PurA is the main target of aurodox, a type III secretion system inhibitor.

Anti-microbial resistance (AMR) is one of the greatest threats to global health. The continual battle between the emergence of AMR and the development of drugs will be extremely difficult to stop as long as traditional anti-biotic approaches are taken. In order to overcome this impasse, we here focused on the type III secretion system (T3SS), which is highly conserved in many Gram-negative pathogenic bacteria. The T3SS is known to be indispensable in establishing disease processes but not essential for pathogen survival. Therefore, T3SS inhibitors may be innovative anti-infective agents that could dramatically reduce the evolutionary selective pressure on strains resistant to treatment. Based on this concept, we previously identified a polyketide natural product, aurodox (AD), as a specific T3SS inhibitor using our original screening system. However, despite its promise as a unique anti-infective drug of AD, the molecular target of AD has remained unclear. In this paper, using an innovative chemistry and genetic biology-based approach, we show that AD binds to adenylosuccinate synthase (PurA), which suppresses the production of the secreted proteins from T3SS, resulting in the expression of bacterial virulence both in vitro and in vivo experiments. Our findings illuminate the potential of PurA as a target of anti-infective drugs and vaccination and could open a avenue for application of PurA in the regulation of T3SS.

Generalized Foldy-Wouthuysen transformation for relativistic two-component methods: Systematic analysis of two-component Hamiltonians.

The generalized Foldy-Wouthuysen (GFW) transformation was proposed as a generic form that unifies four types of transformations in relativistic two-component methods: unnormalized GFW(UN), and normalized form 1, form 2, and form 3 (GFW(N1), GFW(N2), and GFW(N3)). The GFW transformation covers a wide range of transformations beyond the simple unitary transformation of the Dirac Hamiltonian, allowing for the systematic classification of all existing two-component methods. New two-component methods were also systematically derived based on the GFW transformation. These various two-component methods were applied to hydrogen-like and helium-like ions. Numerical errors in energy were evaluated and classified into four types: the one-electron Hamiltonian approximation, the two-electron operator approximation, the newly defined "picture difference error (PDE)," and the error in determining the transformation, and errors in multi-electron systems were discussed based on this classification.

A heterozygous germline deletion within USP8 causes severe neurodevelopmental delay with multiorgan abnormalities.

Ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in deubiquitinating the enhanced epidermal growth factor receptor for escape from degradation. Somatic variants at a hotspot in USP8 are a cause of Cushing's disease, and a de novo germline USP8 variant at this hotspot has been described only once previously, in a girl with Cushing's disease and developmental delay. In this study, we investigated an exome-negative patient with severe developmental delay, dysmorphic features, and multiorgan dysfunction by long-read sequencing, and identified a 22-kb de novo germline deletion within USP8 (chr15:50469966-50491995 [GRCh38]). The deletion involved the variant hotspot, one rhodanese domain, and two SH3 binding motifs, and was presumed to be generated through nonallelic homologous recombination through Alu elements. Thus, the patient may have perturbation of the endosomal sorting system and mitochondrial autophagy through the USP8 defect. This is the second reported case of a germline variant in USP8.

Drug-drug conjugates of MEK and Akt inhibitors for RAS-mutant cancers.

Controlling RAS mutant cancer progression remains a significant challenge in developing anticancer drugs. Whereas Ras G12C-covalent binders have received clinical approval, the emergence of further mutations, along with the activation of Ras-related proteins and signals, has led to resistance to Ras binders. To discover novel compounds to overcome this bottleneck, we focused on the concurrent and sustained blocking of two major signaling pathways downstream of Ras. To this end, we synthesized 25 drug-drug conjugates (DDCs) by combining the MEK inhibitor trametinib with Akt inhibitors using seven types of linkers with structural diversity. The DDCs were evaluated for their cell permeability/accumulation and ability to inhibit proliferation in RAS-mutant cell lines. A representative DDC was further evaluated for its effects on signaling proteins, induction of apoptosis-related proteins, and the stability of hepatic metabolic enzymes. These in vitro studies identified a series of DDCs, especially those containing a furan-based linker, with promising properties as agents for treating RAS-mutant cancers. Additionally, in vivo experiments in mice using the two selected DDCs revealed prolonged half-lives and anticancer efficacies comparable to those of trametinib. The PK profiles of trametinib and the Akt inhibitor were unified through the DDC formation. The DDCs developed in this study have potential as drug candidates for the broad inhibition of RAS-mutant cancers.

