Kazuo Harada

Gram-positive bacteria encode a broad array of ABC transporters that mediate substrate translocation across the cell membrane, with some contributing to their survival under environmental stresses such as antimicrobial exposure. While several of these transporters have been shown to exhibit multidrug efflux activity, the functional roles of many others remain unknown. Here, using an efflux pump screen in the opportunistic human pathogen Streptococcus pneumoniae , we identified a previously uncharacterized type IV ABC transporter (FoeAB) that confers resistance to the antibiotic fosfomycin. We show that purified FoeAB mediates fosfomycin transport in a liposome-reconstituted system and provide evidence that it functions as a multidrug efflux pump with substrate preferences distinct from those of known efflux pumps. Furthermore, we present cryogenic electron microscopy (cryo-EM) structures of FoeAB in inward- and outward-facing states, which reveal conformational changes associated with nucleotide binding and identify residues important for substrate transport. Collectively, these findings expand the known repertoire of antibiotic-exporting ABC transporters in Gram-positive bacteria and provide structural insight into the underlying transport mechanism.

Background: Acute nicotine intoxication can cause cardiac arrest. We report a case of cardiac arrest managed with veno‐arterial extracorporeal membrane oxygenation (VA‐ECMO) alongside serial serum nicotine monitoring. Case Presentation: A 36‐year‐old woman collapsed after ingesting an unknown amount of liquid nicotine, up to 20 g. Emergency medical services found her in cardiac arrest with pulseless electrical activity (PEA). On arrival, she remained in cardiac arrest with PEA/asystole and severe acidosis, and VA‐ECMO was established 82 min after ingestion. Cardiac function gradually improved, and VA‐ECMO was discontinued on Day 3. However, she died on Day 28 from post‐cardiac arrest brain injury. Liquid chromatography–mass spectrometry (LC/MS) revealed an admission serum nicotine concentration of 6.44 μg/mL, declining to 0.057 μg/mL within 18 h. This decline paralleled hemodynamic recovery. Conclusion: Although neurological outcome was unfavorable, VA‐ECMO may provide temporary circulatory support during the toxic phase of massive nicotine intoxication.

Purpose: Comparison of the impurity removal efficiencies of the deproteinization and Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) methods, which are pretreatment methods for drug analysis adopted by many forensic autopsy institutions, was performed. Method: Residual cardiac blood samples were pretreated using deproteinization and QuEChERS methods. The residual amounts of total proteins, total lipids, glucose, galactose, electrolytes, and inorganic elements were measured. We also compared the recovery rates and matrix factors when using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Results: The residual rates of total proteins, total lipids, glucose, galactose, and electrolytes using the deproteinization method were 16%, 75%, 75%, 90%, and 91%, respectively. In contrast, the QuEChERS method showed 1.1%, 11%, 7.6%, 9.4%, and 20%, respectively. The amounts of Mg and Mn in QuEChERS increased compared with those before treatment, but other inorganic elements remained at 9.6–89% during deproteinization and 0.30–17% in the QuEChERS. The recovery rate of metformin was low in QuEChERS; however, no differences were observed in the recovery rates or matrix factors of the other 16 drugs between deproteinization and QuEChERS. Conclusions: This study quantitatively demonstrated that QuEChERS is extremely efficient at removing impurities from blood compared with deproteinization methods. QuEChERS has poor recovery rates for highly polar drugs but does not prevent their detection. The QuEChERS method is superior to the deproteinization method, considering the load of impurities on the analytical instruments.

Seishinrenshiin (SRI), a Kampo formula, is often used for frequent urination. Nelumbo Seed, a crude-drug component of SRI was reported to inhibit bladder smooth muscle contraction using excised rat bladder tissues. However, the active ingredients of Nelumbo Seed have not yet been identified. In this study, we investigated the active ingredients of Nelumbo Seed that inhibit bladder smooth muscle contraction. We obtained liquid chromatography/mass spectrometry profiles of extracts prepared from five types of Nelumbo Seed materials from different production areas and evaluated their inhibitory effects on excised rat bladder smooth muscle contraction. Analysis of these data using orthogonal projections to latent structures revealed neferine (Nef) as the compound with the highest variable influence on projection. Among the Nelumbo Seed materials processed using different methods, the Nef content was the highest in ‘Sekirenshi’, when embryo and pericarp were not removed at all. We determined the effects of administering Nelumbo Seed extracts with different Nef content on frequent-urination model rats. The Nef content in rat plasma was the highest when the Sekirenshi extract was administered, but Nef was not detected when seed embryos were removed. In this model, administration of the Nelumbo Seed extract improved the maximum bladder contraction pressure and bladder contraction interval, albeit not significantly. In particular, Sekirenshi tended to reduce the maximum bladder contraction pressure compared to other Nelumbo Seed. Our results indicate that although Nef in Nelumbo Seed does not clearly improve frequent urination, it might contribute to the improvement of urination disorder. Graphical Abstract

