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In this study, we assessed 80 autopsy samples to investigate the relationships between cause of death and the concentrations of multiple steroids in serum and cerebrospinal fluid (CSF). First, we developed and validated analytical methods to quantify seven steroids (cortisol, cortisone, corticosterone, 11-deoxycortisol, 11-deoxycortiocosterone, progesterone, and testosterone) by using liquid chromatography coupled with electrospray ionization-tandem mass spectrometry. Next, we statistically evaluated the levels of each steroid for six causes of death: hypothermia, traumatic injury, fire fatality, asphyxia, intoxication, and internal disease. We observed that cortisol concentrations in serum and CSF obtained from cadavers who died from hypothermia were significantly higher than those in samples obtained from cadavers who died from the remaining causes of death (P < 0.05). Similarly, corticosterone concentrations obtained from cadavers who died from hypothermia were significantly higher than those in samples from several other causes of death. However, concentrations of the remaining steroids analyzed did not differ significantly among the causes of death. We further elucidated the correlations between steroid concentrations in serum and CSF. Except for 11-deoxycorticosterone and progesterone, steroid concentrations were significantly positively correlated in serum and CSF. Although data on cadaveric steroid concentrations are limited-especially in CSF-values obtained were in the approximate range of the living human data reported to date., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

2-Aminothiazoline-4-carboxylic acid (ATCA), which is produced by the reaction of cyanide with endogenous cystine, is a promising biomarker of cyanide exposure because of its physicochemical stability. Analysis of more stable metabolite than the toxic gas itself is sometimes useful for postmortem diagnosis of gas poisoning. Here, we developed and validated an approach that uses liquid chromatography coupled with electrospray ionization-tandem mass spectrometry for quantifying ATCA in dried blood spot (DBS) samples. The linearity of the calibration curve was good in the concentration range of 20-1500 ng/mL. Our method allows for repeatable and the accurate quantification of ATCA, with intra- and inter assay coefficients of variation of below 7.8 % and below 9.3 %, respectively. In addition, the concentration of ATCA in DBSs remained stable for at least one month when stored at -20°C. Our results indicated that our analytical approach can be used to determine past exposure to higher doses of cyanide. In a comparison of ATCA concentrations in DBSs obtained from cadavers with various causes of death, significantly higher ATCA concentrations were observed in fire victims than in non-fire victims, confirming that fire victims inhale large amounts of cyanide gas. Thus, here we extended the possible uses of DBS for quantification of ATCA to forensic toxicological testing for cyanide poisoning., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: This study aims to evaluate the significance of preoperative magnetic resonance imaging findings to predict subscapularis tear confirmed at arthroscopic surgery., Methods: Sixty-four consecutive shoulders that underwent preoperative magnetic resonance imaging examination and arthroscopic shoulder operations were retrospectively reviewed. Under arthroscopic examination, complete subscapularis tear was defined as a full-thickness tear and incomplete subscapularis tear as tendon detachment larger than 5 mm from the insertion on the joint side., Results: In arthroscopic findings, they were included 11 shoulders with complete subscapularis tear, 13 with incomplete subscapularis tear, and the remaining 28 shoulders without subscapularis tear. Subscapularis discontinuity by axial magnetic resonance imaging had the highest sensitivity and specificity in detecting complete subscapularis tear compared with other magnetic resonance imaging findings. Long head biceps subluxation or dislocation showed significantly higher prevalence in the complete and incomplete subscapularis tear groups than in the group with no tear. Incomplete subscapularis tear groups had a higher incidence of superior subscapularis recess fluid, and this fluid was present in all the shoulders with incomplete subscapularis tear., Conclusions: The presence of subscapularis discontinuity is useful for diagnosis of complete subscapularis tear. In addition, in cases of incomplete subscapularis tear, the presence of superior subscapularis recess fluid had 100% sensitivity. Thus, this finding may be a characteristic diagnosis of subscapularis tear including incomplete tear., Competing Interests: Declaration of competing interest There authors have no commercial or other associations that might pose a conflict of interest in connection with the submitted material., (Copyright © 2021 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.)

