Structural Basis for the Prenylation Reaction of Carbazole-Containing Natural Products Catalyzed by Squalene Synthase-Like Enzymes.

Some enzymes annotated as squalene synthase catalyze the prenylation of carbazole-3,4-quinone-containing substrates in bacterial secondary metabolism. Their reaction mechanisms remain unclear because of their low sequence similarity to well-characterized aromatic substrate prenyltransferases (PTs). We determined the crystal structures of the carbazole PTs, and these revealed that the overall structure is well superposed on those of squalene synthases. In contrast, the stacking interaction between the prenyl donor and acceptor substrates resembles those observed in aromatic substrate PTs. Structural and mutational analyses suggest that the Ile and Asp residues are essential for the hydrophobic and hydrophilic interactions with the carbazole-3,4-quinone moiety of the prenyl acceptor, respectively, and a deprotonation mechanism of an intermediary σ-complex involving a catalytic triad is proposed. Our results provide a structural basis for a new subclass of aromatic substrate PTs.

Structural and Mechanistic Insight into Terpene Synthases that Catalyze the Irregular Non-Head-to-Tail Coupling of Prenyl Substrates.

Terpenoids have diverse structures and thus represent an important class of biologically active natural products. The structural diversity of terpenoids originates from the coupling of prenyl diphosphate substrates, such as isopentenyl diphosphate and dimethylallyl diphosphate. These isoprenyl diphosphates undergo canonical and sequential "head-to-tail" coupling catalyzed by terpene synthases, followed by modifications such as cyclization, hydroxylation, and glycosylation. In recent years, several terpene synthases that catalyze irregular "non-head-to-tail" couplings to afford branched terpenoids have been identified. This minireview describes structural and mechanistic insights into these unusual coupling reactions that provide a new strategy for the structural diversification of natural products.

An Unprecedented Cyclization Mechanism in the Biosynthesis of Carbazole Alkaloids in Streptomyces.

Carquinostatin A (CQS), a potent neuroprotective substance, is a unique carbazole alkaloid with both an ortho-quinone function and an isoprenoid moiety. We identified the entire gene cluster responsible for CQS biosynthesis in Streptomyces exfoliatus through heterologous production of CQS and gene deletion. Biochemical characterization of seven CQS biosynthetic gene products (CqsB1-7) established the total biosynthetic pathway of CQS. Reconstitution of CqsB1 and CqsB2 showed that the synthesis of the carbazole skeleton involves CqsB1-catalyzed decarboxylative condensation of an α-hydroxyl-ß-keto acid intermediate with 3-hydroxybutyryl-ACP followed by CqsB2-catalyzed oxidative cyclization. Based on crystal structures and mutagenesis-based biochemical assays, a detailed mechanism for the unique deprotonation-initiated cyclization catalyzed by CqsB2 is proposed. Finally, analysis of the substrate specificity of the biosynthetic enzymes led to the production of novel carbazoles.

Allosteric activation of cytochrome P450 3A4 by efavirenz facilitates midazolam binding.

1. The purpose of this study is to investigate the heteroactivation mechanism of CYP3A4 by efavirenz, which enhances metabolism of midazolam in vivo, in terms of its binding to CYP3A4 with in vitro spectroscopic methods. 2. Efavirenz exhibited a type II spectral change with binding to CYP3A4 indicating a possible inhibitor. Although dissociation constant (K d) was approximated as 520 µM, efavirenz enhanced binding affinity of midazolam as a co-existing drug with an estimated iK d value of 5.6 µM which is comparable to a clinical concentration. 3. Efavirenz stimulated the formation of 1'-hydroxymidazolam, and the product formation rate (V max) concentration-dependently increased without changing the K m. Besides, an efavirenz analogue, [6-chloro-1,4-dihydro-4-(1-pentynyl)-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one] (efavirenz impurity) slightly facilitated the binding affinity of midazolam in a concentration-dependent manner. These results propose that efavirenz affects midazolam-binding via binding to the peripheral site which is apart from the active site of CYP3A4. 4. A molecular dynamics simulation also suggested the bound-efavirenz was repositioned to effector-binding site. As a consequence, our spectroscopic studies clarified the heteroactivation of CYP3A4 caused by efavirenz with a proper affinity to the peripheral site, and we concluded the method can be a useful tool for characterising the potential for drug-drug interactions.

Design of a Seed-Specific Chimeric Promoter with a Modified Expression Profile to Improve Seed Oil Content.

