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Purpose Quantification of olanzapine (OLZ) and its metabolites such as N-desmethylolanzapine (DM-O), 2-hydroxymethylolanzapine (2H-O) and olanzapine N-oxide (NO-O) in five kinds of human body fluids including whole blood by liquid chromatography (LC)–tandem mass spectrometry (MS/MS) has been presented; the quantification methods were carefully devised and validated using the matrix-matched calibration and standard addition methods. Methods OLZ and its three metabolites were extracted from 40 μL each of body fluids by two-step liquid–liquid separations. The samples and reagents were pre-cooled in a container filled with ice for the extraction because of the thermal instability of OLZ and its three metabolites especially in whole blood. Results The limits of quantification (LOQs) of OLZ and 2H-O were 0.05 ng/mL and those of DM-O and NO-O were 0.15 ng/mL in whole blood and urine, respectively. The concentrations of OLZ and its metabolites in heart whole blood, pericardial fluid, stomach contents, bile and urine were determined for two cadavers and those in whole blood and urine for the other two cadavers. The reduction from NO-O to OLZ was observed at 25 ℃ in whole blood in vitro. Conclusions To our knowledge, this is the first report on the quantification of metabolites of olanzapine in the authentic human body fluids by LC–MS/MS as well as on the confirmation of in vitro reduction from NO-O to OLZ in whole blood that seems to have induced the quick decrease of NO-O.

Polymer nanoparticles have attracted attention as antibacterial materials, but the function of the polymer itself has not yet been clarified sufficiently. To estimate the essential surface properties of antibacterial polymer nanoparticles, herein, we synthesized cationic polystyrene (PSt) nanoparticles via soap-free emulsion polymerization using 2,2'-azobis-[2-(1,3-dimethyl-4,5-dihydro-1

The aim of this study is to investigate the stabilities of the 24 synthetic cannabinoid metabolites (SCMs) in blood and urine at various temperatures from - 30 to 37 ℃ stored for 1-168 days. In addition, experiments of stabilities at lower temperatures and for much longer duration have been performed as described below.The quantification was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The blank blood and urine spiked with SCMs and non-spiked real case (authentic) specimens were incubated at 37 ℃ up to 56 days and at 22, 4 or - 30 ℃ up to 168 days. The non-spiked authentic blood and urine specimens were also stored at - 30 or - 80 ℃ for 1, 3 or 5 years to investigate stabilities during very long time frames.All the 24 SCMs were much more stable in urine than in blood at 37, 22 or 4 ℃. All 24 SCMs spiked into blood or urine were stable at - 30 ℃ for up to 168 days. The 6 SCMs in the authentic specimens exhibited long stabilities at - 30 or - 80 ℃ for 3-5 years. Some tendencies were observed according to the relation between the structures of SCMs and their stabilities.The long-term stabilities of 24 SCMs in spiked samples and those of 6 SCMs in the authentic specimens were examined using LC-MS/MS. SCMs were largely very stable and usable several years after storage at - 30 or - 80 ℃.

Membranous nephropathy (MN) is a disease characterized by deposition of immune complexes on the glomerular basement membrane. More than 10 specific antigens for MN including M-type phospholipase A2 receptor (PLA2R), thrombospondin type-1 domain-containing 7A (THSD7A), exostosin 1/exostosin 2 (EXT1/EXT2) and neural epidermal growth factor-like 1 (NELL1) have so far been identified. Since the clinicopathologic characteristics of each type of MN in Japanese are not well understood, we first examined 107 cases of MN by immunohistochemistry for four antigens (PLA2R, THSD7A, EXT1, and NELL1) (MN-cohort). Of those 107 cases, 40% were PLA2R-positive, 13% were NELL1-positive, 11% were THSD7A-positive, 5% were EXT1-positive, 2% were PLA2R and NELL1-double-positive, and 29% were quadruple-negative. In one case of PLA2R and NELL1-double-positive, the first biopsy showed PLA2R-positive and the second biopsy showed PLA2R and NELL1-double-positive. Of the 16 cases of NELL1-positive, 12.5% had colon cancer, 18.8% had rheumatic diseases treated with bucillamine, and 63% had type 2 diabetes mellitus (T2DM). Next, 34 patients diagnosed with MN who had rheumatoid arthritis (RA) were examined (RA-MN cohort). Of those 34 patients, 79% were NELL1-positive, 6% were PLA2R-positive, and 15% were quadruple-negative. In the RA-MN cohort, 56% had a history of bucillamine use. In conclusion, NELL1-associated MN is a common MN in patients with RA using bucillamine and may also be associated with T2DM.

