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Non-ribosomal peptide synthetases (NRPSs) biosynthesize many pharmaceuticals and virulence factors. The biosynthesis of these natural peptide products from biosynthetic gene clusters depends on complex regulations in bacteria. However, our current knowledge of NRPSs is based on enzymological studies using full NRPS systems and/or a single NRPS domain in heterologous hosts. Chemical and/or biochemical strategies to capture the endogenous activities of NRPSs facilitate studies on NRPS cell biology in bacterial cells. Here, we describe a chemical scaffold for the rapid and selective photoaffinity labelling of NRPSs in purified systems, crude biological samples and living bacterial cells. We synthesized photoaffinity labelling probes coupled with 5′- O-N -(phenylalanyl)sulfamoyladenosine with clickable alkyl diazirine or trifluoromethyl phenyl diazirine. We found that a trifluoromethyl phenyl diazirine-based probe cross-linked the Phe-activating domain of a GrsA-NRPS with high selectivity and sensitivity at shorter ultraviolet (UV) irradiation times (less than 5 min) relative to a prototypical benzophenone-based probe. Our results demonstrated that this quick labelling protocol can prevent damage to proteins and cells caused by long UV irradiation times, providing a mild photoaffinity labelling method for biological samples. This article is part of the theme issue ‘Reactivity and mechanism in chemical and synthetic biology’.

Homeostasis is achieved by balancing cell survival and death. In cancer cells, especially those carrying driver mutations, the processes and signals that promote apoptosis are inhibited, facilitating the survival and proliferation of these dysregulated cells. Apoptosis induction is an important mechanism underlying the therapeutic efficacy of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) for EGFR-mutated non-small cell lung cancer (NSCLC). However, the mechanisms by which EGFR-TKIs induce apoptosis have not been fully elucidated. A deeper understanding of the apoptotic pathways induced by EGFR-TKIs is essential for the developing novel strategies to overcome resistance to EGFR-TKIs or to enhance the initial efficacy through therapeutic synergistic combinations. Recently, therapeutic strategies targeting apoptosis have been developed for cancer. Here, we review the state of knowledge on EGFR-TKI-induced apoptotic pathways and discuss the therapeutic strategies for enhancing EGFR-TKI efficiency. We highlight the great progress achieved with third-generation EGFR-TKIs. In particular, combination therapies of EGFR-TKIs with anti-vascular endothelial growth factor/receptor inhibitors or chemotherapy have emerged as promising therapeutic strategies for patients with EGFR-mutated NSCLC. Nevertheless, further breakthroughs are needed to yield an appropriate standard care for patients with EGFR-mutated NSCLC, which requires gaining a deeper understanding of cancer cell dynamics in response to EGFR-TKIs.

Recent comprehensive analyses of mtDNA and orthogonal RNA-sequencing data revealed that in numerous human cancers, mtDNA copy numbers and mtRNA amounts are significantly reduced, followed by low respiratory gene expression. Under such conditions (called mt-Low), cells encounter severe cell proliferation defects; therefore, they must acquire countermeasures against this fatal disadvantage during malignant transformation. This study elucidated a countermeasure against the mt-Low condition-induced antiproliferative effects in hepatocellular carcinoma (HCC) cells. The mechanism relied on the architectural transcriptional regulator HMGA2, which was preferably expressed in HCC cells of the mt-Low type in vitro and in vivo. Detailed in vitro analyses suggest that HMGA2 regulates insulin-like growth factor binding protein 1 (IGFBP1) expression, leading to AKT activation, which then phosphorylates the cyclin-dependent kinase inhibitor (CKI), P27KIP1, and facilitates its ubiquitin-mediated degradation. Accordingly, intervention in the HMGA2 function by RNAi resulted in an increase in P27KIP1 levels and an induction of senescence-like cell proliferation inhibition in mt-Low-type HCC cells. Conclusively, the HMGA2/IGFBP1/AKT axis has emerged as a countermeasure against P27KIP1 CKI upregulation under mt-Low conditions, thereby circumventing cell proliferation inhibition and supporting the tumorigenic state. Notably, similar to in vitro cell lines, HMGA2 was likely to regulate IGFBP1 expression in HCC in vivo, thereby contributing to poor patient prognosis. Considering the significant number of cases under mt-Low or the threat of CKI upregulation cancer-wide, the axis is noteworthy as a vulnerability of cancer cells or target for tumor-agnostic therapy inducing irreversible cell proliferation inhibition via CKI upregulation in a large population with cancer.

