Fentanyl-Type Antagonist of the µ-Opioid Receptor: Important Role of Axial Chirality in the Active Conformation.

In recent years, synthetic opioids have emerged as a predominant cause of drug-overdose-related fatalities, causing the "opioid crisis." To design safer therapeutic agents, we accidentally discovered µ-opioid receptor (MOR) antagonists based on fentanyl with a relatively uncomplicated chemical composition that potentiates structural modifications. Here, we showed the development of novel atropisomeric fentanyl analogues that exhibit more potent antagonistic activity against MOR than naloxone, a morphinan MOR antagonist. Derivatives displaying stable axial chirality were synthesized based on the amide structure of fentanyl. The aS- and aR-enantiomers exerted antagonistic and agonistic effects on the MOR, respectively, and each atropisomer interacted with the MOR by assuming a distinct binding mode through molecular docking. These findings suggest that introducing atropisomerism into fentanyl may serve as a key feature in the molecular design of future MOR antagonists to help mitigate the opioid crisis.

Synthetic cannabinoid CP-55,940 induces apoptosis in a human skeletal muscle model via regulation of CB1 receptors and L-type Ca2+ channels.

Rhabdomyolysis has been reported in patients who abuse synthetic cannabinoids. However, no studies have yet assessed whether these cases reflect the direct cytotoxicity of synthetic cannabinoids on skeletal muscle, a possibility that the present study sought to address. Specifically, this study investigated the cytotoxicity of the synthetic cannabinoid CP-55,940, a compound that acts equally on both types of cannabinoid receptors (CB1 and CB2), in a human embryonic rhabdomyosarcoma (RD) cell line. Exposure of these cells to CP-55,940 resulted in concentration-dependent decreases in cell viability. These effects were attenuated by pre-incubation with AM251 (30 µM), a selective CB1 receptor antagonist, but not by pre-incubation with AM630 (30 µM), a selective CB2 receptor antagonist. Following treatment with CP-55,940, RD cells exhibited apoptosis, as indicated by the accumulation of annexin-V, activation of caspase-3, and a loss of the mitochondrial membrane potential. Additionally, CP-55,940 treatment of RD cells led to increases in intracellular Ca2+ levels. CP-55,940-induced cell death was significantly attenuated in the absence of extracellular Ca2+, and was partially decreased by pre-incubation with verapamil (5 µM) or diltiazem (5 µM), compounds that block the L-type Ca2+ channel. Our results indicate that the cytotoxicity of CP-55,940 towards RD cells (skeletal muscle cells) is mediated by the CB1 receptor, but not by the CB2 receptor. Our results further suggest that calcium influx through the L-type channel may play an important role in the apoptosis induced by these compounds.

Characteristic Odor of the Japanese Liverwort (Leptolejeunea elliptica).

The volatile components produced by Leptolejeunea elliptica (Lejeuneaceae), which is a liverwort grown on the leaves of tea (Camellia sinensis), were collected and analyzed using headspace solid-phase microextraction-gas chromatography/mass spectrometry (HS-SPME-GC/MS). 1-Ethyl-4-methoxybenzene (1), 1-ethyl-4-hydroxybenzene (2), and 1-acetoxy-4-ethylbenzene (3) were identified as the major components together with several other phenolic compounds, including 1,2-dimethoxy-4-ethylbenzene, and 4-ethylguaiacol in addition to sesquiterpene hydrocarbons, such as α-selinene, ß-selinene, ß-elemene, and ß-caryophyllene. GC/Olfactometry showed the presence of linalool, acetic acid, isovaleric acid, trans-methyl cinnamate, and trans-4,5-epoxy-(2E)-decenal, as the volatile components produced by L. elliptica.

The characteristic smell emitted from two scale insects, Ceroplastes japonicus and Ceroplastes rubens.