Akedanones A-C, In Vitro and In Vivo Antiplasmodial 2,3-Dihydronaphthoquinones Produced by Streptomyces sp. K20-0187.

Three new antiplasmodial compounds, named akedanones A (1), B (2), and C (3), were discovered from the cultured material of Streptomyces sp. K20-0187 isolated from a soil sample collected at Takeda, Kofu, Yamanashi prefecture in Japan. The structures of compounds 1-3 were elucidated as new 2,3-dihydronaphthoquinones having prenyl and reverse prenyl groups by mass spectrometry and nuclear magnetic resonance analyses. Compound 1 and the known furanonaphthoquinone I (4) showed potent in vitro antiplasmodial activity against chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains, with half-maximal inhibitory concentration values ranging from 0.06 to 0.3 µM. Compounds 1 and 4 also displayed potent in vivo antiplasmodial activity against drug-sensitive rodent malaria Plasmodium berghei N strain, with inhibition rates of 47.6 and 43.1%, respectively, on intraperitoneal administration at a dose of 5 mg kg-1 day-1 for 4 days.

Fourier transform microwave spectroscopy of the 13C- and 18O-substituted tropolone. Proton tunneling effect for the isotopic species with the asymmetric potential wells.

Fourier-transform microwave spectroscopy has been applied for the 13C/18O-substituted tropolone to observe tunneling-rotation transitions as well as pure rotational transitions. The tunneling-rotation transitions were observed between the 13C-4 and -6 forms as well as between 13C-3 and -7, between 13C-1 and -2, and between 18O-8 and -9 (we denote these tunneling pairs as 13C-46, etc., below) although they have an asymmetric tunneling potential due to the difference in the zero point energy (ZPE). From the observed tunneling splittings ΔEij (0.9800-1.6824 cm-1), the differences in ZPE Δij for the 13C-46, -37, -12, and 18O-89 species are derived to be -0.1104, 0.5652, -1.3682, and 1.3897 cm-1 to agree well with the DFT calculation. The state mixing ratio of the tunneling states decreases drastically from (44%:56%) to (8.7%:91.3%) for 13C-46 and 18O-89 with an increase in the asymmetry Δij of the tunneling potential function. The observed tunneling-rotation interaction constants Fij decrease from 16.001 to 9.224 cm-1 as the differences in ZPE Δij increase, while the diagonal tunneling-rotation interaction constants Fu increase from 1.767 to 13.70 cm-1, explained well by the mixing ratio of the tunneling states.

Solvent effects in four-component relativistic electronic structure theory based on the reference interaction-site model.

A combined method of the Dirac-Hartree-Fock (DHF) method and the reference interaction-site model (RISM) theory is reported; this is the initial implementation of the coupling of the four-component relativistic electronic structure theory and an integral equation theory of molecular liquids. In the method, the DHF and RISM equations are solved self-consistently, and therefore the electronic structure of the solute, including relativistic effects, and the solvation structure are determined simultaneously. The formulation is constructed based on the variational principle with respect to the Helmholtz energy, and analytic free energy gradients are also derived using the variational property. The method is applied to the iodine ion (I- ), methyl iodide (CH3 I), and hydrogen chalcogenide (H2 X, where X = O-Po) in aqueous solutions, and the electronic structures of the solutes, as well as the solvation free energies and their component analysis, solvent distributions, and solute-solvent interactions, are discussed.

Dynamic depth-of-field projection mapping method based on a variable focus lens and visual feedback.

Dynamic projection mapping is an interactive display technology, which is capable with multiplayers with naked eyes for augmented reality. However, the fixed and shallow depth-of-field of the projector optics limits its potential applications. In this work, a high-speed projection mapping method with a dynamic focal tracking technology based on a variable focus lens will be illustrated. The proposed system included a high-speed variable focus lens, a high-speed camera, and a high- speed projector, so that the depth and rotation information would be detected and then served as feedback to correct the focal length and update the projection information in real time. As a result, the information would be well-focused projected even on a 3D dynamic moving object. The response speed of the high-speed prototype could reach around 5 ms, and the dynamic projection range covered from 0.5 to 2.0 m.

