Penicillin-binding protein-type thioesterases: An emerging family of non-ribosomal peptide cyclases with biocatalytic potentials.

Macrocyclization of peptides reduces conformational flexibilities, potentially leading to improved drug-like properties, such as target specificities and metabolic stabilities. As chemical methodologies often allow side reactions like epimerization and oligomerization, keen attention has been directed toward enzymatic peptide cyclization using peptide cyclases from specialized metabolic pathways. Penicillin-binding protein-type thioesterases (PBP-type TEs) are a recently identified family of peptide cyclases involved in the biosynthesis of non-ribosomal peptides in actinobacteria. PBP-type TEs have undergone intensive investigation due to their outstanding potential as biocatalysts. This review summarizes the rapidly growing knowledge on PBP-type TEs, with special emphasis on their functions, scopes, and structures, and efforts towards their biocatalytic applications.

Bacterial Hydrazine Biosynthetic Pathways Featuring Cupin/Methionyl tRNA Synthetase-like Enzymes.

Nitrogen-Nitrogen (N-N) bond-containing functional groups in natural products and synthetic drugs play significant roles in exerting biological activities. The mechanisms of N-N bond formation in natural organic molecules have garnered increasing attention over the decades. Recent advances have illuminated various enzymatic and nonenzymatic strategies, and our understanding of natural N-N bond construction is rapidly expanding. A group of didomain proteins with zinc-binding cupin/methionyl-tRNA synthetase (MetRS)-like domains, also known as hydrazine synthetases, generates amino acid-based hydrazines, which serve as key biosynthetic precursors of diverse N-N bond-containing functionalities such as hydrazone, diazo, triazene, pyrazole, and pyridazinone groups. In this review, we summarize the current knowledge on hydrazine synthetase mechanisms and the various pathways employing this unique bond-forming machinery.

Chemoenzymatic tandem cyclization for the facile synthesis of bicyclic peptides.

Bicyclic peptides exhibit improved metabolic stabilities and target specificities when compared to their linear or mono-cyclic counterparts; however, efficient and straightforward synthesis remains challenging due to their intricate architectures. Here, we present a highly selective and operationally simple one-pot chemoenzymatic tandem cyclization approach to synthesize bicyclic peptides with small to medium ring sizes. Penicillin-binding protein-type thioesterases (PBP-type TEs) efficiently cyclized azide/alkyne-containing peptides in a head-to-tail manner. Successive copper (I)-catalyzed azide-alkyne cycloaddition generated bicyclic peptides in one-pot, thus omitting the purification of monocyclic intermediates. This chemoenzymatic strategy enabled the facile synthesis of bicyclic peptides bearing hexa-, octa-, and undecapeptidyl head-to-tail cyclic scaffolds.

Actinobacterial chalkophores: the biosynthesis of hazimycins.

Copper is a transition metal element with significant effects on the morphological development and secondary metabolism of actinobacteria. In some microorganisms, copper-binding natural products are employed to modulate copper homeostasis, although their significance in actinobacteria remains largely unknown. Here, we identified the biosynthetic genes of the diisocyanide natural product hazimycin in Kitasatospora purpeofusca HV058, through gene knock-out and heterologous expression. Biochemical analyses revealed that hazimycin A specifically binds to copper, which diminishes its antimicrobial activity. The presence of a set of putative importer/exporter genes surrounding the biosynthetic genes suggested that hazimycin is a chalkophore that modulates the intracellular copper level. A bioinformatic survey of homologous gene cassettes, as well as the identification of two previously unknown hazimycin-producing Streptomyces strains, indicated that the isocyanide-based mechanism of copper homeostasis is prevalent in actinobacteria.

Phylogeny-guided Characterization of Bacterial Hydrazine Biosynthesis Mediated by Cupin/methionyl tRNA Synthetase-like Enzymes.

