Disulfide Bonds of Thyroid Peroxidase Are Critical Elements for Subcellular Localization, Proteasome-Dependent Degradation, and Enzyme Activity.

Background: Congenital hypothyroidism (CH) is caused by mutations in cysteine residues, including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that these disulfide bonds could play an important role in TPO activity. However, to date, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in disulfide bonds formation and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO activity. Methods: Vector plasmid TPO mutants, C655F and C825R, known to occur in CH, were transfected into HEK293 cells. TPO activity and protein expression levels were measured by the Amplex red assay and Western blotting. The same procedure was performed in the presence of MG132 proteasome inhibitor. Subcellular localization was determined using immunocytochemistry and flow cytometry. The locations of all disulfide bonds within TPO were predicted using in silico analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and Western blotting. Results: C655F and C825R showed significantly decreased activity and protein expression compared with the wild type (WT) (p < 0.05). In the presence of the MG132 proteasome inhibitor, the protein expression level of TPO increased to a level comparable with that of the WT without increases in its activity. The degree of subcellular distribution of TPO to the cell surface in the mutants was lower compared with the WT TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO. All TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. Conclusions: All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation, and membrane insertion.

Anaphylaxis and Severe Disseminated Intravascular Coagulation Due to Remdesivir.

A 69-year-old woman suffering with multiple myeloma developed coronavirus disease 2019 (COVID-19). Shortly after administration of remdesivir, she presented with symptoms of facial flushing, wheezing, and hypoxemia. Subsequently, thrombocytopenia and hypofibrinogenemia rapidly manifested, leading to a diagnosis of enhanced fibrinolytic-type disseminated intravascular coagulopathy (DIC). This clinical presentation was considered an immediate hypersensitivity reaction with associated coagulation abnormalities induced by remdesivir. Although remdesivir is generally considered safe and efficacious in the treatment of COVID-19, physicians should remain vigilant regarding the potential for severe adverse events associated with this medication.

HTLV-1 cell-free DNA in plasma as a potential biomarker in HTLV-1 carriers and adult T-cell leukemia-lymphoma.

Viral cell-free DNA (cfDNA) in plasma has been widely evaluated for detecting cancer and monitoring disease in virus-associated tumors. We investigated whether the amount of cfDNA of human T-cell leukemia virus type 1 (HTLV-1) correlates with disease state in adult T-cell leukemia-lymphoma (ATL). HTLV-1 cfDNA in aggressive ATL was significantly higher than that in indolent ATL and asymptomatic carriers. Notably, patients with lymphoma type represented higher HTLV-1 cfDNA amount than chronic and smoldering subtypes, though they had no abnormal lymphocytes in the peripheral blood. HTLV-1 cfDNA can be a universal biomarker that reflects the expansion of ATL clones.

Type II Cryoglobulinemic Membranoproliferative Glomerulonephritis Caused by Mucosa-associated Lymphoid Tissue Lymphoma.

A 67-year-old man complained of lower limb edema with a purpuric skin rash. Laboratory tests revealed proteinuria, elevated serum creatinine levels, and low serum albumin levels. The patient was also positive for cryoglobulin in serum, immunoglobulin (Ig) M gammopathy, hypocomplementemia, and rheumatoid factor. He was negative for anti-hepatitis C virus antibodies. A pathological analysis of the renal tissue revealed membranoproliferative glomerulonephritis, common histological features of cryoglobulinemic vasculitis (CV), and mucosa-associated lymphoid tissue lymphoma invasion. Although hematologic malignancy is a rare cause of type II CV, these clinical findings suggest that mucosa-associated lymphoid tissue lymphoma (MALT) lymphoma may have been the cause in the present case.

Clinical characteristics and human leukocyte antigens in patients with immune checkpoint inhibitor-induced type 1 diabetes and pituitary dysfunction: a single center prospective study.