Previously, we reported that non‐apoptotic cell death was induced in non‐malignant mammary epithelial cells (HMECs) upon loss of anchorage during 48 h incubation in suspension. In this study, we examined HMECs in suspension at an earlier time point and found that most of them lost attachment ability to substrata when replated, although >80% were alive. This suggested that HMECs lost reattachment ability (RA) prior to cell death upon detachment. Concomitant with the loss of RA, a decrease in the levels of β1 and β4 integrin was observed. In sharp contrast, breast cancer cells retained integrin levels, reattached to substrata, and formed colonies after exposure to anchorage loss as efficiently as those maintained under adherent conditions. Such RA of cancer cells is essential for the metastatic process, especially for establishing adhesion contact with ECM in the secondary organ after systemic circulation. Further analysis suggested that sustained levels of β4 integrin, which was mediated by Rac1, was critical for RA after anchorage loss and lung metastasis of breast cancer cells. In the cancer cells, persistent Rac1 activity enhanced escape of β4 integrin from lysosomal degradation depending on actin‐related protein 2/3 and TBC1D2, a GTPase‐activating protein of Rab7 GTPase. Notably, simultaneous high expression of ITGB4 and RAC1 was associated with poor prognosis in patients with breast cancer. Therefore, β4 integrin and Rac1 are attractive therapeutic targets to eliminate RA in cancer cells, thereby preventing the initial step of colonization at the secondary organ during metastasis., Anchorage‐independent reattachment ability (RA) of cells is as important as anchorage‐independent survival or growth for cancer metastasis. The Rac1/beta4 integrin axis has emerged as a critical mediator of RA of breast cancer cells in this study. Notably, simultaneous high expression of RAC1 and ITGB4 was associated with poor prognosis in patients with breast cancer.

Hitachimycin is a macrolactam antibiotic with an (S)-β-phenylalanine (β-Phe) at the starter position of its polyketide skeleton. (S)-β-Phe is formed from l-α-phenylalanine by the phenylananine-2,3-aminomutase HitA in the hitachimycin biosynthetic pathway. In this study, we produced new hitachimycin analogs via mutasynthesis by feeding various (S)-β-Phe analogs to a ΔhitA strain. We obtained six hitachimycin analogs with F at the ortho, meta, or para position and Cl, Br, or a CH3 group at the meta position of the phenyl moiety, as well as two hitachimycin analogs with thienyl substitutions. Furthermore, we carried out a biochemical and structural analysis of HitB, a β-amino acid-selective adenylation enzyme that introduces (S)-β-Phe into the hitachimycin biosynthetic pathway. The KM values of the incorporated (S)-β-Phe analogs and natural (S)-β-Phe were similar. However, the KM values of unincorporated (S)-β-Phe analogs with Br and a CH3 group at the ortho or para position of the phenyl moiety were high, indicating that HitB functions as a gatekeeper to select macrolactam starter units during mutasynthesis. The crystal structure of HitB in complex with (S)-β-3-Br-phenylalanine sulfamoyladenosine (β-m-Br-Phe-SA) revealed that the bulky meta-Br group is accommodated by the conformational flexibility around Phe328, whose side chain is close to the meta position. The aromatic group of β-m-Br-Phe-SA is surrounded by hydrophobic and aromatic residues, which appears to confer the conformational flexibility that enables HitB to accommodate the meta-substituted (S)-β-Phe. The new hitachimycin analogs exhibited different levels of biological activity in HeLa cells and multidrug-sensitive budding yeast, suggesting that they may target different molecules.

We show that salacinol-type α-glucosidase inhibitors are ligand-compatible with the GH 31 family. Salacinol and its 3′-O-benzylated analogs inhibit human lysosomal α-glucosidase at submicromolar levels. Simple structure-activity relationship studies reveal that the salacinol side-chain stereochemistry significantly influences binding to GH31 α-glucosidases.

An important challenge in natural product biosynthesis is the biosynthetic design and production of artificial peptides. One of the most promising strategies is reprogramming adenylation (A) domains to expand the substrate repertoire of nonribosomal peptide synthetases (NRPSs). Therefore, the precise detection of subtle structural changes in the substrate binding pockets of A domains might accelerate their reprogramming. Here we show that an enzyme-linked immunosorbent assay (ELISA) using a combination of small-molecule probes can detect the effects of substrate binding pocket residue substitutions in A-domains. When coupled with a set of aryl acid A-domain variants (total of nine variants), the ELISA can analyze the subtle differences in their active-site architectures. Furthermore, the ELISA-based screening was able to identify the variants with substrate binding pockets that accepted a non-cognate substrate from an original pool of 45. These studies demonstrate that ELISA is a reliable platform for providing insights into the active-site properties of A-domains and can be applied for the reprogramming of NRPS A-domains.

Aryl acids are most commonly found in iron-scavenging siderophores but are not limited to them. The nonribosomal peptide synthetase (NRPS) codes of aryl acids remain poorly elucidated relative to those of amino acids. Here, we defined more precisely the role of active-site residues in aryl acid adenylation domains (A-domains) by gradually grafting the NRPS codes used for salicylic acid (Sal) into an archetypal aryl acid A-domain, EntE [specific for the substrate 2,3-dihydroxybenzoic acid (DHB)]. Enzyme kinetics and modeling studies of these EntE variants demonstrated that the NRPS code residues at positions 236, 240, and 339 collectively regulate the substrate specificity toward DHB and Sal. Furthermore, the EntE variants exhibited the ability to activate the non-native aryl acids 3-hydroxybenzoic acid, 3-aminobenzoic acid, 3-fluorobenzoic acid, and 3-chlorobenzoic acid. These studies enhance our knowledge of the NRPS codes of aryl acids and could be exploited to reprogram aryl acid A-domains for non-native aryl acids.