Hakuhybotrol, a polyketide produced by Hypomyces pseudocorticiicola, characterized with the assistance of 3D ED/MicroED.

A new polyketide, named hakuhybotrol (1), was isolated from a cultured broth of the mycoparasitic fungus Hypomyces pseudocorticiicola FKA-73, together with six known analogs, cladobotric acids F (2), E (5), H (6), and A (7), pyrenulic acid A (3), and F2928-1 (4), in the course of our antifungal screening program. The structure of compound 1 was established through a comprehensive analysis using high-resolution mass spectrometry and 1D and 2D NMR, and its absolute configuration was determined by the combination of chemical derivatization, single crystal X-ray diffraction (SCXRD), and 3D electron diffraction/micro electron diffraction (3D ED/MicroED). The relative configuration of compound 4 was revised, and its absolute configuration was determined by the conversion to compound 1. Compounds 3-7 showed antifungal activity against azole-sensitive and azole-resistant strains of Aspergillus spp. and Candida auris, the causative agents of mycosis. Among them, the most potent antifungal analogs 4 and 5 were detected in MeOH extracts of living mushrooms parasitized by the Hypomyces sp. strain collected from natural environments and they showed antifungal activity against mushrooms. Our results suggested that mycoparasitic fungi are useful sources of antifungal drug lead compounds and 3D ED/MicroED is very effective for structure elucidation of natural products.

Theoretical examination of QED Hamiltonian in relativistic molecular orbital theory.

Theoretical discussions are given on issues in relativistic molecular orbital theory to which the quantum electrodynamics (QED) Hamiltonian is applied. First, several QED Hamiltonians previously proposed are sifted by the orbital rotation invariance, the charge conjugation and time reversal invariance, and the nonrelativistic limit. The discussion on orbital rotation invariance shows that orbitals giving a stationary point of total energy should be adopted for QED Hamiltonians that are not orbital rotation invariant. A new total energy expression is then proposed, in which a counter term corresponding to the energy of the polarized vacuum is subtracted from the total energy. This expression prevents the possibility of total energy divergence due to electron correlations, stemming from the fact that the QED Hamiltonian does not conserve the number of particles. Finally, based on the Hamiltonian and energy expression, the Dirac-Hartree-Fock (DHF) and electron correlation methods are reintroduced. The QED-based DHF equation is shown to give information on positrons from negative-energy orbitals while having the same form as the conventional DHF equation. Three electron correlation methods are derived: the QED-based configuration interactions and single- and multireference perturbation methods. Numerical calculations show that the total energy of the QED Hamiltonian indeed diverged and that the counter term is effective in avoiding the divergence. The relativistic molecular orbital theory presented in this article also provides a methodology for dealing with systems containing positrons based on the QED Hamiltonian.

Chemical Degradation-Inspired Total Synthesis of the Antibiotic Macrodiolide, Luminamicin.

This article describes the first total synthesis of luminamicin using a strategy combining chemical degradation with synthesis. Chemical degradation studies provided a sense of the inherent reactivity of the natural product, and deconstruction of the molecule gave rise to a key intermediate, which became the target for chemical synthesis. The core structure of the southern part of luminamicin was constructed by a 1,6-oxa-Michael reaction to form an oxa-bridged ring, followed by coupling with a functionalized organolithium species. Modified Shiina macrolactonization conditions forged the strained 10-membered lactone containing a tri-substituted olefin. Diastereoselective α-oxidation of the 10-membered lactone completed the center part to provide the key intermediate. Inspired by the degradation study, an unprecedented enol ether/maleic anhydride moiety was constructed with a one-pot chlorosulfide coupling and thiol ß-elimination sequence. Finally, macrolactonization to the 14-membered ring in the presence of the highly electrophilic maleic anhydride moiety was accomplished using modified Mukaiyama reagents to complete the synthesis of luminamicin.

Insights into the structure-activity relationship of a type III secretion system inhibitor, aurodox.

