-3p: 1.92 (1.14-3.23), P=0.011; mir-486-5p: 2.27 (1.41-3.65), P

Objective Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by functional impairment of cilia throughout the body. The early diagnosis of PCD is important for the prevention of long-term sequelae; however, this is often challenging because of the phenotypic heterogeneity of PCD and difficulty in genetic analysis. The majority of PCD patients in Japan are not diagnosed properly. To diagnose PCD more accurately, we developed a targeted next-generation sequencing (NGS) panel. Methods We examined 46 patients (age range, 1–64 years; 23 male and 23 female) who were clinically suspected of PCD. First, mutation hotspots in DNAH5 and DNAI1 were sequenced by the Sanger method. Next, exome sequencing was performed in 32 known PCD genes using our novel NGS panel with the Ion Torrent PGM system. Variant annotation was generated by Ion Reporter Version 5.0 (Life Technologies). Mutations found in the panel were validated by Sanger sequencing. Results Disease-causing gene mutations were found in 10 patients from 7 families: DNAH5 in 4 families, and DNAI1, CCDC40, and RSPH4A in 1 family each. Heterozygous mutations were found in 1 patient. The majority of the mutations found in the present analysis were novel. Conclusion Japanese PCD patients have novel mutations in cilia-related genes. This targeted NGS panel can identify disease-causing mutations in patients with PCD.

This paper describes the development and experimental results of a process control system. The system enabled adjusting film thickness and in-film dopant concentration at the same time on a batch LPCVD machine. By this, anyone could adjust process results easily and exactly in a short time.

Background Platelet (PLT) and plasma transfusion remain the mainstay hemostatic therapy for perioperative bleeding. Several studies have indicated that acquired fibrinogen (FIB) deficiency can be the primary cause of bleeding after cardiac surgery. The aim of this study was to compare hematologic and transfusion profiles between the first-line FIB replacement and PLT transfusion in post–cardiac surgical bleeding. Study Design and Methods In this prospective, randomized, open-label study, 20 adult patients who underwent valve replacement or repair and fulfilled preset visual bleeding scale were randomized to 4 g of FIB or 1 unit of apheresis PLTs. Primary endpoints included hemostatic condition in the surgical field and 24-hour hemostatic product usage. Hematologic data, clinical outcome, and safety data were collected up to the 28th day postoperative visit. Results In patients who received the first-line FIB concentrate (n = 10), the visual bleeding scale improved after intervention, and the incidence of PLT transfusion and total plasma donor exposure were lower compared to the PLT group (n = 10). Postintervention FIB level was statistically higher (209 mg/dL vs. 165 mg/dL) in the FIB group than in the PLT group, but PLT count and prothrombin were lower. There were no statistical differences in the postoperative blood loss and red blood cell transfusion between two groups. Conclusions Our preliminary data indicate that the primary FIB replacement may potentially reduce the incidence of PLT transfusion and the number of donor exposures. Plasma FIB level of 200 mg/dL is attainable with a single dose of 4 g, and this level seems to mitigate bleeding despite moderately decreased thrombin generation.

A light-emitting diode (LED) die and a glass substrate, both of which were coated with a few-micrometers-thick silver layer, was successfully direct-bonded above 200°C in air without any pressure. In this silver direct bonding, the following three factors are essential: 1) keeping surfaces of silver layers clean; 2) bonding with a suitable solvent that can be reduced and has a boiling point near the sintering temperature; and 3) avoiding excessive heating. Oxygen concentration in a sintering atmosphere has a great impact on bonding. The surface of a silver layer shows roughening by abnormal grain growth as increasing oxygen resulting in increase of bonding strength. The abnormal grain growth on the surface of silver effectively contributes to the increase of junction area. Sintering at too high temperature, however, causes the formation of large voids, which migrated towards silver/glass interfaces resulting in the reduction of bonding strength. An LED die bonded at 240°C for 2 h can maintain high bonding strength through a heat run test at 350°C for 2 h. Furthermore, transient thermal resistance of an LED die bonded with the silver direct bonding is lower than that of an LED die bonded by traditional eutectic bonding with Au-Sn alloy. This bonding method can realize high thermal conductivity with low-temperature pressure-less process and can contribute to high-power semiconductor devices.

