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Abstract Background Fentanyl is widely used as an analgesic and sedative for patients with severe burn injuries in intensive care units. However, pharmacokinetic (PK) data for fentanyl, particularly for continuous intravenous infusion during the acute phase of burn injuries, are limited. Here, we report the clinical course and changes in blood fentanyl concentrations during the acute phase in a patient with severe burns treated with continuous intravenous infusion of fentanyl. Case presentation A woman in her 40s, with burns caused by a gas cylinder explosion, was transported to our hospital. The patient had burn wounds on face, neck, shoulders, and all four extremities, with a total burn area of 39.0%. For pain relief, the patient received a continuous infusion of 0.01 mg/mL fentanyl (20–30 µg/h) with a target blood concentration of 1.0–1.5 ng/mL, but continued to suffer from pain due to burning during the acute phase. We measured the blood fentanyl concentrations and found that all concentrations obtained during the acute phase were subtherapeutic. Notably, during the burn shock stage, blood concentrations of fentanyl were 0.50 ng/mL on day 1 and 0.66 ng/mL on day 2, indicating that the blood concentration did not rise sufficiently for the dosage. From days 0 to 2, the patient was administered a massive fluid load for burn shock. After the burn shock stage resolved, fentanyl concentrations gradually approached the target range, and the pain rating scale improved, even though the fentanyl administration rate remained unchanged (30 µg/h). Conclusions Major changes in the fluid volumes of body compartments that occur with large burns might increase the volume of fentanyl distribution, thereby lowering its concentration when a standard dose is administered. Our findings indicate that the PK of fentanyl in patients with severe burns can be substantially affected, especially during the shock phase, implying the importance of titrating analgesics for clinical efficacy in the acute phase.

Abstract Cisplatin (CDDP)-induced nephrotoxicity is a common dose-limiting toxicity, and diuretics are often administered to prevent nephrotoxicity. However, the efficacy and optimal administration of diuretics in preventing CDDP-induced nephrotoxicity remain to be established. This study aimed to evaluate the efficacy of combining furosemide and mannitol to prevent CDDP-induced nephrotoxicity. This was a post-hoc analysis of pooled data from a multicenter, retrospective, observational study, including 396 patients who received one or two diuretics for CDDP-based chemotherapy, compared using propensity score matching. Multivariate logistic regression analyses were used to identify risk factors for nephrotoxicity. There was no significant difference in the incidence of nephrotoxicity between the two groups (22.2% vs. 28.3%, P = 0.416). Hypertension, CDDP dose ≥ 75 mg/m2, and no magnesium supplementation were identified as risk factors for nephrotoxicity, whereas the use of diuretics was not found to be a risk factor. The combination of furosemide and mannitol showed no advantage over a single diuretic in preventing CDDP-induced nephrotoxicity. The renal function of patients receiving CDDP-based chemotherapy (≥ 75 mg/m2) and that of those with hypertension should be carefully monitored. Magnesium supplementation is important for these patients.

Abstract Epidermal growth factor receptor (EGFR) inhibitors frequently cause severe skin rash as a side effect, which is a critical burden for patients who continuously receive drug treatments. Several recent clinical trials have shown that vitamin K is effective against these side effects; however, the underlying mechanisms remain unclear. EGFR inhibitors induce C–C motif chemokine ligand 5 (CCL5) in dermopathy. We hypothesized that menahydroquinone-4 (MKH), the active form of menaquinone-4 (MK-4, vitamin K2(20)), supplied by biosynthesis or external delivery, is essential for the suppressive effect on CCL5. The aim of this study was to explore the underlying mechanisms governing the relieving effects of MKH against skin rashes caused by EGFR inhibitors. The responses generated by EGFR inhibitors and the effect of MKH derivatives (two ester derivatives and MK-4) on them were evaluated using human skin cell lines (HaCaT and HSC-1). EGFR inhibitors downregulated UbiA prenyltransferase domain-containing protein-1 (UBIAD1, MKH synthetase) expression and MKH biosynthesis. Knockdown of UBIAD1 or γ-glutamyl carboxylase and treatment with warfarin upregulated CCL5 expression. MKH derivatives suppressed the CCL5 expression induced by EGFR inhibitors. Our data strongly suggest that MKH is involved in suppressing CCL5 expression and alleviating the skin damage caused by EGFR inhibitors.

