Molecular characterization of Rft1, an ER membrane protein associated with congenital disorder of glycosylation RFT1-CDG.

The oligosaccharide needed for protein N-glycosylation is assembled on a lipid carrier via a multi-step pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 Congenital Disorders of Glycosylation (CDGs) are associated with this pathway, including RFT1-CDG which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what essential role Rft1 plays in N-glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multi-spanning membrane protein located in the ER, with its N and C-termini facing the cytoplasm. It is not N-glycosylated. The majority of RFT1-CDG mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1's ability to support N-glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.

Zinc chloride-catalyzed Grignard addition reaction of aromatic nitriles.

In the alkyl addition reaction of aromatic nitriles using Grignard reagents, ketones are formed after hydrolysis. However, this addition reaction is often slow compared to that using reactive organolithium(i) reagents. In this study, we improved the reaction by using zinc(ii)ates, which are generated in situ using Grignard reagents and zinc chloride (ZnCl2) as a catalyst. As a result, the corresponding ketones and amines were obtained via hydrolysis and reduction, respectively, in good yields under mild reaction conditions. Scale-up reactions are also demonstrated. Interestingly, using a catalytic amount of ZnCl2 was more effective than using a stoichiometric amount of zinc(ii)ates. Possible transition states are proposed on the basis of the active zinc(ii)ate species, and DFT calculations were carried out to elucidate a plausible reaction mechanism.

Iridium-Catalyzed [2 + 2 + 2] Cycloaddition of Bithiophen-Linked Diynes with Nitriles: Scope and Mechanistic Study with Quantum Chemical Calculation.

We report a simple and atom-efficient method for the synthesis of bithiophene-fused isoquinolines by iridium-catalyzed [2 + 2 + 2] cycloaddition of bithiophene-linked diynes with nitriles. All three structural isomers of bithiophene-linked diynes underwent [2 + 2 + 2] cycloaddition, and the trend in the reactivity for cycloaddition was diyne 1 = diyne 3 > diyne 2. Dibenzothiophene-linked diyne also reacted with nitriles to form a variety of cycloadducts. Cycloaddition of bithiophene-linked diynes with alkynes and an isocyanate formed naphthodithiophenes and a 2-pyridone derivative, respectively. Cycloadducts bearing a 2-aminopyridine moiety and benzothiophene rings showed intense fluorescence at around 530 nm and gave a fluorescence quantum yield of 0.44. Furthermore, quantum chemical calculations provided insight into the origin of the difference in reactivity of three bithiophene-linked diynes. The different reactivities of the three diynes 1-3 are believed to originate from the step where an iridacyclopentadiene reacts with a coordinated nitrile to form azairidabicyclo[3.2.0]heptatriene. HOMOs of iridacyclopentadiene play a decisive role in this step.

Excitation configuration analysis for divide-and-conquer excited-state calculation method using dynamical polarizability.

The authors previously developed a divide-and-conquer (DC)-based non-local excited-state calculation method for large systems using dynamical polarizability [Nakai and Yoshikawa, J. Chem. Phys. 146, 124123 (2017)]. This method evaluates the excitation energies and oscillator strengths using information on the dynamical polarizability poles. This article proposes a novel analysis of the previously developed method to obtain further configuration information on excited states, including excitation and de-excitation coefficients of each excitation configuration. Numerical applications to simple molecules, such as ethylene, hydrogen molecule, ammonia, and pyridazine, confirmed that the proposed analysis could accurately reproduce the excitation and de-excitation coefficients. The combination with the DC scheme enables both the local and non-local excited states of large systems with an excited nature to be treated.

Rikkosan's Short-Term Analgesic Effect on Burning Mouth Syndrome: A Single-Arm Cohort Study.

Burning mouth syndrome (BMS) is a chronic oral pain disorder. There is a theory that BMS is a form of nociplastic pain. A standard treatment for BMS has not yet been established. Kampo medicine is a traditional oriental medicine. The purpose of this study is to evaluate the effectiveness of Rikkosan-a traditional Japanese herbal medicine (Kampo)-in the treatment of BMS. A single-center retrospective study was conducted on 20 patients who were diagnosed with BMS and treated with Rikkosan alone (total daily dose; 7.5 g) three times daily for approximately 4 weeks (29.5 ± 6.5 days). Rikkosan was dissolved in hot water and taken internally. They had an average age of 63 years, and 90% were being treated for other illnesses, but their medication status was the same during this study period, except for Rikkosan. No adverse events were observed in patients. Numerical rating scale (NRS) or visual analog scale (VAS)/10 scores decreased significantly between the time of the initiation of Rikkosan and one month after (-2.1 ± 1.2, p < 0.05). Rikkosan has a short-term effect of reducing NRS by two levels in BMS patients.