Background: No anti-adhesive materials are currently in clinical use for orthopaedic surgery. We developed a hyperdry amniotic membrane (HD-AM) for easy storage and transplantation as amniotic membrane. The purpose of this study was to examine the application of HD-AM to reduce peritendinous adhesions without impairing tendon healing., Methods: We randomly divided 3 digits (2nd, 3rd, and 4th digits) from each rabbit into three groups: a tendon repair group; a tendon repair with HD-AM group (HD-AM group); and a control group (cast only). The effects of HD-AM on peritendinous adhesions and tendon healing were examined using microscopic, histological, and mechanical analyses in a rabbit flexor digitorum profundus tendon model., Results: Adhesions on macroscopic evaluation of the tendon repair site were significantly smaller in the HD-AM group than in the tendon repair group. Little adhesion formation or foreign body reactions were seen by on histologic evaluation in the HD-AM group. Range of motion following tendon repair was significantly better in the HD-AM group than in the tendon repair group. Maximal tensile strength required to pull the tendon from the site of adhesion was significantly smaller in the HD-AM group than in the tendon repair group. As for tendon repair site, no significant difference was seen between the tendon repair and HD-AM groups., Conclusions: HD-AM prevented peritendinous adhesion macroscopically, pathologically, and mechanically without impairing the sutured tendon. HD-AM has already been clinically applied in neurosurgery, ophthalmology, and otolaryngology, and clinical application as an anti-adhesive materials may be achieved in the future., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2021 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.)

Ferroptosis was recently defined as a novel type of programmed cell death depending on iron and lipid peroxidation. It is biologically different from other types of cell death such as apoptosis. While the involvement of ferroptosis in cancer, patient and animal model have been intensely studied, ferroptosis in human motor neuron model is still clearly unknown. Here we carefully assessed ferroptosis using human iPS cell-derived motor neuron (hiMNs). We found that almost all hiMNs died by the treatment of glutathione peroxidase 4 (GPX4) inhibitors. Importantly, the cell death was rescued by one antioxidant, vitamin E acetate, iron chelators and lipid peroxidase inhibitors with high dynamic ranges. Finally, these data clearly indicated that ferroptosis constitutively occurs in hiMNs, suggesting the possibility that it might play a biologically and pathologically important roles in motor neuron death such as motor neuron disease (MND)/Amyotrophic lateral sclerosis (ALS)., Competing Interests: Declaration of competing interest H.O. is a paid member of the Scientific Advisory Board of San Bio Co., Ltd and K Pharma, Inc. M.Y. is a scientific advisor of K Pharma, Inc. The other authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: In total knee arthroplasty (TKA) with posterior condylar osteotomy using anatomical landmarks, predicting the final flexion gap is impossible, as it differs with the presence or absence of the posterior cruciate ligament. We compared the predicted flexion gap, based on pre-femoral posterior condylar osteotomy measurements, with the postsurgical final flexion gap in cruciate-retaining (CR) and posterior-stabilized (PS) TKA., Methods: One hundred knees of patients with osteoarthritis were included: 35 underwent CR, and 65 PS TKA. Distal femoral and proximal tibial osteotomy using the measured resection technique was performed. An anterior and posterior femoral osteotomy guide was set parallel to the surgical epicondylar axis, and the predicted flexion gap was measured using a seesaw tensor attached to the guide. After all procedures, the final component gap in flexion was measured using a similar seesaw tensor at the patella reduction position and was compared with the predicted gap., Results: The correlation coefficients for predicted vs. final component gap were 0.45 (P < 0.05) in CR and 0.82 (P < 0.001) in PS. The mean differences between predicted and final gaps were 1.8 mm for CR and 1.0 mm for PS. In 34.3% of CR cases, the gap difference was more than 2 mm., Conclusion: It is possible to predict the final flexion gap before femoral posterior condylar osteotomy, with a strong correlation observed between predicted and final component gaps in PS TKA. However, in