This research identified a marine fungal isolate, Aspergillus sp. strain GAD7, which produces an acidic and sulfated extracellular polysaccharide (EPS) with notable anticoagulant and antioxidant properties. Six fungal strains from the Egyptian Mediterranean Sea were screened for EPS production, with Aspergillus sp. strain GAD7 (EPS-AG7) being the most potent, yielding ~5.19 ± 0.017 g/L. EPS-AG7 was characterized using UV-Vis and FTIR analyses, revealing high carbohydrate (87.5%) and sulfate (24%) contents. HPLC and GC-MS analyses determined that EPS-AG7 is a heterogeneous acidic polysaccharide with an average molecular weight (Mw¯) of ~7.34 × 103 Da, composed of mannose, glucose, arabinose, galacturonic acid, galactose, and lyxose in a molar ratio of 6.6:3.9:1.8:1.3:1.1:1.0, linked through α- and β-glycosidic linkages as confirmed by NMR analysis. EPS-AG7 adopted a triple helix-like conformation, as evidenced by UV-Vis (Congo Red experiment) and circular dichroism (CD) studies. This helical arrangement demonstrated stability under various experimental conditions, including concentration, ionic strength, temperature, and lipid interactions. EPS-AG7 exhibited significant anticoagulant activity, doubling blood coagulation time at a concentration of 3.0 mg/mL, and showed significant antioxidant activity, with scavenging activities reaching up to 85.90% and 58.64% in DPPH and ABTS+ assays at 5.0 mg/mL, and EC50 values of 1.40 mg/mL and 3.80 mg/mL, respectively. These findings highlight the potential of EPS-AG7 for therapeutic applications due to its potent biological activities.

Abstract Background In Japan, the use of risperidone in combination with adrenaline is contraindicated, except in cases of anaphylaxis. Therefore, there is limited clinical evidence regarding the interaction of these two drugs. Here, we report the clinical course of a case of adrenaline-resistant anaphylactic shock induced by a contrast medium injection after a risperidone overdose. Case presentation A man in his 30s was transported to our hospital after attempting suicide by taking 10 mg of risperidone and jumping from a height of 10 m. To determine the location and severity of his injuries, he was injected with an iodinated contrast medium, after which he developed generalized erythema and hypotension and was diagnosed with anaphylactic shock. A 0.5 mg dose of adrenaline was administered with no improvement, followed by another 0.5 mg dose that did not change his blood pressure. After infusion of a sodium bicarbonate solution (8.4%), administration of fresh frozen plasma, and additional administration of adrenaline (0.6–1.2 µg/min), his blood pressure improved, and he recovered from the anaphylactic shock. Conclusions This was a rare case of a risperidone overdose followed by adrenaline-resistant anaphylactic shock. The resistance is likely associated with the high blood concentration of risperidone. Our findings indicate that the potential for decreased adrenergic responsiveness should be considered in patients undergoing risperidone treatment in the event of anaphylactic shock.

Active regulation of chirality, or handedness, is crucial for life. Here, the authors describe assays to characterise proteome chirality in vivo and show that errors degrade enzyme activity triggering tumour formation and reduced lifespan.

ABSTRACT The chemokine fractalkine (FKN) is produced by various cell types, including osteoblasts and endothelial cells in bone tissue, and signals through a sole receptor, CX3CR1, which is expressed on monocytes/macrophages, including osteoclast precursors (OCPs). However, the direct effects of FKN signaling on osteoclast lineage cells under homeostatic noninflammatory conditions remain unclear. Here, we report that FKN regulates mouse OCP survival and primes OCPs for subsequent osteoclast differentiation. Wild‐type but not CX3CR1‐deficient OCPs grown on immobilized FKN showed enhanced osteoclast formation following receptor activator of NF‐κB ligand (RANKL) stimulation, with increased expression of osteoclast differentiation markers. Interestingly, the growth of OCPs on immobilized FKN increased the expression of Cx3cr1 and Tnfrsf11a (Rank) transcripts, but following RANKL stimulation, OCPs rapidly downregulated Cx3cr1 expression. Consistently, anti‐FKN monoclonal antibody (mAb) treatment attenuated RANKL‐induced osteoclast formation on immobilized FKN before, but not during, RANKL stimulation. CX3CR1 and RANK proteins were highly expressed on bone marrow‐derived CD11bhigh CD115+ OCPs. Growth on immobilized FKN prior to RANKL stimulation also increased CD11bhigh CD115+ OCP number and their survival and differentiation potential. In a RANKL‐based mouse model of bone loss, anti‐FKN mAb pretreatment significantly inhibited RANKL‐dependent bone loss. Thus, blocking the FKN‐CX3CR1 axis could represent a therapeutic option in noninflammatory bone loss diseases. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

The auditory brainstem response (ABR) is a scalp recording of potentials produced by sound stimulation, and is commonly used as an indicator of auditory function. However, the ABR threshold, which is the lowest audible sound pressure, cannot be objectively determined since it is determined visually using a measurer, and this has been a problem for several decades. Although various algorithms have been developed to objectively determine ABR thresholds, they remain lacking in terms of accuracy, efficiency, and convenience. Accordingly, we proposed an improved algorithm based on the mutual covariance at adjacent sound pressure levels. An ideal ABR waveform with clearly defined waves I–V was created; moreover, using this waveform as a standard template, the experimentally obtained ABR waveform was inspected for disturbances based on mutual covariance. The ABR testing was repeated if the value was below the established cross-covariance reference value. Our proposed method allowed more efficient objective determination of ABR thresholds and a smaller burden on experimental animals.