Orofacial Granulomatosis among Pediatric Patients Well Controlled by Corticosteroid Treatment: A Rare Case Series.

Orofacial granulomatosis (OFG) is a rare disease entity characterized by nonnecrotizing granulomatous inflammation in the oral and maxillofacial regions, typically characterized by recurrent or persistent edema, primarily in the lips and occasionally in the gingiva. OFG is often associated with Crohn's disease and sarcoidosis, and an accurate diagnosis requires systemic examination of patients. Pediatric patients possess unique oral conditions where dental plaque rapidly forms, especially during tooth replacement due to tooth crowding. Moreover, controlling oral hygiene can be challenging, rendering it difficult to distinguish plaque-induced gingivitis from nonplaque-induced gingivitis. We elucidate the reports of pediatric patients who developed OFG in the lips and/or gingiva alone, which was well controlled through corticosteroid treatment. The patients demonstrated recurrent lips and/or gingival swelling with redness, which failed to improve despite oral health care and treatment with antibiotics and/or corticosteroid ointment. Incision biopsy was performed, which demonstrated granulomatous inflammation. Further systemic examination ruled out Crohn's disease and sarcoidosis and confirmed OFG diagnosis. Corticosteroid treatment orally or through gargling was administered to the patients, which provided improvement of symptoms after 1 month. As OFG may be associated with intractable diseases, monitoring the patient regularly is crucial. Pediatric patients with OFG require a collaborative approach with pediatricians and pediatric dentists to manage their oral and overall health.

Molecular characterization of Rft1, an ER membrane protein associated with congenital disorder of glycosylation RFT1-CDG.

The oligosaccharide needed for protein N-glycosylation is assembled on a lipid carrier via a multi-step pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 Congenital Disorders of Glycosylation (CDGs) are associated with this pathway, including RFT1-CDG which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what essential role Rft1 plays in N-glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multi-spanning membrane protein located in the ER, with its N and C-termini facing the cytoplasm. It is not N-glycosylated. The majority of RFT1-CDG mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1's ability to support N-glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.

Expression analysis of zinc-metabolizing enzymes in the saliva as a new method of evaluating zinc content in the body: two case reports and a review of the literature.

BACKGROUND: The activity level of alkaline phosphatase, a zinc-requiring enzyme in the serum, is used to indicate zinc nutritional status; however, it does not correlate with serum zinc levels or subjective symptoms of taste disorder in many cases. Hence, this study focused on the total activity of alkaline phosphatase, a zinc-requiring enzyme. The total alkaline phosphatasa activity level in the saliva was measured before and after zinc supplementation, and the results were compared with serum zinc levels. CASE PRESENTATION: This study included patients with hypozincemia, specifically a patient with zinc-deficient taste disorder (patient 1: a 69-year-old Japanese woman) and a patient with glossodynia with zinc deficiency (patient 2: an 82-year-old Japanese woman). Saliva samples were collected, and blood tests were performed before and after zinc supplementation. Subjective symptoms and serum zinc levels were simultaneously evaluated. Zinc supplementation was performed using zinc acetate hydrate or Polaprezinc. CONCLUSIONS: Total alkaline phosphatase activity levels were found to be associated with serum zinc levels and subjective symptoms. A further study with a higher number of patients is necessary to confirm whether total alkaline phosphatase activity levels more accurately reflect the amounts of zinc in the body than serum zinc levels.

Leveraging the Stereochemical Complexity of Octahedral Diastereomeric-at-Metal Catalysts to Unlock Regio-, Diastereo-, and Enantioselectivity in Alcohol-Mediated C-C Couplings via Hydrogen Transfer.

Experimental and computational studies illuminating the factors that guide metal-centered stereogenicity and, therefrom, selectivity in transfer hydrogenative carbonyl additions of alcohol proelectrophiles catalyzed by chiral-at-metal-and-ligand octahedral d6 metal ions, iridium(III) and ruthenium(II), are described. To augment or invert regio-, diastereo-, and enantioselectivity, predominantly one from among as many as 15 diastereomeric-at-metal complexes is required. For iridium(III) catalysts, cyclometalation assists in defining the metal stereocenter, and for ruthenium(II) catalysts, iodide counterions play a key role. Whereas classical strategies to promote selectivity in metal catalysis aim for high-symmetry transition states, well-defined low-symmetry transition states can unlock selectivities that are otherwise difficult to achieve or inaccessible.