CR, more than 30% of the cases showed unexpectedly large final flexion gaps., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The DEAD-box family of RNA helicases plays essential roles in both transcriptional and translational mRNA degradation; they unwind short double-stranded RNA by breaking the RNA-RNA interactions. Two DEAD-box RNA helicases, eukaryotic translation initiation factor 4A3 (eIF4A3) and DEAD-box helicase 3 (DDX3X), show high homology in the ATP-binding region and are considered key molecules for cancer progression. Several small molecules that target eIF4A3 and DDX3X have been reported to inhibit cancer cell growth; however, more potent compounds are required for cancer therapeutics, and there is a critical need for high-throughput assays to screen for RNA helicase inhibitors. In this study, we developed novel fluorescence resonance energy transfer-based high-throughput RNA helicase assays for eIF4A3 and DDX3X. Using these assays, we identified several eIF4A3 allosteric inhibitors whose inhibitory effect on eIF4A3 ATPase showed a strong correlation with inhibitory effect on helicase activity. From 102 compounds that exhibited eIF4A3 ATPase inhibition, we identified a selective DDX3X inhibitor, C1, which showed stronger inhibition of DDX3X than of eIF4A3. Small-molecule helicase inhibitors can be valuable for clarifying the molecular machinery of DEAD-box RNA helicases. The high-throughput quantitative assays established here should facilitate the evaluation of the helicase inhibitory activity of compounds., Competing Interests: Declaration of competing interest In accordance with the journal’s policy regarding conflicts of interest: SN, MN, MI, TI, MIto, TT, MT, SE, DC, MO, YI, TK, AN, and SAr are/were employees of Takeda Pharmaceutical Co. Ltd., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The objective of the present study is to demonstrate that a newly developed selective c-Fos/activator protein (AP)-1 inhibitor, T-5224, inhibits the expression of matrix metalloproteinases (MMPs) in human articular chondrocytes, and prevents cartilage destruction in an osteoarthritis (OA)-induced mouse model. First, we examined the effect of T-5224 on MMP and inflammatory cytokine expression by real-time polymerase chain reaction in human articular chondrocytes. We created an OA model by destabilization of the medial meniscus (DMM) in mice. T-5224 was orally administered once a day and the OA pathology was assessed by histological, immunohistochemical, and micro-computed tomography (CT) analyses. T-5224 inhibited the mRNA expression levels of MMP-1, 3, and 13, and interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6 in IL-1-stimulated human chondrocytes. Oral administration of T-5224 to OA-induced mice prevented cartilage destruction. The histological scores for OA were significantly better in the T-5224-treated group than the vehicle-treated group. Type X collagen and MMP-13 were not increased in the T-5224-treated group by immunohistochemical staining. Micro-CT analysis showed mild but apparent osteophyte development in the femoral condyle and antero-medial aspect of the tibia in the vehicle-treated group but not in the T-5224-treated group. Taken together, specific inhibition of c-Fos/AP-1 and the resulting inhibition of the transactivation of a broad spectrum of downstream MMPs, along with inflammatory cytokines, effectively prevented cartilage destruction and osteophyte formation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is still one of the major causes of cancer-related death. Kinetochore-associated protein 2 (KNTC2) is specifically upregulated in tumor tissues of HCC patients and recognized as a potential candidate target for the treatment of HCC. However, the relationship between KNTC2 and in vivo tumor growth of HCC is not yet fully understood. Here we encapsulated KNTC2 siRNAs into a lipid nanoparticle (LNP) and investigated their knockdown activity, target engagement marker, anti-tumor activity and hepatotoxicity in an orthotopic HCC model mice of Hep3B-luc cells. Single i.v. administration of KNTC2 siRNA-LNP specifically suppressed the expression levels of both human KNTC2 mRNA and mouse Kntc2 mRNA in tumor tissues. Phosphorylation levels of histone H3 (HH3) at serine 10 in tumor tissues were increased by KNTC2 siRNA-LNP. Repeated administration of KNTC2 siRNA-LNP (twice a week) specifically inhibited the growth of tumor tissues without increasing the plasma AST and ALT levels. Their growth inhibitory activities