Cupin/methionyl-tRNA synthetase (MetRS)-like didomain enzymes catalyze nitrogen-nitrogen (N-N) bond formation between Nω-hydroxylamines and amino acids to generate hydrazines, key biosynthetic intermediates of various natural products containing N-N bonds. While the combination of these two building blocks leads to the creation of diverse hydrazine products, the full extent of their structural diversity remains largely unknown. To explore this, we herein conducted phylogeny-guided genome-mining of related hydrazine biosynthetic pathways consisting of two enzymes: flavin-dependent Nω-hydroxylating monooxygenases (NMOs) that produce Nω-hydroxylamine precursors and cupin/MetRS-like enzymes that couple the Nω-hydroxylamines with amino acids via N-N bonds. A phylogenetic analysis identified the largely unexplored sequence spaces of these enzyme families. The biochemical characterization of NMOs demonstrated their capabilities to produce various Nω-hydroxylamines, including those previously not known as precursors of N-N bonds. Furthermore, the characterization of cupin/MetRS-like enzymes identified five new hydrazine products with novel combinations of building blocks, including one containing non-amino acid building blocks: 1,3-diaminopropane and putrescine. This study substantially expanded the variety of N-N bond forming pathways mediated by cupin/MetRS-like enzymes.

Validation of Wistar-Kyoto rats kept in solitary housing as an animal model for depression using voxel-based morphometry.

Major depressive disorder is a common psychiatric condition often resistant to medication. The Wistar-Kyoto (WKY) rat has been suggested as an animal model of depression; however, it is still challenging to translate results from animal models into humans. Solitary housing is a mild stress paradigm that can simulate the environment of depressive patients with limited social activity due to symptoms. We used voxel-based morphometry to associate the solitary-housed WKY (sWKY) rat model with data from previous human studies and validated our results with behavioural studies. As a result, atrophy in sWKY rats was detected in the ventral hippocampus, caudate putamen, lateral septum, cerebellar vermis, and cerebellar nuclei (p < 0.05, corrected for family-wise error rate). Locomotor behaviour was negatively correlated with habenula volume and positively correlated with atrophy of the cerebellar vermis. In addition, sWKY rats showed depletion of sucrose consumption not after reward habituation but without reward habituation. Although the application of sWKY rats in a study of anhedonia might be limited, we observed some similarities between the regions of brain atrophy in sWKY rats and humans with depression, supporting the translation of sWKY rat studies to humans.

Association between prior-night sleep and next-day fatigue in older adults: a daily diary study.

BACKGROUND: Fatigue is known as an element of frailty. Sleep problems (e.g., short sleep duration and low sleep quality) can increase fatigue, but the day-to-day relationship between sleep and fatigue has not been studied well in older adults. Using a daily diary method, this study examined the within- and between-person associations between sleep and fatigue in older adults. METHODS: The study recruited 56 Japanese community dwellers (age: 82-86 years; female: 37.5%). Participants responded to a daily diary questionnaire at the end of each day. Over seven days, time in bed and satisfaction were measured after waking up, whereas fatigue was assessed before going to bed. We included person-level covariates (demographic factors, and physical and mental health) and day-level covariates (time in study, and positive and negative emotions). Multilevel models were estimated to examine within- and between-person associations. RESULTS: At the within-person level, on days following short and long time in bed and days following low levels of sleep satisfaction, individuals felt higher levels of fatigue compared with usual days. At the between-person level, no statistically significant differences in fatigue were observed between individuals with long and short time in bed. CONCLUSIONS: The findings suggest that prior-day sleep is associated with next-day fatigue in older adults. Long and short sleep duration and low sleep quality can lead to fatigue. Considering that sleep is a modifiable health behavior, appropriate management of sleep behavior may reduce fatigue.

Neutrophil Count in Severe Burns Is Useful for Predicting Prognosis.

Background: Burn injuries, particularly extensive severe burns, often have a fatal prognosis. However, many prognostic predictors are based on changes in the clinical course of treatment, and no prognostic predictors can be estimated in the early phases of injury. Using the Burn Index (BI) for evaluations requires familiarity with daily burn treatment, such as being able to evaluate the change from a second degree burn to a third degree burn appropriately. We sought to find a simpler and more quantitative prognostic prediction index.Methods We hypothesized that, in addition to the current prognostic predictors, the number of neutrophils in severe burns may correlate with the prognosis, and analyzed its usefulness. The neutrophil and white blood cell counts were measured within 48 hours of injury in 35 burn patients who required inpatient treatment at our own institution. Mann-Whitney test was used to determine the significant of differences between the Survivor and Non-survivor groups. Results: Compared to the Survivor group, neutrophil (P = 0.038) and white blood cell counts were increased significantly in the Non-survivor group (P = 0.004). Neutrophil counts and white blood cell counts correlated positively with the length of hospital stay, total body surface area, Prognostic Burn Index (PBI), and BI. The BI and PBI correlated with patient prognosis, as did neutrophil and white blood cell counts. Conclusion: These results suggested that neutrophil and white blood cell counts in the early phases of burn injuries might be another factor in the prognosis of burn patients in addition to the current predictors.