Nonribosomal peptide synthetases (NRPSs) are complex multi-modular enzymes containing catalytic domains responsible for the loading and incorporation of amino acids into natural products. These unique molecular factories can produce peptides with nonproteinogenic d-amino acids in which the epimerization (E) domain catalyzes the conversion of l-amino acids to d-amino acids, but its mechanism remains not fully understood. Here, we describe the development of pantetheine crosslinking probes that mimic the natural substrate l-Phe of the initiation module of tyrocidine synthetase, TycA, to elucidate and study the catalytic residues of the E domain. Mechanism-based crosslinking assays and MALDI-TOF MS were used to identify both H743 and E882 as the crosslinking site residues, demonstrating their roles as catalytic bases. Mutagenesis studies further validated these results and allowed the comparison of reactivity between the catalytic residues, concluding that glutamate acts as the dominant nucleophile in the crosslinking reaction, resembling the deprotonation of the Cα-H of amino acids in the epimerization reaction. The crosslinking probes employed in these studies provide new tools for studying the molecular details of E domains, as well as the potential to study C domains. In particular, they would elucidate key information for how these domains function and interact with their substrates in nature, further enhancing the knowledge needed to assist combinatorial biosynthetic efforts of NRPS systems to produce novel compounds.

Structured summary Background A characteristic feature of SARS-CoV-2 is its ability to transmit from pre- or asymptomatic patients, complicating the tracing of infection pathways and causing outbreaks. Despite several reports that whole genome sequencing (WGS) and haplotype networks are useful for epidemiologic analysis, little is known about their use in nosocomial infections. Aim We aimed to demonstrate the advantages of genetic epidemiology in identifying the link in nosocomial infection by comparing single nucleotide variations (SNVs) of isolates from patients associated with an outbreak in Showa University Hospital. Methods We used specimens from 32 patients in whom COVID-19 had been diagnosed using clinical reverse transcription-polymerase chain reaction tests. RNA of SARS-CoV-2 from specimens was reverse-transcribed and analysed using WGS. SNVs were extracted and used for lineage determination, phylogenetic tree analysis, and median-joining analysis. Findings The lineage of SARS-CoV-2 that was associated with outbreak in Showa University Hospital was B.1.1.214, which was consistent with that found in the Kanto metropolitan area during the same period. Consistent with canonical epidemiological observations, haplotype network analysis was successful for the classification of patients. Additionally, phylogenetic tree analysis revealed three independent introductions of the virus into the hospital during the outbreak. Further, median-joining analysis indicated that four patients were directly infected by any of the others in the same cluster. Conclusion Genetic epidemiology with WGS and haplotype networks is useful for tracing transmission and optimizing prevention strategies in nosocomial outbreaks.

A major challenge in fluorescence imaging experiments, which are essential to determine protein activity, expression, and localization, is the penetration of small-molecule probes through the outer membrane permeability barrier of bacteria. Here, we describe a novel strategy for small-molecule probe-based fluorescence protein labeling and imaging in the Gram-negative bacterium

The transition metal dichalcogenide $\mathrm{Pd}{\mathrm{Te}}_{2}$ has attractive features based on its classification as a type-II Dirac semimetal and the occurrence of type-I superconductivity, providing a platform for discussion of a topological superconductor. Our recent work revealed that type-I superconductivity persists up to pressures of $\ensuremath{\sim}2.5$ GPa and the superconducting transition temperature ${T}_{\mathrm{c}}$ reaches a maximum at around 1 GPa, which is inconsistent with the theoretical prediction. To understand its nonmonotonic variation and investigate superconductivity at higher pressures, we performed structural analysis by x-ray diffraction at room temperature below 8 GPa and electrical resistivity measurements at low temperatures from 1 to 8 GPa. With regard to the superconductivity beyond 1 GPa, the monotonic decrease in ${T}_{\mathrm{c}}$ is reproduced without any noticeable anomalies; ${T}_{\mathrm{c}}$ changes from 1.8 K at 1 GPa to 0.82 K at 5.5 GPa with $d{T}_{\mathrm{c}}/dP\ensuremath{\sim}\ensuremath{-}0.22$ K/GPa. The crystal structure with space group $P\overline{3}m1$ is stable in the pressure range we examined. On the other hand, the normalized pressure-strain analysis (finite strain analysis) indicates that the compressibility changes around 1 GPa, suggesting that a Lifshitz transition occurs. We here discuss the effect of pressure on the superconducting and structural properties based on the comparison of these experimental results.

