De Novo Discovery of Pseudo-Natural Prenylated Macrocyclic Peptide Ligands.

Prenylation of peptides is widely observed in the secondary metabolites of diverse organisms, granting peptides unique chemical properties distinct from proteinogenic amino acids. Discovery of prenylated peptide agents has largely relied on isolation or genome mining of naturally occurring molecules. To devise a platform technology for de novo discovery of artificial prenylated peptides targeting a protein of choice, here we have integrated the thioether-macrocyclic peptide (teMP) library construction/selection technology, so-called RaPID (Random nonstandard Peptides Integrated Discovery) system, with a Trp-C3-prenyltransferase KgpF involved in the biosynthesis of a prenylated natural product. This unique enzyme exhibited remarkably broad substrate tolerance, capable of modifying various Trp-containing teMPs to install a prenylated residue with tricyclic constrained structure. We constructed a vast library of prenylated teMPs and subjected it to in vitro selection against a phosphoglycerate mutase. This selection platform has led to the identification of a pseudo-natural prenylated teMP inhibiting the target enzyme with an IC50 of 30 nM. Importantly, the prenylation was essential for the inhibitory activity, enhanced serum stability, and cellular uptake of the peptide, highlighting the benefits of peptide prenylation. This work showcases the de novo discovery platform for pseudo-natural prenylated peptides, which is readily applicable to other drug targets.

A Compact Reprogrammed Genetic Code for De Novo Discovery of Proteolytically Stable Thiopeptides.

Thiopeptides make up a group of structurally complex peptidic natural products holding promise in bioengineering applications. The previously established thiopeptide/mRNA display platform enables de novo discovery of natural product-like thiopeptides with designed bioactivities. However, in contrast to natural thiopeptides, the discovered structures are composed predominantly of proteinogenic amino acids, which results in low metabolic stability in many cases. Here, we redevelop the platform and demonstrate that the utilization of compact reprogrammed genetic codes in mRNA display libraries can lead to the discovery of thiopeptides predominantly composed of nonproteinogenic structural elements. We demonstrate the feasibility of our designs by conducting affinity selections against Traf2- and NCK-interacting kinase (TNIK). The experiment identified a series of thiopeptides with high affinity to the target protein (the best KD = 2.1 nM) and kinase inhibitory activity (the best IC50 = 0.15 µM). The discovered compounds, which bore as many as 15 nonproteinogenic amino acids in an 18-residue macrocycle, demonstrated high metabolic stability in human serum with a half-life of up to 99 h. An X-ray cocrystal structure of TNIK in complex with a discovered thiopeptide revealed how nonproteinogenic building blocks facilitate the target engagement and orchestrate the folding of the thiopeptide into a noncanonical conformation. Altogether, the established platform takes a step toward the discovery of thiopeptides with high metabolic stability for early drug discovery applications.

Co-infection with Neisseria mucosa in a patient with tuberculous otitis media.

Tuberculous otitis media (TOM) is a rare manifestation caused by Mycobacterium tuberculosis with low incidence rates among extrapulmonary tuberculosis cases. Diagnosis is often delayed because of the presence of several clinical manifestations and the high prevalence of secondary bacterial infections. Few reports have attributed secondary bacterial infections in patients with TOM to commensal Neisseria. Thus, understanding the pathogenic mechanisms and clinical features of commensal Neisseria is important, considering its recent presentation as an infection-causing pathogen. Neisseria mucosa is a commensal inhabitant in humans and is generally considered non-pathogenic but can cause infection in rare cases. Here, we report an atypical secondary infection caused by Neisseria mucosa in an 81-year-old woman with TOM being treated for pulmonary tuberculosis. Direct purulent otorrhea smear microscopy revealed no acid-fast bacilli using Ziehl-Neelsen staining, whereas the phagocytosis of gram-negative cocci by white blood cells was confirmed using Gram staining. Otorrhea culture revealed the growth of N. mucosa. Subsequently, M. tuberculosis infection in the otorrhea was identified using a culture-based method. Vigilance is critical for the early detection of TOM to prevent further complications. This report raises awareness regarding TOM and provides insight into the pathogenicity of N. mucosa in otitis media.