Correlates of delirium care competency among shift leader nurses in acute medical wards in Japan: A cross-sectional study.

AIM: This study identified the correlates of delirium care competency among shift leader nurses in acute medical wards in Japan. METHODS: A cross-sectional study was conducted from November 2019 to February 2020. We sent request letters to a random sample of 381 general acute care hospitals in Japan. Of these, 68 agreed to participate and distributed 735 self-administered questionnaires to shift leader nurses working in their acute medical wards. The questionnaire included the Self-rated Delirium Care Competency Scale for Shift Leader Nurses in Acute Medical Wards (DCSL-M), developed by the authors. It gathered data on the respondents' demographics and delirium care competency, and investigated a total of 25 variables. We calculated descriptive statistics and examined associations between delirium care competency and the demographics using multiple logistic regression analysis. RESULTS: Of the total, 301 (40.9%) questionnaires were returned. Delirium care competency was high among shift leader nurses who: (1) had experience as a clinical practice preceptor for nursing students; (2) had experience attending training related to dementia or delirium care; (3) worked at a hospital/ward that charged additional medical fees for dementia care; and (4) had access to consulting psychiatrists to refer patients with delirium. CONCLUSIONS: The results suggest the need for efforts to improve delirium care competency among shift leader nurses who work in hospitals that do not charge additional medical fees for dementia care or that do not have consulting psychiatrists to refer patients with delirium.

Carrier Protein Mediated Formation of the Dihydropyridazinone Ring in Actinopyridazinone Biosynthesis.

Heterocycles with nitrogen-nitrogen (N-N) bonds are privileged building blocks of synthetic drugs. They are also found in natural products, although the biosynthetic logic behind them is poorly understood. Actinopyridazinones produced by Streptomyces sp. MSD090630SC-05 possess unique dihydropyridazinone rings that have been studied as core nuclei in several approved synthetic therapeutics. Herein, we performed gene knockouts and in vitro biochemical experiments to elucidate the major steps in actinopyridazinone biosynthesis, including the unprecedented carrier protein mediated machinery for dihydropyridazinone formation.

Chemo-Enzymatic Synthesis of Non-ribosomal Macrolactams by a Penicillin-Binding Protein-Type Thioesterase.

Penicillin-binding protein-type thioesterases (PBP-type TEs) are an emerging family of non-ribosomal peptide cyclases. PBP-type TEs exhibit distinct substrate scopes from the well-exploited ribosomal peptide cyclases and traditional non-ribosomal peptide cyclases. Their unique properties, as well as their stand-alone nature, highlight PBP-type TEs as valuable candidates for development as biocatalysts for peptide macrocyclization. Here in this chapter, we describe the scheme for the chemoenzymatic synthesis of non-ribosomal macrolactam by SurE, a representative member of PBP-type TEs.

Streamlined Chemoenzymatic Synthesis of Cyclic Peptides by Non-ribosomal Peptide Cyclases.

Macrocyclization improves the pharmaceutical properties of peptides; however, regio- and chemoselective intramolecular cyclizations remain challenging. Here we developed a streamlined chemoenzymatic approach to synthesize cyclic peptides by exploiting non-ribosomal peptide (NRP) cyclases. Linear peptides linked to the resin through a C-terminal diol ester functionality are synthesized on a solid support, to circumvent the installation of leaving groups to the peptidic substrates in the liquid phase which often triggers undesirable epimerization. Cleavage of the resin-bound peptides yielded the diol esters with sufficient purity to be readily cyclized in a head-to-tail manner by SurE, a representative penicillin-binding protein-type thioesterase (PBP-type TE). Explorations of homologous wild-type enzymes as well as rational protein engineering have broadened the scope of the enzymatic macrolactamization. This method will potentially accelerate the exploitation of NRP cyclases as biocatalysts.