Although 4,5-didehydroguadiscine (12a), an alkaloid with potent melanogenesis-inhibitory activity isolated from Hornschuchia obliqua (Annonaceae), consists of an aporphine nucleus with an aromatized B-ring, to date, it has not been utilized as a template for structure-activity relationship (SAR) studies of pharmacological activities because of its exceptional structure. Accordingly, herein, five analogs (12b-12f) of 12a and five benzylisoquinoline analogs (13b-13f) lacking the C11a-C11b bond of 12b-12f were prepared. The inhibitory effects of 12b-12f and 13b-13f on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells were examined and compared with those of 12a. Melanogenesis-inhibitory activities of 12b-12f were the same as that of 12a, whereas the melanogenesis-inhibitory activities of 13b-13f were significantly inferior to those of 12a and 12b-12f. These results suggest that the C11a-C11b bond plays an essential role in the melanogenesis-inhibitory activities of 12a-12e.

In Japan, during the coronavirus disease 2019 (COVID-19) pandemic, patients with non-hypoxia are recommended to recuperate at home or in pre-hospital facilities. However, it was observed that unexpected hypoxia may occur and become severe subsequently in patients whose symptoms were initially expected to improve naturally. The aim of this study is to validate biomarkers that can predict at an early stage the emergence of hypoxia in COVID-19 patients without hypoxia.We retrospectively enrolled 193 patients with COVID-19, excluding patients with hypoxia and severe disease from the onset. Participants were classified into two groups according to the emergence of hypoxia during the clinical course, and the laboratory data were compared to identify biomarkers that could predict early the emergence of hypoxia.The areas under the curve for serum cystatin C (CysC) and C-reactive protein (CRP) levels for the emergence of hypoxia during the clinical course were higher than those for other biomarkers (CysC, 0.84 and CRP, 0.83). Multivariate analysis showed that high serum CysC and CRP levels were associated with the emergence of hypoxia during the clinical course.Elevated serum CysC and CRP levels were associated with the emergence of hypoxia during the clinical course in COVID-19 patients without hypoxia. These findings may help determine the need for hospitalization in initially non-hypoxic COVID-19 patients.

The presence of phase-transition in hydrogen (H2) at around 560 megapascal (MPa) and room temperature was clarified by Raman and x-ray diffraction studies on both pure H2 and graphite-H2 mixture. H2 is intercalated into the nano-space of graphite, which lowers the transition pressure and temporally expands the size of the honeycomb lattice of graphite under pressure up to 600 MPa. It is supposed that is caused by a gas-liquid phase-transition. According to the peak analysis for Q1(J) mode, the ortho-para conversion of H2 gradually begins to appear after the phase-transition pressure even at room temperature, while peak separation is difficult to achieve under pressure above 1.6 gigapascal (GPa) because of significant overlapping of the peak intensities. Because we have missed the ortho-para conversion which could be observed in only such a small pressure range, the fluid phase at room temperature was full of mystery.

Nonribosomal peptide (NRP) natural products are among the most promising resources for drug discovery and development, owing to their wide range of biological activities and therapeutic applications. These peptide metabolites are biosynthesized by large multienzyme machinery known as NRP synthetases (NRPSs). The structural complexity of a number of NRPs poses an enormous challenge in their synthesis. A major issue in this field is reprogramming NRPS machineries to allow the biosynthetic production of artificial peptides. NRPS adenylation (A) domains are responsible for the incorporation of a wide variety of amino acids and can be considered as reprogramming sites; therefore, advanced methods to accelerate the functional prediction and assessment of A-domains are required. This Concept article demonstrates that activity-based protein profiling of NRPSs offers a simple, rapid, and robust analytical platform for A-domains and provides insights into enzyme-substrate candidates and active-site microenvironments. It also describes the background associated with the development and application of a method to analyze endogenous NRPS machinery in its natural environment.

The red maple and sugar maple (Acer rubrum and A. saccharum, respectively) contain acertannins (ginnalins and maplexins), galloylated derivatives of 1,5-anhydro- d -glucitol (1,5-AG, 1). These compounds have a variety of potential medicinal properties and we have shown that some of them promote the expression of ceramide synthase 3. We now report on the beneficial effects of ginnalin B, (6-O-galloyl-1,5-AG, 5), leading to acceleration of skin metabolism and reduction of the turnover time. Ginnalin B dose-dependently increased the relative amount of keratin 10, keratin 1, and filaggrin gene, with maximal increase of 1.7-, 2.9, and 5.2-fold at 100 μM, respectively. The validation study showed that it had superior capacity to induce multiple stages of keratinocyte differentiation and significantly elevated the immunostaining site of keratin 10 and filaggrin in a 3-dimensional cultured human skin model, by 1.2 and 2.8-fold, respectively. Furthermore, ginnalin B caused the arrest of proliferation at the G0/G1 phase but it did not induce apoptotic cell death in normal human keratinocytes. Molecular studies revealed that ginnalin B up-regulated the levels of NOTCH1 and a concomitant increase p21 expression. Ginnalin B, therefore, represents a new class of promising functional and medical cosmetic compound and it could contribute to the maintenance of homeostasis of the epidermis.