The volatile components emitted from two scale insects, Ceroplastes japonicus and Ceroplastes rubens, were identified using GC-MS analysis. The major volatile components of the solvent extract from C. japonicus were α-humulene (35.8%) and δ-cadinene (17.0%), while those of C. rubens were ß-selinene (10.3%) and ß-elemene (5.1%). In GC/olfactometry, linalool, butyric acid, 3-methylbutyric acid, 2-methylbutyric acid, and vanillin were identified as the odor-active components of the extract from C. japonicus, in addition to trace amounts of trans-4,5-epoxy-(2E)-decenal, 4-methyl-(3E)-hexenoic acid, and phenylacetic acid. With regard to C. rubens, trans-4,5-epoxy-(2E)-decenal, 3-methylbutyric acid, and phenylacetic acid were identified as the odor-active components. Besides, decan-1,4-olide (γ-decalactone) with milky cherry-like note and 3-hydroxy-4,5-dimethylfuran-2(5H)-one (sotolone) with brown sugar-like note were also detected as the characteristic cherry-like sweet-and-sour note of these two scale insects. ABBREVIATIONS: GC: Gas chromatography; GC/O: gas chromatography/olfactometry.

Oesophagobronchial perforations after placement of an oesophageal self-expanding metallic stent.

An oesophageal fully covered self-expanding metallic stent (SEMS) was placed in a 54-year-old Japanese man to relieve dysphagia owing to a stage cT1bN3M1c lung adenocarcinoma. High expression of programmed cell death-ligand 1 was microscopically confirmed, and pembrolizumab was subsequently administered. Several days later, the patient was hospitalized with septic shock, and severe mediastinitis and pneumonia caused by oesophageal SEMS-induced oesophageal and bronchial perforations were observed. Thoracoscopic surgery was performed to drain the mediastinal abscess, and an additional oesophageal SEMS was placed to close the oesophageal perforation. The patient gradually recovered from the potentially fatal infection, and the SEMS was retrieved after confirming perforation closure. We re-initiated pembrolizumab administration, and the patient responded well. The present report reveals the potential risk and effectiveness of SEMS, especially when administered with immune checkpoint inhibitors.

[Present Conditions of Marijuana Regulation in USA: Medical and Recreational Use].

In most countries marijuana is regulated by the Single Convention on Narcotic Drugs. In Japan marijuana use is illegal under the Marijuana Control Law. In USA, marijuana is also classified as a schedule I drug, which is the most stringent regulation category under federal law. On the other hand, California became the first state to legalize marijuana for medical uses in 1996. Since then, several other US states have approved marijuana for medical or recreational use. However, marijuana remains completely illegal in most states, while some allow only cannabidiol (CBD) extracted from marijuana for medical use. In June 2018, the US Food and Drug Administration approved Epidiolex, the first marijuana-derived drug, containing purified CBD, to treat certain rare childhood seizure syndromes. Therefore the situation surrounding control of marijuana in USA is complex. Recently, a definite trend toward reconsidering marijuana regulation has been seen around the world, which could have a major impact on marijuana policy in Japan. In this review, we investigated existing medical and recreational marijuana laws in various US states, with a focus on California, which approved recreational use in 2018. Here, we describe the current state of marijuana regulation in terms of both medical and recreational use. In addition, we discuss public safety issues associated with marijuana, including crime, traffic accidents, and emergency department visits from possible marijuana exposure, as well as generated tax revenues, from official marijuana-related reports in Colorado, which legalized marijuana use in 2012.

[Dependence and Cytotoxicity of Components of Cannabis].