Abstract Background Patients with lung cancer who are treated with carboplatin-based chemotherapy regimens often experience chemotherapy-induced nausea and vomiting (CINV). However, knowledge on the effect of regimen and cofactors on the risk of CINV is limited. This study aimed to analyze and compare the incidence of CINV between lung cancer patients undergoing carboplatin plus pemetrexed (CBDCA+PEM) and those undergoing carboplatin plus paclitaxel (CBDCA+PTX) chemotherapy. Methods Pooled data of 240 patients from two prospective observational studies were compared using propensity score matching. Separate multivariate logistic regression analyses were used to identify risk factors for nausea and vomiting following chemotherapy. Results Delayed nausea was significantly more common in patients treated with CBDCA+PEM than in those treated with CBDCA+PTX (51.1% vs. 36.2%, P = 0.04), but the incidence of vomiting did not significantly differ between the two groups (23.4% vs. 14.9%, P = 0.14). The occurrence of CINV peaked on day 4 in the CBDCA+PTX group and on day 5 in the CBDCA+PEM group. Multivariate analysis showed that female sex, younger age, and CBDCA+PEM regimen were independent risk factors for delayed nausea, while female sex was an independent risk factor for delayed vomiting. Conclusions The CBDCA + PEM regimen has a higher risk of causing delayed nausea than the CBDCA + PTX regimen, and aggressive antiemetic prophylaxis should be offered to patients treated with CBDCA + PEM.

Abstract Background The substantial increase in the use of expensive anticancer drugs has been accompanied by an increase in the amount of disposing residual liquid from drug preparations. Many Western countries, including the United States, have implemented drug vial optimization (DVO) to prevent the waste of anticancer drugs and have reported the reductions in the total drug costs. This study was designed to estimate the expected reduction in spending on anticancer drugs by Japanese cancer hospitals when DVO was implemented instead of individual preparations and to test the effectiveness of this approach. Methods We investigated the doses of drugs used and quantity specifications for individually prepared vials for patients who received anticancer drug treatment in December 2017 at the Outpatient Treatment Center of the National Cancer Center Hospital East. Based on these findings, we calculated the total quantity of each drug used on a given day, and the minimum cost for preparation of the number of specified combinations corresponding to the total cost (DVO preparation). Based on the differences in these two costs, we estimated the economic impact of implementing DVO. Results While the cost for anticancer drugs for the 1-month study period was US$3,305,595 (US$1 = \110) for individual preparations, the estimated cost for DVO preparations was US$3,092,955, equivalent to a reduction of US$212,640. Conclusions Based on these study results, implementation of DVO-based preparation of injectable anticancer drugs in Japan in 2017 would have resulted in saving approximately US$460 million. This calculation revealed the need for the Japanese government to modify the methods employed to calculate drug costs in the insurance system and develop policies for the proper and optimal use of medical resources.

Abstract Background Lactate is a well-known marker to estimate prognosis after cardiac surgery and critically ill patients. The liver and kidney have a major role in lactate metabolism; however, there was less characterized about the change of lactate and threshold to predict in-hospital mortality in dialysis-dependent patients undertaking cardiac surgery. We conducted this retrospective observational study to characterize when and how lactate values after cardiac surgery affected in-hospital mortality. Methods This two-center retrospective study included dialysis-dependent patients who underwent cardiac surgery with a cardiopulmonary bypass from January 2014 to December 2018. Lactate values were collected at three points: at ICU admission (T1), the maximum level of lactate within 24 h postoperatively (T2), and 24 h after ICU admission (T3). We determined hyperlactatemia as more than 2 mmol/L following previous studies. Results We enrolled 122 dialysis-dependent patients. The mean age was 73 ± 8 years and hyperlactatemia was observed in 100 patients (81.9%). In-hospital mortality was 11.4%. Univariate analysis and area under curve in ROC suggested that T2 lactate was the most significantly associated with in-hospital mortality (AUC = 0.845). Multivariate logistic analysis showed a significant association between in-hospital mortality when patients showed early peak lactate levels of > 4.5 mmol/L after ICU admission (adjusted OR 8.35; 95% CI: 1.44–57.13). Conclusions In dialysis-dependent patients after cardiac surgery, the early-onset of a maximum arterial lactate concentration of > 4.5 mmol/L was significantly associated with in-hospital mortality.