Increasing the yield of plant oil is an important objective to meet the demand for sustainable resources and energy. Some attempts to enhance the expression of genes involved in oil synthesis in seeds have succeeded in increasing oil content. In many cases, the promoters of seed-storage protein genes have been used as seed-specific promoters. However, conventional promoters are developmentally regulated and their expression periods are limited. We constructed a chimeric promoter that starts to express in the early stage of seed development, and high-level expression is retained until the later stage by connecting the promoters of the biotin carboxyl carrier protein 2 (BCCP2) gene encoding the BCCP2 subunit of acetyl-CoA carboxylase and the fatty acid elongase 1 (FAE1) gene from Arabidopsis. The constructed promoter was ligated upstream of the TAG1 gene encoding diacylglycerol acyltransferase 1 and introduced into Arabidopsis. Seeds from transgenic plants carrying AtTAG1 under the control of the chimeric promoter showed increased oil content (up by 18⁻73%) compared with wild-type seeds. The novel expression profile of the chimeric promoter showed that this could be a promising strategy to manipulate the content of seed-storage oils and other compounds.

Microchip electrophoresis utilizing an in situ photopolymerized Phos-tag binding polyacrylamide gel for specific entrapment and analysis of phosphorylated compounds.

A method was developed for the specific entrapment and separation of phosphorylated compounds using a Phos-tag polyacrylamide gel fabricated at the channel crossing point of a microfluidic electrophoresis chip. The channel intersection of the poly(methyl methacrylate)-made microchip was filled with a solution comprising acrylamide, N,N-methylene-bis-acrylamide, Phos-tag acrylamide, and 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], which functioned as a photocatalytic initiator. In situ polymerization at the channel crossing point was performed by irradiation with a UV LED laser beam. The fabricated Phos-tag gel (100 × 100 × 30 µm) contains ca. 20 fmol of the Phos-tag group and therefore could entrap phosphorylated compounds at the femtomolar level. The electrophoretically trapped phosphorylated compounds were released from the gel by switching the voltage to deliver high concentrations of phosphate and EDTA in a background electrolyte. The broad sample band eluted from the gel was effectively reconcentrated at the boundary of a pH junction generated by sodium ions delivered from the outlet reservoir. The reconcentrated sample components were then separated and fluorometrically detected at the end of the separation channel. Under the optimized conditions, the phosphorylated compounds were concentrated by a factor of 100-fold, and the peak resolution was comparable to that obtained by pinched injection. This method was successfully utilized to preconcentrate and analyze phosphorylated peptides in a complex peptide mixture.

Structure and Mechanism of the Monoterpene Cyclolavandulyl Diphosphate Synthase that Catalyzes Consecutive Condensation and Cyclization.

We report the three-dimensional structure of cyclolavandulyl diphosphate (CLPP) synthase (CLDS), which consecutively catalyzes the condensation of two molecules of dimethylallyl diphosphate (DMAPP) followed by cyclization to form a cyclic monoterpene, CLPP. The structures of apo-CLDS and CLDS in complex with Tris, pyrophosphate, and Mg2+ ion were refined at 2.00 Šresolution and 1.73 Šresolution, respectively. CLDS adopts a typical fold for cis-prenyl synthases and forms a homo-dimeric structure. An in vitro reaction using a regiospecifically 2 H-substituted DMAPP substrate revealed the intramolecular proton transfer mechanism of the CLDS reaction. The CLDS structure and structure-based mutagenesis provide mechanistic insights into this unprecedented terpene synthase. The combination of structural and mechanistic insights advances the knowledge of intricate terpene synthase-catalyzed reactions.

Multi-Dimensional Dynamics of Psychological Health Disparities under the COVID-19 in Japan: Fairness/Justice in Socio-Economic and Ethico-Political Factors.

This article addresses citizens' psychological health disparities in pandemic-stricken Japan from the perspective of positive psychology with a collective/political perspective. Our analysis of three internet surveys in 2020 and 2021 in Japan indicates most people's well-being declined continuously during this period, while some people's well-being increased. As previous studies of health inequality proved about physical health, the objective income/assets level has influenced psychological inequality. This paper demonstrated this relation in Japan, although it is often mentioned as an egalitarian country with comparatively better health conditions. Moreover, psychological levels and changes have been associated with biological, natural environmental, cultural, and social factors. Social factors include economic, societal-community, and political factors, such as income/assets, stratification, general trust, and fairness/justice. Accordingly, multi-dimensional disparities are related to psychological health disparity; tackling the disparities along the multi-layered strata is desirable. Furthermore, subjective perception of fairness/justice is significantly associated with the level of psychological health and mitigating its decrease. Thus, fairness and justice are found to be dynamic and protective factors against the decline of psychological health. While relatively little literature on health inequality analyzes fairness/justice philosophically, this paper highlights these together with income/assets by clarifying the significance of multi-dimensional factors: natural environmental, cultural, socioeconomic, and political.