Neokotalanol (5) is a sulfonium-type α-glucosidase inhibitor isolated from the traditional Ayurvedic medicine “Salacia.” Its potency against maltase-glucoamylase was 2000-fold stronger than acarbose. Despite 5 having been recognized as the most active among this series of sulfonium salts, a facile and effective synthetic protocol leading to 5 has not been established to date because of the difficulty in selecting and designing a protected key intermediate. In this study, an appropriately protected epoxide (β-20) was successfully designed and diastereoselectively synthesized from the easily accessible d-galactose (18). By use of β-20, S-alkylation of sulfides (7b) was successfully proceeded in a highly diastereoselective manner to afford 5 in a good total yield (11%, via 14 steps), which was superior to the three previously reported sequences (∼1% via 15 steps, ∼0.5% via 18 steps, ∼2% via 14 steps).

Adenylation (A) domains act as the gatekeepers of non-ribosomal peptide synthetases (NRPSs), ensuring the activation and thioesterification of the correct amino acid/aryl acid building blocks. Aryl acid building blocks are most commonly observed in iron-chelating siderophores, but are not limited to them. Very little is known about the reprogramming of aryl acid A-domains. We show that a single asparagine-to-glycine mutation in an aryl acid A-domain leads to an enzyme that tolerates a wide range of non-native aryl acids. The engineered catalyst is capable of activating non-native aryl acids functionalized with nitro, cyano, bromo, and iodo groups, even though no enzymatic activity of wild-type enzyme was observed toward these substrates. Co-crystal structures with non-hydrolysable aryl-AMP analogues revealed the origins of this expansion of substrate promiscuity, highlighting an enlargement of the substrate binding pocket of the enzyme. Our findings may be exploited to produce diversified aryl acid containing natural products and serve as a template for further directed evolution in combinatorial biosynthesis.

Several retrospective studies have shown that the antitumor efficacy of capecitabine-containing chemotherapy decreases when co-administered with a proton pump inhibitor (PPI). Although a reduction in capecitabine absorption by PPIs was proposed as the underlying mechanism, the effects of PPIs on capecitabine pharmacokinetics remain unclear. We prospectively examined the effects of rabeprazole on the pharmacokinetics of capecitabine and its metabolites. We enrolled patients administered adjuvant capecitabine plus oxaliplatin (CapeOX) for postoperative colorectal cancer (CRC) patients and metastatic CRC patients receiving CapeOX with/without bevacizumab. Patients receiving a PPI before registration were allocated to the rabeprazole group, and the PPI was changed to rabeprazole (20 mg/day) at least 1 week before the initiation of capecitabine treatment. On day 1, oral capecitabine (1000 mg/m2) was administered 1 h after rabeprazole intake. Oxaliplatin (and bevacizumab) administration on day 1 was shifted to day 2 for pharmacokinetic analysis of the first capecitabine dose. Plasma concentrations of capecitabine, 5′-deoxy-5-fluorocytidine, 5′-deoxy-5-fluorouridine, and 5-fluorouracil were analyzed by high-performance liquid chromatography. Effects of rabeprazole on inhibition of cell proliferation by each capecitabine metabolite were examined with colon cancer cells (COLO205 and HCT116). Five and 9 patients enrolled between September 2017 and July 2018 were allocated to rabeprazole and control groups, respectively. No significant effects of rabeprazole on area under the plasma concentration–time curve divided by capecitabine dose for capecitabine and its three metabolites were observed. Rabeprazole did not affect the proliferation inhibition of colon cancer cells by the respective capecitabine metabolites. Rabeprazole does not affect capecitabine pharmacokinetics.

We report magnetic properties in the random spinel magnet CoGa2O4. Rietveld analysis of the x-ray diffraction profile for CoGa2O4 reveals that the Co and Ga ions are distributed randomly in the tetrahedral A-sites and octahedral B-sites in the cubic spinel structure. CoGa2O4 exhibits a spin-glass transition at TSG = 8.2 K that is confirmed by measurements of the dc- and ac-susceptibilities and thermoremanent magnetization (TRM) that develops below TSG. From the frequency dependence of the freezing temperature Tf for CoGa2O4, it is indicated that the relaxation time follows a Vogel-Fulcher law. Magnetic entropy is considerably reduced, probably because magnetic cluster formation developed even at T > TSG. The relaxation rate of TRM is considerably enhanced at TSG and decays rapidly above and below TSG. The time course of TRM is reproduced by non-exponential relaxation forms, such as a stretched exponential (Kohlrausch) as well as Ogielski and Weron relaxation forms. This behavior is displayed universally in glass systems, and the characteristic parameters associated with these functions were reasonable., 34 pages, 14 figures, 3 table

Integrable systems have provided various insights into physical phenomena and mathematics. The way of constructing many-body integrable systems is limited to few ansatzes for the Lax pair, except for highly inventive findings of conserved quantities. Machine learning techniques have recently been applied to broad physics fields and proven powerful for building non-trivial transformations and potential functions. We here propose a machine learning approach to a systematic construction of classical integrable systems. Given the Hamiltonian or samples in latent space, our neural network simultaneously learns the corresponding natural Hamiltonian in real space and the canonical transformation between the latent space and the real space variables. We also propose a loss function for building integrable systems and demonstrate successful unsupervised learning for the Toda lattice. Our approach enables exploring new integrable systems without any prior knowledge about the canonical transformation or any ansatz for the Lax pair.

