Protocol for detecting threonine deaminase activity in fission yeast cell lysates.

Threonine deaminase catalyzes the first step of isoleucine biosynthesis from threonine. In this protocol, we describe the process of measuring the enzymatic activity of threonine deaminase in the fission yeast cell lysate, which is catalyzed by Tda1. First, we describe the process of preparing cell lysates from fission yeast cell cultures. Subsequently, we explain how to measure the threonine deaminase activity using threonine or serine as a substrate. For complete details on the use and execution of this protocol, please refer to Sasaki et al. (2022).1.

Etiology, Severity, Audiogram Type, and Device Usage in Patients with Unilateral Moderate to Profound Sensorineural Hearing Loss in Japan.

BACKGROUND: Few studies have reported on the etiology, severity, or device usage of unilateral sensorineural hearing loss (UHL) compared to bilateral hearing loss. Therefore, this study investigated the characteristics of UHL in adults and children. METHODS: We performed a survey using questionnaires for secondary and tertiary otolaryngology institutions. RESULTS: We included 15,981 patients (1549 children and 14,432 adults) from 196 institutions with otolaryngology residency programs and 2844 patients (336 children and 2508 adults) from 27 institutions with board members of the Japan Audiology Society. The latter submitted audiological data. Among children, most diagnoses were made at age 0. Approximately half of them had profound hearing loss, and 37 children (2.2%) used hearing devices. Among adults, the number of cases increased with age, but decreased when people reached their 80s and 90s. More than half of them had moderate hearing loss. Sudden sensorineural hearing loss was the most common cause of UHL of all ages; 4.4% of UHL patients used hearing devices, and most of the device users (98.6%) selected a conventional hearing aid. CONCLUSIONS: Hearing aid use is limited in children and adults with UHL in Japan. There could be many candidates with UHL for intervention such as a cochlear implant.

A set of vectors and strains for chromosomal integration in fission yeast.

The expression of heterologous genes is an important technique in yeast genetics. In fission yeast, the leu1 and ura4 genes have been used mainly as selectable markers for heterologous expression. To expand the repertoire of selection markers available for heterologous expression of genes, here we developed new host-vector systems employing lys1 and arg3. By employing genome editing with the CRISPR/Cas9 system, we isolated several alleles of lys1 and arg3, each having a critical mutation in the ORF region. In parallel, we developed a set of vectors that complement the amino acid auxotrophy of lys1 and arg3 mutants when integrated into each locus. Using these vectors in combination with the previously developed integration vector pDUAL, we successfully observed the localization of three proteins in a cell simultaneously by fusing them with different fluorescent proteins. Thus, these vectors enable combinatorial expression of heterologous genes, which addresses increasingly diverse experimental challenges.

FK506-binding protein, FKBP12, promotes serine utilization and negatively regulates threonine deaminase in fission yeast.

FK506-binding protein with a molecular weight of 12 kDa (FKBP12) is a receptor of the immunosuppressive drugs, FK506 and rapamycin. The physiological functions of FKBP12 remain ambiguous because of its nonessentiality and multifunctionality. Here, we show that FKBP12 promotes the utilization of serine as a nitrogen source and regulates the isoleucine biosynthetic pathway in fission yeast. In screening for small molecules that inhibit serine assimilation, we found that the growth of fission yeast cells in medium supplemented with serine as the sole nitrogen source, but not in glutamate-supplemented medium, was suppressed by FKBP12 inhibitors. Knockout of FKBP12 phenocopied the action of these compounds in serine-supplemented medium. Metabolome analyses and genetic screens identified the threonine deaminase, Tda1, to be regulated downstream of FKBP12. Genetic and biochemical analyses unveiled the negative regulation of Tda1 by FKBP12. Our findings reveal new roles of FKBP12 in amino acid biosynthesis and nitrogen metabolism homeostasis.

Ferrichrome, a fungal-type siderophore, confers high ammonium tolerance to fission yeast.