Cannabis use among the younger population in Japan has been steadily increasing. The aim of the present review is to highlight recent knowledge regarding the molecular mechanisms of action and health risks associated with cannabis and synthetic cannabinoid consumption. We investigated the effects of Δ9-tetrahydrocannabinol (THC) and synthetic cannabinoids on place conditioning in ICR mice. Both Δ9-THC and synthetic cannabinoids produce a significant conditioned place preference. These rewarding effects were completely suppressed by the cannabinoid CB1 receptor type antagonist AM251. The cytotoxicological effects of Δ9-THC and synthetic cannabinoids were also characterized in the limbic forebrain of mice in primary culture in vitro. Δ9-THC and synthetic cannabinoids caused cell death in a dose-dependent manner. The rank order of cytotoxicological potency was synthetic cannabinoids>Δ9-THC and related to the agonistic activities of the CB1 receptor. A recent review on the harmful effects of cannabis use in humans reported that behavioral impairments, especially in terms of attention, memory, and complex information-processing ability, can last for many weeks after cessation of cannabis use among heavy users. In addition, cannabis use could be a risk factor for drug dependence and later psychosis among adolescents. The results of animal and human studies suggest that CB1 receptors play an important role in the expression of harmful effects of cannabis and synthetic cannabinoid use. Moreover, concern regarding increasing concentrations of Δ9-THC in cannabis in many countries has been noted, because more potent cannabis may be associated with worse adverse effects.

Characteristic Scent from the Tahitian Liverwort, Cyathodium foetidissimum.

The volatile components of the Tahitian liverwort Cyathodium foetidissimum was analyzed using headspace solid phase micro-extraction (SPME) and GC-MS. Three volatile components, 4-methoxystyrene (24.4%), 3,4-dimethoxystyrene (28.7%), and skatole (15.9%) were identified as the major components from the fresh C. foetidissimum, along with several aliphatic aldehydes, n-octanal, n-nonanal, and n-decanal. However, (E)-2-nonenal recognized as aged malodor was not identified. In GC-O analysis, 2-aminoacetophenone was detected as one of the minor components with a strong aging note. In fact, C. foetidissimum showed the characteristic aging odor reminiscent the damp smell from old chest of drawers, or the civet like note with very strong feces and urine odor. The mixture consisted of 4-methoxystyrene, 3,4-dimethoxystyrene, and skatole in the detected ratio showed the sedative effect on CNV (contingent negative variation) measurement.

Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.

Comparative Study on Volatile Compounds of Alpinia japonica and Elettaria cardamomum.

The volatile compounds obtained from the ether extracts, headspace gases and steam distillates of Alpinia japonica and Elettaria cardamomum were analyzed by GC/MS. Both species were rich sources of naturally rare fenchane-type monoterpenoids, fenchene, fenchone, fenchyl alcohol and its acetate, together with 1,8-cineole. The distributions of volatile sesquiterpenoids were very poor in both species. Chiralities of fenchone in A. japonica and E. cardamomum were 99% of (1S,4R)-(+)-form. Camphor in A. japonica is composed of a mixture of (1R,4R)-(+)-form (94.3%) and (1S,4S)-(-)-form (5.7%). On the other hand, E. cardamomum produced only (1R,4R)-(+)-camphor (99%).

Volatile Compounds from the Different Organs of Houttuynia cordata and Litsea cubeba (L. citriodora).

The volatile compounds obtained from the different organs of Houttuynia cordata (Saururaceae) and Litsea cubeba (Lauraceae) were analyzed by Gas Chromatography/Mass Spectrometry (GC/MS), Headspace Solid Phase Micro Extraction-Gas Chromatography/Mass Spectrometry (HS-SPME-GC/MS), and GC/olfactometry (GC/O). The major component of all parts of H. cordata is assigned as 4-tridecanone. Each organ produces myrcene as the major monoterpenoid. The major monoterpene in the rhizomes and roots was ß-pinene instead of myrcene. 1-Decanal which was responsible for the unpleasant odor of this plant, was the predominant polyketide in both leaves and stems. The presence of 1-decanal was very poor in flowers, stem collected in summer, rhizomes, and roots. GC/MS analyses were very simple in case of the crude extracts of flowers. The content of sesquiterpenoids was extremely poor. (8Z)-Heptadecene, geranial, and neral were detected as the major components in Litsea cubeba. Odor-contributing components by GC/O analysis of the ether extract of the fresh flowers of L. cubeba were neral and geranial which played an important role in sweet-lemon fragrance of the flowers. The role of a high content of (8Z)-heptadecene was still unknown but it might play a significant role in the dispersion of the volatile monoterpene hydrocarbons and aldehydes. The flower volatiles of the Japanese L. cubeba were chemically quite different from those of the Chinese same species.