The quantification of parent molecules of pyrethroids tetramethrin and resmethrin in human specimens by a mass spectrometry (MS) technique has not been reported yet. A woman in her 60s was found dead in a wasteland. At the scene, an empty beer can and a spray for insecticides containing tetramethrin and resmethrin were found. Therefore, the concentrations of tetramethrin and resmethrin in postmortem specimens and the methanol solution used for rinsing the inside of the beer can were determined using liquid chromatography (LC)–tandem mass spectrometry (MS/MS). The quantification method by LC–MS/MS for intact parent molecules of tetramethrin and resmethrin in whole blood and urine has been devised and validated in this work. The method was applied to the quantification of tetramethrin and resmethrin in whole blood, urine and stomach contents obtained from a cadaver at autopsy. The limits of detection of tetramethrin and resmethrin were 0.06 and 0.03 ng/mL; limits of quantification were 0.2 and 0.1 ng/mL in blood and urine, respectively. The concentrations of tetramethrin of the deceased were 11.1 ± 1.2 and 0.425 ± 0.017 ng/mL for stomach contents and urine, respectively; the concentration of resmethrin in stomach contents was 1.77 ± 0.18 ng/mL. The tetramethrin and resmethrin were unstable in blood and urine at room temperature; they should be kept at not higher than 4 ℃. To our knowledge, this is the first report for quantification of unchanged tetramethrin and resmethrin in human specimens obtained in a fatal case.

Multipoint lock-and-key particle assembly, consisting of lock particles with multiple anisotropic dents and rod-shaped particles as key particles, is developed for colloidal modeling application. The lock particles were connected with each other at a key particle as their joint in the presence of depletants, forming rigid colloidal molecules imitating rigid polymers (e.g., polymers containing aromatic rings and intramolecular hydrogen bonds). A single-particle level observation was conducted to visualize the colloidal polymerization of the particle assembly. Motion trajectories of the lock particles observed by optical microscopy indicated that the particle diffusivity was dramatically lowered when the lock particle connected with another one, suggesting that particle diffusion was suppressed by particle assembly formation. Because the kinetic and regioselectivity of colloidal polymerization are assumed to be analogous to those at the atomic scale, the proposed lock-and-key assembly can be a promising colloidal model for atomic-scale polymers associated with their micro-Brownian motion.

Mesoporous silica shells were formed on nonporous spherical silica cores during the sol–gel reaction to elucidate the mechanism for the generation of secondary particles that disturb the efficient growth of mesoporous shells on the cores. Sodium bromide (NaBr) was used as a typical electrolyte for the sol–gel reaction to increase the ionic strength of the reactant solution, which effectively suppressed the generation of secondary particles during the reaction wherein a uniform mesoporous shell was formed on the spherical core. The number of secondary particles (N2nd) generated at an ethanol/water weight ratio of 0.53 was plotted against the Debye–Hückel parameter κ to quantitatively understand the Debye screening effect on secondary particle generation. Parameter κa, where a is the average radius of the secondary particles finally obtained in the silica coating, expresses the trend in N2nd at different concentrations of ammonia and NaBr. N2nd was much lower than that expected theoretically from the variation of secondary particle sizes at a constant Debye–Hückel parameter. A similar correlation with κa was observed at the high and low ethanol/water weight ratios of 0.63 and 0.53, respectively, with different hydrolysis rate constants. The good correlation between N2nd and κa revealed that controlling the ionic strength of the silica coating is an effective approach to suppress the generation of secondary particles for designing mesoporous shells with thicknesses appropriate for their application as high-performance liquid chromatography column packing materials.

Background Behçet’s disease (BD) is a systemic inflammatory vasculitis with both autoimmune and autoinflammatory properties. Renal involvement in BD and its spontaneous remission have been rare. We herein describe a case of parallel disease activity of BD with entero and renal involvements, followed by a spontaneous remission without corticosteroid treatment. Case presentation A 54-year-old woman who had a 4-year history of BD, maintained with colchicine treatment, suffered abdominal pain, hemorrhagic stool and diarrhea. Physical examination revealed strong tenderness in the entire abdomen. Laboratory test results showed increased levels of inflammation, and a computed tomography scan revealed edematous intestinal wall thickening with ascites. Blood and stool cultures showed no specific findings. Since she was suspected to have developed panperitonitis with acute enterocolitis, she started treatment with an antibacterial agent under bowel rest. Her abdominal symptoms gradually improved, while diarrhea and high levels of inflammatory reaction persisted. Colonoscopy revealed discontinuous abnormal mucosal vascular patterns and ulcerations in the whole colon except for the rectum, and histological analyses of the intestine demonstrated transmural mucosal infiltration of inflammatory cells without epithelioid granuloma or amyloid deposition. Based on these findings, she was diagnosed with entero BD. Meanwhile, pedal edema appeared during her hospitalization. Urinalysis results were consistent with nephrotic syndrome, thus a renal biopsy was performed. Light microscopy showed no obvious glomerular and interstitial abnormalities, whereas electron microscopy revealed foot process effacement without immune complex deposition or fibrillary structure, compatible with minimal change disease (MCD). Only with conservative therapy, her proteinuria decreased, followed by a complete remission in 3 weeks from the onset of edema. The coincident episode of MCD was finally diagnosed as renal BD that paralleled disease activity to entero BD. She started adalimumab administration, resulting in the further improvement of diarrhea and inflammatory levels. Conclusions This is the first report to demonstrate MCD as renal involvement of BD along with the disease activity of entero BD.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is pathologically characterized by focal fibrinoid necrosis, in which ANCA-mediated neutrophil extracellular trap (NET) formation and subsequent endothelial cell necrosis occur. Cyclophilin D (CypD) plays an important role in mediation of cell necrosis and inflammation via the opening of mitochondrial permeability transition pores. This study was undertaken to examine the role of CypD in AAV pathogenesis.We assessed the role and mechanism of CypD in ANCA-stimulated neutrophils in vitro by immunostaining and electron microscopy observation. We performed a comprehensive RNA-sequencing analysis on ANCA-treated murine neutrophils. To investigate the role of CypD in vivo, we assessed disease features in CypD-knockout mice and wild-type mice using 2 different murine AAV models: anti-myeloperoxidase IgG transfer-induced AAV and spontaneous AAV.In vitro experiments showed that pharmacologic and genetic inhibition of CypD suppressed ANCA-induced NET formation via the suppression of reactive oxygen species and cytochrome c release from the mitochondria. RNA-sequencing analyses in ANCA-treated murine neutrophils revealed the involvement of inflammatory responses, with CypD deficiency reducing ANCA-induced alterations in gene expression. Furthermore, analyses of upstream regulators revealed the relevance of intracellular calcium (CypD activator) and cyclosporin (CypD inhibitor) in ANCA stimulation, indicating that the CypD-dependent opening of mitochondrial permeability transition pores is associated with ANCA-induced neutrophil activation and NETosis. In both AAV mouse models, the genetic deletion of CypD ameliorated crescentic glomerulonephritis via the inhibition of CypD-dependent neutrophil and endothelial necrosis.CypD targeting is a novel and specific therapeutic strategy for AAV via the resolution of necrotizing vasculitis.

