1,3-Butadiynyl sulfide-based compact trialkyne platform molecule for sequential assembly of three azides.

A compact trialkyne platform with a silyl-protected 1,3-butadiynyl sulfide moiety and a terminal alkyne group has been developed for sequential regioselective transition metal-catalyzed triazole formation reactions with three azides. This method enabled the facile construction of a low-molecular-weight triazole library and the synthesis of middle-molecular-weight trifunctional probes for protein modification.

Common and rare genetic variants predisposing females to unexplained recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is a major reproductive health issue with multifactorial causes, affecting 2.6% of all pregnancies worldwide. Nearly half of the RPL cases lack clinically identifiable causes (e.g., antiphospholipid syndrome, uterine anomalies, and parental chromosomal abnormalities), referred to as unexplained RPL (uRPL). Here, we perform a genome-wide association study focusing on uRPL in 1,728 cases and 24,315 female controls of Japanese ancestry. We detect significant associations in the major histocompatibility complex (MHC) region at 6p21 (lead variant=rs9263738; P = 1.4 × 10-10; odds ratio [OR] = 1.51 [95% CI: 1.33-1.72]; risk allele frequency = 0.871). The MHC associations are fine-mapped to the classical HLA alleles, HLA-C*12:02, HLA-B*52:01, and HLA-DRB1*15:02 (P = 1.1 × 10-10, 1.5 × 10-10, and 1.2 × 10-9, respectively), which constitute a population-specific common long-range haplotype with a protective effect (P = 2.8 × 10-10; OR = 0.65 [95% CI: 0.57-0.75]; haplotype frequency=0.108). Genome-wide copy-number variation (CNV) calling demonstrates rare predicted loss-of-function (pLoF) variants of the cadherin-11 gene (CDH11) conferring the risk of uRPL (P = 1.3 × 10-4; OR = 3.29 [95% CI: 1.78-5.76]). Our study highlights the importance of reproductive immunology and rare variants in the uRPL etiology.

Progress on the Synthesis of the Aromathecin Family of Compounds: An Overview.

We present a systematic review of the methods developed for the synthesis of the aromathecin family of compounds (benz[6,7]indolizino[1,2-b]quinolin-11(13H)-ones) and their derivatives. These methods can be broadly classified into four categories based on the construction of pentacyclic structures: Category 1: by constructing a pyridone moiety (D-ring) on the pyrroloquinoline ring (A/B/C-ring), Category 2: by constructing a pyridine moiety (B-ring) on the pyrroloisoquinolone ring (C/D/E-ring), Category 3: by constructing an indolizidinone moiety (C/D-ring) in a tandem reaction, and Category 4: by constructing a pyrrolidine moiety (C-ring) on the isoquinolone ring (D/E-ring).

Characteristics of the patients consulted with emergency medicine physicians at a large-scale COVID-19 vaccination center: Prospective observational study.

OBJECTIVE: We aimed to clarify the characteristics of patients consulted by the medical staff with emergency medicine (EM) physicians after vaccination and EM physicians transferred to an outside hospital. DESIGN: The Japanese Self-Defense Force established a large-scale coronavirus disease 2019 (COVID-19) vaccination center. Overall, 1,306,928 citizens received the Moderna vaccine, which targeted the first and second vaccinations between May 24, 2021 and November 30, 2021. EM physicians were always available in the emergency room (ER). The medical staff could consult the patients with EM physicians; however, the criteria were ambiguous. We conducted signal detection analysis on the patients who experienced adverse events to detect characteristics. RESULTS: Of the 3,312 patients experienced adverse events after vaccination, the medical staff consulted 344 with EM physicians. The patients whose respiratory rate and systolic blood pressure (BP) were more than 18 per minute and 162 mmHg, respectively, were considerably consulted. In addition, the patients whose systolic BP was more than 186.5 mmHg were transferred to an outside hospital. No patients were seriously ill or died after being transferred to an outside hospital. CONCLUSIONS: The medical staff consulted the patients with a high respiratory rate or BP with EM physicians. In addition to BP, the respiratory rate would also be necessary as a finding that suggests a patient's severity after vaccination. Therefore, it appears safer that EM physicians are always available to ensure the recipients' safety when running a new large-scale vaccination center against unknown diseases, such as COVID-19.