Differentiation and Molecular Properties of Mesenchymal Stem Cells Derived from Murine Induced Pluripotent Stem Cells Derived on Gelatin or Collagen.

The generation of induced-pluripotential stem cells- (iPSCs-) derived mesenchymal stem cells (iMSCs) is an attractive and promising approach for preparing large, uniform batches of applicable MSCs that can serve as an alternative cell source of primary MSCs. Appropriate culture surfaces may influence their growth and differentiation potentials during iMSC derivation. The present study compared molecular properties and differentiation potential of derived mouse iPS-MSCs by deriving on gelatin or collagen-coated surfaces. The cells were derived by a one-step method and expressed CD73 and CD90, but CD105 was downregulated in iMSCs cultured only on gelatin-coated plates with increasing numbers of passages. A pairwise scatter analysis revealed similar expression of MSC-specific genes in iMSCs derived on gelatin and on collagen surfaces as well as in primary mouse bone marrow MSCs. Deriving iMSCs on gelatin and collagen dictated their osteogenic and adipose differentiation potentials, respectively. Derived iMSCs on gelatin upregulated Bmp2 and Lif prior to induction of osteogenic or adipose differentiation, while PPARγ was upregulated by deriving on collagen. Our results suggest that extracellular matrix components such as gelatin biases generated iMSC differentiation potential towards adipose or bone tissue in their derivation process via up- or downregulation of these master genes.

Intra- and extracellular plasminogen activator inhibitor-1 regulate effect of vitronectin against radiation-induced endothelial cell death.

Plasminogen activator inhibitor-1 (PAI-1) is induced by radiation resulting in endothelial cell impairment, potentially leading to multiple organ failure. Vitronectin (VN) is a 75-kDa glycoprotein (VN75) cleaved into two forms (VN75 or VN65/10) by furin, which is regulated by intracellular PAI-1. VN protects against radiation-induced endothelial cell death, but the mechanisms involved in VN processing and its interactions with intra- and extracellular PAI-1 remain unclear. We examined these processes in cells in vitro using recombinant proteins or overexpression of VN and PAI-1 genes, including furin-susceptible (T381) and furin-resistant VN (A381). VN processing was analyzed using a mutant PAI-1 with relatively weaker binding to VN. VN function was evaluated by survival of radiation-damaged endothelial cells. Wild-type, but not mutant PAI-1 inhibited furin-dependent VN processing. Gene transfer revealed that furin-susceptible VN was processed more than the furin-resistant form, but processing of both was inhibited by PAI-1 overexpression. Intracellular PAI-1 formed a complex with VN75 (T381) in cells and media, and the VN75 form was secreted preferentially. Only VN75 protected against radiation-induced endothelial cell death, in which its effect was abolished by wild-type but not mutant PAI-1. These findings indicate that intracellular PAI-1 inhibits VN processing and protects against radiation-induced endothelial cell death.

[Zinc- and tin-induced apoptotic mechanisms in immune system and cranial nerve system].