PURPOSE: Immune checkpoint inhibitor (ICI) induced type 1 diabetes (T1D) and pituitary dysfunction are life-threatening adverse events, yet there is little clinical data available. We aimed to investigate the clinical characteristics of patients with these adverse events and report their human leukocyte antigen (HLA) profile to determine its relevance. METHODS: This is a single-center prospective study. We enrolled patients with cancers who were administered ICI and diagnosed as ICI induced T1D (ICI-T1D) and pituitary dysfunction (ICI-PD). Clinical data and extracted DNA from blood samples were collected. HLA typing was performed using next-generation sequencing. We compared our results with those previously reported in healthy controls and investigated the correlation between HLA and the occurrence of ICI-T1D and ICI-PD. RESULTS: We identified 914 patients treated with ICI in our facility from 1st September, 2017 to 30th June, 2022. Six of these patients developed T1D and 15 developed pituitary dysfunction. The duration from the initiation of ICI treatment to the onset of T1D or pituitary dysfunction averaged 492 ± 196 days and 191 ± 169 days. Among the six patients with T1D, two were positive for anti-GAD antibody. The frequencies of HLA-DR11, -Cw10, -B61, -DRB1*11:01, and -C*03:04 were significantly higher in patients with ICI-T1D than in controls. The frequencies of HLA-DR15 and -DRB*15:02 were significantly higher in patients with ICI-PD than in controls. CONCLUSION: This study revealed the clinical characteristics of ICI-T1D and ICI-PD and the association between specific HLAs and these adverse events.

Diffuse Large B-cell Lymphoma Involving an Abundant Infiltration of T Follicular Helper Cells.

A 76-year-old man presented with skin plaque and splenic nodules, and diffuse large B-cell lymphoma (DLBCL) with infiltration of T-cells was suspected based on the skin lesions. The disease showed indolent clinical behavior for three months, when systemic lymphadenopathy rapidly evolved. An inguinal lymph node biopsy revealed DLBCL with abundant infiltration of T follicular helper (TFH) cells. A polymerase chain reaction-based analysis of immunoglobulin variable heavy chain showed that the skin, splenic nodules, and inguinal lymph node shared the same clone. This case indicates that the dysregulated infiltration of TFH cells in the tumor microenvironment accelerates the lymphomagenesis and progression of DLBCL.

Ventricular fibrillation immediately after the treatment of Graves' disease coexisting with atypical angina and long QT syndrome: a case report.

BACKGROUND: Palpitations due to Graves' disease are often caused by supraventricular arrhythmia. However, in rare cases, the background of coronary artery disease, genetic abnormalities, or channel abnormalities can cause ventricular fibrillation, which is a lethal arrhythmia. Here, we report a case of ventricular fibrillation after administration of beta-blockers early in the course of treatment for Graves' disease coexisting with atypical angina and long QT syndrome. CASE PRESENTATION: A 48-year-old man consulted a local general physician for chest discomfort and palpitations for approximately 2 weeks. He was diagnosed with Graves' disease and treated with thiamazole 15 mg, bisoprolol 1.25 mg, and nitroglycerin 0.3 mg. The patient continued to experience chest discomfort the next day and visited our hospital. The patient was treated with landiolol 0.125 mg/kg/min for heart rate control, and 20 min later, electrocardiography showed a change from the R-on-T phenomenon to ventricular fibrillation. After cardiopulmonary resumption and improvement of thyroid function, a stress test was performed, which revealed coronary angina and long QT syndrome. An implantable cardioverter defibrillator (ICD) was implanted in the patient for secondary prevention. Since then, no fatal arrhythmia has been observed to date. CONCLUSIONS: When beta-blockers are administered to patients with Graves' disease who have severe chest symptoms, fatal arrhythmias are possible. ICD implantation should be considered for the secondary prevention of fatal arrhythmias.

[HLA-haploidentical peripheral blood stem cell transplantation with post-transplantation cyclophosphamide for adult T-cell leukemia].

Recently, allogeneic peripheral blood stem cell transplantation from human leukocyte antigen (HLA)-haploidentical donors using post-transplantation cyclophosphamide (PTCY-haploPBSCT) has become available in clinical practice. However, the efficacy of PTCY in adult T-cell leukemia (ATL) is not fully established yet. In this study, we retrospectively examined data of seven patients who underwent PTCY-haploPBSCT. The overall survival rate at 100 days after transplantation was 85.7%, and the 1-year overall survival rate was 68.6%. The cumulative incidence of relapse at 1 year was 31.4%, whereas the 1-year nonrelapse mortality was 17.1%. The cumulative incidence of grade III-IV acute graft-versus-host disease (GVHD) on day 100 was 14.3%, and the incidence of chronic GVHD at 1 year was 33.3%. These results suggest that PTCY-haploPBSCT can be a viable option even in patients with ATL. Further accumulation of knowledge and improvement of transplantation outcomes are warranted in the future.