Parechovirus-A3 (PeV-A3), first reported in 2004 in Japan, is an emerging pathogen that causes sepsis and meningoencephalitis in neonates and young infants. Although PeV-A3 has been identified worldwide, its epidemiological characteristics differ by region. To investigate the molecular evolution and epidemiology of PeV-A3, we performed genetic analyses of 131 PeV-A3 strains from the years 1997–2019 in Niigata, Japan. During 2016–2019, annual numbers remained steady, in contrast to the PeV-A3 epidemic interval of every 2–3 years that was observed in Japan from 2006. Bayesian evolutionary analysis of the complete viral protein 1 region revealed alternate dominant clusters during years of PeV-A3 epidemics. The branch including the oldest and first isolated PeV-A3 strains in Japan has been disrupted since 2001. The year of PeV-A3 emergence was estimated to be 1991. Continuous surveillance with genetic analyses of different regions will improve understanding of PeV-A3 epidemiology worldwide.

Background Enterovirus D68 (EV-D68) causes asthma-like respiratory infection in children. Several EV-D68 outbreaks have been reported worldwide since the largest outbreak occurred in the United States in 2014. We experienced an accumulation of pediatric cases with asthma-like respiratory illness in Niigata, Japan, in 2018. Study design To determine whether EV-D68 was responsible for the case accumulation, this prospective observational study evaluated children hospitalized in 1 of 8 hospitals with asthma-like respiratory illness in Niigata, Japan, during October and November 2018. Diagnoses were made by EV-D68-specific RT-PCR using nasopharyngeal samples. The clade was identified by sequence analyses, and a phylogenetic tree was created. To evaluate seasonal variation, data from pediatric cases with asthma-like respiratory illness in 2018 were retrospectively analyzed. Results In 2018, 114 children were hospitalized with asthma-like respiratory illness in October and November, and 47 nasopharyngeal samples were collected. EV-D68 was detected in 22/47 (47%) patients during the study period. The phylogenetic tree revealed that all strains belonged to the clade B3 branch, which has been detected worldwide every 2 years since 2014. Conclusions EV-D68 was the associated pathogen for asthma-like respiratory illness in children in Japan in 2018. Clade B3, the dominant clade in outbreaks worldwide, was responsible for the outbreak. Detection and detailed virologic analysis of EV-D68 is important as part of worldwide surveillance, as it will aid in understanding the epidemiologic characteristics of EV-D68 infection.

In this study, solid tissues such as the lung, liver, kidney and urine were highlighted to profile the AB-PINACA in vivo metabolites in a fatal abuse case, although such metabolite analysis is usually made with urine specimens. We compared the relative peak intensities of in vivo metabolites of AB-PINACA in lung, liver, kidney and urine specimens collected at the autopsy of its abuser with its in vitro metabolites in human hepatocytes. The metabolites of AB-PINACA in tissues were extracted after homogenization. The urine specimen and portions of the extracted metabolites from tissues were firstly hydrolyzed with β-glucuronidase, and the metabolites were extracted. For in vitro experiment, AB-PINACA was incubated with human hepatocytes for 3 h to produce its metabolites. The identification of the in vivo and in vitro metabolites was performed using liquid chromatography (LC)–high-resolution Orbitrap-tandem mass spectrometry (MS-MS), and the relative intensities of these metabolites were measured using low resolution LC–quadrupole-ion trap-MS-MS. Thirteen metabolites of AB-PINACA were characterized in vivo in several human specimens and in in vitro human hepatocytes. They were produced by the terminal amide hydrolysis to carboxylic acid, hydroxylation, carbonyl formation and/or glucuronidation. The most detectable metabolite in the hepatocytes, lung or liver was the one produced by the terminal amide hydrolysis, whereas the top metabolite in the kidney or urine was the one produced by hydroxylation or carbonyl formation on the pentyl side chain after the terminal amide hydrolysis, respectively. At least 12 metabolites of AB-PINACA were detected in authentic human lung, liver or kidney specimen from a cadaver. It is concluded that the postmortem metabolite profiling of AB-PINACA can be fulfilled with solid tissues, and the lung and kidney were most recommendable especially when urine specimen is not available.