Microorganisms and plants produce siderophores, which function to transport environmental iron into cells as well as participate in cellular iron use and deposition. Their biological functions are diverse although their role in primary metabolism is poorly understood. Ferrichrome is a fungal-type siderophore synthesized by nonribosomal peptide synthetase (NRPS). Herein we show that ferrichrome induces adaptive growth of fission yeast on high ammonium media. Ammonium is a preferred nitrogen source as it suppresses uptake and catabolism of less preferred nitrogen sources such as leucine through a mechanism called nitrogen catabolite repression (NCR). Therefore, the growth of fission yeast mutant cells with leucine auxotrophy is suppressed in the presence of high concentrations of ammonium. This growth suppression was canceled by ferrichrome in a manner dependent on the amino acid transporter Cat1. Additionally, growth retardation of wild-type cells by excess ammonium was exacerbated by deleting the NRPS gene sib1, which is responsible for the biosynthesis of ferrichrome, suggesting that intrinsically produced ferrichrome functions in suppressing the metabolic action of ammonium. Furthermore, ferrichrome facilitated the growth of both wild-type and sib1-deficient cells under low glucose conditions. These results suggest that intracellular iron regulates primary metabolism, including NCR, which is mediated by siderophores.

Genome-based discovery and total synthesis of janustatins, potent cytotoxins from a plant-associated bacterium.

Host-associated bacteria are increasingly being recognized as underexplored sources of bioactive natural products with unprecedented chemical scaffolds. A recently identified example is the plant-root-associated marine bacterium Gynuella sunshinyii of the chemically underexplored order Oceanospirillales. Its genome contains at least 22 biosynthetic gene clusters, suggesting a rich and mostly uncharacterized specialized metabolism. Here, in silico chemical prediction of a non-canonical polyketide synthase cluster has led to the discovery of janustatins, structurally unprecedented polyketide alkaloids with potent cytotoxicity that are produced in minute quantities. A combination of MS and two-dimensional NMR experiments, density functional theory calculations of 13C chemical shifts and semiquantitative interpretation of transverse rotating-frame Overhauser effect spectroscopy data were conducted to determine the relative configuration, which enabled the total synthesis of both enantiomers and assignment of the absolute configuration. Janustatins feature a previously unknown pyridodihydropyranone heterocycle and an unusual biological activity consisting of delayed, synchronized cell death at subnanomolar concentrations.

Anhydrous Fast Proton Transport Boosted by the Hydrogen Bond Network in a Dense Oxide-Ion Array of α-MoO3.

Developing high-power battery chemistry is an urgent task to buffer fluctuating renewable energies and achieve a sustainable and flexible power supply. Owing to the small size of the proton and its ultrahigh mobility in water via the Grotthuss mechanism, aqueous proton batteries are an attractive candidate for high-power energy storage devices. Grotthuss proton transfer is ultrafast owing to the hydrogen-bonded networks of water molecules. In this work, similar continuous hydrogen bond networks in a dense oxide-ion array of solid α-MoO3 are discovered, which facilitate the anhydrous proton transport even without structural water. The fast proton transfer and accumulation that occurs during (de)intercalation in α-MoO3 is unveiled using both experiments and first-principles calculations. Coupled with a zinc anode and a superconcentrated Zn2+ /H+ electrolyte, the proton-transport mechanism in anhydrous hydrogen-bonded networks realizes an aqueous MoO3 -Zn battery with large capacity, long life, and fast charge-discharge abilities.

Differential Biosynthesis and Roles of Two Ferrichrome-Type Siderophores, ASP2397/AS2488053 and Ferricrocin, in Acremonium persicinum.

Ferrichromes are a family of fungal siderophores with cyclic hexapeptide structures. Most fungi produce one or two ferrichrome-type siderophores. Acremonium persicinum MF-347833 produces ferrichrome-like potent Trojan horse antifungal antibiotics ASP2397 and AS2488053, the aluminum- and iron-chelating forms of AS2488059, respectively. Here, we show by gene sequencing followed by gene deletion experiments that A. persicinum MF-347833 possesses two nonribosomal peptide synthetase genes responsible for AS2488059 and ferricrocin assembly. AS2488059 was produced under iron starvation conditions and excreted into the media to serve as a defense metabolite and probably an iron courier. In contrast, ferricrocin was produced under iron-replete conditions and retained inside the cells, likely serving as an iron-sequestering molecule. Notably, the phylogenetic analyses suggest the different evolutionary origin of AS2488059 from that of conventional ferrichrome-type siderophores. Harnessing two ferrichrome-type siderophores with distinct biological properties may give A. persicinum a competitive advantage for surviving the natural environment.