Boron Trifluoride-Mediated Domino Dehydration/Electrophilic Cyclization of Silylalkynols Leading to 2,3-Fused Tricyclic Benzofulvenes.

A BF3-mediated domino dehydration/electrophilic cyclization of silylalkynols to form 2,3-fused tricyclic benzofulvenes was achieved. In the latter step, in situ generated BF3·OH2 enables the electrophilic activation of alkynes. The predominant Z-selectivity of the reaction is also discussed.

Comparison between hangeshashinto and dexamethasone for IL-1α and ß-defensin 1 production by human oral keratinocytes.

OBJECTIVE: Human ß-defensin 1 (hBD-1) is a antimicrobial peptide that is constantly secreted by oral tissues. Hangeshashinto (HST), a traditional Japanese medicine, has been reported to be effective against stomatitis. This study aimed to clarify the profile of HST by comparing the system of production of interleukin-1α (IL-1α) and hBD-1 in human oral mucosal epithelial cells with dexamethasone (DEX), a steroid used for the treatment of stomatitis. METHODS: Human oral keratinocytes (HOK) were treated with HST, DEX, or HST components (baicalein, baicalin, berberine, and glycyrrhizin) for 24 h, and subsequently cultured for 24 h with or without Pam3CSK4 or lipopolysaccharide (LPS). The cell supernatants, total RNA, and intracellular proteins were collected, and changes in IL-1α and hBD-1 protein production and gene expression were evaluated using ELISA and RT-PCR. The phosphorylation of NF-kB and the cell proliferative ability of HOK were evaluated by western blotting and XTT assay, respectively. RESULTS: DEX (0.01-10 µM) significantly suppressed IL-1α and hBD-1 production induced by either Pam3CSK4 or LPS, and also decreased cell growth. In contrast, HST inhibited Pam3CSK4- and LPS-induced IL-1α production at a concentration range of 12.5-100 µg/mL without affecting the cell proliferative capacity and hBD-1 production of HOK. Baicalein and baicalin, which are flavonoid ingredients of HST, showed anti-IL-1α production. CONCLUSION: HST may be useful as a therapeutic agent for stomatitis and other inflammatory diseases of the oral cavity.

Exploratory Study of the Relationship between an Oral Fungal Swab Test and Patient Blood Test Data.

Our understanding of the relationship between oral Candida and systemic conditions has significantly increased recently, which this study aims to extend further by investigating the risks of oral candidiasis. A total of 314 patients were involved in this study and underwent an oral swab test at the Department of Oral Medicine, Hokkaido University Hospital, between January and December 2021. Data were collected on age, sex, white and red blood cell counts, Hb, total protein, vitamin B12, as well as serum albumin, iron, copper, and zinc levels. The clinical fungus samples were swabbed to identify those with Candida species using a MALDI Biotyper, then applied analysis of covariance and multivariant logistic regression analysis. It was possible to assess the oral swab test results without considering the difference between sex (p = 0.946). The oral swab test results were associated with aging (odds ratio: 1.03) and serum albumin levels (odds ratio: 0.32). In summary, the results of our study suggest a relationship between aging and oral candidiasis and offer in-depth insights into how to prevent or treat oral candidiasis onset.

Potential relationship between the dosage of prednisolone and delayed healing at tooth extraction: A retrospective study.

Background/purpose: Delayed healing of the extraction socket is not uncommon when tooth extraction is performed on patients taking prednisolone. This study aimed to identify specific dosage of prednisolone and factors associated with delayed healing of the extraction socket in patients taking prednisolone. Materials and methods: This single-center retrospective study included 80 patients who underwent tooth extraction under local anesthesia and were taking prednisolone orally. Patients were divided into the nondelayed healing group (n = 50) and delayed healing group (n = 30), and their background and dosage of prednisolone were compared. Results: The dosage of prednisolone was significantly higher in the delayed healing group than in the nondelayed healing group. A receiver operating characteristics curve analysis resulted in moderate accuracy when the cutoff value was set at 8.0, with 67% sensitivity, 76% specificity, and 0.765 area under the curve. The multivariate logistic regression analysis revealed that prednisolone dosage >8.0 mg/day (odds ratio [OR], 10.8; 95% confidence interval [CI], 2.79-41.6) and osteosclerotic changes beyond the alveolar bone around the tooth to be extracted (OR, 10.3; 95% CI, 2.81-37.8) in X-ray imaging had significant effects on delayed healing. Conclusion: The results of this study suggested that delayed healing following tooth extractions in patients taking prednisolone was related to a dosage of 8.0 mg/day or higher and osteosclerotic changes.