Three Distinct Reporter Systems of Hepatitis E Virus and Their Utility as Drug Screening Platforms.

The hepatitis E virus (HEV) is increasingly acknowledged as the primary cause of acute hepatitis. While most HEV infections are self-limiting, cases of chronic infection and fulminant hepatitis necessitate the administration of anti-HEV medications. However, there is a lack of specific antiviral drugs designed for HEV, and the currently available drug (ribavirin) has been associated with significant adverse effects. The development of innovative antiviral drugs involves targeting distinct steps within the viral life cycle: the early step (attachment and internalization), middle step (translation and RNA replication), and late step (virus particle formation and virion release). We recently established three HEV reporter systems, each covering one or two of these steps. Using these reporter systems, we identified various potential drug candidates that target different steps of the HEV life cycle. Through rigorous in vitro testing using our robust cell culture system with the genotype 3 HEV strain (JE03-1760F/P10), we confirmed the efficacy of these drugs, when used alone or in combination with existing anti-HEV drugs. This underscores their significance in the quest for an effective anti-HEV treatment. In the present review, we discuss the development of the three reporter systems, their applications in drug screening, and their potential to advance our understanding of the incompletely elucidated HEV life cycle.

Development of a HiBiT-tagged reporter hepatitis E virus and its utility as an antiviral drug screening platform.

Previously, we developed an infectious hepatitis E virus (HEV) harboring the nanoKAZ gene in the hypervariable region of the open reading frame 1 (ORF1) of the HEV3b (JE03-1760F/P10) genome and demonstrated the usefulness for screening anti-HEV drugs that inhibit the early infection process. In the present study, we constructed another reporter HEV (HEV3b-HiBiT) by placing a minimized HiBiT tag derived from NanoLuc luciferase at the 3'-end of the viral capsid (ORF2) coding sequence. It replicated efficiently in PLC/PRF/5 cells, produced membrane-associated particles identical to those of the parental virus, and was genetically stable and infectious. The HiBiT tag was fused to both secreted ORF2s (ORF2s-HiBiT) and ORF2c capsid protein (ORF2c-HiBiT). The ORF2c-HiBiT formed membrane-associated HEV particles (eHEV3b-HiBiT). By treating these particles with digitonin, we demonstrated that the HiBiT tag was expressed on the surface of capsid and was present inside the lipid membrane. To simplify the measurement of luciferase activity and provide a more convenient screening platform, we constructed an ORF2s-defective mutant (HEV3b-HiBiT/ΔORF2s) in which the secreted ORF2s are suppressed. We used this system to evaluate the effects of introducing small interfering RNAs and treatment with an inhibitor or accelerator of exosomal release on HEV egress and demonstrated that the effects on virus release can readily be analyzed. Therefore, HEV3b-HiBiT and HEV3b-HiBiT/ΔORF2s reporters may be useful for investigating the virus life cycle and can serve as a more convenient screening platform to search for candidate drugs targeting the late stage of HEV infection such as particle formation and release. IMPORTANCE The construction of recombinant infectious viruses harboring a stable luminescence reporter gene is essential for investigations of the viral life cycle, such as viral replication and pathogenesis, and the development of novel antiviral drugs. However, it is difficult to maintain the stability of a large foreign gene inserted into the viral genome. In the present study, we successfully generated a recombinant HEV harboring the 11-amino acid HiBiT tag in the ORF2 coding region and demonstrated the infectivity, efficient virus growth, particle morphology, and genetic stability, suggesting that this recombinant HEV is useful for in vitro assays. Furthermore, this system can serve as a more convenient screening platform for anti-HEV drugs. Thus, an infectious recombinant HEV is a powerful approach not only for elucidating the molecular mechanisms of the viral life cycle but also for the screening and development of novel antiviral agents.

Effect of intravenous fluid therapy for acute alcohol intoxication on length of time from arrival at the emergency department until awakening: A prospective observational cohort study.