Machine learning approach to predict subtypes of primary aldosteronism is helpful to estimate indication of adrenal vein sampling.

INTRODUCTION: Primary aldosteronism (PA) is a common disease. Especially in unilateral PA (UPA), the risk of cardiovascular disease is high and proper localization is important. Adrenal vein sampling (AVS) is commonly used to localize PA, but its availability is limited. Therefore, it is important to predict the unilateral or bilateral PA and to choose the appropriate cases for AVS or watchful observation. AIM: The purpose of this study is to develop a model using machine learning to predict bilateral or unilateral PA to extract cases for AVS or watchful observation. METHODS: We retrospectively analyzed 154 patients diagnosed with PA and who underwent AVS at our hospital between January 2010 and June 2021. Based on machine learning, we determined predictors of PA subtypes diagnosis from the results of blood and loading tests. RESULTS: The accuracy of the machine learning was 88% and the top predictors of the UPA were plasma aldosterone concentration after the saline infusion test, aldosterone to renin ratio after the captopril challenge test, serum potassium and aldosterone-to-renin ratio. By using these factors, the accuracy, sensitivity, specificity and the area under the curve (AUC) were 91%, 70%, 99% and 0.91, respectively. Furthermore, we examined the surgical outcomes of UPA and found that the group diagnosed as unilateral by the predictors showed improvement in clinical findings, while the group diagnosed as bilateral by the predictors showed no improvement. CONCLUSION: Our predictive model based on machine learning can support to choose the performance of adrenal vein sampling or watchful observation.

A case of diabetic ketoacidosis in a patient with COVID-19 and newly diagnosed type 1 diabetes.

To improve severe ketoacidosis with COVID-19, insulin treatment, invasive mechanical ventilation therapy, and continuous hemodiafiltration with sodium bicarbonate infusion were effective.

Asymmetric epoxidation of allylic alcohols catalyzed by vanadium-binaphthylbishydroxamic Acid complex.

A vanadium-binaphthylbishydroxamic acid (BBHA) complex-catalyzed asymmetric epoxidation of allylic alcohols is described. The optically active binaphthyl-based ligands BBHA 2a and 2b were synthesized from (S)-1,1'-binaphthyl-2,2'-dicarboxylic acid and N-substituted-O-trimethylsilyl (TMS)-protected hydroxylamines via a one-pot, three-step procedure. The epoxidations of 2,3,3-trisubstituted allylic alcohols using the vanadium complex of 2a were easily performed in toluene with a TBHP water solution to afford (2R)-epoxy alcohols in good to excellent enantioselectivities.

Non-phosphorylated FTY720 induces apoptosis of human microglia by activating SREBP2.

A synthetic analog of sphingosine named FTY720 (Fingolimod), phosphorylated by sphingosine kinase-2, interacts with sphingosine-1-phosphate (S1P) receptors expressed on various cells. FTY720 suppresses the disease activity of multiple sclerosis (MS) chiefly by inhibiting S1P-dependent egress of autoreactive T lymphocytes from secondary lymphoid organs, and possibly by exerting anti-inflammatory and neuroprotective effects directly on brain cells. However, at present, biological effects of FTY720 on human microglia are largely unknown. We studied FTY720-mediated apoptosis of a human microglia cell line HMO6. The exposure of HMO6 cells to non-phosphorylated FTY720 (FTY720-non-P) induced apoptosis in a dose-dependent manner with IC50 of 10.6 ± 2.0 µM, accompanied by the cleavage of caspase-7 and caspase-3 but not of caspase-9. The apoptosis was inhibited by Z-DQMD-FMK, a caspase-3 inhibitor, but not by Pertussis toxin, a Gi protein inhibitor, suramin, a S1P3/S1P5 inhibitor, or W123, a S1P1 competitive antagonist, although HMO6 expressed S1P1, S1P2, and S1P3. Furthermore, both phosphorylated FTY720 (FTY720-P) and SEW2871, S1P1 selective agonists, did not induce apoptosis of HMO6. Genome-wide gene expression profiling and molecular network analysis indicated activation of transcriptional regulation by sterol regulatory element-binding protein (SREBP) in FTY720-non-P-treated HMO6 cells. Western blot verified activation of SREBP2 in these cells, and apoptosis was enhanced by pretreatment with simvastatin, an activator of SREBP2, and by overexpression of the N-terminal fragment of SREBP2. These observations suggest that FTY720-non-P-induced apoptosis of HMO6 human microglia is independent of S1P receptor binding, and positively regulated by the SREBP2-dependent proapoptotic signaling pathway.