Butyrate, Valerate, and Niacin Ameliorate Anaphylaxis by Suppressing IgE-Dependent Mast Cell Activation: Roles of GPR109A, PGE2, and Epigenetic Regulation.

Short-chain fatty acids (SCFAs) are produced by the intestinal microbiota during the fermentation of dietary fibers as secondary metabolites. Several recent studies reported that SCFAs modulate the development and function of immune-related cells. However, the molecular mechanisms by which SCFAs regulate mast cells (MCs) remain unclear. In the current study, we analyzed the function and gene expression of mouse MCs in the presence of SCFAs in vitro and in vivo. We found that the oral administration of valerate or butyrate ameliorated passive systemic anaphylaxis and passive cutaneous anaphylaxis in mice. The majority of SCFAs, particularly propionate, butyrate, valerate, and isovalerate, suppressed the IgE-mediated degranulation of bone marrow-derived MCs, which were eliminated by the Gi protein inhibitor pertussis toxin and by the knockdown of Gpr109a. A treatment with the HDAC inhibitor trichostatin A also suppressed IgE-mediated MC activation and reduced the surface expression level of FcεRI on MCs. Acetylsalicylic acid and indomethacin attenuated the suppressive effects of SCFAs on degranulation. The degranulation degree was significantly reduced by PGE2 but not by PGD2. Furthermore, SCFAs enhanced PGE2 release from stimulated MCs. The SCFA-mediated amelioration of anaphylaxis was exacerbated by COX inhibitors and an EP3 antagonist, but not by an EP4 antagonist. The administration of niacin, a ligand of GPR109A, alleviated the symptoms of passive cutaneous anaphylaxis, which was inhibited by cyclooxygenase inhibitors and the EP3 antagonist. We conclude that SCFAs suppress IgE-mediated activation of MCs in vivo and in vitro involving GPR109A, PGE2, and epigenetic regulation.

Association of excess visceral fat and severe illness in hospitalized COVID-19 patients in Japan: a retrospective cohort study.

BACKGROUND/OBJECTIVES: Obesity, defined by body mass index (BMI), is a well-known risk factor for the severity of coronavirus disease 2019 (COVID-19). Adipose tissue distribution has also been implicated as an important factor in the body's response to infection, and excess visceral fat (VF), which is prevalent in Japanese, may contribute significantly to the severity. Therefore, this study aimed to evaluate the association of obesity and VF with COVID-19 severe illness in Japan. SUBJECTS/METHODS: This retrospective cohort study involved 550 COVID-19 patients admitted to a tertiary care hospital with BMI and body composition data, including VF. The primary endpoint was severe illness, including death, due to COVID-19 during hospitalization. Logistic regression analysis was applied to examine the quartiles of BMI and VF on severe illness after adjusting for covariates such as age, sex, subcutaneous fat, paraspinal muscle radiodensity, and comorbidities affecting VF (COPD, cancer within 5 years, immunosuppressive agent use). RESULTS: The median age was 56.0 years; 71.8% were males. During hospitalization, 82 (14.9%) experienced COVID-19 severe illness. In the multivariate logistic regression analysis, Q4 of BMI was not significantly associated with severe illness compared to Q1 of BMI (OR 1.03; 95% CI 0.37-2.86; p = 0.95). Conversely, Q3 and Q4 of VF showed a higher risk for severe illness compared to Q1 of VF (OR 2.68; 95% CI 1.01-7.11; p = 0.04, OR 3.66; 95% CI 1.30-10.26; p = 0.01, respectively). Stratified analysis by BMI and adjusted for covariates showed the positive association of VF with severe illness only in the BMI < 25 kg/m2 group. CONCLUSIONS: High BMI was not an independent risk factor for COVID-19 severe illness in hospitalized patients in Japan, whereas excess VF significantly influenced severe illness, especially in patients with a BMI < 25 kg/m2.

[The past and future of occupational health nursing research in Japan].