Aim: To evaluate the association of intravenous fluid (IVF) therapy on the length of time from arrival at the emergency department (ED) until awakening in cases of acute alcohol intoxication. Methods: This single-center, prospective, observational study was conducted in the ED of the Self-Defense Forces Central Hospital during October 1, 2018 to July 31, 2019. Patients with 1,000 mL bolus of lactated Ringer's solution and those without bolus were compared. The primary outcome was the length of time until awakening. Secondary outcomes were the length of stay in the ED and occurrence of conditions requiring extra care. Predictors of the occurrence of any event requiring extra care were identified. Results: We included 201 patients, of whom 109 received IVF and 92 did not. No significant difference existed in the baseline characteristics between the groups. The median length of time until awakening did not significantly differ between the groups (P = 0.77). Multivariable regression analysis adjusted by age, sex, hemoglobin, blood alcohol concentration, and initial Glasgow Coma Scale (GCS) score demonstrated that the regression coefficient of IVF for length of time until awakening was -9.55 (95% confidence interval [CI], -36.2 to 17.2). Hemoglobin (regression coefficient, 10.1; 95% CI, 0.38-19.9) and initial GCS score (regression coefficient, -7.51; 95% CI, -10.8 to -4.21) were significantly associated with length of time. Conclusion: IVF therapy was not associated with the length of time until awakening in patients with acute alcohol intoxication in the ED. Routine IVF administration was unnecessary.

Novel Approach to the Construction of Fused Indolizine Scaffolds: Synthesis of Rosettacin and the Aromathecin Family of Compounds.

Camptothecin-like compounds are actively employed as anticancer drugs in clinical treatments. The aromathecin family of compounds, which contains the same indazolidine core structure as the camptothecin family of compounds, is also expected to display promising anticancer activity. Therefore, the development of a suitable and scalable synthetic method of aromathecin synthesis is of great research interest. In this study, we report the development of a new synthetic approach for constructing the pentacyclic scaffold of the aromathecin family by forming the indolizidine moiety after synthesizing the isoquinolone moiety. Thermal cyclization of 2-alkynylbenzaldehyde oxime to the isoquinoline N-oxide, followed by a Reissert-Henze-type reaction, forms the key strategy in this isoquinolone synthesis. Under the optimum reaction conditions for the Reissert-Henze-type reaction step, microwave irradiation-assisted heating of the purified N-oxide in acetic anhydride at 50 °C reduced the formation of the 4-acetoxyisoquinoline byproduct to deliver the desired isoquinolone at a 73% yield after just 3.5 h. The eight-step sequence employed afforded rosettacin (simplest member of the aromathecin family) at a 23.8% overall yield. The synthesis of rosettacin analogs was achieved by applying the developed strategy and may be generally applicable to the production of other fused indolizidine compounds.

Synthesis of Functionalized Dibenzoazacyclooctynes by a Decomplexation Method for Dibenzo-Fused Cyclooctyne-Cobalt Complexes.

A concise route for dibenzoazacyclooctynes (DIBACs) synthesis was developed based on Pictet-Spengler reaction and a novel cobalt decomplexation method established for dibenzo-fused cyclooctyne-cobalt complexes. The method allowed for the facile preparation of functionalized DIBACs, including bisDIBAC, which served as an efficient bisreactive linker for protein modification via the double-click reaction.

Inhibition of pancreatic cancer-cell growth and metastasis in vivo by a pyrazole compound characterized as a cell-migration inhibitor by an in vitro chemotaxis assay.

Pancreatic cancer is recalcitrant to treatment as it is highly metastatic and rapidly progressive. While observing the behavior of human pancreatic BxPC-3 cells using an optical assay device called TAXIScan, we found that several synthetic pyrazole and pyrimidine derivatives inhibited cell migration. One such compound, 14-100, inhibited metastasis of fluorescence-labeled BxPC-3 cells, which were transplanted into the pancreas of nude mice as a subcutaneously grown cancer fragment. Surprisingly, despite its low cytotoxicity, the compound also showed an inhibitory effect on cancer cell proliferation in vivo, suggesting that the compound alters cancer cell characteristics needed to grow in situ. Single-cell RNA-sequencing revealed changes in gene expression associated with metastasis, angiogenesis, inflammation, and epithelial-mesenchymal transition. These data suggest that the compound 14-100 could be a good drug candidate against pancreatic cancer.

Incidence and Risk Factors of Immediate Hypersensitivity Reactions and Immunization Stress-Related Responses With COVID-19 mRNA Vaccine.