Psychological Examination of Political Philosophies: Interrelationship Among Citizenship, Justice, and Well-Being in Japan.

This paper examines assumptions concerning the relationship between citizenship, justice, and well-being, based on representative political philosophies, including egoism, utilitarianism, libertarianism, liberalism, and communitarianism. A previous paper raised the possibility of an inter-disciplinary framework for collaboration between psychology and political philosophy. This study picks up that thread and attempts to actualize a collaborative research effort based on a framework grounded in positive political psychology. The first part of this study reflects on the methodology situated between empirical psychology and philosophy in reference to the debates caused by psychological and philosophical situationism. In response to its criticism against virtue ethics, the possibility of reconstructing it on empirical psychology has paradoxically emerged. Similarly, this study validates assumptions on political philosophies employing the psychological method concerning well-being. Accordingly, the central part examines the plausibility of the assumptions by empirical evidence obtained from two internet surveys (2020, N = 5000; 2021, N = 6885) in Japan. The relationships between citizenship, justice, and well-being are the most substantial in the communitarian assumption. The exploratory factor analysis of the two surveys illuminates that the correlations between citizenship, justice, and well-being (or political well-being) are substantial. This relationship denies the egoism assumption. Moreover, almost all correlations between the three are higher based on virtue-related indicators than hedonic ones. These findings are not in tune with the utilitarian assumption and are most congruent to the communitarian assumption. In addition, citizenship and justice correlate more with political well-being than overall well-being. As these are more directly associated with political well-being in the communitarian assumption, this result aligns with the assumption. Furthermore, the positive relationship between disparity elimination and well-being fits the liberal rather than the libertarian assumption. Nevertheless, the substantial correlation between ethical justice and well-being is higher by virtue-related indicators than hedonic indicators, suggesting distributive justice is associated with the ethical dimension. Again, this fits the communitarian assumption rather than the liberal assumption. Thus, philosophical psychology empirically verifies the interdependence of the three conceptions and the relative plausibility of the communitarian assumption. Moreover, as the relationship between the three is essential for political philosophies, the result increases the reliability of communitarianism.

Political Philosophies and Positive Political Psychology: Inter-Disciplinary Framework for the Common Good.

This manuscript explores the relationship between positive psychology and political philosophy, revealing an inter-disciplinary approach that speaks to the concerns of the common good. Since positive psychology has been expanding its reach into social and political spheres, its relationship to philosophical arguments has been worthy of exploration. Positive psychology is associated with utilitarianism, and aspects of hedonic psychology. However, an alternative concept of eudaimonic well-being has enabled this psychology to have links to other political philosophies. Therefore, this manuscript provides an overview of contemporary political philosophies: first, it discusses the debate between liberalism and communitarianism, and secondly, it summarizes the subsequent developments of liberal perfectionism, capability approach, and deliberative democracy. Then, the configuration of these political philosophies is indicated by the figure of two axes of "individual/collective" and "ethical/non-ethical." The following section compiles the inter-relationships between the conceptions of citizenship, justice, and well-being, regarding the main political philosophies: egoism, utilitarianism, libertarianism, liberalism, communitarianism, and conservatism. Utilitarianism is associated with happiness, while liberalism and libertarianism rely on the concept of rights, which is almost equal to the idea of justice. Accordingly, utilitarianism is a philosophy of well-being, while liberalism and libertarianism are philosophies of justice. However, there is little connection between well-being and justice in these philosophies because the two kinds of philosophies are incompatible. The latter kind criticizes the former because the maximization of happiness can infringe on people's rights. Moreover, these philosophies do not particularly value citizenship. In contrast, communitarianism is intrinsically the political philosophy of citizenship most attuned to increasing well-being, and it can connect an idea of justice with well-being. The final part offers a framework to develop an inter-disciplinary collaboration. Positive psychology can provide the empirical basis of the two axes above concerning political philosophies. On the other hand, the correspondence makes the character of political philosophies clearer. While libertarianism and liberalism correspond to psychology as usual, utilitarianism and communitarianism correspond to positive psychology, and the latter can be regarded as positive political philosophies. This recognition leads to the interdisciplinary framework, enabling multi-disciplinary collaboration, including work with the social sciences, which could benefit the common good.

Recent Advances in the Biosynthesis of Carbazoles Produced by Actinomycetes.