Characterization of the surugamide biosynthetic gene cluster of TUA-NKU25, a Streptomyces diastaticus strain isolated from Kusaya, and its effects on salt-dependent growth.

Kusaya, a traditional Japanese fermented fish product, is known for its high preservability, as it contains natural antibiotics derived from microorganisms, and therefore molds and yeasts do not colonize it easily. In this study, the Streptomyces diastaticus strain TUA-NKU25 was isolated from Kusaya, and its growth as well as the production of antibiotics were investigated. Strain TUA-NKU25 showed advantageous growth characteristics in the presence, but not in the absence, of sodium chloride (NaCl). Antimicrobial assay, high-performance liquid chromatography, and electrospray ionization-mass spectrometry analysis showed that this strain produced surugamide A and uncharacterized antimicrobial compound(s) during growth in the presence of NaCl, suggesting that the biosynthesis of these compounds was upregulated by NaCl. Draft genomic analysis revealed that strain TUA-NKU25 possesses a surugamide biosynthetic gene cluster (sur BGC), although it is incomplete, lacking surB/surC. Phylogenetic analysis of strain TUA-NKU25 and surugamide-producing Streptomyces showed that sur BGC formed a clade distinct from other known groups.

New azodyrecins identified by a genome mining-directed reactivity-based screening.

Only a few azoxy natural products have been identified despite their intriguing biological activities. Azodyrecins D-G, four new analogs of aliphatic azoxides, were identified from two Streptomyces species by a reactivity-based screening that targets azoxy bonds. A biological activity evaluation demonstrated that the double bond in the alkyl side chain is important for the cytotoxicity of azodyrecins. An in vitro assay elucidated the tailoring step of azodyrecin biosynthesis, which is mediated by the S-adenosylmethionine (SAM)-dependent methyltransferase Ady1. This study paves the way for the targeted isolation of aliphatic azoxy natural products through a genome-mining approach and further investigations of their biosynthetic mechanisms.

Molecular Basis for Enzymatic Aziridine Formation via Sulfate Elimination.

Natural products containing an aziridine ring, such as mitomycin C and azinomycin B, exhibit antitumor activities by alkylating DNA via their aziridine rings; however, the biosynthetic mechanisms underlying the formation of these rings have not yet been elucidated. We herein investigated the biosynthesis of vazabitide A, the structure of which is similar to that of azinomycin B, and demonstrated that Vzb10/11, with no similarities to known enzymes, catalyzed the formation of the aziridine ring via sulfate elimination. To elucidate the detailed reaction mechanism, crystallization of Vzb10/11 and the homologous enzyme, AziU3/U2, in the biosynthesis of azinomycin B was attempted, and the structure of AziU3/U2, which had a new protein fold overall, was successfully determined. The structural analysis revealed that these enzymes adjusted the dihedral angle between the amino group and the adjacent sulfate group of the substrate to almost 180° and enhanced the nucleophilicity of the C6-amino group temporarily, facilitating the SN2-like reaction to form the aziridine ring. The present study reports for the first time the molecular basis for aziridine ring formation.

A Natural Dihydropyridazinone Scaffold Generated from a Unique Substrate for a Hydrazine-Forming Enzyme.

Nitrogen-nitrogen bond-containing functional groups are rare, but they are found in a considerably wide class of natural products. Recent clarifications of the biosynthetic routes for such functional groups shed light onto overlooked biosynthetic genes distributed across the bacterial kingdom, highlighting the presence of yet-to-be identified natural products with peculiar functional groups. Here, the genome-mining approach targeting a unique hydrazine-forming gene led to the discovery of actinopyridazinones A (1) and B (2), the first natural products with dihydropyridazinone rings. The structure of actinopyridazinone A was unambiguously established by total synthesis. Biosynthetic studies unveiled the structural diversity of natural hydrazines derived from this family of N-N bond-forming enzymes.

Current educational concepts and techniques related to impression making of removable complete dentures.

The purpose of the present study is to gain an understanding of the current state of pre-clinical education for procedures of impression making. In 2019, a survey questionnaire was emailed to the senior professors and department heads of 29 Japanese dental colleges. The response rate was 100%. This cross-sectional survey clarified the impression making for removable complete denture fabrication in Japanese dental schools' education.