Abstract Background Inappropriate dosing of direct oral anticoagulants (DOACs) has been associated with clinical safety and efficacy; however, little is known about clinical data associated with an inappropriate DOAC dosing in Japan. In addition, there is no report in which the appropriateness of DOAC dosing between prescription for inpatients and for outpatients was examined. In this study, we aimed to investigate the prevalence and factors associated in the inappropriate dosing of DOACs in patients with atrial fibrillation (AF). Methods The retrospective cohort study was conducted at a single Japanese university hospital. Both inpatients and outpatients, who were diagnosed with AF and for whom treatment with either dabigatran, rivaroxaban, apixaban, or edoxaban was initiated between April 1, 2014 and March 31, 2018, were enrolled in the study. Appropriateness of DOAC dosing was assessed according to the manufacturer’s labeling recommendations (dose reduction criteria) of each DOAC. Inappropriate reduced dose, namely, underdosing, was defined as prescription of a reduced dose of DOAC despite the patient not meeting the dose reduction criteria. Inappropriate standard dose, namely, overdosing, was defined as prescription of a standard dose of DOAC despite the patient meeting the dose reduction criteria. Inappropriate DOAC dosing was defined as a deviation of the recommended dose (both underdosing and overdosing). Results A total of 316 patients (dabigatran, 28; rivaroxaban, 107; apixaban, 116; and edoxaban, 65) were included, with a median (interquartile range) age of 75 (66–81) years and 62.3% male. DOACs were prescribed at an appropriate standard dose in 39.2% of patients, an appropriate reduced dose in 36.7%, an inappropriate standard dose in 2.5%, and an inappropriate reduced dose in 19.3%. Multivariate analysis revealed that the inappropriate dosing of DOACs was significantly associated with prescriptions for outpatients (vs. inpatients; odds ratio [OR] 2.87, 95% confidence interval [CI] 1.53–5.62, p

The locomotor system is highly bilateral at the macroscopic level. Homochirality of biological molecules is fully compatible with the bilateral body. However, whether and how single-handed cells contribute to the bilateral locomotor system is obscure. Here, exploiting the small number of cells in the swimming tadpole larva of the ascidian Ciona, we analyzed morphology of the tail at cellular and subcellular scales. Quantitative phase-contrast X-ray tomographic microscopy revealed a high-density midline structure ventral to the notochord in the tail. Muscle cell nuclei on each side of the notochord were roughly bilaterally aligned. However, fluorescence microscopy detected left-right asymmetry of myofibril inclination relative to the longitudinal axis of the tail. Zernike phase-contrast X-ray tomographic microscopy revealed the presence of left-handed helices of myofibrils in muscle cells on both sides. Therefore, the locomotor system of ascidian larvae harbors symmetry-breaking left-handed helical cells, while maintaining bilaterally symmetrical cell alignment. These results suggest that bilateral animals can override cellular homochirality to generate the bilateral locomotor systems at the supracellular scale.

NCYM, a cis-antisense gene of MYCN, encodes a Homininae-specific protein that promotes the aggressiveness of human tumors. Newly evolved genes from non-genic regions are known as de novo genes, and NCYM was the first de novo gene whose oncogenic functions were validated in vivo. Targeting NCYM using drugs is a potential strategy for cancer therapy; however, the NCYM structure must be determined before drug design. In this study, we employed vacuum-ultraviolet circular dichroism to evaluate the secondary structure of NCYM. The SUMO-tagged NCYM and the isolated SUMO tag in both hydrogenated and perdeuterated forms were synthesized and purified in a cell-free in vitro system, and vacuum-ultraviolet circular dichroism spectra were measured. Significant differences between the tagged NCYM and the isolated tag were evident in the wavelength range of 190–240 nm. The circular dichroism spectral data combined with a neural network system enabled to predict the secondary structure of NCYM at the amino acid level. The 129-residue tag consists of α-helices (approximately 14%) and β-strands (approximately 29%), which corresponded to the values calculated from the atomic structure of the tag. The 238-residue tagged NCYM contained approximately 17% α-helices and 27% β-strands. The location of the secondary structure predicted using the neural network revealed that these secondary structures were enriched in the Homininae-specific region of NCYM. Deuteration of NCYM altered the secondary structure at D90 from an α-helix to another structure other than α-helix and β-strand although this change was within the experimental error range. All four nonsynonymous single-nucleotide polymorphisms (SNPs) in human populations were in this region, and the amino acid alteration in SNP N52S enhanced Myc-nick production. The D90N mutation in NCYM promoted NCYM-mediated MYCN stabilization. Our results reveal the secondary structure of NCYM and demonstrated that the Homininae-specific domain of NCYM is responsible for MYCN stabilization.