Regularly ordered polymer nanoparticle (PNP) assemblies incorporating gold nanoparticle (Au NP) clusters into the PNP interstices were fabricated by a simultaneous deposition of PNPs and Au NPs on a glass substrate. Monodisperse PNPs with an average size of 66 nm were employed as a template in the co-assembly to create the sub-100 nm periodic Au nanostructures on the substrate. First, mono-layering of PNP array with incorporation of 14 nm Au NPs was performed by a drop-casting to examine the number ratio of Au NPs to PNPs for multi-layering. Absorption spectra of the mono-layered co-assemblies of PNPs and Au NPs were employed to characterize the clustered state of Au NPs in the interstices of mono-layered PNPs. The number ratio suitable for homogeneous incorporation of Au NPs clustered in the interstice was found to be ranged from 6 to 8 in the characterization. Then, multi-layered co-assemblies of PNPs and clustered Au NPs were fabricated by a vertical deposition method with the Au NP number ratio of 8 to PNPs. Lifting rate of the substrate on which the PNPs were deposited was varied in the vertical deposition method to tune the film thickness of NP co-assembly. A decrease in the lifting rate to 1 μm/s could thicken the film to 0.71 μm corresponding to 13 layers of PNPs, resulting in the fabrication of periodic structures of Au NP clusters with a high packing density. Signal-to-noise ratio in the Raman measurement using p-mercaptobenzoic acid as a target molecule was successfully enhanced by multi-layering of the co-assembly, indicating that Au NP clusters were homogeneously incorporated into the interstices of PNPs in the co-assemblies.

Enterovirus D68 (EV-D68) causes a range of clinical manifestations, including asthma-like illness, severe respiratory disease, and acute flaccid myelitis. EV-D68 has caused worldwide outbreaks since 2014 and is now recognized as a reemerging infection in many countries. EV-D68-specific PCR assays are widely used for the diagnosis of EV-D68 infection; however, assay sensitivity is a concern because of genetic changes in recently circulated EV-D68. To address this, we summarized EV-D68 sequences from previously reported world outbreaks from 2014 through 2020 on GenBank, and found several mutations at the primer and probe binding sites of the existing EV-D68-specific PCR assays. Subsequently, we designed two novel assays corresponding to the recently reported EV-D68 sequences: an EV-D68-specific real-time and seminested PCR. In an analysis of 22 EV-D68 confirmed cases during a recent EV-D68 outbreak in Japan, the new real-time PCR had higher sensitivity than the existing assay (100% versus 45%, P

In this paper, reinforcement of dielectric nanocomposite was performed for polyvinyl fluoride (PVDF) as a matrix, which is a practical requirement for foldable and flexible electric energy devices. A fibrous nanofiller, 2,2,6,6-Tetramethylpiperidine 1-oxyl (TEMPO)-oxidized cellulose nanofiber (TOCN) was employed as a nanofiller for the PVDF nanocomposite incorporating highly dielectric barium titanate nanoparticles (BT NPs). Prior to the fabrication of nanocomposite films, a chemical treatment by HCl and a surface-modification with trifluoropropyl group were applied to TOCN and BT nanoparticles, respectively. Their treatments brought about both nanofillers well-dispersed in a polar aprotic solvent for PVDF. A low percentage incorporation of the treated TOCNs up to 0.5 wt% was found to be sufficient for enhancement on the mechanical strength of nanocomposites at a high content of surface-modified BT NPs (30 vol%). The permittivity of the nanocomposites was in the range of 12 to 14 that is twice higher than that in the PVDF film. The PVDF nanocomposites at the treated TOCN loading of 0.3 to 0.5 wt% exhibited superior mechanical strength to the PVDF film. The ternary combination of PVDF, the treated TOCN, and the surface-modified BT NPs showed an improvement on tensile stiffness with maintaining the intrinsic high dielectric properties of nanocomposite films. The present nanocomposite can be a promising ternary system for an advanced design of polymer dielectrics with excellent energy storage properties.