Toward the Creation of Induced Pluripotent Small (iPS) Molecules: Establishment of a Modular Synthetic Strategy for the Heronamide C-type Polyene Macrolactams and Their Conformational and Reactivity Analysis.

A highly modular synthetic strategy for the heronamide C-type polyene macrolactams was established by synthesizing 8-deoxyheronamide C (2). The developed strategy enabled not only the total synthesis of 8-deoxyheronamide C (2) but also the unified synthesis of four heronamide-like molecules named "heronamidoids" (5-8). Conformational and reactivity analysis of the heronamidoids clarified that (1) the C19 stereochemistry mainly affected the conformation of the amide linkage, resulting in the change of alignment of two polyene units and reactivity toward photochemical [6π + 6π] cycloaddition, and (2) the C8,C9-diol moiety is important for the conversion to the heronamide A-type skeleton from the heronamide C skeleton.

Design, Synthesis, and Antifungal Activity of 16,17-Dihydroheronamide C and ent-Heronamide C.

16,17-Dihydroheronamide C (8) and ent-heronamide C (ent-1) were designed as probes for the mode-of-action analysis of heronamide C (1). These molecules were synthesized by utilizing a highly modular strategy developed in the preceding paper. The evaluation of the antifungal activity of these compounds revealed the exceptional importance of the C16-C17 double bond for the antifungal activity of heronamide C and the existence of chiral recognition between heronamide C (1) and cell membrane components.

Infrafloccular approach effectively prevents hearing complication in microvascular decompression surgery for hemifacial spasm.

OBJECTIVE: The infrafloccular approach in microvascular decompression (MVD) surgery for hemifacial spasm (HFS) was investigated for the prevention of hearing complications. METHODS: Retrospective analysis of 136 patients who underwent MVD for HFS in 2019. The infrafloccular approach for MVD was adopted to resolve the symptom and protect hearing function. Postoperative hearing function was evaluated subjectively and objectively within 30 postoperative days, and later mainly subjectively. Postoperative condition of mastoid air cells based on the CT scan of the following day was also recorded. RESULTS: No final postoperative subjective hearing impairment was found in all patients. Mastoid air cells were opened in 105 patients, and subjective hearing impairment was recorded in 57 patients within 30 postoperative days, but all improved later. Increases in thresholds of greater than 10 dB in air conduction were observed in 28 patients, including 26 of transient conductive hearing loss (increases in the threshold of less than or equal to 10 dB). The other 2 patients with increases in the threshold of greater than 10 dB in both air and bone conduction had improvement confirmed by later audiometry. CONCLUSIONS: Infrafloccular approach in MVD for HFS provides a good hearing outcome. Inclusion of postoperative hearing conditions in the later period is ideal for a precise hearing evaluation.

Marine natural products targeting the eukaryotic cell membrane.

The cell membrane, with high fluidity and alternative curvatures, maintains the robust integrity to distinguish inner and outer space of cells or organelles. Lipids are the main components of the cell membrane, but their functions are largely unknown. Even the visualization of lipids is not straightforward since modification of lipids often hampers its correct physical properties. Many natural products target cell membranes, some of which are used as pharmaceuticals and/or research tools. They show specific recognition on lipids, and thus exhibit desired pharmacological effects and unique biological phenotypes. This review is a catalog of marine natural products that target eukaryotic cell membranes. Chemical structures, biological activities, and molecular mechanisms are summarized. I hope that this review will be helpful for readers to notice the potential of marine natural products in the exploration of the function of lipids and the druggability of eukaryotic cell membranes.

Healthcare resource utilization and economic burden of antifungal management in patients with hematologic malignancy in Japan: a retrospective database study.