were consistent with knockdown activities. These data strongly indicated that KNTC2 is a promising target for the treatment of HCC and that phosphorylated HH3 at serine 10 is one of the target engagement markers for KNTC2., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Linezolid is an effective antibiotic against most gram-positive bacteria including drug-resistant strains such as methicillin-resistant Staphylococcus aureus. Although linezolid therapy is known to result in thrombocytopenia, dosage adjustment or therapeutic drug monitoring of linezolid is not generally necessary. In this report, however, we describe the case of a 79-year-old woman with recurrent methicillin-resistant S. aureus osteomyelitis that was successfully treated via surgery and combination therapy using linezolid and rifampicin under therapeutic drug monitoring for maintaining an appropriate serum linezolid concentration. The patient underwent surgery for the removal of the artificial left knee joint and placement of vancomycin-impregnated bone cement beads against methicillin-resistant S. aureus after total left knee implant arthroplasty for osteoarthritis. We also initiated linezolid administration at a conventional dose of 600 mg/h at 12-h intervals, but reduced it to 300 mg/h at 12-h intervals on day 9 because of a decrease in platelet count and an increase in serum linezolid trough concentration. However, when the infection exacerbated, we again increased the linezolid dose to 600 mg/h at 12-h intervals and performed combination therapy with rifampicin, considering their synergistic effects and the control of serum linezolid trough concentration via drug interaction. Methicillin-resistant S. aureus infection improved without reducing the dose of or discontinuing linezolid. The findings in the present case suggest that therapeutic drug monitoring could be useful for ensuring the therapeutic efficacy and safety of combination therapy even in patients with osteomyelitis who require long-term antibiotic administration., (Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Lumbar disc disease (LDD) is one of the most common musculoskeletal disorders, and accompanies intervertebral disc degeneration. CILP encodes cartilage intermediate layer protein, which is highly associated with LDD. Moreover, CILP inhibits transcriptional activation of cartilage matrix genes in nucleus pulposus (NP) cells in vitro by binding to TGF-β1 and inhibiting the phosphorylation of Smads. However, the aetiology and mechanism of pathogenesis of LDD in vivo are unknown. To demonstrate the role of CILP in LDD in vivo, we generated transgenic mice that express CILP specifically in the intervertebral disc tissues and assessed whether CILP exacerbates disc degeneration. Degeneration of the intervertebral discs was assessed using magnetic resonance imaging (MRI) and histology. The level of phosphorylation of Smad2/3 in intervertebral discs was measured to determine whether overexpressed CILP suppressed TGF-beta signalling. Although the macroscopic skeletal phenotype of transgenic mice appeared normal, histological findings revealed significant degeneration of lumbar discs. MRI analysis of the lumbar intervertebral discs indicated a significantly lower signal intensity of the nucleus pulposus where CILP was overexpressed. Intervertebral disc degeneration was also observed. The number of phosphorylation of Smad2/3 immuno-positive cells in the NP significantly was decreased in CILP transgenic mice compared with normal mice. In summary, overexpression of CILP in the NP promotes disc degeneration, indicating that CILP plays a direct role in the pathogenesis of LDD., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Oestrogen is known to play a cardioprotective role in cardiovascular diseases, as demonstrated in a number of animal studies. However, few human studies have investigated sex-based differences with regard to cardiac glucose uptake using (18)F-fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT)., Methods: Therefore, we evaluated healthy male and female subjects who underwent FDG-PET/CT examination to determine whether there was a sex-related difference in cardiac glucose uptake with age., Results: In females, the prevalence of maximal FDG uptake (PET score 2) demonstrated a convex pattern with ageing, and it peaked at age 51-60 years in the females, gradually decreasing to a minimum at age >70 years. In contrast, the prevalence of maximal FDG uptake by age in males was a mirror image of that in