The aim of this study is to characterize the metabolites of EAM-2201 in human hepatocytes obtained in vitro and those in liver and urine specimens obtained in vivo from the autopsy of an EAM-2201 abuser. EAM-2201 was incubated with human hepatocytes for 3 h in a CO2 incubator and the metabolites of EAM-2201 were produced. The human liver specimen was homogenized and the metabolites were extracted. The urine specimen was hydrolyzed first with β-glucuronidase and the metabolites were extracted. The tentative detection and identification of those metabolites were performed using liquid chromatography (LC)–quadrupole-ion trap tandem mass spectrometry (MS/MS) and high-resolution LC–Orbitrap-MS/MS, respectively. Twelve metabolites of EAM-2201 could be characterized in hepatocytes, liver and urine. They were produced by defluorination, hydroxylation, carboxylation, dehydrogenation, N-dealkylation and/or glucuronidation of EAM-2201. The most detectable metabolite obtained from the in vitro hepatocyte incubation was that monohydroxylated at the 4-ethylnaphthalene moiety of EAM-2201 after dehydrogenation. However, only a trace amount of the same metabolite was detected in the liver tissue obtained postmortem from an EAM-2201 abuser. In the liver tissue, the highest metabolite was 5-carboxypentyl derivative of EAM-2201, which was monohydroxylated at the 4-ethylnaphthalene moiety, followed by monohydroxylpentyl and monohydroxy-4-ethylnaphthalenyl derivative of EAM-2201 without dehydrogenation. Twelve metabolites of a synthetic cannabinoid, EAM-2201, were tentatively identified in human hepatocytes in vitro, and in human liver and urine specimens in vivo for the first time. There was a distinct difference in metabolism profile between the in vitro and in vivo results.

A simple and high-throughput analytical method for determining clitidine in Paralepistopsis acromelalga using hydrophilic interaction liquid chromatography–tandem mass spectrometry (LC–MS/MS) was established. To determine clitidine in the mushrooms, a simple procedure including dilution with methanol solution and filtering by cartridge was employed just before quantification by LC–MS/MS for high-throughput analysis. In this report, concentrations of clitidine in mushrooms were determined by the standard addition method. The present established method was successfully applied to the analysis of fruit bodies of P. acromelalga, which were obtained from five different locations in Japan. Results on concentrations of clitidine in each stem and cap of P. acromelalga specimens tested showed that their concentrations were quite different, not only between stems and caps, but also among locations and strains; the concentrations of clitidine in stems and caps ranged from 1.41 to 9.30 mg/g and 3.17 to 14.4 mg/g, respectively. This is the first report to present a detailed quantitative analysis of clitidine by MS and the distribution of clitidine in stems and caps of P. acromelalga. This analytical method for clitidine was thought to be useful in P. acromelalga poisoning cases to identify the causative toxic mushroom.

Colloidal molecules created by clustering monodisperse particles are a significant model system of atoms and molecules in colloidal scale. To attain modelling of complex or high-molecular-weight molecules such as hyper-branched polymers and proteins, it is required to develop a new assembling system which can prepare three-dimensionally designed colloidal molecules. In this study, we proposed an approach for complex colloidal molecules created by association between golf ball-like particles and spherical particles via depletion interaction. Several golf ball-like particles joined together, which formed joined particle assemblies at the spherical particles acting as their joints in a depletant solution. The number of golf ball-like particles in a particle assembly was increased by increasing the concentration of the depletant, leading to the formation of branched colloidal chains of the golf ball-like particles. Because the golf ball-like particles in the colloidal chain flexibly rotated at their joints, the conformation of the chains varied in the depletant solution. The present results demonstrated that the golf-ball like particle that can be joined to form colloidal macromolecules with high association number and high flexibility will be a promising building block for complex colloidal model systems.

Thrombomodulin (TM), which is predominantly expressed on the endothelium, plays an important role in maintaining vascular homeostasis by regulating the coagulation system. Intravascular injury and inflammation are complicated physiological processes that are induced by injured endothelium-mediated pro-coagulant signaling, necrotic endothelial- and blood cell-derived damage-associated molecular patterns (DAMPs), and DAMP-mediated inflammation. During the hypercoagulable state after endothelial injury, TM is released into the intravascular space by proteolytic cleavage of the endothelium component. Recombinant TM (rTM) is clinically applied to patients with disseminated intravascular coagulation, resulting in protection from tissue injury. Recent studies have revealed that rTM functions as an inflammatory regulator beyond hemostasis through various molecular mechanisms. More specifically, rTM neutralizes DAMPs, including histones and high mobility group box 1 (HMGB1), suppresses excessive activation of the complement system, physiologically protects the endothelium, and influences both innate and acquired immunity. Neutrophil extracellular traps (NETs) promote immunothrombosis by orchestrating platelets to enclose infectious invaders as part of the innate immune system, but excessive immunothrombosis can cause intravascular injury. However, rTM can directly and indirectly regulate NET formation. Furthermore, rTM interacts with mediators of acquired immunity to resolve vascular inflammation. So far, rTM has shown good efficacy in suppressing inflammation in various experimental models, including thrombotic microangiopathy, sterile inflammatory disorders, autoimmune diseases, and sepsis. Thus, rTM has the potential to become a novel tool to regulate intravascular injury via pleiotropic effects.