Predictive factors for tooth loss in older adults vary according to occlusal support: A 6-year longitudinal survey from the SONIC study.

OBJECTIVES: The aim of this cohort study among community-dwelling older adults aged over 70 years was to investigate the influence of occlusal support on tooth loss, and to determine predictive factors for tooth loss for each occlusal support category using multilevel analyses. METHODS: Participants were 812 older adults who completed the baseline survey and the follow-up survey 6 years later. The Eichner index was used to evaluate occlusal support status. A generalized estimating equation (GEE) logistic regression analysis was used to examine the influence of occlusal support status on tooth loss while adjusting for various factors at individual and tooth levels. Similar analyses were separately performed in each Eichner class to determine predictive factors for tooth loss. RESULTS: The GEE showed that a decline in occlusal support increased the risk of tooth loss (Eichner A: reference category, Eichner B: odds ratio (OR) = 1.96, p < 0.001, Eichner C: OR = 3.04, p < 0.001). Stratified analysis showed that deeper periodontal pockets and abutment teeth for fixed partial dentures were significantly associated with tooth loss, regardless of occlusal support. In Eichner A, the presence of an opposing tooth was advantageous in protecting the tooth, and a missing adjacent tooth was not significantly associated with tooth loss. An opposing tooth was associated with the risk of tooth loss in Eichner B, and a missing adjacent tooth was significantly associated with tooth loss in Eichner B and C. CONCLUSIONS: A decline in occlusal support accelerated tooth loss. Predictive factors for tooth loss varied depending on occlusal support status. CLINICAL SIGNIFICANCE: Occlusal support is an important factor in preventing tooth loss. Dentition conditions such as missing adjacent teeth and the presence of opposing teeth increase the risk of tooth loss in participants with poor occlusal support. Dental personnel need to carefully examine dentition conditions to assess the risk of occlusal collapse.

Sweep imaging with Fourier transform as a tool with MRI for evaluating the effect of teriparatide on cortical bone formation in an ovariectomized rat model.

BACKGROUND: Teriparatide (TPTD) is a drug for osteoporosis that promotes bone formation and improves bone quality. However, the effects of TPTD on cortical bone are not well understood. Sweep imaging with Fourier transform (SWIFT) has been reported as a useful tool for evaluating bound water of cortical bone, but it has yet to be used to investigate the effects of TPTD on cortical bone. This study aimed to evaluate the consequences of the effect of TPTD on cortical bone formation using SWIFT. METHODS: Twelve-week-old female Sprague-Dawley rats (n = 36) were reared after ovariectomy to create a postmenopausal osteoporosis model. They were divided into two groups: the TPTD and non-TPTD groups. Rats were euthanized at 4, 12, and 24 weeks after initiating TPTD treatment. Tibial bones were evaluated using magnetic resonance imaging (MRI) and bone histomorphometry. In MRI, proton density-weighted imaging (PDWI) and SWIFT imaging were performed. The signal-to-noise ratio (SNR) was calculated for each method. The same area evaluated by MRI was then used to calculate the bone formation rate by bone histomorphometry. Measurements were compared using the Mann-Whitney U-test, and a P-value of < 0.05 was considered significant. RESULTS: PDWI-SNR was not significantly different between the two groups at any time point (P = 0.589, 0.394, and 0.394 at 4, 12, and 24 weeks, respectively). Contrarily, SWIFT-SNR was significantly higher in the TPTD group than in the non-TPTD group at 4 weeks after initiating treatment, but it was not significantly different at 12 and 24 weeks (P = 0.009, 0.937, and 0.818 at 4, 12, and 24 weeks, respectively). The bone formation rate assessed by histomorphometry was significantly higher in the TPTD group than in the non-TPTD group at all timepoints (P < 0.05, all weeks). In particular, at 4 weeks, the bone formation rate was markedly higher in the TPTD group than in the non-TPTD group (P = 0.028, 1.98 ± 0.33 vs. 0.09 ± 0.05 µm3/µm2/day). CONCLUSIONS: SWIFT could detect increased signals of bound water, reflecting the effect of TPTD on the cortical bone. The signal detected by SWIFT reflects a marked increase in the cortical bone formation rate.