females, i.e. it formed a concave pattern with a nadir at 61-70 years, followed by an increase in the prevalence. These findings suggest that female hearts depend more on glucose as an energy substrate as they age, however, efficient glucose uptake is attenuated with increasing age. In contrast, the male heart sustains its glucose uptake capacity at age >70 years., Conclusion: This characteristic sex-based difference in cardiac glucose uptake might be related to the female predominance of Takotsubo cardiomyopathy., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Ion suppression can negatively affect the performance characteristics of LC/ESI-MS/MS based methods, and we wished to identify sources of ion suppression in an assay for quantitating sirolimus in human whole blood. We first compared the peak areas of sirolimus and ascomycin added to human blood samples treated with and without extraction using octadecyl silyl (ODS)-silica gel after protein precipitation, and we found that water-soluble compounds cause the ion suppression for both drugs. Post-column infusion studies indicated that compounds retained in the sample after ODS extraction and protein precipitation caused ion suppression. MS analysis of these compounds suggested they were hydroxyl group-possessing phosphocholines, and this was confirmed using purified lysophosphatidylcholine variants. Therefore, we included a HybridSPE treatment step after the ODS extraction into the preanalytical workflow to remove phosphocholines, and this successfully eliminated the observed ion suppression for determining sirolimus concentration in human whole blood by LC/ESI-MS/MS. Sirolimus is a highly lipophilic molecule, and this study demonstrates the impact that preanalytical extraction and purification steps can have on a laboratory's ability to accurately detect and quantitate this and other lipophilic drugs., (Copyright © 2011 Elsevier B.V. All rights reserved.)

For effective screening by biopanning, we propose a new affinity screening method utilizing protein-coupled liposomes (proteoliposomes) as adsorbents. With multilamellar vesicles (MLVs) composed of dipalmitoylphosphatidylcholine (DPPC): dicetylphosphate (DCP) = 10: 1 (molar ratio), adsorption of nonspecific phage VCSM13 to the liposomes without any blocking was comparable to that on polystyrene tube wall coated with blocking protein. Phages displaying octapeptides specific to an anti-peptide antibody against a peptide antigen (FVNQHLCK) were screened from an octapeptide-displayed phage library by biopanning utilizing liposomes coupled with the antibody (AB-MLVs) or a conventional immunotube coated with the antibody (AB-tube). After four rounds of biopanning, all selected phages displayed homological peptides to the antigen peptide by use of AB-MLVs, while only 15% of the selected phages displayed homological peptides in the conventional biopanning. The octapeptide selected by AB-MLVs against the anti-peptide antibody showed comparable binding affinity, which were determined by the competitive ELISA and an immunoaffinity chromatography, to that of the peptide antigen. Thus, protein-coupled liposomes are useful as adsorbents for screening from combinatorial phage libraries.

For study of the molecular basis of regulation of proteasome gene expression, we isolated the gene encoding the alpha-type HC8 subunit of the human proteasome. About 2.3 kb of the 5' flanking region of this gene was tested for promoter function by chloramphenicol acetyltransferase assay. This analysis revealed that CAAT and TATA boxes, but not a GC box, are essential for its promoter activity. These results differed from previous findings that the genes for the alpha-type HC3 and beta-type HC5 subunits of the human proteasome have a TATA-less promoter and that two or three GC boxes function as the promoter sequences (Tamura, T. et al. (1994) J. Mol. Biol. 244, 1117-1124). We mapped the HC8 gene at q23 on human chromosome 14, which differs from the chromosomal locations of nine other proteasomal subunit genes mapped so far.

C16- and C18- platelet-activating factor (PAF) at 10(-6) M enhanced the uptake of [14C]aminopyrine (AP) by isolated guinea pig parietal cells. This increase was inhibited by a PAF antagonist CV-6209. In a medium with a low calcium (Ca2+) concentration (2 uM), this increase was not observed, which indicates that the increase of AP uptake by PAF is dependent on extracellular Ca2+. PAF and lyso-PAF showed no effect on AP uptake in the presence of histamine or carbachol.