Human parechovirus 3 (HPeV-3) and enteroviruses (EV) are commonly detected viruses in febrile neonates and young infants and are usually diagnosed by PCR. However, in this population, data on detection rates for samples from different anatomical sites are limited.To determine PCR detection rates for HPeV-3 and EVs in serum and cerebrospinal fluid (CSF) samples from febrile neonates and young infants.This prospective study identified viruses in serum and CSF samples collected from febrile neonates and young infants (age4 months) in Niigata, Japan, during 2014-2018. HPeV-3 or EV infection was defined as a positive quantitative real-time PCR result for the virus in serum or CSF. Genotypes were identified by sequence analyses of the viral protein 1 region.Among 216 patients, we identified 56 HPeV-3-infected (26 %) and 48 EV-infected patients (22 %). All (56/56; 100 %) HPeV-3-infected patients had a positive PCR result for serum, and 49/56 (88 %) had a positive result for CSF. In EV-infected patients, 40/48 (83 %) were positive for serum, and 34/48 (71 %) were positive for CSF, and 22/48 (46 %) were positive for serum (n = 14) or CSF (n = 8). If only a CSF sample had been obtained, 7 (12 %) HPeV-3 infections and 14 (29 %) EV infections would have been undiagnosed. Detection rates in serum and CSF differed by genotype in EV-infected patients.Viral RNA detection rates differed between serum and CSF in HPeV-3- and EV-infected neonates/infants. Combined evaluation of serum and CSF samples is important for accurate viral diagnosis in this population.

AB-FUBINACA M3 was reported to be a major metabolite of the drug, but its in vivo concentration in authentic human solid tissues has not been quantified yet. Another metabolite AB-FUBINACA M4 did not receive much attention previously and also has not been quantified yet in any authentic human specimens. The aims of this study are to establish a sensitive method for quantification of M3 and M4 in solid tissues and to compare the metabolite profile of AB-FUBINACA in authentic human specimens in vivo with that produced by human hepatocytes in vitro. The quantification was performed by liquid chromatography (LC)–quadrupole-ion trap-tandem mass spectrometry (MS-MS), and the characterization by LC–quadrupole Orbitrap MS-MS The limits of quantification of M3 were 10 pg/mL and 60 pg/g, and those of M4 were 100 pg/mL and 600 pg/g in urine and tissues, respectively. In the present work, M3 and M4 were identified and quantified in human lung, liver and kidney obtained from a cadaver for the first time; the concentrations of M3 were 226, 255, 202 and 155 pg/mL or g, and those of M4 14,400, 768, 637 and 1,390 pg/mL or g in urine, lung, liver and kidney, respectively. The peak intensity profiles of seven metabolites in these specimens were compared with that produced by human hepatocytes; the top three metabolites in urine specimen were completely different from those of hepatocytes. M3 was reported as the predominant metabolite in several previous works and M4 was listed as a minor metabolite in only one work, but, in this work, M4 has been found to be the major metabolite in all of the authentic urine, lung, liver and kidney specimens. The M3 plus M4 metabolites in lung or kidney were found most recommendable to prove AB-FUBINACA consumption, when urine specimen is lacking.

Background and Aims Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a necrotizing vasculitis that affects systemic organs. Although the standard immunosuppressive therapy in AAV could control disease activity, these existing treatments have no effect to regulate the necrosis, which could cause the persistent organ damage. ANCA binds to neutrophils, leading to neutrophil extracellular (NETs) formation. The formed NETs induce vascular injury and become auto-antigens against ANCA, resulting in an auto-amplification loop of necrosis and inflammation. NETs work as innate immunity and simultaneously undergo programmed cell necrosis. However, the signaling pathway of ANCA-mediated NETs remains elusive. Cyclophilin D (CypD) regulates program necrosis via the opening of mitochondrial permeability transition pores (mPTP) with the consequence of mitochondrial ROS release. The Inhibition or genetic deletion of CypD has been reportedly to protect organ damage in brain and kidney ischemia-reperfusion injury mice. Herein, we hypothesized that CypD would be involved in ANCA-mediated NETs pathway and the pathogenesis of AAV. Method Animal experiment; Autoimmune vasculitis was induced in 10 week-old CypD(+/+) or CypD deficient (-/-) mice by the intravenous injection with anti-mouse MPO serum. At 24 hours after injection, we evaluated the renal and lung involvements. The severity of glomerulonephritis was assessed by the levels of BUN, haematuria, histological findings and TUNEL positivity. The lung injury was evaluated by histopathological alveolar haemorrhage scores and TUNEL positivity. The mPTP related necrosis in tissues was identified by immunostaining against cytchrome C. The NETs-forming neutrophils in peripheral bloods were detected as Sytox Green (SG)-positive neutrophils by flow cytometry (FCM). In vitro experiment; We investigated whether CypD inhibitor and genetic ablation suppresses ANCA-mediated NETs. Healthy human neutrophils were incubated with ANCA-IgG derived from AAV patients with pre-treatment of CypD inhibitor (cyclosporine A) or PBS for 4 hours. Next, WT or CypD (-/-) mice neutrophils were cultured with anti mouse MPO antibody for 4 hours. The NETs induction was assessed as Sytox green positive neutrophil using FCM and live imaging. Results The amount of haematuria, histological severity including glomerulonephritis and pulmonary haemorrhage in CypD(-/-) mice significantly decreased compared to those in CypD(+/+) mice. Furthermore, TUNEL and cytochrome C positivity of tissues in CypD(-/-) mice was lower than that of CypD(+/+) mice, respectively. The level of BUN in AAV-induced CypD (-/-) mice was significantly lower than that of AAV-induced CypD(+/+) mice (92.4 ± 41.8 vs 36.8 ± 21.5 mg/ dL, p In vitro experiments, CypD inhibitor suppressed ANCA-induced NETs formation (Control; 0.45 ± 0.14 vs CypD inhibitor; 0.16 ± 0.04%, p Conclusion Our data indicate that the inhibition of CypD might ameliorate autoimmune vasculitis via the suppression of programmed NETs formation.