Background and Objectives: Remdesivir (RDV) is the first antiviral agent approved in Japan for the treatment of coronavirus disease 2019 (COVID-19). The aim of our study was to assess the efficacy and safety of RDV treatment in mildly to moderately ill patients with COVID-19. Materials and Methods: A single-center, retrospective study was performed in Fukuoka University Chikushi Hospital. Patients admitted to our hospital from June to October 2021 for RDV treatment against COVID-19 were enrolled. The primary end point was clinical status on days 10 and 14, using a 6-point ordinal scale ranging from death (category 6) to discharge (category 1). Adverse events were assessed and graded using the Japanese version of Common Terminology Criteria for Adverse Events (CTCAE) v5.0. Results: In total, 47 COVID-19 patients receiving RDV treatment were assessed during the study period. Thirty-four (72.3%) out of 47 patients required oxygen therapy. Out of these 34 patients, 30 (88.2%) showed a 2-point clinical improvement on day 14 after RDV was initiated. Serum alanine aminotransferase levels were elevated in three patients (6.4%) (CTCAE Grade 3) and neutropenia was detected in one patient (2.1%) out of the 47 patients. Conclusions: RDV may be highly effective, with good safety profiles, in patients with COVID-19 requiring oxygen therapy.

Glycated human serum albumin (gHSA) undergoes conformational changes and unfolding events caused by free radicals. The glycation process results in a reduced ability of albumin to act as an endogenous scavenger and transporter protein in diabetes mellitus type 2 (T2DM) patients. Astaxanthin (ASX) in native form and complexed with metal ions (Cu2+ and Zn2+) has been shown to prevent gHSA from experiencing unfolding events. Furthermore, it improves protein stability of gHSA and human serum albumin (HSA) as it is shown through molecular dynamics studies. In this study, the ASX/ASX-metal ion complexes were reacted with both HSA/gHSA and analyzed with electronic paramagnetic resonance (EPR) spectroscopy, rheology and zeta sizer (particle size and zeta potential) analysis, circular dichroism (CD) spectroscopy and UV-Vis spectrophotometer measurements, as well as molecular electrostatic potential (MEP) and molecular docking calculations. The addition of metal ions to ASX improves its ability to act as an antioxidant and both ASX or ASX-metal ion complexes maintain HSA and gHSA stability while performing their functions.

An x-ray phase tomographic microscope has been developed by optimizing the optical system for 9-keV synchrotron x rays at BL37XU, SPring-8, Japan. The system consists of a full-field x-ray imaging microscope and a Talbot interferometer. In contrast with our previous system, a Fresnel zone plate for the microscope objective and transmission gratings of the interferometer was designed to improve the refraction sensitivity, with a larger field of view and a higher spatial resolution, utilizing a 28-m space available at BL37XU. An x-ray phase tomographic microscope with a field of view of 375 µm and a spatial resolution of 560 nm was thus constructed, even with a considerably low x-ray dosage. By applying this tomographic microscope to auditory bone specimens of young mice, it was shown that soft tissues in addition to bone tissue could be observed simultaneously without serious radiation damage.

The antimicrobial peptide magainin 2 (M2) interacts with and induces structural damage in bacterial cell membranes. Although extensive biophysical studies have revealed the interaction mechanism between M2 and membranes, the mechanism of membrane-mediated oligomerization of M2 is controversial. Here, we measured the synchrotron-radiation circular dichroism and linear dichroism (LD) spectra of M2 in dipalmitoyl-phosphatidylglycerol lipid membranes in lipid-to-peptide (L/P) molar ratios from 0–26 to characterize the conformation and orientation of M2 on the membrane. The results showed that M2 changed from random coil to α-helix structures via an intermediate state with increasing L/P ratio. Singular value decomposition analysis supported the presence of the intermediate state, and global fitting analysis revealed that M2 monomers with an α-helix structure assembled and transformed into M2 oligomers with a β-strand-rich structure in the intermediate state. In addition, LD spectra showed the presence of β-strand structures in the intermediate state, disclosing their orientations on the membrane surface. Furthermore, fluorescence spectroscopy showed that the formation of β-strand oligomers destabilized the membrane structure and induced the leakage of calcein molecules entrapped in the membrane. These results suggest that the formation of β-strand oligomers of M2 plays a crucial role in the disruption of the cell membrane.

Abstract Bone marrow mesenchymal stem and progenitor cells (BM-MSPCs) maintain homeostasis of bone tissue by providing osteoblasts. Although several markers have been identified for labeling of MSPCs, these labeled cells still contain non-BM-MSPC populations. Studies have suggested that MSPCs are observed as leptin receptor (LepR)-positive cells, whereas osteoblasts can be classified as positive for Runx2, a master regulator for osteoblastogenesis. Here, we demonstrate, using Runx2-GFP reporter mice, that the LepR-labeled population contains Runx2-GFPlow sub-population, which possesses higher fibroblastic colony-forming units (CFUs) and mesensphere capacity, criteria for assessing stem cell activity, than the Runx2-GFP− population. In response to parathyroid hormone (PTH), a bone anabolic hormone, LepR+Runx2-GFPlow cells increase Runx2 expression and form multilayered structures near the bone surface. Subsequently, the multilayered cells express Osterix and Type I collagen α, resulting in generation of mature osteoblasts. Therefore, our results indicate that Runx2 is weakly expressed in the LepR+ population without osteoblastic commitment, and the LepR+Runx2-GFPlow stromal cells sit atop the BM stromal hierarchy.