BACKGROUND: With the implementation of mass vaccination campaigns against COVID-19, the safety of vaccine needs to be evaluated. OBJECTIVE: We aimed to assess the incidence and risk factors for immediate hypersensitivity reactions (IHSR) and immunization stress-related responses (ISRR) with the Moderna COVID-19 vaccine. METHODS: This nested case-control study included recipients who received the Moderna vaccine at a mass vaccination center, Japan. Recipients with IHSR and ISRR were designated as cases 1 and 2, respectively. Controls 1 and 2 were selected from recipients without IHSR or ISRR and matched (1 case: 4 controls) with cases 1 and cases 2, respectively. Conditional logistic regression analysis was used to identify risk factors associated with IHSR and ISRR. RESULTS: Of the 614,151 vaccine recipients who received 1,201,688 vaccine doses, 306 recipients (cases 1) and 2478 recipients (cases 2) showed 318 events of IHSR and 2558 events of ISRR, respectively. The incidence rates per million doses were estimated as IHSR: 266 cases, ISRR: 2129 cases, anaphylaxis: 2 cases, and vasovagal syncope: 72 cases. Risk factors associated with IHSR included female, asthma, atopic dermatitis, thyroid diseases, and a history of allergy; for ISRR, the risk factors were younger age, female, asthma, thyroid diseases, mental disorders, and a history of allergy and vasovagal reflex. CONCLUSION: In the mass vaccination settings, the Moderna vaccine can be used safely owing to the low incidence rates of IHSR and anaphylaxis. However, providers should be aware of the occurrence of ISRR. Although recipients with risk factors are associated with slightly increased risks of IHSR and ISRR, this is not of sufficient magnitude to warrant special measures regarding their vaccination.

Development of Recombinant Infectious Hepatitis E Virus Harboring the nanoKAZ Gene and Its Application in Drug Screening.

Hepatitis E virus (HEV) is a quasi-enveloped virus with a single-stranded positive-sense RNA genome belonging to the family Hepeviridae. Studies of the molecular aspects of HEV and drug screening have benefited from the discovery of bioluminescent reporter genes. However, the stability of large foreign genes is difficult to maintain after insertion into the viral genome. Currently, ribavirin is used to treat HEV-infected patients who require antiviral therapy. This has several major drawbacks. Thus, the development of novel anti-HEV drugs is of great importance. We developed a system consisting of recombinant infectious HEV harboring a small luciferase gene (nanoKAZ) in the hypervariable region (HVR) of the open reading frame 1 (ORF1) (HEV-nanoKAZ). It replicated efficiently in cultured cells, was genetically stable, and had morphological characteristics similar to those of the parental virus. Both membrane-associated (eHEV-nanoKAZ) and membrane-unassociated (neHEV-nanoKAZ) particles were infectious. HEV particles circulating in the bloodstream and attaching to hepatocytes in HEV-infected patients are membrane-associated; thus, eHEV-nanoKAZ was applied in drug screening. The eHEV-nanoKAZ system covers at least the inhibitor of HEV entry and inhibitor of HEV RNA replication. Four drugs with anti-HEV activity were identified. Their effectiveness in cultured cells was confirmed in naive and HEV-producing PLC/PRF/5 cells. Two hit drugs (azithromycin and ritonavir) strongly inhibited HEV production in culture supernatants, as well as intracellular expression of ORF2 protein, and may therefore be candidate novel anti-HEV drugs. The HEV-nanoKAZ system was developed and applied in drug screening and is expected to be useful for investigating the HEV life cycle. IMPORTANCE Bioluminescent reporter viruses are essential tools in molecular virological research. They have been widely used to investigate viral life cycles and in the development of antiviral drugs. For drug screening, the use of a bioluminescent reporter virus helps shorten the time required to perform the assay. A system, consisting of recombinant infectious HEV harboring the nanoKAZ gene in the HVR of ORF1 (HEV-nanoKAZ), was developed in this study and was successfully applied to drug screening in which four hit drugs with anti-HEV activity were identified. The results of this study provide evidence supporting the use of this system in more variable HEV studies. In addition, both forms of viral particles (eHEV-nanoKAZ and neHEV-nanoKAZ) are infectious, which will enable their application in HEV studies requiring both forms of viral particles, such as in the investigation of unknown HEV receptors and the elucidation of host factors important for HEV entry.