OBJECTIVES: This study aimed to review the trends in "occupational health nursing research" conducted in Japan and determine research areas that need strengthening eventually. METHODS: All articles published in Ichushi Web since 1903 were searched using the keywords "occupational health nursing" and "research." The 486 papers identified were extracted and categorized according to type and publication year. The research content of 194 original and review papers was categorized according to whether they addressed practical methods and abilities. RESULTS AND DISCUSSION: According to publication type, out of the 486 papers, 230 (47.3%) were conference proceedings, whereas 194 (39.9%) were original and review papers. Among practical methods-related papers, 107 (55.2%), 86 (44.3%), and 1 (0.5%) were on general, health, and work environment management, respectively, whereas none addressed work management and occupational health education. Among practical ability-related papers, 125 (64.4%), 23 (11.9%), and 46 (23.7%) were on knowledge, technology, and competency, respectively. The overview of annual trends showed that the papers of all types have increased progressively. While conference proceedings accounted for 80% of the papers until 1992, recent proportions have shown a decrease to < 50%. Conversely, original papers accounted for < 20% of the papers early on, but observed a progressive increase, with recent proportions of > 40%. Regarding content, almost no papers addressed work environment management, work management, and occupational health education. Additionally, practical ability-related papers highlighted that although some research on technologies and competencies has been conducted, some areas remain inadequately addressed. CONCLUSIONS: In Japan, occupational health nursing papers have been steadily increasing since the first conference proceeding was published in 1980. Among papers addressing practical methods, those related to general management were most common, of which occupational health management accounted for approximately 80%, indicating efforts aimed at the development of occupational health nursing through research. Moreover, limited papers addressed work environment management, work management, and occupational health education, suggesting the need for future research. Among papers addressing practical ability, > 60% were related to knowledge, with research on competencies was only > 20%, suggesting the need for further research eventually.

Deep Learning-Driven Library Design for the De Novo Discovery of Bioactive Thiopeptides.

Broad substrate tolerance of ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic enzymes has allowed numerous strategies for RiPP engineering. However, despite relaxed specificities, exact substrate preferences of RiPP enzymes are often difficult to pinpoint. Thus, when designing combinatorial libraries of RiPP precursors, balancing the compound diversity with the substrate fitness can be challenging. Here, we employed a deep learning model to streamline the design of mRNA display libraries. Using an in vitro reconstituted thiopeptide biosynthesis platform, we performed mRNA display-based profiling of substrate fitness for the biosynthetic pathway involving five enzymes to train an accurate deep learning model. We then utilized the model to design optimal mRNA libraries and demonstrated their utility in affinity selections against IRAK4 kinase and the TLR10 cell surface receptor. The selections led to the discovery of potent thiopeptide ligands against both target proteins (KD up to 1.3 nM for the best compound against IRAK4 and 300 nM for TLR10). The IRAK4-targeting compounds also inhibited the kinase at single-digit µM concentrations in vitro, exhibited efficient internalization into HEK293H cells, and suppressed NF-kB-mediated signaling in cells. Altogether, the developed approach streamlines the discovery of pseudonatural RiPPs with de novo designed biological activities and favorable pharmacological properties.

Switching Prenyl Donor Specificities of Cyanobactin Prenyltransferases.

Prenyltransferases in cyanobactin biosynthesis are of growing interest as peptide alkylation biocatalysts, but their prenylation modes characterized so far have been limited to dimethylallylation (C5) or geranylation (C10). Here we engaged in structure-guided engineering of the prenyl-binding pocket of a His-C2-geranyltransferase LimF to modulate its prenylation mode. Contraction of the pocket by a single mutation led to a His-C2-dimethylallyltransferase. More importantly, pocket expansion by a double mutation successfully repurposed LimF for farnesylation (C15), which is an unprecedented mode in this family. Furthermore, the obtained knowledge of the essential residues to construct the farnesyl-binding pocket has allowed for rational design of a Tyr-O-farnesyltransferase by a triple mutation of a Tyr-O-dimethylallyltransferase PagF. These results provide an approach to manipulate the prenyl specificity of cyanobactin prenyltransferases, broadening the chemical space covered by this class of enzymes and expanding the toolbox of peptide alkylation biocatalysts.

Ribosomal Synthesis of Peptides Bearing Noncanonical Backbone Structures via Chemical Posttranslational Modifications.

Noncanonical peptide backbone structures, such as heterocycles and non-α-amino acids, are characteristic building blocks present in peptidic natural products. To achieve ribosomal synthesis of designer peptides bearing such noncanonical backbone structures, we have devised translation-compatible precursor residues and their chemical posttranslational modification processes. In this chapter, we describe the detailed procedures for the in vitro translation of peptides containing the precursor residues by means of genetic code reprogramming technology and posttranslational generation of objective noncanonical backbone structures.