A new application of vacuum-ultraviolet circular dichroism (VUVCD), which enables the measurement of CD spectra in the vacuum-ultraviolet region (140–200 nm), for the assignment of the absolute configurations of bromoallenes is described. Bromoallene moieties are found in natural products obtained from many marine organisms. To date, the absolute configuration of bromoallenes has been assigned almost exclusively with Lowe’s rule, which is based on specific rotation. However, exceptions to Lowe’s rule have been reported arising from the presence of other substituents with large specific rotations. For the unambiguous assignment of the absolute configuration of the bromoallene moiety with its characteristic absorption wavelength at 180–190 nm due to the π–π* transition, VUVCD was applied to four pairs of bromoallene diastereomers prepared by modifying the synthetic scheme of omaezallene. The VUVCD spectra clearly showed positive or negative Cotton effects around 180–190 nm according to the configuration of the bromoallene employed, revealing the potential of VUVCD for determining absolute stereochemistry.

OBJECTIVE:The aim of this study was to examine the association between medication adherence and illness perceptions, and to explore the factors associated with poor medication adherence in atrial fibrillation (AF) patients receiving direct oral anticoagulants (DOACs) in a real-world clinical setting. METHODS:An observational cross-sectional pilot study was conducted at a single Japanese university hospital. One hundred and twenty-nine patients who were diagnosed with AF and who were taking DOACs were recruited from outpatients between January 4th and April 25th, 2017. We evaluated medication adherence to DOACs using the Morisky Medication Adherence Scale-8 (MMAS-8) and illness perceptions using the Brief Illness Perception Questionnaire (BIPQ). The patients' characteristics and clinical data were collected from electronic medical records. RESULTS:Ninety-nine (76.7%) patients (male, n = 74; mean age, 71.4±9.8 years) participated in this study. According to the MMAS-8, 21 (21.2%) of the patients were classified into the poor adherence group (MMAS-8 score of

Smoking is a common risk factor for both chronic obstructive pulmonary disease (COPD) and osteoporosis. In patients with COPD, severe emphysema is a risk factor for vertebral fracture; however, the effects of smoking or emphysema on bone health remain largely unknown. We report bone deterioration in a mouse model of emphysema induced by nose-only cigarette smoke (CS) exposure. Unexpectedly, short-term exposure for 4-weeks decreased bone turnover and increased bone volume in mice. However, prolonged exposure for 20- and 40-weeks reversed the effects from suppression to promotion of bone resorption. This long-term CS exposure increased osteoclast number and impaired bone growth, while it increased bone volume. Strikingly, long-term CS exposure deteriorated bone quality of the lumbar vertebrae as illustrated by disorientation of collagen fibers and the biological apatite c-axis. This animal model may provide a better understanding of the mechanisms underlying the deterioration of bone quality in pulmonary emphysema caused by smoking.

A plant-derived Enterococcus mundtii 15-1A, that has been previously isolated from Brassica rapa L. subsp. nipposinica (L.H. Bailey) Hanelt var. linearifolia by our group, possesses two kinds of l-lactate dehydrogenase (l-LDH): LDH-1 and LDH-2. LDH-1 was activated under low concentration of fluctose-1,6-bisphosphate (FBP) at both pH 5.5 and 7.5. Although LDH-2 was also activated under the low concentration of FBP at pH 5.5, a high concentration of FBP is necessary to activate it at pH 7.5. The present study shows the crystal structures of the acidophilic LDH-2 in a complex with and without FBP and NADH. Although the tertiary structure of the ligands-bound LDH-2 is similar to that of the active form of other bacterial l-LDHs, the structure without the ligands is different from that of any other previously determined l-LDHs. Major structural alterations between the two structures of LDH-2 were observed at two regions in one subunit. At the N-terminal parts of the two regions, the ligands-bound form takes an α-helical structure, while the form without ligands displays more disordered and extended structures. A vacuum-ultraviolet circular dichroism analysis showed that the α-helix content of LDH-2 in solution is approximately 30% at pH 7.5, which is close to that in the crystal structure of the form without ligands. A D241N mutant of LDH-2, which was created by us to easily form an α-helix at one of the two parts, exhibited catalytic activity even in the absence of FBP at both pH 5.5 and 7.5.