Structurally diverse carbazole alkaloids are valuable due to their pharmaceutical properties and have been isolated from nature. Experimental knowledge on carbazole biosynthesis is limited. The latest development of in silico analysis of the biosynthetic gene clusters for bacterial carbazoles has allowed studies on the biosynthesis of a carbazole skeleton, which was established by sequential enzyme-coupling reactions associated with an unprecedented carbazole synthase, a thiamine-dependent enzyme, and a ketosynthase-like enzyme. This review describes the carbazole biosynthetic mechanism, which includes a key step in enzymatic formation of a tricyclic carbazole skeleton, followed by modifications such as prenylation and hydroxylation in the skeleton.

Extending the piezoelectric transducer bandwidth of an optical interferometer by suppressing resonance using a high dimensional IIR filter implemented on an FPGA.

This paper considers the application of Field Programmable Gate Array (FPGA)-based infinite impulse response (IIR) filtering to increase the usable bandwidth of a piezoelectric transducer used in optical phase locking. We experimentally perform system identification of the interferometer with the cross-correlation method integrated on the controller hardware. Our model is then used to implement an inverse filter designed to suppress the low frequency resonant modes of the piezoelectric transducer. This filter is realized as a 24th-order IIR filter on the FPGA, while the total input-output delay is kept at 350 ns. The combination of the inverse filter and the piezoelectric transducer works as a nearly flat response position actuator, allowing us to use a proportional-integral (PI) control in order to achieve stability of the closed-loop system with significant improvements over a non-filtered PI control. Finally, because this controller is completely digital, it is straightforward to reproduce. Our control scheme is suitable for many experiments that require highly accurate control of flexible structures.

High-Salt Intake Ameliorates Hyperglycemia and Insulin Resistance in WBN/Kob-Leprfa/fa Rats: A New Model of Type 2 Diabetes Mellitus.

High-salt intake is a major risk factor for developing hypertension in type 2 diabetes mellitus, but its effects on glucose homeostasis are controversial. We previously found that high-salt intake induces severe hypertension in WBN/Kob diabetic fatty (WBKDF) rats. In the present study, we examined the effects of a high-salt intake on glucose homeostasis in WBKDF rats. Male WBKDF rats and age-matched Wistar rats at 6 weeks of age were each divided into two groups and fed either a normal-sodium (NS, 0.26%) diet or high-sodium (HS, 8%) diet for 7 weeks. Systolic blood pressure and urine volume were increased in WBKDF-HS and Wistar-HS. Body weight gain and food consumption were comparable between NS and HS in both strains. Plasma and urine glucose levels were significantly increased in WBKDF-NS but not in WBKDF-HS. HOMA-IR in WBKDF-HS was significantly lower compared with that in WBKDF-NS. The high plasma adiponectin level in WBKDF-NS compared with that in Wistar-NS was further enhanced in WBKDF-HS. Glycogen deposits and fat droplets in the livers of WBKDF-HS were reduced compared with those of WBKDF-NS. The present study demonstrated that HS intake ameliorated hyperglycemia and insulin resistance in WBKDF rats, which may be due to increased plasma levels of adiponectin.

Objective image analysis of non-magnifying image-enhanced endoscopy for diagnosis of small depressed early gastric cancers.

Background Gastric cancers (GC) after H. pylori eradication are difficult to diagnose even by magnifying narrow-band imaging (NBI) or blue laser imaging (BLI) endoscopy. Little is known with regard to non-magnifying (NM)-NBI/BLI for early GC so we examined the efficacy of NM-NBI/BLI for early GC diagnosis. Methods We retrospectively analyzed the images of 29 small (≤ 1 cm) intramucosal GC that had been treated with endoscopic submucosal dissection and 137 benign depressed lesions (BDLs). The brightness and shape of the GCs and BDLs by NM-NBI/BLI were assessed with ImageJ software. Results The NBI/BLI-index, which indicates the brightness of NBI/BLI for visualization, was significantly higher in GC than BDLs in both the H. pylori -infected ( P  = 0.009) and -eradicated group ( P  < 0.0001), indicating that GC exhibited brighter colors than the normal surrounding mucosa. The C-index, which refers to the circularity of the lesion, was also significantly higher in GC than BDLs in both H. pylori -infected ( P  = 0.006) and -eradicated cases ( P  = 0.004). Based on receiver-operating characteristic curve analysis, cutoff values for the NBI/BLI- and C-indices for GC were 1.04 and 0.58 in the H. pylori -infected cases, and 0.98 and 0.64 in the H. pylori -eradicated cases. With the reference value of the NBI/BLI-index set at ≥ 0.69 with the C-index at ≥ 0.21 in the H. pylori -infected and the NBI/BLI-index at ≥ 0.80 with the C-index at ≥ 0.32 in the H. pylori -eradicated cases, both the sensitivity and negative predictive value for early GC were 100 %. A high NBI/BLI-index tended to be associated with a wide length of the intervening part histologically in the H. pylori -eradicated cases ( P  = 0.09). Conclusions The small depressed-type early GC had brighter color and rounder shape compared to BDLs in both H. pylori -infected and -eradicated cases. The NBI/BLI- and C-indices calculated by the image analysis may facilitate identification of small depressed-type GC.