Structural basis of tRNAPro acceptor stem recognition by a bacterial trans-editing domain.

High fidelity tRNA aminoacylation by aminoacyl-tRNA synthetases is essential for cell viability. ProXp-ala is a trans-editing protein that is present in all three domains of life and is responsible for hydrolyzing mischarged Ala-tRNAPro and preventing mistranslation of proline codons. Previous studies have shown that, like bacterial prolyl-tRNA synthetase, Caulobacter crescentus ProXp-ala recognizes the unique C1:G72 terminal base pair of the tRNAPro acceptor stem, helping to ensure deacylation of Ala-tRNAPro but not Ala-tRNAAla. The structural basis for C1:G72 recognition by ProXp-ala is still unknown and was investigated here. NMR spectroscopy, binding, and activity assays revealed two conserved residues, K50 and R80, that likely interact with the first base pair, stabilizing the initial protein-RNA encounter complex. Modeling studies are consistent with direct interaction between R80 and the major groove of G72. A third key contact between A76 of tRNAPro and K45 of ProXp-ala was essential for binding and accommodating the CCA-3' end in the active site. We also demonstrated the essential role that the 2'OH of A76 plays in catalysis. Eukaryotic ProXp-ala proteins recognize the same acceptor stem positions as their bacterial counterparts, albeit with different nucleotide base identities. ProXp-ala is encoded in some human pathogens; thus, these results have the potential to inform new antibiotic drug design.

Label-free quantification of passive membrane permeability of cyclic peptides across lipid bilayers: penetration speed of cyclosporin A across lipid bilayers.

Cyclic peptides that passively penetrate cell membranes are under active investigation in drug discovery research. PAMPA (Parallel Artificial Membrane Permeability Assay) and Caco-2 assay are mainly used for permeability measurements in these studies. However, permeability rates across the artificial membrane and the cell monolayer used for these assays are intrinsically different from the ones across pure lipid bilayers. There are also membrane permeability assays for peptides using reconstructed lipid bilayers, but they require labeling for detection, and the absolute membrane permeability of the natural peptides themselves could not be determined. Here, we constructed a lipid bilayer permeability assay and realized the first label-free measurements of the lipid bilayer permeability of cyclic peptides. Quantitative permeability values across lipid bilayers were determined for model cyclic hexapeptides and an important natural product, cyclosporin A (CsA). The obtained quantitative permeability values will provide new and advanced knowledge about the passive permeability of cyclic peptides.

Reactivity and mechanism in chemical and synthetic biology.

Physical organic chemistry and mechanistic thinking provide a strong intellectual framework for understanding the chemical logic of evolvable informational macromolecules and metabolic transformations in living organisms. These concepts have also led to numerous successes in designing and applying tools to delineate biological function in health and disease, chemical ecology and possible alternative chemistries employed by extraterrestrial life. A symposium at the 2020 Pacifichem meeting was scheduled in December 2020 to discuss designing and exploiting expanded genetic alphabets, methods to understand the biosynthesis of natural products and re-engineering primary metabolism in bacteria. The COVID-19 pandemic led to postponement of in-person discussions, with the symposium eventually being held on 20-21 December 2021 as an online event. This issue is a written record of work presented on biosynthetic pathways and enzyme catalysis, engineering microorganisms with new metabolic capabilities, and the synthesis of non-canonical, nucleobases for medical applications and for studies of alternate chemistries for living organisms. The variety of opinion pieces, reviews and original research articles provide a starting point for innovations that clarify how complex biological systems emerge from the rules of chemical reactivity and mechanism. This article is part of the themed issue 'Reactivity and mechanism in chemical and synthetic biology'.

Senescence in brain pericytes attenuates blood-brain barrier function in vitro: A comparison of serially passaged and isolated pericytes from aged rat brains.