OBJECTIVE: To examine treatment patterns of real-world antifungal management of patients at high risk of invasive fungal infections (IFI) and evaluate healthcare resource utilization and costs associated with antifungal management of IFIs in Japan. METHODS: This retrospective, observational study extracted data from a hospital-based claims database for patients in Japan who either (a) underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT), or (b) were hospitalized with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) and received chemotherapy during the study period of January 2010 to January 2019. RESULTS: 863 patients were included in the allo-HSCT cohort and 4498 patients were included in the AML/MDS cohort. In the allo-HSCT cohort, 91% received more than one antifungal drug during the index hospitalization. In the AML/MDS cohort, approximately 50% received more than one antifungal drug during the index hospitalization. For both the allo-HSCT and AML/MDS cohorts, about 90% of the total cost was attributed to inpatient costs. Of note, both the total cost (the total inpatient and outpatient cost) and the index hospitalization costs were higher in patients treated with multiple antifungal drugs than in those treated with a single antifungal drug during the index hospitalization. Despite being at high IFI risk, 12% of the patients in the AML/MDS cohort did not receive antifungal drugs during the index hospitalization. CONCLUSIONS: Most patients with hematologic malignancy and high IFI risk underwent complicated antifungal management requiring use of multiple drugs, and accounted for high healthcare resource utilization and costs.

Amphiol, an Antifungal Fungal Pigment from Pseudogymnoascus sp. PF1464.

An antifungal metabolite, designated amphiol (1), was isolated from the culture broth of the fungus Pseudogymnoascus sp. PF1464. It exists as a mixture of inseparable tautomers, an acetal form and a keto form. The chemical structure was determined by spectroscopic analyses and chemical derivatization. Amphiol (1) showed antifungal but not antibacterial activities, while yeast mutant cells lacking ergosterol biosynthetic genes were less sensitive, implying a fungal specific, membrane-related mechanism of action.

Genomic and Targeted Approaches Unveil the Cell Membrane as a Major Target of the Antifungal Cytotoxin Amantelide A.

Amantelide A, a polyhydroxylated macrolide isolated from a marine cyanobacterium, displays broad-spectrum activity against mammalian cells, bacterial pathogens, and marine fungi. We conducted comprehensive mechanistic studies to identify the molecular targets and pathways affected by amantelide A. Our investigations relied on chemical structure similarities with compounds of known mechanisms, yeast knockout mutants, yeast chemogenomic profiling, and direct biochemical and biophysical methods. We established that amantelide A exerts its antifungal action by binding to ergosterol-containing membranes followed by pore formation and cell death, a mechanism partially shared with polyene antifungals. Binding assays demonstrated that amantelide A also binds to membranes containing epicholesterol or mammalian cholesterol, thus suggesting that the cytotoxicity to mammalian cells might be due to its affinity to cholesterol-containing membranes. However, membrane interactions were not completely dependent on sterols. Yeast chemogenomic profiling suggested additional direct or indirect effects on actin. Accordingly, we performed actin polymerization assays, which suggested that amantelide A also promotes actin polymerization in cell-free systems. However, the C-33 acetoxy derivative amantelide B showed a similar effect on actin dynamics in vitro but no significant activity against yeast. Overall, these studies suggest that the membrane effects are the most functionally relevant for amantelide A mechanism of action.

Retro-aza-Michael reaction of an o-aminophenol adduct in protic solvents inspired by natural products.

α,ß-Unsaturated carbonyls are reactive group often found in bioactive small molecules. Their non-specific reaction with biomolecules can be the cause of the low efficacy and unexpected side-effects of the molecule. Accordingly, unprotected α,ß-unsaturated carbonyls are not often found in drugs. Here, we report that o-aminophenol is a new masking group of α,ß-unsaturated ketone, which is inspired by natural products saccharothriolides. o-Aminophenol adduct of α,ß-unsaturated ketone, but not those of α,ß-unsaturated amide or ester, undergoes a retro-Michael reaction to yield o-aminophenol and the Michael acceptor. This reaction was observed only in protic solvents, such as MeOH and aqueous MeOH. In contrast, o-anisidine was not eliminated from its Michael adduct. o-Aminophenol may be a promising masking tool of highly-reactive bioactive α,ß-unsaturated carbonyl compounds.