Photoreactions of bicyclic aziridines with alkenes and alkynes: a novel synthetic methodology for 8-azabicyclo[3.2.1]octane derivatives.

The photochemical C-C bond cleavage of bicyclic aziridines 7 and subsequent [3 + 2] cycloaddition with electron-deficient alkenes and alkynes afforded the novel head-to-head adducts selectively and efficiently. The adducts contain the naturally occurring 8-azabicyclo[3.2.1]octane skeleton (e.g. tropane alkaloids). The aziridine 8 fused with a 6-membered ring also afforded the cycloadducts but in poor yields. The methylaziridine 9 reacted with an electron-deficient alkene, affording the head-to-tail adduct 23 in addition to head-to-head adducts 22a and 22b. The photoreactions of bicyclic aziridines with alkenes and alkynes indicate a similar behavior to that of aziridines with a linear chain.

Metal triflate-catalyzed cationic benzylation and allylation of 1,3-dicarbonyl compounds.

The rare earth metal and hafnium triflate-catalyzed secondary benzylation and allylation of 1,3-diketones, ketoesters, and ketoamides are described. The procedure was carried out under non-anhydrous conditions. Various 1-phenylethyl cations were generated from substituted 1-phenylethanols using 0.5 mol % of the metal triflates in CH3NO2. The cations reacted with 1,3-diketones and ketoesters to give benzylated products in high yields. Following the GC analysis, the reaction conditions were easily optimized by the selection of catalysts based on the Lewis acidity of the triflates and reaction temperature. A tertiary-alkylated diketone and a corresponding ketoester were also benzylated to afford products with a quaternary carbon atom in 57-84% yield. The ketoamide reactions required stronger Lewis acids than those used in the diketone and ketoester reactions. The reactions of benzylic alcohols possessing various substituents on the aromatic ring and dibenzoylmethane (2b) as a diketone were examined in the presence of Hf(OTf)4. Electron-rich benzylic alcohols reacted with 2b in 86-96% yield, and electron-deficient alcohol gave the desired product in 79-65% yield. Despite possessing a strong electron-withdrawing group, the reaction of 1-(4-nitrophenyl)ethanol gave the corresponding product in 61% yield. It was also possible to use allylic alcohols directly for the allylation of diketone 2b. The catalyst can be recovered by water extraction and reused up to five times.

A convenient synthesis of immunosuppressive agent FTY720 using the petasis reaction.

A convenient synthesis of immunosuppressive agent FTY720 (1) using the Petasis reaction was developed. 4-Octylbenzaldehyde (9) was converted into 1-ethenyl-4-octylbenzene (11) by two-step synthesis. Hydroboration of 11 using catecholborane and hydrolysis gave (E)-2-(4-octylphenyl)vinylboronic acid (4). The Petasis reaction of 4, dihydroxyacetone (3), and benzylamine following catalytic hydrogenation afforded FTY720 (1).

Secondary benzylation using benzyl alcohols catalyzed by lanthanoid, scandium, and hafnium triflate.

The combination of a secondary benzyl alcohol and a metal triflate (e.g., La, Yb, Sc, and Hf triflate) in nitromethane was a highly effective secondary-benzylation system. Secondary benzylation of carbon (aromatic compounds, olefins, an enol acetate), nitrogen (amide derivatives), and oxygen (alcohols) nucleophiles was carried out with a secondary benzyl alcohol and 0.01-1 mol % of a metal triflate in the presence of water. Secondary benzyl alcohols and nucleophiles bearing acid-sensitive functional groups (e.g., tert-butyldimethylsilyloxy and acetoxy groups and methyl and benzyl esters) could be used for alkylation. Hf(OTf)4 was the most active catalyst for this alkylation, and trifluoromethanesulfonic acid (triflic acid, TfOH) was also a good catalyst. The catalytic activity of metal triflates and TfOH increased in the order La(OTf)3 < Yb(OTf)3 < TfOH < Sc(OTf)3 < Hf(OTf)4. A mechanistic study was also performed. The reaction of 1-phenylethanol (4a) in the presence of Sc(OTf)3 in nitromethane gave an equilibrium mixture of 4a and bis(1-phenylethyl) ether (54). Addition of a carbon nucleophile to the equilibrium mixture gave alkylated product in high yield.