Aging is associated with the dysfunction of the blood-brain barrier (BBB), which comprises brain microvessel endothelial cells (BMECs), astrocytes, and pericytes. Pericytes are present at intervals along the walls of the brain capillaries and play a key role in maintaining BBB integrity. Accumulation of senescent cells and the senescence-associated secretory phenotype (SASP) in the brain facilitate the development of age-related neurodegenerative diseases with BBB dysfunction. However, the ability of pericytes to support BBB integrity and their correlation with cellular senescence or aging remain unknown. Here, we investigated cellular senescence in pericytes focusing on its impact on BBB function using BBB models comprising intact BMECs co-cultured with senescent pericytes, which were obtained through a serial passage or isolated from 18-month-old rats. To assess BBB function, transendothelial electrical resistance (TEER) and permeability of sodium fluorescein (Na-F) were studied. Both serially passaged pericytes (in passage 4, 7, and 10) and aged pericytes isolated from 18-month-old rats showed decreased TEER and enhanced permeability of BMECs to Na-F compared to that of normal pericytes (passage 2 or young). Furthermore, serially passaged and aged pericytes showed characteristic features of cellular senescence, including increased ß-galactosidase activity, cell cycle arrest, enhanced expression of mRNA, and SASP factors. However, the senescence-induced mRNA expression profile of pericyte markers varied between serially passaged and aged pericytes. Hence, in vitro serial passages and isolation from naturally aged rodents differently influenced genetic and biochemical features of senescent brain pericytes. We conclude that senescent brain pericytes can induce BBB dysfunction and those isolated from aged rodents retain the senescence-specific properties. Our findings provide an alternative tool to investigate the senescence in brain pericytes in vitro.

Effectiveness of remdesivir with corticosteroids for COVID-19 patients in intensive care unit: A hospital-based observational study.

The effectiveness of remdesivir on survival in coronavirus disease 2019 (COVID-19), especially in cases treated in the intensive care unit (ICU), is controversial. We investigated the effectiveness of remdesivir with corticosteroids on the survival of COVID-19 patients in a real ICU clinical practice. For laboratory-confirmed COVID-19 patients admitted to the ICU of a tertiary hospital in Tokyo (April 2020-November 2021) and who received corticosteroids, the effectiveness of remdesivir for survival, stratified by interval length (within 9 or 10+ days), was retrospectively analyzed using Cox regression model. A total of 168 patients were included: 35 with no remdesivir use (control), 96 with remdesivir use within 9 days, and 37 with remdesivir use with an interval of 10+ days. In-hospital mortality was 45.7%, 10.4%, and 16.2%, respectively. After adjusting for possible covariates including comorbidities, laboratory data, oxygen demand, or level of pneumonia, remdesivir use within 9 days from symptom onset reduced mortality risk (hazard ratio [HR]: 0.10; 95% confidence interval (CI): 0.025-0.428) compared to the control group. However, remdesivir use with an interval of 10+ days showed no significant association with mortality (HR: 0.42; 95% CI: 0.117-1.524). Among COVID-19 patients who received corticosteroids in ICU, remdesivir use within 9 days from symptom onset was associated with reduced in-hospital mortality risk.

Discovery, biochemical characterization, and bioengineering of cyanobactin prenyltransferases.

Prenylation is a post-translational modification (PTM) widely found in primary and secondary metabolism. This modification can enhance the lipophilicity of molecules, enabling them to interact with lipid membranes more effectively. The prenylation of peptides is often carried out by cyanobactin prenyltransferases (PTases) from cyanobacteria. These enzymes are of interest due to their ability to add prenyl groups to unmodified peptides, thus making them more effective therapeutics through the subsequent acquisition of increased membrane permeability and bioavailability. Herein we review the current knowledge of cyanobactin PTases, focusing on their discovery, biochemistry, and bioengineering, and highlight the potential application of them as peptide alkylation biocatalysts to generate peptide therapeutics.

Digital-Based Policy and Health Promotion Policy in Japan, the Republic of Korea, Singapore, and Thailand: A Scoping Review of Policy Paths to Healthy Aging.

People are living longer, and our life has become more digital. Hence, the benefits from digital technology, including economic growth, increasing labor productivity, and ensuring health equity in the face of an aging population emerged as a vital topic for countries around the world. Japan, the Republic of Korea (ROK), Singapore, and Thailand are in the top ten rankings in terms of information and communication technology (ICT) development within the Asia Pacific Region and all are facing challenges of population aging. Well-designed national ICT policy and health promotion policies enabled the countries to make significant progress and development in terms of digitalization and healthy aging. This paper aims to answer questions regarding digitization and health promotion: when it started, how it is going, what are the achievements, and what it holds for the future, considering healthy aging and digitalization by reviewing the national ICT policy and health promotion policies of Japan, Korea, Singapore, and Thailand. This paper is expected to help readers build a comprehensive understanding of each country's journey towards building a healthy aging digital society. Furthermore, we hope this paper can be a source for countries to exchange experiences and learn from each other with a joint goal of building a healthy aging digital society.