The gatekeeping adenylation (A) domain of the non-ribosomal peptide synthetase (NRPS) selectively incorporates specific proteinogenic/non-proteinogenic amino acid into a growing peptide chain. The EntE of the enterobactin NRPS is a discrete aryl acid A-domain with 2,3-dihydroxybenzoic acid (DHB) substrate specificity. Reprogrammed EntE N235G variant possesses an enlarged substrate recognition site, and is capable of accepting non-native aryl acids. Biochemical characterization of this unique substrate recognition site should provide a better understanding of activi-site microenvironments. Here, we synthesized a non-hydrolysable adenylate analogue with 2-aminobenzoic acid (2-ABA), 3-aminobenzoic acid (3-ABA), and 4-aminobenzoic acid (4-ABA) and used them to calculate the apparent inhibition constants (Kiapp.). Dose-response experiments using 3-ABA-sulfamoyladenosine (AMS) provided Kiapp. values of 596 nM for wild-type EntE and 2.4 nM for the N235G variants. These results suggest that 3-amino group of benzoic acid plays an important role in substrate recognition by the N235G variant. These findings would help designing aryl acid substrates with substituents at the 2- and 3-positions.

Four chain-extended analogs (12a–12d) and two related de-O-sulfonated analogs (13a and 13c) by introducing alkyl groups (a: R = C3H7, b R = C6H13, c: R = C8H17, d: R = C10H21) to the side chains of salacinol (1), a natural α-glucosidase inhibitor from Ayurvedic traditional medicine “Salacia”, were synthesized. The α-glucosidase inhibitory activities of all the synthesized analogs were evaluated in vitro. Against human intestinal maltase, the inhibitory activities of 12a and 13a with seven-carbon side chain were equal to that of 1. In contrast, analogs (12b–12d, and 13c) exhibited higher level of inhibitory activity against the same enzyme than 1 and had equal or higher potency than those of the clinically used anti-diabetics, voglibose, acarbose, and miglitol. Thus, elongation of the side chains of 1 was effective for specifically increasing the inhibitory activity against human intestinal maltase.

The Dirac semimetal PdTe$_2$ was recently reported to be a type-I superconductor ($T_c = $1.64 K, $��_0 H_c (0) = 13.6$ mT) with unusual superconductivity of the surface sheath. We here report a high-pressure study, $p \leq 2.5$ GPa, of the superconducting phase diagram extracted from ac-susceptibility and transport measurements on single crystalline samples. $T_c (p)$ shows a pronounced non-monotonous variation with a maximum $T_c = $1.91 K around 0.91 GPa, followed by a gradual decrease to 1.27 K at 2.5 GPa. The critical field of bulk superconductivity in the limit $T \rightarrow 0$, $H_c(0,p)$, follows a similar trend and consequently the $H_c(T,p)$-curves under pressure collapse on a single curve: $H_c(T,p)=H_c(0,p)[1-(T/T_c(p))^2]$. Surface superconductivity is robust under pressure as demonstrated by the large superconducting screening signal that persists for applied dc-fields $H_a > H_c$. Surprisingly, for $p \geq 1.41$ GPa the superconducting transition temperature at the surface $T_c^S$ is larger than $T_c$ of the bulk. Therefore surface superconductivity may possibly have a non-trivial nature and is connected to the topological surface states detected by ARPES. We compare the measured pressure variation of $T_c$ with recent results from band structure calculations and discuss the importance of a Van Hove singularity., manuscript 9 pages with 8 figures + supplemental material 3 pages with 6 figures

Acyltransferases (ATs) are responsible for the selection and incorporation of acyl building blocks in the biosynthesis of various polyketide natural products. The trans-AT modular polyketide synthases have a discrete trans-acting AT for the loading of an acyl unit onto the acyl carrier protein (ACP) located within each module. Despite the importance of protein-protein interactions between ATs and ACPs in trans-AT assembly lines, the dynamic actions of ACPs and trans-acting ATs remain largely uncharacterized because of the inherently transient nature of ACP-enzyme interactions. Herein, we report the crystal structure of the AT-ACP complex of disorazole trans-AT polyketide synthase. We used a bromoacetamide pantetheine cross-linking probe in combination with a Cys mutation to trap the transient AT-ACP complex, allowing the determination of the crystal structure of the disorazole AT-ACP complex at 2.03 Å resolution. On the basis of the cross-linked AT-ACP complex structure, ACP residues recognized by trans-acting AT were identified and validated by mutational studies, which demonstrated that the disorazole AT recognizes the loop 1 and helix III' residues of disorazole ACP. The disorazole AT-ACP complex structure presents a foundation for defining the dynamic processes associated with trans-acting ATs and provides detailed mechanistic insights into their ability to recognize ACPs.