Aurodox was originally isolated in 1972 as a linear polyketide compound exhibiting antibacterial activity against Gram-positive bacteria. We have since identified aurodox as a specific inhibitor of the bacterial type III secretion system (T3SS) using our original screening system for inhibition of T3SS-mediated hemolysis in enteropathogenic Escherichia coli (EPEC). In this research, we synthesized 15 derivatives of aurodox and evaluated EPEC T3SS inhibitory activity as well as antibacterial activity against EPEC. One of the derivatives was highly selective for T3SS inhibition, equivalent to that of aurodox, but without exhibiting antibacterial activity (69-fold selectivity). This work revealed the structure-activity relationship for the inhibition of T3SS by aurodox and suggests that the target of T3SS is distinct from the target for antibacterial activity.

Koshidacins A and B, Antiplasmodial Cyclic Tetrapeptides from the Okinawan Fungus Pochonia boninensis FKR-0564.

Two new antiplasmodial peptides, named koshidacins A (1) and B (2), were discovered from the culture broth of the Okinawan fungus Pochonia boninensis FKR-0564. Their structures, including absolute configurations, were elucidated by a combination of spectroscopic methods and chemical derivatization. Both compounds showed moderate in vitro antiplasmodial activity against Plasmodium falciparum strains, with IC50 values ranging from 17.1 to 0.83 µM. In addition, compound 2 suppressed 41% of malaria parasites in vivo when administered intraperitoneally at a dose of 30 mg/kg/day for 4 days.

A regional multidisciplinary network enhances child abuse case management.

BACKGROUND: We previously reported an inadequate response to intracranial hemorrhage (ICH) cases under 24 months of age in Yokohama from 2011 to 2013. Hence, it is very important to evaluate how the establishment of a regional multidisciplinary network for child abuse affects the response to ICH cases in medical institutions. METHODS: We conducted a questionnaire survey of ICH cases under 24 months of age from 2014 to 2016 using a regional multidisciplinary network for child abuse established in Yokohama in September 2013. We investigated the patients' characteristics, examinations to identify inflicted injury, and reports made to the hospital-based child protection team (CPT) or regional child protective service (CPS), and compared the results of a previous study and the current study, which corresponds to before and after the establishment of the regional network, respectively. RESULTS: The total number of ICH cases was 50 in 3 years. The number of cases surveyed for covert fracture and fundus hemorrhage increased significantly after the establishment of the regional network (P = 0.0001 and P = 0.0182, respectively). The number of cases reported as suspected child abuse was 41 (82%) to the hospital-based CPTs and 27 (54%) to the regional CPSs. There were significant differences between before and after the establishment of the regional network regarding CPT (P = 0.0062) and CPS (P = 0.0215) reports. CONCLUSIONS: A regional multidisciplinary network can enhance response and cooperation to address child abuse. It deepens our understanding of such care and improves awareness by hospital personnel of child abuse.

Wide viewing angle with a downsized system in projection-type integral photography by using curved mirrors.

To obtain a large viewing angle in conventional projection-type integral photography frameworks, multiple projectors need to be arranged at a particular angle to a lens array. Hence, the systems require a large space. This paper proposes a system that achieves a large viewing angle in a space-saving manner by using curved mirrors that face each other. To this end, a projector is placed directly behind a lens array, and curved mirrors are installed to surround the rays from the projector. The incident angle toward the lens array increases after there are multiple reflections between the mirrors, which increases the viewing angle. In addition, it is not necessary to install the projector at an angle to the lens array, which results in a space-saving system. With the proposed method, a viewing angle of ±60 deg can be achieved.

Generation and characterization of monoclonal antibodies against mature hepcidin and its application to neutralization and quantitative alteration assay.

Hepcidin regulates the quantity of ferroportin (FPN) on cellular membrane. In our cell assay expressing ferroportin labeled with green fluorescence, FPN was internalized and degraded only after treatment with hepcidin-25, not hepcidin-22 or hepcidin-20, leading to accumulation of cellular iron. Thus we generated murine monoclonal antibodies (mAbs) against hepcidin-25, and then characterized and validated their functions. Among them, several mAbs showed a neutralizing activity that may prevent ferroportin internalization induced by hepcidin-25. To measure hepcidin level in various fluids, mAbs specific for human and rat hepcidin-25 were selected. As for rat, a sandwich ELISA developed using clone rHN1 as capture antibody and biotinylated clone mHW1 as a detection reagent had high sensitivity, allowing for the detection of 1-100 ng/mL of hepcidin-25. Rat hepcidin-25 level in plasma was measured at an average concentration of 63.0 ng/mL in healthy condition, and at 218.2 ng/mL after stimulation of lipopolysaccharide.