This review explains the mechanisms of apoptosis related to the impacts of zinc deficiency and organotin exposure on the immune and central nervous systems. In the immune systems, both zinc deficiency and trialkyltin exposure lead to severe thymic atrophy and affect T-lymphocyte development through apoptosis of double positive stage pre-T-cells(CD4+/CD8+) in the cortex region. Their apoptosis are caused mainly through decrease in Bcl-2 expression, activation of ROS production/release, oxidative stress, mitochondrial cytochrome c release and activation of caspase cascade, with increases in glucocorticoids in zinc deficiency, without the involvement of glucocorticoid in organotin exposure In the central nervous system, both zinc deficiency and trialkyltin exposure reduce learning, memory and sensory functions through neuronal apoptosis caused by activation of ROS production/release, release of pro-apoptotic factors such as cytochrome c or apoptosis-inducing factor(AIF), with Fe excessive accumulation leading to ROS production and with depletion of hippocampus Zn (mossy fiber Zn) causing various Ca2+ channel disorder of synapse in the hippocampus, and with excessive accumulation of Ca through cAMP-dependent Ca(2+)-channel disorder by excessive PTH and cAMP excessive production in the olfactory systems such as olfactory epithelium and olfactory bulb.

[Biological functions of tin and disease].

Tin generates a wide variety of biological functions due to its chemical character. In this article, the modes of the biological functions of tin(especially organotin compounds) are reviewed, with special emphasis on the connection with the immune system, brain nervous system and endocrine system, on the basis of our data. To sum up this article, the biological functions of organotin compounds appear to be due to the following several processes: (1) their incorporation into the cells in vesicle form through fusion or in a similar manner to their incorporation in cationic form; (2) transport to and accumulation in the regions of the Golgi apparatus and endoplasmic reticulum (ER), but not to or in the plasma membrane or nucleus because of their hydrophobicity; (3) inhibition of intracellular phospholipid transport between organelles due to impairment of the structures and functions of the Golgi apparatus and ER; (4) inhibition of the membrane-mediated signal transduction system leading to DNA synthesis via phospholipid turnover and Ca2+ mobilization, as in cell proliferation systems; (5) disturbance of the trace element balance and the localization of certain elements; (6) disorders of membrane-mediated Ca2+ homeostasis via various channel functions including Zn modulation on the plasma and organelle membranes, and protein phosphorylation, as in the signal transduction systems of memory and olfaction; (7) necrosis or apoptosis in vivo or toxic cell death in vitro.

Volatile Components Emitted from the Liverwort Marchantia paleacea subsp. diptera.

The volatile components from the thalloid liverwort, Marchantia paleacea subsp. diptera were investigated by HS-SPME-GC-MS analysis. The monocyclic monoterpene aldehyde, perillaldehyde was identified for the first time as the major component and its content was about 50% of the volatiles, along with ß-pinene, limonene, ß-caryophyllene, α-selinene and ß-selinene as minor volatiles. Using MD (Multi-dimensional) GC-MS analysis equipped with a chiral column as the second column, the chirality was determined of both perillaldehyde and limonene, which was considered as the precursor of perillaldehyde. Both compounds were (S)-(-)-enantiomers (over 99.0 %) and (R)-enantiomers (less than 0.5 %). This is the first report of the existence of perillaldehyde in liverworts.

Characteristics of three-dimensional prospectively isolated mouse bone marrow mesenchymal stem/stromal cell aggregates on nanoculture plates.

Three-dimensional (3-D) aggregate culturing is useful for investigating the functional properties of mesenchymal stem/stromal cells (MSCs). For 3-D MSC analysis, however, pre-expansion of MSCs with two-dimensional (2-D) monolayer culturing must first be performed, which might abolish their endogenous properties. To avoid the need for 2-D expansion, we used prospectively isolated mouse bone marrow (BM)-MSCs and examined the differences in the biological properties of 2-D and 3-D MSC cultures. The BM-MSCs self-assembled into aggregates on nanoculture plates (NCP) that have nanoimprinted patterns with a low-cellular binding texture. The 3-D MSCs proliferated at the same rate as 2-D-cultured cells by only diffusion culture and secreted higher levels of pro-angiogenic factors such as vascular endothelial growth factor and hepatocyte growth factor (HGF). Conditioned medium from 3-D MSC cultures promoted more capillary formation than that of 2-D MSCs in an in vitro tube formation assay. Matrigel-implanted 3-D MSC aggregates tended to induce angiogenesis in host mice. The 3-D culturing on NCP induced alpha-fetoprotein (AFP) expression in MSCs without the application of AFP- or endodermal-inducible factors, possibly via an HGF-autocrine mechanism, and maintained their differentiation ability for adipocytes, osteocytes, and chondrocytes. Prospectively isolated mouse BM-MSCs expressed low/negative stemness-related genes including Oct3/4, Nanog, and Sox2, which were not enhanced by NCP-based 3-D culturing, suggesting that some of these cells differentiate into meso-endodermal layer cells. Culturing of prospectively isolated MSCs on NCP in 3-D allows the analysis of the biological properties of more closely endogenous BM-MSCs and might contribute to tissue engineering and repair.