[The chemistry of pericyclic reactions and their application to syntheses of heterocyclic compounds].

Diels-Alder reactions of benzylidenecyanomethyl-1,3-benzothiazoles 17 and -1,3-benzoxazoles 18 as 1-aza-1,3-butadienes are described. The dienes 17 and 18 featuring stabilized imine moieties in the form of heteroaromatic rings react with both electron-deficient and electron-rich dienophiles to give corresponding cycloadducts regioselectively. The cycloadditions of the intramolecular systems 34c,d and 35c,d proceeded smoothly via the exo-transition state, stereoselectively affording polycyclic compounds 36c,d and 37c,d in good to excellent yields. The diene systems of 17 and 18 were extended to dienes 19a-c with ester groups at diene-4-positions. Dienes 19a-c exhibited high Diels-Alder reactivities with electron-rich alkenes. Dienes 19a-c also reacted with allyl alcohols 55-58 in the presence of stanoxane catalyst 53 to give cycloadducts 59-62 via transesterification and intramolecular cycloaddition. Although alpha-alkoxycarbonylnitrones 64 have been very attractive nitrones for the syntheses of amino acids, the nitrones 64 exist as equilibrating mixtures of (E)-64 and (Z)-64. To solve this problem, three methods were explored: 1) sequential transesterification and intermolecular cycloaddition of nitrones 64 with allyl alcohols; 2) use of chiral and geometry-fixed nitrone 84; and 3) selective activation of (Z)-64 by Eu(fod)3. These methods were applied to syntheses of nikkomycins, clavalanine, and beta-substituted alpha-amino acids. The reactions of photoinduced carbonyl ylides from alpha,beta-unsaturated gamma,delta-epoxy nitriles were studied. Direct irradiation (lambda = 254 nm) of (E)-129 led selectively to products arising from the carbonyl ylide XXV or the carbene intermediate XXVI. The carbonyl ylides generated from (E)-129, (E)-139, and (Z)-143 were trapped with MeOH in the presence of amine, affording the corresponding acetals in moderate yields (Schemes 42 and 43). Photocyclization reactions of delta-hydroxyalkyl epoxy nitriles 148a-e led to spiro acetals arising from the carbonyl ylides (Scheme 45). The photoinduced carbonyl ylides from the epoxy dinitriles 158 and 160-163 underwent 1,3-dipolar cycloaddition with enol ethers, leading to a tetrahydrofuran system (Schemes 49 and 50, Table 14). Electrocyclization of 3-butadienylindoles 184 to intermediary dihydrocarbazoles XXXII followed by elimination of MeOH gave 3-oxyganated carbazoles 185, which were transformed to carbazole alkaloids hyellazole 168, 4-demethoxycarbazomycin B 170 and carazostatin 171, respectively. Claisen rearrangement of 3-(1-amino-1-vinyloxy) indolines derived from 3-hydroxyindolines 192 and amide acetal 193 gave indol-4-ylacetamides 194, which was reduced to afford 4-(2-aminoethyl) indoles 198, which has a framework of biologically active 4-substituted indole compounds. Claisen rearrangement of 3-allyloxyindoles produced in situ by condensation of indolin-3-ones 202 with allyl alcohols 203 and 206-211 gave 2-allylindolin-3-ones 204, 205 and 212-220. The domino reactions, Horner-Wadsworth-Emmons olefination of 2-allyloxyindole 233, isomerization, and Claisen rearrangement produced 3-allylindolin-2-one 234, which was derivatized to 3a-allylpyrrolo [2,3-b] indole alkaloid, flustramine C 221. Reverse aromatic Cope rearrangement of 2-allyl-3-indolidene acetonitriles 241-243, formed by Horner-Wadsworth-Emmons reaction of 2-allylindolin-3-ones 238-240, afforded indoles 244-246.