Quantum Chemical Study of the Cycloaddition Reaction of Tropone with 1,1-Diethoxyethene Catalyzed by B(C6F5)3 or BPh3.

Cycloaddition reaction of tropone with 1,1-diethoxyethene catalyzed by Lewis acid (LA), B(C6F5)3 or BPh3, was examined by using ωB97X-D-level density functional theory (DFT) calculations. In the absence of LA, the reaction proceeds in a stepwise fashion to form two chemical bonds, first between the C2 atom in tropone and the C2 atom in ethene and then between the C5 atom in the former and the C1 atom in the latter. When B(C6F5)3 is attached to the O atom in tropone, the C5 atom in tropone is attacked preferentially by the C1 atom in ethene in the second stage. The attack of the O atom in tropone is shown to be less likely; thus, the [4 + 2] addition is favored in the B(C6F5)3-catalyzed reaction. In contrast, the attack of the O atom in the BPh3-attached tropone to the C1 atom in ethene is preferred over the attack of the C5 atom, indicating that the [8 + 2] cycloaddition instead of the [4 + 2] cycloaddition proceeds in the BPh3-catalyzed reaction. Whether the C1 atom in ethene is attacked by C5 or by O in the second bond formation step is shown in this study to be governed mainly by the nucleophilicity of σ-lone pair electrons of the carbonyl O atom of tropone in the presence of LA. These results are consistent with the experiments reported by Li and Yamamoto.

Chiral-at-Ruthenium-SEGPHOS Catalysts Display Diastereomer-Dependent Regioselectivity: Enantioselective Isoprene-Mediated Carbonyl tert-Prenylation via Halide Counterion Effects.

The first correlation between metal-centered stereogenicity and regioselectivity in a catalytic process is described. Alternate pseudo-diastereomeric chiral-at-ruthenium complexes of the type RuX(CO)[η3-prenyl][(S)-SEGPHOS] form in a halide-dependent manner and display divergent regioselectivity in catalytic C-C couplings of isoprene to alcohol proelectrophiles via hydrogen autotransfer. Whereas the chloride-bound ruthenium-SEGPHOS complex prefers a trans-relationship between the halide and carbonyl ligands and delivers products of carbonyl sec-prenylation, the iodide-bound ruthenium-SEGPHOS complex prefers a cis-relationship between the halide and carbonyl ligands and delivers products of carbonyl tert-prenylation. The chloride- and iodide-bound ruthenium-SEGPHOS complexes were characterized in solution and solid phase by 31P NMR and X-ray diffraction. Density functional theory calculations of the iodide-bound catalyst implicate a Curtin-Hammett-type scenario in which the transition states for aldehyde coordination from an equilibrating mixture of sec- and tert-prenylruthenium complexes are rate- and product-determining. Thus, control of metal-centered diastereoselectivity has unlocked the first catalytically enantioselective isoprene-mediated carbonyl tert-prenylations.

Unveiling controlling factors of the S0/S1 minimum-energy conical intersection (3): Frozen orbital analysis based on the spin-flip theory.

Conical intersections (CIs), which indicate the crossing of two or more adiabatic electronic states, are crucial in the mechanisms of photophysical, photochemical, and photobiological processes. Although various geometries and energy levels have been reported using quantum chemical calculations, the systematic interpretation of the minimum energy CI (MECI) geometries is unclear. A previous study [Nakai et al., J. Phys. Chem. A 122, 8905 (2018)] performed frozen orbital analysis (FZOA) based on time-dependent density functional theory (TDDFT) at the MECI formed between the ground and first electronic excited states (S0/S1 MECI), thereby inductively clarifying two controlling factors. However, one of the factors that the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) energy gap became close to the HOMO-LUMO Coulomb integral was not valid in the case of spin-flip TDDFT (SF-TDDFT), which is frequently used as a means of the geometry optimization of MECI [Inamori et al., J. Chem. Phys. 152, 144108 (2020)]. This study revisited the controlling factors using FZOA for the SF-TDDFT method. Based on spin-adopted configurations within a minimum active space, the S0-S1 excitation energy is approximately represented by the HOMO and LUMO energy gap ΔεHL, a contribution from Coulomb integrals JHL″ and that from the HOMO-LUMO exchange integral KHL″. Furthermore, numerical applications of the revised formula at the SF-TDDFT method confirmed the control factors of S0/S1 MECI.