The disease burden of mucormycosis in Japan: results from a systematic literature review and retrospective database study.

OBJECTIVE: To evaluate outcomes, healthcare resource utilization, and costs associated with mucormycosis in inpatient settings in Japan. METHODS: We performed a systematic literature review, followed by a retrospective database study using hospital health claims for patients in Japan hospitalized with a diagnosis of mucormycosis between January 2010 and January 2019. Outcomes assessed included duration of index hospitalization; index stay mortality; hospital readmission within 30, 60, and 90 days after index hospitalization discharge; drug/treatment utilization and patterns; number of patients examined for mucormycosis during the index hospitalization; and index stay inpatient costs. RESULTS: From our systematic literature review of articles describing 133 patients with mucormycosis, mortality in the index hospitalization was 55.6%. From our database study of 126 patients hospitalized for mucormycosis, mortality during the index hospitalization was 35.7% and mean index stay duration was 94 days. Hematologic malignancies were the most common risk factor in the literature review and the most common comorbidity in the database study. During the index stay, 39 patients (31.0%) received liposomal amphotericin B (L-AMB) treatment and 74 patients (58.7%) received other antifungal treatments. Median total inpatient costs for the index hospitalization were equivalent to approximately US$60,945, including US$29,283 in drug costs. CONCLUSIONS: This study investigated the healthcare resource utilization and cost of medical resources caused by mucormycosis in Japan. The drug costs for antifungal treatments comprised about half of total inpatient costs. Mucormycosis leads to high mortality, high healthcare resource utilization, and high costs.

Serine catabolism produces ROS, sensitizes cells to actin dysfunction, and suppresses cell growth in fission yeast.

Serine is an essential component in organisms as a building block of biomolecules, a precursor of metabolites, an allosteric regulator of an enzyme, etc. This amino acid is thought to be a key metabolite in human diseases including cancers and infectious diseases. To understand the consequence of serine catabolism, we screened natural products to identify a fungal metabolite chaetoglobosin D (ChD) as a specific inhibitor of fission yeast cell growth when cultivated with serine as a sole nitrogen source. ChD targets actin, and actin mutant cells showed severe growth defect on serine medium. ROS accumulated in cells when cultivated in serine medium, while actin mutant cells showed increased sensitivity to oxidative stress. ROS production is a new aspect of serine metabolism, which might be involved in disease progression, and actin could be the drug target for curing serine-dependent symptoms.

Chemical diversity and mode of action of natural products targeting lipids in the eukaryotic cell membrane.

Covering: up to 2019Nature furnishes bioactive compounds (natural products) with complex chemical structures, yet with simple, sophisticated molecular mechanisms. When natural products exhibit their activities in cells or bodies, they first have to bind or react with a target molecule in/on the cell. The cell membrane is a major target for bioactive compounds. Recently, our understanding of the molecular mechanism of interactions between natural products and membrane lipids progressed with the aid of newly-developed analytical methods. New technology reconnects old compounds with membrane lipids, while new membrane-targeting molecules are being discovered through the screening for antimicrobial potential of natural products. This review article focuses on natural products that bind to eukaryotic membrane lipids, and includes clinically important molecules and key research tools. The chemical diversity of membrane-targeting natural products and the molecular basis of lipid recognition are described. The history of how their mechanism was unveiled, and how these natural products are used in research are also mentioned.

HPO32- as a building unit for sodium-ion battery cathodes: 3.1 V operation of Na2-xFe(HPO3)2 (0 < x < 1).

We report Na2Fe(HPO3)2 as the first HPO32--based iron(ii) cathode material for sodium-ion batteries, which delivers a reversible capacity of approximately 100 mA h g-1 at an average reaction voltage of 3.1 V. In situ X-ray diffraction and ex situ57Fe Mössbauer spectroscopy clarify reversible (de)sodiation associated with the Fe3+/Fe2+ redox reaction.