Characteristic Volatile Components of Trifoliate Orange Peel (Poncirus trifoliata).

The volatile components of the peel of trifoliate orange {Poncirus trifoliata (L.) Raf.}, family Rutaceae, were investigated using SAFE technique after solvent extraction. Limonene was the most abundant component in the peel aroma extract, followed by myrcene, trans-D-ocimcne, -indole, P-caryophyllenc, (ME,6E)-ra-famescne, germacrene D, and j-phcllandrcne. In this study, the single sulfur- and nitrogen-containing compound, 4-methyl-5-vinylthiazole, and two macrocyclic lactones, cyclododecanolide and (7Z,IOZ,13Z)-hexadecatrien-16-olide, were identified as citrus aroma components for thefirst time. As aresultof AEDA for the polar fraction of the aroma extract, indole, ethyl octanoate and those macrocyclic lactones with musky notes were found to be responsible f or the characteristic aroma profile of the peel of trifoliate orange. The enantiomeric distributions of the four odor-active components, linalool, P-citronellol, ethyl 2-methylbutanoate, and 2-methylbutanoic acid, were also determined by means of multidimensional chiral GC/MS.

Odor-Active Components of Luo Han Guo (Siraitia grosvenorii).

The volatile components -of the dried fruit of Luo Han Guo (Siraitia grosvenorii Swingle) belonging to the family Cucurbitaeae were analyzed by AROMASCOPE® technique using MonoTrap® DCC 18 as an absorbent. A total of 124 volatile components were identified from the headspace aroma solvent extract. The major components were ethanol, butan-l-ol, pentanal, 2-methylbutanal, hexanal, furfural, pent-3-en-2-one, acetic acid, propionic acid, 3- methylbutanoic acid, hexadecanoic acid, and so on. Among them, acetic acid, 3-methylbutanoic acid, and 3-hydroxy-4,5-dimethylfuran-2(5H)-one (sotolon) strongly contributed to the overall aroma of the fruit. Besides, sotolon and 5-ethyl-3-hydroxy-4-methylfuran-2(5H)-one (maple furanone) were responsible for - the characteristic molasses-like aroma of the fruit.

Estimation of secondary measles transmission from a healthcare worker in a hospital setting.

Measles among healthcare workers (HCWs) is associated with a significant risk of nosocomial transmission to susceptible patients. When a measles case occurs in the healthcare setting, most guidelines recommend exhaustive measures. To evaluate the effects of measures against measles transmission in the healthcare setting precisely, it is essential to determine whether secondary transmission generally occurs. This study describes, for the first time, the actual secondary transmission rate for a measles-infected HCW in a ward with no special air ventilation capacity. The routine treatment of a number of immunocompromised patients occurs in this ward, and thus patients as well as HCWs have a thorough understanding and practice of standard and extended precautions. Our paired serum sample study revealed that none of the people in the ward exposed to the HCW at the catarrhal stage over a period of 4 days exhibited elevated levels of antibodies against measles. We suggest that strict adherence to standard and expanded precautions among patients and HCWs may be effective for preventing the transmission of a highly airborne disease, such as measles.