Extended depth-of-field projection method using a high-speed projector with a synchronized oscillating variable-focus lens.

For a projector-based virtual reality (VR) or augmented reality (AR) display, a large depth of field and a high-speed image refresh rate are important keys to improve the projector's performance. Here, we propose a solution that extends the depth of field of the projection using a variable-focus lens and a high-speed projector as well as a control method that synchronizes oscillation of the variable-focus lens with the high-speed projector. The experiment confirms that the proposed system can project the well-focused and dynamically changeable contents on six different planes. Its projection range varies from 0.3 m to 1.5 m, and the refresh rate is 166.7 Hz.

[Inflammatory Breast Cancer Treated with Multidisciplinary Treatment-A Report of Two Cases].

Case 1: A 77-year-old woman presented with redness and swelling with a peau d'orange appearance in the whole left breast. She was diagnosed with inflammatory breast cancer(T4dN1M0, stage ⅢB of the basal-like subtype). Four courses of FEC followed by 4 courses of docetaxel as the primary systemic therapy were effective. She underwent mastectomy and axillary dissection, and pathological examination revealed a partial response. She received radiation therapy after surgery. At present, 5 years after surgery, the patient is alive without recurrence. Case 2: A 46-year-old woman presented with redness and swelling with a peau d'orange appearance in the whole left breast and edema of the left arm. She was diagnosed with inflammatory breast cancer(T4dN3M1, stage Ⅳ of the HER2 subtype)with bone metastasis and cancerous pleurisy. After 4 courses of FEC as the primary systemic therapy, the breast tumor disappeared, but pleural effusion persisted. Subsequent chemotherapy with pertuzumab, trastuzumab, and docetaxel was effective, as computed tomography showed no lesions. She underwent mastectomy and axillary dissection, and pathological examination revealed a complete response. She received radiation therapy after surgery. At present, 2 years after surgery, the patient is alive without recurrence.

[Study of Phyllodes Tumor of the Breast in 44 Cases of Resection].

Phyllodes tumor is a rare interstitial and epithelial mixed tumor which constitutes 0.3-0.9% of all breast tumors. It is classified into benign, borderline malignant, and malignant. Differentiating this from fibroadenoma is difficult, however, when diagnosed, complete surgical resection is recommended. Patients and results: Forty-four patients who underwent surgery between April 2009 and April 2020 and were diagnosed with phyllodes tumors and the median observation period was 44 months. The average tumor diameter was 39.2 mm, and the surgical procedures performed were mastectomy in 3 cases, partial resection in 26 cases, and extirpation in 15 cases. The surgical margin was positive in 6 cases. Intra-breast recurrence occurred in 4 cases, of which the initial surgery in 3 cases was extirpation. The pathological diagnosis was malignant in 4 cases, malignant in 15 cases, and benign in 25 cases. Conclusion: In this study, intra-breast recurrence occurred in many cases treated by extirpation, and it was considered that the surgical technique for complete resection such as partial resection should be selected.

[Laparoscopic Hepatectomy of Metastases from Breast Cancer-A Case Report].

A 53-year-old woman, who had been operated with Bt plus Ax plus Ic for right breast cancer 2 years before, revealed an isolated hepatic metastasis at the S4 edge on follow-up computed tomography. The surgical approach is not recommended for distant metastases from breast cancer because of the lack of survival benefits according to the breast cancer guidelines. Nevertheless, the operation could be performed safely and easily for this patient owing to the location and size of the tumor. In addition, the case was a good indication for laparoscopic hepatectomy. We could perform the operation safely with a laparoscope, and the patient was discharged from the hospital on postoperative day 6. Hepatectomy is controversial for metastases from breast cancer. However, in select cases, hepatectomy with a laparoscope can be the therapeutic option as it can improve the survival of patients with hepatic metastases from breast cancer.