The main cause of unsuccessful peripheral radial artery catheterization using traditional palpation is imprecisely locating the arterial center. The authors evaluated factors causing disparities between the arterial centers determined by palpation versus ultrasound. The authors applied them to create and test a novel catheterization training program.The arterial central axis was determined by ultrasound and palpation in 350 adults. Potential independent predictors of disparity included sex, body mass index, pulse pressure, transverse arterial diameter, subcutaneous arterial depth, chronic hypertension, and experience as an anesthesiologist (less than 3 vs. greater than or equal to 3 yr). Using the results, the authors developed a radial artery catheterization training program. It was tested by enrolling 20 first-year interns, randomized to a training or control group. The time to successful insertion was the primary outcome measure. The success rate and time required for catheterization by palpation were evaluated in 100 adult patients per group.Independent predictors of central axis disparity were pulse pressure, subcutaneous radial artery depth, years of experience, and chronic hypertension. Training improved the catheterization time (training group 56 ± 2 s vs. control group 109 ± 2 s; difference -53 ± 3 s; 95% CI, -70 to -36 s; P0.0001) and total success rate (training group 83 of 100 attempts, 83%; 95% CI, 75 to 90 vs. control group 57 of 100, 57%; 95% CI, 47 to 66; odds ratio, 3.7; 95% CI, 2.7 to 5.1).Misjudging the central axis position of the radial artery is common with a weak pulse and/or deep artery. The authors' program, which focused on both these issues, shortened the time for palpation-guided catheterization and improved success.

We developed the palm size (optical unit: 73[mm]×102[mm]×66[mm]) and light weight (total weight with electrical controller: 1.7[kg]) middle infrared (wavelength range: 8[μm]-14[μm]) 2-dimensional spectroscopy for UAV (Unmanned Air Vehicle) like drone. And we successfully demonstrated the flights with the developed hyperspectral camera mounted on the multi-copter so-called drone in 15/Sep./2015 at Kagawa prefecture in Japan. We had proposed 2 dimensional imaging type Fourier spectroscopy that was the near-common path temporal phase-shift interferometer. We install the variable phase shifter onto optical Fourier transform plane of infinity corrected imaging optical systems. The variable phase shifter was configured with a movable mirror and a fixed mirror. The movable mirror was actuated by the impact drive piezo-electric device (stroke: 4.5[mm], resolution: 0.01[μm], maker: Technohands Co.,Ltd., type:XDT50-45, price: around 1,000USD). We realized the wavefront division type and near common path interferometry that has strong robustness against mechanical vibrations. Without anti-mechanical vibration systems, the palm-size Fourier spectroscopy was realized. And we were able to utilize the small and low-cost middle infrared camera that was the micro borometer array (un-cooled VOxMicroborometer, pixel array: 336×256, pixel pitch: 17[μm], frame rate 60[Hz], maker: FLIR, type: Quark 336, price: around 5,000USD). And this apparatus was able to be operated by single board computer (Raspberry Pi.). Thus, total cost was less than 10,000 USD. We joined with KAMOME-PJ (Kanagawa Advanced MOdule for Material Evaluation Project) with DRONE FACTORY Corp., KUUSATSU Corp., Fuji Imvac Inc. And we successfully obtained the middle infrared spectroscopic imaging with multi-copter drone.

BACKGROUND The activated clotting time (ACT) is widely used for monitoring heparin anticoagulation during cardiac surgery. Celite-based ACT values are prolonged when aprotinin is administered. MDCO-2010, a novel serine protease inhibitor, is currently being evaluated as a possible alternative to aprotinin. Therefore, we evaluated the in vitro effects of this novel agent on ACT values using 3 different point-of-care instruments with kaolin or celite as an activator. METHODS The study was performed in 2 parts. In the first part, blood samples were obtained from 15 healthy volunteers. Samples were pipetted into small Eppendorf tubes and 2 concentrations of the MDCO-2010 (100 and 500 nM, final concentration) alone or with heparin (1.2 or 2.4 U/mL) were added. ACTs were measured using Helena (celite), Hemochron (kaolin), and Medtronic (kaolin) devices. In the second part of the study, blood samples were obtained intraoperatively, at 5 time points, from 15 patients undergoing cardiopulmonary bypass. MDCO-2010 at a final concentration of 100 or 500 nM was added and ACT testing was performed as before. Additional coagulation tests included prothrombin time, activated partial thromboplastin time, fibrinogen, antithrombin, prothrombin, and anti-Xa levels. RESULTS Addition of MDCO-2010 concentration-dependently prolonged ACTs in volunteers' and patients' blood samples regardless of the ACT activator or device used. In volunteer samples (no heparin) and in patient samples (baseline and intensive care unit) percent changes in ACTs due to MDCO-2010 were on average 3.1 ± 1.8 times higher (95% confidence interval 2.6-3.6; P < 0.001) for the celite-based Helena device compared with either Hemochron or Medtronic devices. CONCLUSION MDCO-2010 causes less ACT prolongation with kaolin than with celite activation.