In Vitro and in Silico Studies of Potential Coronavirus-Specific 3C-Like Protease Inhibitors.

We investigated similar compounds to ebselen and tideglusib, which exhibit strong activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), using Molecular ACCess System (MACCS) keys. Four candidate compounds were identified. One of them, phenyl-benzothiazol-3-one, showed coronavirus-specific 3C-like (3CL) protease inhibitory activity. The results indicated that a similarity score above 0.81 is a good indicator of activity for ebselen-and-tideglusib-like compounds. Subsequently, we simulated the ring-cleavage Michael reaction of ebselen at the Se center, which is responsible for its 3CL protease inhibitory activity, and determined the activation free energy of the reaction. The results showed that reaction simulation is a useful tool for estimating the activity of inhibitory compounds that undergo Michael addition reactions with the relevant cysteine S atom of 3CL proteases.

A Novel Boron Lipid to Modify Liposomal Surfaces for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a cancer treatment with clinically demonstrated efficacy using boronophenylalanine (BPA) and sodium mercaptododecaborate (BSH). However, tumor tissue selectivity of BSH and retention of BPA in tumor cells is a constant problem. To ensure boron accumulation and retention in tumor tissues, we designed a novel polyethylene glycol (PEG)-based boron-containing lipid (PBL) and examined the potency of delivery of boron using novel PBL-containing liposomes, facilitated by the enhanced permeability and retention (EPR) effect. PBL was synthesized by the reaction of distearoylphosphoethanolamine and BSH linked by PEG with Michael addition while liposomes modified using PBL were prepared from the mixed lipid at a constant molar ratio. In this manner, novel boron liposomes featuring BSH in the liposomal surfaces, instead of being encapsulated in the inner aqueous phase or incorporated in the lipid bilayer membrane, were prepared. These PBL liposomes also carry additional payload capacity for more boron compounds (or anticancer agents) in their inner aqueous phase. The findings demonstrated that PBL liposomes are promising candidates to effect suitable boron accumulation for BNCT.

[Development of New Synthetic Methods for Carbazole Compounds Aimed at Drug Discovery and Exploratory Research on Pharmaceutical Materials].

We are developing the synthesis of biologically interesting carbazole compounds, including natural products by tandem cyclic reactions. In this report, we describe the new synthesis of carbazole-1,4-quinones as follows; 1) the synthesis of carbazole-1,4-quinones using a tandem ring closing metathesis (RCM) -dehydrogenation reaction, 2) a novel one-pot synthesis of carbazole-1,4-quinone by consecutive Pd-catalyzed cyclocarbonylation, desilylation, and oxidation reactions. Two new synthetic strategies were applied to the synthesis of carbazole-1,4-quinone alkaloids and ellipticine quinones, and then the antiproliferative activity against HCT-116 and HL-60 cells of the synthesized compounds were evaluated.

Startup of pilot-scale single-stage nitrogen removal using anammox and partial nitritation (SNAP) reactor for waste brine treatment using marine anammox bacteria.

This study is the first to demonstrate the startup of a pilot-scale single-stage nitrogen removal using anammox and partial nitritation (SNAP) reactor utilizing marine anammox bacteria. A complete mixing type reactor, continuously fed with waste brine obtained from a natural gas plant (salinity 3%, NH4+-N 130-180 mg/L) and having an effective volume of 2 m3, achieved stable operation at temperatures of 20-30°C with a maximum nitrogen removal rate of 1.43 kg-N/m3/day. During the startup process, along with a small amount of seed sludge, granular sludge was additionally inoculated as a biomass carrier for the enrichment of ammonia oxidizing bacteria (AOB), followed by the enrichment of anammox bacteria. A mesh screen equipped at the outlet of the reactor facilitated the successful sludge retention in the reactor. Analysis of bacterial community composition indicated that Candidatus Scalindua was successfully enriched in the pilot SNAP reactor. These methods for stable sludge retention in the reactor greatly contributed to the startup of the first pilot-scale SNAP reactor using marine anammox bacteria.

Snakebite and local envenomation by Boiruna maculata treated without antivenom.