With the increasing interest in scarce proteins, reducing the sample volume for circular dichroism (CD) spectroscopy has become desirable. Demagnification of the incident beam size is required to reduce the sample volume for CD spectroscopy detecting transmitted light passed through the sample. In this study, the beam size was demagnified using a focal mirror, and small-capacity sample cells were developed in an attempt to reduce the sample volume. The original beam size was 6 × 6 mm2; we successfully converged it to a size of 25 × 25 μm2 using the Schwarzschild objective (SO). The new sample cell and SO allowed the required sample volume to be reduced to 1/10 (15 → 1.5 μL), when using a 15 μm path length cell. By adopting a smaller sample cell, further sample reduction could be achieved. By using the SO system, the secondary structural contents of the lysine-36 trimethylated histone H3 protein were analyzed. The trimethylation induced the increment of helix structures and decrement of unordered structures. These structural alterations may play a role in regulating cellular function(s), such as DNA damage repair processes.

Osteoprotegerin (OPG), a decoy receptor for receptor activator of NF-κB ligand (RANKL), antagonizes RANKL's osteoclastogenic function in bone. We previously demonstrated that systemic administration of lipopolysaccharide (LPS) to mice elevates OPG levels and reduces RANKL levels in peripheral blood. Here, we show that mice infected with Salmonella, Staphylococcus, Mycobacteria or influenza virus also show elevated serum OPG levels. We then asked whether OPG upregulation following microbial invasion had an effect outside of bone. To do so, we treated mice with LPS and observed OPG production in pancreas, especially in β-cells of pancreatic islets. Insulin release following LPS administration was enhanced in mice lacking OPG, suggesting that OPG inhibits insulin secretion under acute inflammatory conditions. Consistently, treatment of MIN6 pancreatic β-cells with OPG decreased their insulin secretion following glucose stimulation in the presence of LPS. Finally, our findings suggest that LPS-induced OPG upregulation is mediated in part by activator protein (AP)-1 family transcription factors, particularly Fos proteins. Overall, we report that acute microbial infection elevates serum OPG, which maintains β-cell homeostasis by restricting glucose-stimulated insulin secretion, possibly preventing microbe-induced exhaustion of β-cell secretory capacity.

A novel uracil-DNA degrading protein factor (termed UDE) was identified in Drosophila melanogaster with no significant structural and functional homology to other uracil-DNA binding or processing factors. Determination of the 3D structure of UDE is excepted to provide key information on the description of the molecular mechanism of action of UDE catalysis, as well as in general uracil-recognition and nuclease action. Towards this long-term aim, the random library ESPRIT technology was applied to the novel protein UDE to overcome problems in identifying soluble expressing constructs given the absence of precise information on domain content and arrangement. Nine constructs of UDE were chosen to decipher structural and functional relationships. Vacuum ultraviolet circular dichroism (VUVCD) spectroscopy was performed to define the secondary structure content and location within UDE and its truncated variants. The quantitative analysis demonstrated exclusive α-helical content for the full-length protein, which is preserved in the truncated constructs. Arrangement of α-helical bundles within the truncated protein segments suggested new domain boundaries which differ from the conserved motifs determined by sequence-based alignment of UDE homologues. Here we demonstrate that the combination of ESPRIT and VUVCD spectroscopy provides a new structural description of UDE and confirms that the truncated constructs are useful for further detailed functional studies.

Despite the recent advances in the treatment of multiple myeloma (MM), MM patients with high-risk cytogenetic changes such as t(4;14) translocation or deletion of chromosome 17 still have extremely poor prognoses. With the goal of helping these high-risk MM patients, we previously developed a novel phthalimide derivative, TC11. Here we report the further characterization of TC11 including anti-myeloma effects in vitro and in vivo, a pharmacokinetic study in mice, and anti-osteoclastogenic activity. Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous tumors in human myeloma-bearing SCID mice. Immunohistochemical analyses showed that TC11 induced apoptosis of MM cells in vivo. In the pharmacokinetic analyses, the Cmax was 2.1 μM at 1 h after the injection of TC11, with 1.2 h as the half-life. TC11 significantly inhibited the differentiation and function of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts in mouse osteoclast cultures using M-CSF and RANKL. We also revealed that TC11 induced the apoptosis of myeloma cells accompanied by α-tubulin fragmentation. In addition, TC11 and lenalidomide, another phthalimide derivative, directly bound to nucleophosmin 1 (NPM1), whose role in MM is unknown. Thus, through multiple molecular interactions, TC11 is a potentially effective drug for high-risk MM patients with bone lesions. The present results suggest the possibility of the further development of novel thalidomide derivatives by drug designing.