We experienced a curious fatal case, in which a male in his 20s self-administered zolpidem intravenously. The victim was found dead lying on floor of his apartment room, with a tourniquet band and new injection marks on his right forearm. Nearby the body, a medical disposal syringe containing small-volume solution dissolving crushed zolpidem tablets was found. The postmortem interval was estimated at about two days. The direct cause of his death was judged as asphyxia due to the aspiration of stomach contents into the trachea and bronchi. The specimens dealt with were body fluids and solid tissues including femoral vein blood, right and left heart blood, pericardial fluid, urine, bile, stomach contents, the brain, lung, heart muscle, liver, spleen, kidney, pancreas and skeletal muscle. For the extractions of zolpidem, zolpidem phenyl-4-carboxylic acid, deuterated internal standards zolpidem-d7 and zolpidem phenyl-4-carboxylic acid-d4, a modified QuEChERS method was used, followed by the analysis by liquid chromatography–tandem mass spectrometry. Because this study included various kinds of human matrices with quite different properties, the standard addition method was most preferable to overcome the matrix effects and recovery rates, and also did not need to use blank human matrices for validation experiments. The concentration of zolpidem and its phenyl-4-carboxylic acid metabolite in various specimens tested were generally extreme higher than those of reported fatal cases, supporting that the victim had died of intravenous zolpidem injection. The concentrations of zolpidem in femoral vein blood and right and left heart blood specimens in the present case were 9.55, 28.5 and 46.9 μg/mL, respectively, which far exceeded estimated fatal levels. The present study also showed the postmortem distribution/redistribution of zolpidem and its phenyl-4-carboxylic acid metabolite in 15 body fluid and solid tissue specimens including stomach contents. Although a number of published literatures dealt with zolpidem poisoning cases due to oral ingestion of the drug, this is the first report on fatal intravenous zolpidem injection case and postmortem distribution of zolpidem and its predominant metabolite.

A synthetic cannabinoid 5F-NNEI and its metabolites in authentic human specimens have not been reported yet. The aim of this study is, firstly, to establish a sensitive quantification method of 5F-NNEI in human serum and urine specimens, and, secondly, the characterization of its metabolites in authentic urine specimens obtained from three individuals. These compounds were extracted from β-glucuronide-hydrolyzed and unhydrolyzed urine and/or serum specimens via liquid-liquid extraction. The identification and quantification were performed using liquid chromatography (LC)–QTRAP tandem mass spectrometry (MS/MS), and the characterization of the metabolites was done using LC–high-resolution-MS/MS. The limits of detection for unchanged 5F-NNEI were 6 pg/mL with the quantification range of 10–2000 pg/mL in serum, and 3 pg/mL with the quantification range of 5–1000 pg/mL in urine. The devised method was applied to quantify 5F-NNEI in authentic serum and/or urine specimens from three individuals. The levels of 5F-NNEI were 11.6 and 3680 pg/mL in serum specimens of cases 1 and 2, respectively, and were 5.07 and 7.90 pg/mL in urine specimens of cases 2 and 3, respectively. Several metabolites of 5F-NNEI produced by amide hydrolysis, defluorination, hydroxylation, carboxylation and/or glucuronization were tentatively identified in the urine specimens by the LC–high-resolution-MS/MS. A synthetic cannabinoid, 5F-NNEI, was identified and quantified in its unchanged form, and its several metabolites were tentatively identified in authentic specimens obtained from three individuals, for the first time to our knowledge.

OBJECTIVE The purpose was to explore the presence of myeloperoxidase (MPO)-deoxyribonucleic acid (DNA) complex as a surrogate marker of neutrophil extracellular traps (NETs) in the middle ear fluid, and to clarify the correlation between its quantifiable level and hearing outcome in patients with otitis media associated with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). STUDY DESIGN Prospective study. SETTING Tertiary referral center. PATIENTS Nine AAV patients presenting with otitis media. INTERVENTION Collection of the fluid samples from middle ear. MAIN OUTCOME MEASURE The quantifiable levels of MPO-DNA complex using an enzyme-linked immunosorbent assay. RESULTS The quantifiable levels of MPO-DNA complex in patients with AAV were significantly higher than those in controls (p