Post-translational backbone-acyl shift yields natural product-like peptides bearing hydroxyhydrocarbon units.

Hydroxyhydrocarbon (Hhc) moieties in the backbone of peptidic natural products can exert a substantial influence on the bioactivities of the products, making Hhc units an attractive class of building blocks for de novo peptides. However, despite advances in in vitro genetic code reprogramming, the ribosomal incorporation of Hhc units remains challenging. Here we report a method for in vitro ribosomal synthesis of natural-product-like peptides bearing Hhc units. A series of azide/hydroxy acids were designed as chemical precursors of Hhc units and incorporated into the nascent peptide chain by means of genetic code reprogramming. Post-translational reduction of the azide induced an O-to-N acyl shift to rearrange the peptide backbone. This method allows for one-pot ribosomal synthesis of designer macrocycles bearing various ß-, γ- and δ-type Hhc units. We also report the synthesis of a statine-containing peptidomimetic inhibitor of ß-secretase 1 as a showcase example.

Solid-Phase-Based Synthesis of Lactazole-Like Thiopeptides.

A strategy for the synthesis of de novo discovered lactazole-like thiopeptides is reported. The approach revolves around a convergent and scalable preparation of the central triheterocyclic amino acid and its utilization in Fmoc solid-phase peptide synthesis for modular peptide chain assembly. A technique for preparing C-terminally functionalized thiopeptides for biological studies is also described. The syntheses of 11 TNIK-inhibitor thiopeptides and 6 of their derivatives in multimilligram quantities highlight the practical utility of the developed protocols.

De Novo Discovery of Thiopeptide Pseudo-natural Products Acting as Potent and Selective TNIK Kinase Inhibitors.

Bioengineering of ribosomally synthesized and post-translationally modified peptides (RiPPs) is an emerging approach to explore the diversity of pseudo-natural product structures for drug discovery purposes. However, despite the initial advances in this area, bioactivity reprogramming of multienzyme RiPP biosynthetic pathways remains a major challenge. Here, we report a platform for de novo discovery of functional thiopeptides based on reengineered biosynthesis of lactazole A, a RiPP natural product assembled by five biosynthetic enzymes. The platform combines in vitro biosynthesis of lactazole-like thiopeptides and mRNA display to prepare and screen large (≥1012) combinatorial libraries of pseudo-natural products. We demonstrate the utility of the developed protocols in an affinity selection against Traf2- and NCK-interacting kinase (TNIK), a protein involved in several cancers, which yielded a plethora of candidate thiopeptides. Of the 11 synthesized compounds, 9 had high affinities for the target kinase (best KD = 1.2 nM) and 10 inhibited its enzymatic activity (best Ki = 3 nM). X-ray structural analysis of the TNIK/thiopeptide interaction revealed the unique mode of substrate-competitive inhibition exhibited by two of the discovered compounds. The thiopeptides internalized to the cytosol of HEK293H cells as efficiently as the known cell-penetrating peptide Tat (4-6 µM). Accordingly, the most potent compound, TP15, inhibited TNIK in HCT116 cells. Altogether, our platform enables the exploration of pseudo-natural thiopeptides with favorable pharmacological properties in drug discovery applications.

Experimental and Theoretical Heterodyne-Detected Sum Frequency Generation Spectroscopy of Isotopically Pure and Diluted Water Surfaces.

The χ(2) (second-order nonlinear optical susceptibility) spectrum of the water surface has been a matter of debate for a few decades. Here, we report that we experimentally measured the isotopic dilution dependence of the χ(2) spectrum and theoretically reproduced it by employing the quantum/classical mixed approach with a new idea to subtract an artifact. The present theoretical framework allows for clarifying the effects of the intramolecular, intermolecular, and Fermi resonance couplings on the OH-stretch vibrational spectra of water at the surface as well as in the bulk.