A facile and highly diastereoselective approach toward the synthesis of potent salacinol-type α-glucosidase inhibitors, originally isolated from plants of the genus "Salacia", was developed using the S-alkylation of thiosugars with epoxides in HFIP (∼90%, dr, α/β = ∼ 26/1). The dr ratio of the product was significantly improved by the protocol as compared to that of the conventional S-alkylation of thiosugars (dr, α/β = ∼ 8/1). The protocol could be used for gram scale synthesis of the desired compounds. The 3'-O-benzylated salacinol analogs, which are the most potent in vitro inhibitors to date, were synthesized and evaluated in vivo; all analogs suppressed blood glucose levels in maltose-loaded mice, at levels comparable to those of the antidiabetic agent, voglibose.

Structural and activity studies have revealed the dynamic and transient actions of carrier protein (CP) activity in primary and secondary metabolic pathways. CP-mediated interactions play a central role in nonribosomal peptide biosynthesis, serving as covalent tethers for amino acid and aryl acid substrates and enabling the growth of peptide intermediates. Strategies are therefore required to study protein-protein interactions efficiently. Herein, we describe activity-based probes used to demonstrate the protein-protein interactions between aryl CP (ArCP) and aryl acid adenylation (A) domains as well as the substrate specificities of aryl acid A-domains. When coupled with in-gel fluorescence imaging, this strategy allows visualization of the protein-protein interactions required to recognize and transfer the substrate to the partner ArCP. This technique has potential for the analysis of protein-protein interactions within these biosynthetic enzymes at the molecular level and for use in development of the combinatorial biosynthesis of new nonribosomal peptides.

The first total syntheses of the geranylated pyranocoumarins, mameasins C (1) and D (2), aromatase inhibitors isolated from the flowers of Mammea siamensis, were accomplished in five steps, starting from phloroglucinol 3. In this strategy, the characteristic pyran ring-fused coumarin core of 1 and 2 was effectively constructed by Friedel-Crafts acylation of 3, followed by Reformatsky reaction of the resultant ketone to give a key coumarin intermediate 9. Compound 9 was converted to targets 1 and 2 in a stepwise manner by successive C-acylation and O-geranylation, followed by a [1,3]-sigmatropic geranyl shift. Furthermore, screening of intermediates obtained in the synthetic pathway to 1 and 2 revealed that de-geranylated pyranocoumarins (10 and 11) show superior aromatase inhibitory activity as compared to the natural products 1 and 2.

Long-chain acyl-coenzyme A synthetases (ACSLs) are a family of enzymes that convert free long-chain fatty acids into their acyl-coenzyme A (CoA) forms. ACSL4, belonging to the ACSL family, shows a preferential use of arachidonic acid (AA) as its substrate and plays a role in the remodeling of AA-containing phospholipids by incorporating free AA. However, little is known about the roles of ACSL4 in inflammatory responses. Here, we assessed the roles of ACSL4 on the effector functions of bone marrow-derived macrophages (BMDMs) obtained from mice lacking ACSL4. Liquid chromatography-tandem mass spectrometry analysis revealed that various highly unsaturated fatty acid (HUFA)-derived fatty acyl-CoA species were markedly decreased in the BMDMs obtained from ACSL4-deficient mice compared with those in the BMDMs obtained from wild-type mice. BMDMs from ACSL4-deficient mice also showed a reduced incorporation of HUFA into phosphatidylcholines. The stimulation of BMDMs with lipopolysaccharide (LPS) elicited the release of prostaglandins (PGs), such as PGE2, PGD2 and PGF2α, and the production of these mediators was significantly enhanced by ACSL4 deficiency. In contrast, neither the LPS-induced release of cytokines, such as IL-6 and IL-10, nor the endocytosis of zymosan or dextran was affected by ACSL4 deficiency. These results suggest that ACSL4 has a crucial role in the maintenance of HUFA composition of certain phospholipid species and in the incorporation of free AA into the phospholipids in LPS-stimulated macrophages. ACSL4 dysfunction may facilitate inflammatory responses by an enhanced eicosanoid storm.