４年間のＡ大学成人看護学実習における学生の看護技術経験のデータより実際の技術経験状況を明らかにし、成人看護学領域内でどのように構造的に教授していくか教育方略について検討する。【研究方法】調査対象は、Ａ大学看護学部成人看護学実習看護技術経験表とした。技術経験表の各項目を単純集計し、パイロットスタディから本調査第３期の４年間の継続調査の累積計について経験頻度（率）を算出した。事前に研究者の所属する研究機関の研究倫理審査委員会より承認を得た後に実施した。【結果】４年間の継続調査における成人看護学実習終了後の学生289名の技術経験表を分析した。技術経験表から学内演習項目を除外し50％以上経験している項目数の方が経験していない項目数より多い大項目は６つであった。患者・状況特性による項目のうち50％以上経験している項目は、学内演習項目を除外した項目とほぼ同様の結果だった。学内演習項目を除外し50％以上経験していない項目は、排泄援助技術、活動・休息援助技術、呼吸循環を整える技術、褥瘡管理技術、与薬の技術、症状・生体機能管理技術、安全管理技術の７つだった。【考察】現在、臨地実習では学生が多くの看護技術を経験することは難しい。卒後研修である新人看護職員研修も視野に入れ、学士課程において卒業時に担保できる看護技術項目の精選が必要である。そして、看護技術経験の場でもある成人看護学領域における講義や学内演習の充実が求められる。臨地実習では、確実に実施できる看護技術をこれまでの指導者・教員との連携など教育的配慮のもと機会を逃すことなく、学習体験の意味づけを継続していくことが必要である。４年間の研究結果を踏まえ、A大学のDPや成人看護学の独自性を活かした到達目標と臨地実習病院の特徴を踏まえ、現状に合った看護技術内容を精選し、教授していくことが求められる。, To determine the actual condition for nursing-skill experiences in adult nursing clinical practices of the students of College A, and to examine how to systematically teach students in the adult nursing education program at the university.[Method] We investigated the results from questionnairs completed by students of the nursing department in College A. We simply tallied the number of each item from the nursing skill experience questionnaire, and worked out the frequency of the experience based on the accumulation in the four-year study. This study was conducted after obtaining the approval from the Research Ethics Review Board of the affi liated research institutions. [Result] We analyzed the skill experience questionnaire of 289 students who finished the clinical practicum for adult nursing in four years. There are thirteen items. We discovered that in off -campus clinical practicum there were six items in which more than 50% of students had those experiences. Items regarding characteristics of patients and situations in which 50% of students had experience were almost the same number as those who attended off-campus clinical practicum.[Consideration] Today, it is diffi cult for students to experience many nursing-skills in clinical practicum. We should carefully select the nursing-skills and secure them in the university program, and consider the training program for new nurses after their graduation. We also should develop improved lectures in adult the nursing education program and on-campus clinical practicum. We should continuously conduct clinical practicum for students to acquire nursingskills in cooperation with the present trainers and teachers. In the light of the four-year study, Diploma Policy of University A and the attainment target of adult nursing education, we need to teach the best nursing-skills suited to the present conditions.

Japanese cedar (Cryptomeria japonica) pollinosis (JCP), affecting more than a quarter of the Japanese population, is a significant public health problem, due to its negative impact on daily activity. JCP patients have used the four-stage daily pollen deposition information based on the pollen monitoring over 20 years. However, the procedure for monitoring pollen was recently changed dramatically, to hourly average pollen concentration monitoring. In that type of monitoring, JCP patients cannot identify pollen exposure level because the relationship between hourly average pollen concentration and daily pollen deposition is unclear. Based on the parallel monitoring of concentration and deposition counts that we performed in Niigata prefecture, Eastern Japan, we found that the relationship between the daily pollen deposition (pollen cm−2 day−1) and the daily-average pollen concentration (pollen m−3) calculated from hourly average pollen concentration was not only statistically significant but also consistent with the aerodynamic properties of pollen. Using the relationship, we proposed new range criteria of hourly average pollen concentrations corresponding to the four stages of pollen deposition. Additionally, the conversion of pollen deposition to pollen concentration made the long-term trend analysis of the daily-average pollen concentration possible in this study area, and an increasing trend was identified at one site.

Urine is the most suitable specimen to collect from individuals because of noninvasiveness and relatively large volumes obtainable. In authentic urine specimens, however, synthetic cannabinoids having the structures of quinolinyl ester indoles, such as 5F-PB-22, PB-22 and BB-22, in unchanged forms as well as their metabolites, have not been quantified yet. Therefore, the aim of this study was to establish a sensitive analytical method for the quantification of 5F-PB-22 and its three metabolites 5F-PB-22 3-carboxyindole, PB-22 N-5-hydroxypentyl and PB-22 N-pentanoic acid in authentic urine samples in four cases. These compounds were extracted from β-glucuronide-hydrolyzed and unhydrolyzed urine via liquid-liquid extraction. The identification and quantification were performed using the QTRAP type of a liquid chromatography–tandem mass spectrometer. The limits of detection were 3–30 pg/mL and their summed quantitation range was 10–10,000 pg/mL. The devised method was applied to quantify these compounds in authentic urine specimens obtained from four individuals. The levels of 5F-PB-22 were 5.1, 13.6, 94.7 and 470 pg/mL; those of 5F-PB-22 3-carboxyindole were 8.25, 3.39, 23.2 and 880 ng/mL; and those of PB-22 N-pentanoic acid were 12.0, 57.4, 959 and 2090 pg/mL, respectively, in four unhydrolyzed urine samples, and the levels of PB-22 N-5-hydroxypentyl could be quantified as 29.9 and 131 pg/mL in two unhydrolyzed urine samples. The 5F-PB-22 and its metabolites PB-22 N-5-hydroxypentyl have been detected from authentic human urine samples for the first time. Also, this is the first report dealing with the quantification of the three metabolites in human urine samples.