Purpose: Fire victims often inhale hydrogen cyanide (HCN) gas in addition to carbon monoxide. This study aimed to investigate the current prevalence of HCN inhalation among fire victims and assess the contribution of HCN as a toxic factor in fire-related deaths. Methods: The study included 29 cases of fire-related deaths, where autopsies were conducted at the Department of Legal Medicine, Osaka University, from April 2014 to March 2020. No resuscitation was performed before death was confirmed and blood samples were obtained from both the left and right cardiac chambers. Blood cyanide concentrations were measured. Additionally, a physiologically based pharmacokinetic model, as described by Stamyr et al. (Arch Toxicol 89:1287–1296, 2015), was used to simulate the time course of blood concentration changes for different inhaled HCN concentrations. The inhaled HCN concentration and inhalation time that minimized the difference between the measured and simulated blood concentrations were calculated. Results: Cyanide was detected in the cardiac blood of 76.3% of cases. In all instances, left cardiac blood concentrations were higher than those in the right cardiac blood. The simulations using the physiologically based pharmacokinetic model revealed eight cases where the inhaled HCN concentration exceeded 5000 ppm, with an inhalation time of less than 0.5 min. Conclusions: Many fire victims inhaled HCN gas, and in a few cases, it appears that death occurred rapidly after inhalation of high HCN concentrations. These findings suggest that the contribution of cyanide gas to fire-related deaths warrants closer examination.

Deuterated molecules are of growing interest because of the specific characteristics of deuterium, such as stronger C–D bonds being stronger than C–H bonds. Polyethylene glycols (PEGs) are widely utilized in scientific fields (e.g., drug discovery and material sciences) as linkers and for the improvement of various properties (solubility in water, stability, etc.) of mother compounds. Therefore, deuterated PEGs can be used as novel tools for drug discovery. Although the H/D exchange reaction (deuteration) is a powerful and straightforward method to produce deuterated compounds, the deuteration of PEGs bearing many unactivated C(sp3)–H bonds has not been developed. Herein, we report the photocatalytic deuteration of multiple PEGs using tetra-n-butylammonium decatungstate (TBADT) and D2O as inexpensive deuterium sources. This deuteration can be adapted to PEG derivatives bearing various substituents ((hetero)aryl, benzoyl, alkyl, etc.) at the terminal oxygen atoms of PEGs. The deuteration efficiencies of the alpha-oxy C(sp3)–H bonds at the terminal positions of the PEGs were strongly influenced by the substituents. These reactivities were elucidated by density functional theory calculations of the reaction barriers towards the formation of radical intermediates, induced by the excited state of TBADT and the PEG substrate. In addition, the applicability of deuterated PEGs to internal standard experiments and Raman spectroscopy was demonstrated.

The fungal genus Trichoderma is a rich source of structurally diverse secondary metabolites with remarkable pharmaceutical properties. The chemical constituents and anticancer activities of the marine-derived fungus Trichoderma lixii have never been investigated. In this study, a bioactivity-guided investigation led to the isolation of eleven compounds, including trichodermamide A (1), trichodermamide B (2), aspergillazine A (3), DC1149B (4), ergosterol peroxide (5), cerebrosides D/C (6/7), 5-hydroxy-2,3-dimethyl-7-methoxychromone (8), nafuredin A (9), and harzianumols E/F (10/11). Their structures were identified by using various spectroscopic techniques and compared to those in the literature. Notably, compounds 2 and 5–11 were reported for the first time from this species. Evaluation of the anticancer activities of all isolated compounds was carried out. Compounds 2, 4, and 9 were the most active antiproliferative compounds against three cancer cell lines (human myeloma KMS-11, colorectal HT-29, and pancreas PANC-1). Intriguingly, compound 4 exhibited anti-austerity activity with an IC₅₀ of 22.43 μM against PANC-1 cancer cells under glucose starvation conditions, while compound 2 did not.

Preparation of drug metabolites at the milligram scale is essential for determining the structure and toxicity of drug metabolites. However, their preparation using recombinant proteins and human liver microsomes (HLM) is often difficult because of technical and ethical issues. Reproducing human drug metabolism in food-derived microorganisms may be useful for overcoming these challenges. In this study, we identified an unknown metabolite of the anaesthetic drug lidocaine, which is metabolised by HLM. By screening for lidocaine metabolic activity in five types of foods (blue cheese, shiitake mushroom, natto, yoghurt, and dry yeast), we found that bacteria isolated from natto reproduced the lidocaine metabolic reaction that occurs in HLM. A fraction containing the unknown lidocaine metabolite was prepared through mass cultivation of a Bacillus subtilis standard strain, ethyl acetate extraction, open column chromatography, and HPLC purification. We identified the unknown metabolite as 3-(2,6-dimethylphenyl)-1-ethyl-2-methyl-4-imidazolidinone using NMR. Our results showed that food-derived microorganisms can produce large amounts of human drug metabolites via large-scale cultivation. Additionally, food microorganisms that can reproduce drug metabolism in humans can be used to examine drug metabolites at a low cost and without ethical issues.