BACKGROUND: When snake breeders are bitten by rare snakes, deciding whether to administer snake antivenom can be challenging. CASE PRESENTATION: A 50-year-old man was bitten on the right finger by Boiruna maculata. The next day, his right upper limb exhibited pronounced local manifestations of envenomation. At the first consultation, a dark purple bleeding spot and a necrotic site were present under the fang marks at the bitten finger and his affected limb showed extensive swelling and redness. Snake antivenom was not administered because it was difficult to identify the snake and obtain the antivenom. We performed the pressure immobilization technique to his limb. The patient's symptoms peaked in severity on the second day of illness. He was discharged with marked improvement. CONCLUSIONS: We have experienced a case of snakebite envenomation by Boiruna maculata.

Total synthesis of pyrrolo[2,3-c]quinoline alkaloid: trigonoine B.

The first total synthesis of the pyrrolo[2,3-c]quinoline alkaloid trigonoine B (1) was accomplished via a six-step sequence involving the construction of an N-substituted 4-aminopyrrolo[2,3-c]quinoline framework via electrocyclization of 2-(pyrrol-3-yl)benzene containing a carbodiimide moiety as a 2-azahexatriene system. The employed six-step sequence afforded trigonoine B (1) in 9.2% overall yield. The described route could be employed for the preparation of various N-substituted 4-aminopyrroloquinolines with various biological activities.

The Capsid (ORF2) Protein of Hepatitis E Virus in Feces Is C-Terminally Truncated.

The hepatitis E virus (HEV) is a causative agent of hepatitis E. HEV virions in circulating blood and culture media are quasi-enveloped, while those in feces are nonenveloped. The capsid (ORF2) protein associated with an enveloped HEV virion is reported to comprise the translation product of leucine 14/methionine 16 to 660 (C-terminal end). However, the nature of the ORF2 protein associated with fecal HEV remains unclear. In the present study, we compared the molecular size of the ORF2 protein among fecal HEV, cell-culture-generated HEV (HEVcc), and detergent-treated protease-digested HEVcc. The ORF2 proteins associated with fecal HEV were C-terminally truncated and showed the same size as those of the detergent-treated protease-digested HEVcc virions (60 kDa), in contrast to those of the HEVcc (68 kDa). The structure prediction of the ORF2 protein (in line with previous studies) demonstrated that the C-terminal region (54 amino acids) of an ORF2 protein is in flux, suggesting that proteases target this region. The nonenveloped nondigested HEV structure prediction indicates that the C-terminal region of the ORF2 protein moves to the surface of the virion and is unnecessary for HEV infection. Our findings clarify the maturation of nonenveloped HEV and will be useful for studies on the HEV lifecycle.

Intravenous Immunoglobulin G Modulates the Expression of Sepsis-Induced Coagulopathy Factors and Increases Serum IgM Levels: A Prospective, Single-Center Intervention Study.

Sepsis and sepsis-related multiple organ failure are major causes of mortality in intensive care unit (ICU) settings. This study aimed to determine the effect of intravenous immunoglobulin G (IVIgG) on different types of immunoglobulin and anti-coagulant factor types in sepsis patients. A single-center observational study of patients with sepsis, severe sepsis, or septic shock was conducted from August 2008 to March 2013. Patients were divided into the IVIgG (immunoglobulin G [IgG] <870 mg/dL; lower normal range) and non-IVIgG (IgG ≥870 mg/dL) groups. The IVIgG group received IVIgG for three days, and other standard medications. Serial measurements were taken of serum IgG, immunoglobulin A (IgA), immunoglobulin M (IgM), total plasminogen activator inhibitor 1 (tPAI-1), and protein C. Patients in the IVIgG treatment group had significantly higher serum IgM level on Days 4 and 7 than on Day 1, but no significant changes in IgM levels were observed in patients in the non-IVIgG group. Patients in the IVIgG treatment had lower tPAI-1 levels on Days 4 and 7 than on Day 1 and increased protein C levels on Day 7 compared to those on Days 1 and 4. There were no significant differences in tPAI-1 levels or protein C levels in the non-IVIgG group, although a similar trend was observed. IVIgG administration increased patients' serum IgM and protein C levels and decreased their serum tPAI-1 levels. IVIgG has potential application for preventing sepsis-induced coagulopathy and disseminated intravascular coagulation.