Interplay of diruthenium catalyst in controlling enantioselective propargylic substitution reactions with visible light-generated alkyl radicals.

Transition metal-catalyzed enantioselective free radical substitution reactions have recently attracted attention as convenient and important building tools in synthetic chemistry, although construction of stereogenic carbon centers at the propargylic position of propargylic alcohols by reactions with free radicals remains unchallenged. Here we present a strategy to control enantioselective propargylic substitution reactions with alkyl radicals under photoredox conditions by applying dual photoredox and diruthenium catalytic system, where the photoredox catalyst generates alkyl radicals from 4-alkyl-1,4-dihydropyridines, and the diruthenium core with a chiral ligand traps propargylic alcohols and alkyl radicals to guide enantioselective alkylation at the propargylic position, leading to high yields of propargylic alkylated products containing a quaternary stereogenic carbon center at the propargylic position with a high enantioselectivity. The result described in this paper provides the successful example of transition metal-catalyzed enantioselective propargylic substitution reactions with free alkyl radicals.

Iridium-Catalyzed Branch-Selective Hydroalkylation of Simple Alkenes with Malonic Amides and Malonic Esters.

We report the iridium-catalyzed branch-selective hydroalkylation of simple alkenes such as aliphatic alkenes and aromatic alkenes with malonic amides and malonic esters under neutral reaction conditions. A variety of aliphatic alkenes and aromatic alkenes bearing bromine, chlorine, ester, 2-thienylcarboxylate, silyl, and phthalimide groups were all found to be suitable for this hydroalkylation. The combination of this method with Krapcho dealkoxycarbonylation realized a one-pot synthesis of ß-substituted amide and ester from ß-amide ester and malonic ester. The hydroalkylated products derived from malonic amides are suitable for further transformation. The finely tuned reaction conditions realized the selective transformation of hydroalkylated products to 1,3-diamines or monoamides with the same reagent. Deuterium labeling experiments and measurement of the kinetic isotope effect indicated that the catalytic cycle involves a reversible step and cleavage of the C-H bond is not a rate-determining step. Density functional theory calculations provided insight into the reaction mechanism, where the carboiridation step is followed by C-H reductive elimination.

Effect of Propargylic Substituents on Enantioselectivity and Reactivity in Ruthenium-Catalyzed Propargylic Substitution Reactions: A DFT Study.

We recently proposed a transition-state model for asymmetric propargylic substitution reactions of propargylic alcohols catalyzed by optically active thiolate-bridged diruthenium complexes [Chem. - Asian J.2021, 16, 3760-3766]. In the present study, we further examined the effects of propargylic substituents on both enantioselectivity and reactivity in the propargylic substitution reactions via ωB97X-D-level density functional theory (DFT) calculations. When the propargylic alcohol bears a methyl group at the propargylic position, we obtained results that contrast with the result of our previous study on propargylic alcohols without methyl groups. This result indicates that methyl group substitution at the propargylic position reverses the stereoselectivity. Substitution of a trifluoromethyl group for a methyl group was suggested to result in higher enantioselectivity. The obtained results are consistent with the experimental study on enantioselective propargylic phosphinylation reactions reported by our group.

Ethyl loflazepate as a treatment for patients with idiopathic and psychogenic taste disorder.

BACKGROUND: Ethyl loflazepate (EL) is a benzodiazepine derivative that has been reported to activate the gustatory cortex. Our department routinely uses EL as a first-line treatment for idiopathic and psychogenic taste disorders, although little has been reported in the literature with respect to patient outcomes, so we conducted a retrospective study examining its safety and efficacy. METHODS: Between 2008 and 2020, 49 patients (14 males and 35 females; mean age, 62.1 years) were diagnosed with taste disorders and received EL as their only treatment for > 14 days. Severity of taste disorder was evaluated using the paper disc method by Sakai et al., and treatment efficacy was evaluated using the Visual Analog Scale, wherein patients gave subjective ratings for their symptoms (reductions by > 50% after administration of EL for 4 weeks were defined as improvements). RESULTS: Results showed that the improvement rates for patients with idiopathic and psychogenic taste disorders were 55 and 70%, respectively. Additionally, the majority (78%) improved within 2 weeks, and side effects were mild (seven cases with drowsiness and one case with dizziness). CONCLUSIONS: We conclude that EL is an appropriate first-line medication for patients with idiopathic and psychogenic taste disorders.