Rotational thromboelastometry (ROTEM®)-based FIBTEM is used perioperatively to assess the extent of fibrin polymerization in whole blood. In FIBTEM, cytochalasin D eliminates the contribution of platelets to whole blood clotting, but changing levels in fibrin(ogen) and erythrocytes may differently affect clot formation. Because dynamic changes of hematocrit are not reflected in plasma fibrinogen measurements, we hypothesized that the lack of erythrocytes in isolated plasma measurements would affect the relationship between the Clauss method and whole blood-based FIBTEM during cardiac surgery. Therefore, in the current study we investigated the influence of perioperative hematocrit changes on FIBTEM and fibrinogen measurements.Blood samples were collected from 6 consenting healthy volunteers. FIBTEM tests were run before and after serial in vitro dilutions of whole blood with saline or autologous plasma (5:1, 2:1, and 1:1 v/v). We then evaluated the relationship between FIBTEM-maximal clot firmness (MCF) and the Clauss fibrinogen method in relation to hematocrit values before and after cardiac surgery. Pearson correlation coefficients were determined between laboratory test results and ROTEM variables.Upon in vitro hematocrit reduction, FIBTEM-MCF was progressively decreased depending on the extent of saline dilution, but it was increased by 31% after 1:1 volume replacement with autologous plasma (P0.05). In samples from cardiac patients (150 measurements in 50 patients), the overall correlation coefficient between FIBTEM-MCF and plasma fibrinogen was 0.80 (P0.001). In hemodiluted blood samples (during surgery or at intensive care unit), FIBTEM-MCF 10 mm corresponded to plasma fibrinogen levels of 200 mg/dL. In the subgroup analysis (n = 50 each), according to hematocrit levels (25%, ≥25% to 30%, ≥30%), plasma fibrinogen levels of 200 mg/dL corresponded to 11 mm, 10 mm, and 8 mm of FIBTEM-MCF, respectively. The correlation between FIBTEM-MCF and plasma fibrinogen was higher at lower hematocrit (25%) than at higher hematocrit (30%) (r = 0.88 and 0.67, respectively).Perioperative changes in hematocrit affect the correlation between plasma fibrinogen levels and FIBTEM-MCF values. The higher correlation between FIBTEM-MCF and plasma fibrinogen with lower hematocrit (25%) indicates that FIBTEM is a practical method to determine the need for fibrinogen replacement in bleeding patients who typically develop perioperative anemia.

A paste composed of micrometer-sized Ag particles and an organic solvent formed a soft porous bonding layer under atmospheric conditions at 200°C and above. Using this Ag paste for die-bonding of a light-emitting diode (LED) resulted in excellent thermal resistance and reliability. Ag/Ag interactions are thought to be responsible for internal bonding, and the Ag paste does not bond strongly to other materials. To ameliorate this, polymer particles were added to the Ag paste. The flow starting temperature of the polymer did not hinder the sintering of Ag particles, and the melted polymer particles served as an adhesive. The polymer-containing Ag paste had a lower specific resistance than that of the conventional conductive adhesives, so could be treated using low-temperature processes. The presence of polymer improved the die-shearing strength during the bonding of LED die to the plastic substrate.

Micrometer-sized Ag particles adsorb substantial oxygen above 200 °C. A dispersion of these particles in alcohol can be successfully used as a die-attach material by sintering the Ag particles to form a soft porous bonding layer. Since the low- temperature sintering of micrometer-sized Ag particles is unlikely to involve the nanometer size effect associated with melting, it was considered that the low-temperature sintering between Ag layers of micrometer thickness would occur in air. The contact surface of a light emitting diode die and a glass substrate were both coated with an Ag sputtered layer of 2 μm in thickness. The Ag-Ag direct bonding proceeded by sintering above 200 °C in air. The bonding strength of the Ag-Ag direct bonding exceeded that of the low-temperature sintering of micrometer-sized Ag particles, and the bonding strength depended on oxygen concentration in the sintering atmosphere. On the Ag sputtered surface of the glass substrate, a substantial morphology change was observed by heating the Ag sputtered layer in air. It was supposed that this morphology change was caused by abnormal grain growth of Ag in the Ag sputtered surface. It is concluded that low-temperature sintering of Ag materials is accelerated by the abnormal grain growth of Ag with oxygen adsorption near the Ag surface.