Clusters of GM1 gangliosides act as platforms for conformational transition of monomeric, unstructured amyloid β (Aβ) to its toxic β-structured aggregates. We have previously shown that Aβ(1–40) accommodated on the hydrophobic/hydrophilic interface of lyso-GM1 or GM1 micelles assumes α-helical structures under ganglioside-excess conditions. For better understanding of the mechanisms underlying the α-to-β conformational transition of Aβ on GM1 clusters, we performed spectroscopic characterization of Aβ(1–40) titrated with GM1. It was revealed that the thioflavin T- (ThT-) reactive β-structure is more populated in Aβ(1–40) under conditions where the Aβ(1–40) density on GM1 micelles is high. Under this circumstance, the C-terminal hydrophobic anchor Val39-Val40 shows two distinct conformational states that are reactive with ThT, while such Aβ species were not generated by smaller lyso-GM1 micelles. These findings suggest that GM1 clusters promote specific Aβ-Aβ interactions through their C-termini coupled with formation of the ThT-reactive β-structure depending on sizes and curvatures of the clusters.

Various receptors on cell surface recognize specific extracellular molecules and trigger signal transduction altering gene expression in the nucleus. Gain or loss-of-function mutations of one molecule have shown to affect alternative signaling pathways with a poorly understood mechanism. In Toll-like receptor (TLR) 4 signaling, which branches into MyD88- and TRAM-dependent pathways upon lipopolysaccharide (LPS) stimulation, we investigated the gain or loss-of-function mutations of MyD88. We predict, using a computational model built on the perturbation-response approach and the law of mass conservation, that removal and addition of MyD88 in TLR4 activation, enhances and impairs, respectively, the alternative TRAM-dependent pathway through signaling flux redistribution (SFR) at pathway branches. To verify SFR, we treated MyD88-deficient macrophages with LPS and observed enhancement of TRAM-dependent pathway based on increased IRF3 phosphorylation and induction of Cxcl10 and Ifit2. Furthermore, increasing the amount of MyD88 in cultured cells showed decreased TRAM binding to TLR4. Investigating another TLR4 pathway junction, from TRIF to TRAF6, RIP1 and TBK1, the removal of MyD88-dependent TRAF6 increased expression of TRAM-dependent Cxcl10 and Ifit2. Thus, we demonstrate that SFR is a novel mechanism for enhanced activation of alternative pathways when molecules at pathway junctions are removed. Our data suggest that SFR may enlighten hitherto unexplainable intracellular signaling alterations in genetic diseases where gain or loss-of-function mutations are observed.

Developing long bones alter their shape while maintaining uniform cortical thickness via coordinated activity of bone)forming osteoblasts and bone-resorbing osteoclasts at periosteal and endosteal surfaces, a process we designate trans-pairing. Two types of trans-pairing shift cortical bone in opposite orientations: peri-forming trans-pairing (peri-t-p) increases bone marrow space and endo-forming trans-pairing (endo-t-p) decreases it, via paired activity of bone resorption and formation across the cortex. Here, we focused on endo-t-p in growing bones. Analysis of endo-t-p activity in the cortex of mouse fibulae revealed osteoclasts under the periosteum compressed by muscles, and expression of RANKL in periosteal cells of the cambium layer. Furthermore, mature osteoblasts were localized on the endosteum, while preosteoblasts were at the periosteum and within cortical canals. X-ray tomographic microscopy revealed the presence of cortical canals more closely associated with endo- than with peri-t-p. Sciatic nerve transection followed by muscle atrophy and unloading induced circumferential endo-t-p with concomitant spread of cortical canals. Such canals likely supply the endosteum with preosteoblasts from the periosteum under endo-t-p, allowing bone shape to change in response to mechanical stress or nerve injury. [ABSTRACT FROM AUTHOR]

Toxin-antitoxin (TA) systems consist of a toxin inhibiting essential cellular functions (such as DNA, RNA and protein synthesis), and its cognate antitoxin neutralizing the toxicity. Recently, we identified a TA system termed TsbA/TsbT in the Staphylococcus aureus genome. The induction of the tsbT gene in Escherichia coli halted both DNA and RNA synthesis, reduced supercoiled plasmid and resulted in increasingly relaxed DNA. These results suggested that DNA gyrase was the target of TsbT. In addition, TsbT inhibited both E. coli and S. aureus DNA gyrase activity and induced linearization of plasmid DNA in vitro. Taken together, these results demonstrate that the TsbT toxin targets DNA gyrase in vivo. Sitedirected mutagenesis experiments showed that the E27 and D37 residues in TsbT are critical for toxicity. Secondary structure prediction combining the analysis of vacuum-ultraviolet circular-dichroism spectroscopy and neural network method demonstrated that the 22nd-32nd residues of TsbT form an a-helix structure, and that the E27 residue is located around the centre of the a-helix segment. These findings give new insights not only into S. aureus TA systems, but also into bacterial toxins targeting DNA topoisomerases. [ABSTRACT FROM AUTHOR]