AIM To analyse the long-term prognostic impact of circulating tumour cells (CTCs) in gastric cancer patients who underwent surgery. METHODS A 7.5-mL peripheral vein blood sample was obtained from each patient with treatment-negative gastric adenocarcinoma before surgery. OBP-401, a telomerase-specific, replication-selective, oncolytic adenoviral agent carrying the green fluorescent protein gene, was used to label CTCs. Correlations between the number of CTCs and clinical end points were evaluated. RESULTS The median follow-up period of the surviving patients with gastric cancer was 60 mo. The CTC number tended to increase concomitantly with disease progression. The overall survival of patients with more than five CTCs in 7.5-mL of peripheral blood was lower than that of patients with five or less CTCs, although the difference was not significant (P = 0.183). A significant difference in relapse-free survival was found between patients with more than five and those with five or less CTCs (P = 0.034). CONCLUSION A lower number of CTCs was correlated with higher relapse-free survival rates in patients. Detection of CTCs using OBP-401 may be useful for predicting prognosis in gastric cancer.

Andrimid (Adm) synthase, which belongs to the type-II system of enzymes, produces Adm in Pantoea agglomerans. The adm biosynthetic gene cluster lacks canonical acyltransferases (ATs) to load the malonyl group to acyl carrier proteins (ACPs), suggesting that a malonyl-CoA: ACP transacylase (MCAT) from the producer organism's fatty acid synthase (FAS) complex provides the essential acyltransferase (AT) activity in Adm biosynthesis. Here we report that the MCAT is essential for catalysis of the transacylation of malonate from malonyl-CoA to AdmA polyketide synthase (PKS) ACP in vitro. Furthermore, a catalytic self-malonylation of the AdmA PKS ACP was not observed in reactions without MCAT, while many type II PKS ACPs are capable of catalyzing the self-acylation process. This lack of self-malonylation was validated by amino acid sequence analysis of the AdmA PKS ACP and the type II PKS ACPs. These results show that the MCAT from the producer organism's FAS complex provides the missing AT activity in trans, suggesting a functional protein-protein interaction between the fatty acid and polyketide synthases in the Adm assembly line.

Detection of circulating tumor cells (CTCs) in peripheral blood is useful for estimating the prognosis of patients with cancer. We previously reported the detection of CTCs by OBP-401, a telomerase-specific, replication-selective, oncolytic adenoviral agent carrying the green fluorescent protein (GFP) gene. We demonstrated that the number of large (L)-GFP+ cells (≥7.735 µm in diameter) in peripheral blood samples correlated significantly with the prognosis of treatment-naïve gastric cancer patients, whereas the number of small (S)-GFP+ cells (

Mitochondria are multifunctional organelles; they have been implicated in various aspects of tumorigenesis. In this study, we investigated a novel role of the basal electron transport chain (ETC) activity in cell proliferation by inhibiting mitochondrial replication and transcription (mtR/T) using pharmacological and genetic interventions, which depleted mitochondrial DNA/RNA, thereby inducing ETC deficiency. Interestingly, mtR/T inhibition did not decrease ATP levels despite deficiency in ETC activity in different cell types, including MDA-MB-231 breast cancer cells, but it severely impeded cell cycle progression, specifically progression during G2 and/or M phases in the cancer cells. Under these conditions, the expression of a group of cell cycle regulators was downregulated without affecting the growth signaling pathway. Further analysis suggested that the transcriptional network organized by E2F1 was significantly affected because of the downregulation of E2F1 in response to ETC deficiency, which eventually resulted in the suppression of cell proliferation. Thus, in this study, the E2F1-mediated ETC-dependent mechanism has emerged as the regulatory mechanism of cell cycle progression. In addition to E2F1, FOXM1 and BMYB were also downregulated, which contributed specifically to the defects in G2 and/or M phase progression. Thus, ETC-deficient cancer cells lost their growing ability, including their tumorigenic potential in vivo. ETC deficiency abolished the production of reactive oxygen species (ROS) from the mitochondria and a mitochondria-targeted antioxidant mimicked the deficiency, thereby suggesting that ETC activity signaled through ROS production. In conclusion, this novel coupling between ETC activity and cell cycle progression may be an important mechanism for coordinating cell proliferation and metabolism.

We describe a proof-of-concept study of a competitive enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) with active-site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold. A biotin functionality immobilizes the probes onto a streptavidin-coated solid support. Dissociation constants were determined with a series of ligands, including enzyme substrates and a library of sulfamoyloxy-linked aminoacyl/aryl-AMP analogues. As it enables direct readout of protein-ligand interaction, the competitive ELISA technique provided information on comparative structure- activity relationships and insights into the enzyme active-site architecture of NRPS A-domains. These studies indicate that the ELISA technique can accelerate the discovery of small-molecule inhibitors of the A-domains with new scaffolds that perturb the production of NRPS-related virulence factors.

The recent discovery that an ERK signaling modulator [ACA-28 (2a)] preferentially kills human melanoma cell lines by inducing ERK-dependent apoptosis has generated significant interest in the field of anti-cancer therapy. In the first SAR study on 2a, here, we successfully developed candidates (2b, 2c) both of which induce more potent and selective apoptosis towards ERK-active melanoma cells than 2a, thus revealing the structural basis for inducing the ERK-dependent apoptosis and proposing the therapeutic prospect of these candidates against ERK-dependent cancers represented by melanoma.