Toll-like receptor-dependent production of IL-12p40 causes chronic enterocolitis in myeloid cell-specific Stat3-deficient mice.

Stat3 plays an essential role in IL-10 signaling pathways. A myeloid cell-specific deletion of Stat3 resulted in inflammatory cytokine production and development of chronic enterocolitis with enhanced Th1 responses in mice. In this study, we analyzed the mechanism by which a Stat3 deficiency in myeloid cells led to the induction of chronic enterocolitis in vivo. Even in the absence of Stat1, which is essential for IFN-gamma signaling pathways, Stat3 mutant mice developed chronic enterocolitis. TNF-alpha/Stat3 double-mutant mice developed severe chronic enterocolitis with enhanced Th1 cell development. IL-12p40/Stat3 double-mutant mice, however, showed normal Th1 responses and no inflammatory change in the colon. RAG2/Stat3 double-mutant mice did not develop enterocolitis, either. These findings indicate that overproduction of IL-12p40, which induces potent Th1 responses, is essential for the development of chronic enterocolitis in Stat3 mutant mice. Furthermore, enterocolitis was significantly improved and IFN-gamma production by T cells was reduced in TLR4/Stat3 double-mutant mice, indicating that TLR4-mediated recognition of microbial components triggers aberrant IL-12p40 production by myeloid cells, leading to the development of enterocolitis. Thus, this study clearly established a sequential innate and acquired immune mechanism for the development of Th1-dependent enterocolitis.

Enhanced expression of programmed death-1 (PD-1)/PD-L1 in salivary glands of patients with Sjögren's syndrome.

OBJECTIVE: Programmed death-1 (PD-1) mediates a negative signal and introduces tolerance for lymphocytes. Dysfunction of the PD-1 pathway is thought to result in autoimmune diseases such as rheumatoid arthritis (RA). To investigate the role of the PD-1/PD-L system in the pathology of Sjögren's syndrome (SS), we examined the expression of PD-1 and its ligand PD-L1 in salivary lymphocytes and salivary glands from patients with SS. METHODS: Flow cytometry analysis was used to determine expression of PD-1 in SS salivary lymphocytes. Intracellular staining of interleukin 10 (IL-10) was performed after stimulation with PMA and ionomycin. Indirect immunohistochemistry was used to investigate the expression of PD-1 and PD-L1. RESULTS: The mean fluorescence intensity of PD-1 expression in SS salivary lymphocytes was significantly higher than that from healthy controls and patients with RA or systemic lupus erythematosus. PD-1-positive SS salivary lymphocytes expressed IL-10 intracellularly upon PMA/ionomycin stimulation. Immunohistochemical analysis showed that PD-1 was expressed on infiltrating lymphocytes in salivary gland from 52% of SS patients, and PD-L1 was expressed on ductal and acinar epithelial cells from 68% of SS patients. In vitro analysis using HSG cells revealed that PD-L1 was induced by interferon-gamma but not by tumor necrosis factor-alpha and IL-1beta. CONCLUSION: PD-1 is expressed on T lymphocytes and PD-L1 on epithelial cells from inflamed salivary glands of patients with SS, which suggests that dysfunction of the PD-1/PD-L1 pathway may be related to tolerance for lymphocytes, which causes SS.

CD4+ PD-1+ T cells accumulate as unique anergic cells in rheumatoid arthritis synovial fluid.

OBJECTIVE: The PD-1 receptor, whose deficiency in mice causes autoimmune diseases such as arthritis, is considered to be a negative regulator of activated T cells and to play a crucial role in peripheral tolerance. To clarify the involvement of the PD-1 system in rheumatoid arthritis (RA), we investigated PD-1 expression on synovial fluid (SF) T cells from patients with RA. METHODS: FACS analysis for PD-1 was performed on SF T cells from 44 patients with RA and 6 with osteoarthritis (OA), and also on peripheral blood (PB) T cells from 12 RA patients and 7 healthy controls. Two-color analysis of cell surface PD-1 expression and the intracellular concentration of cytokine production was used to investigate CD4+ T cells from SF of patients with RA and PB from controls. RESULTS: Scarcely any PD-1 expression was detected on control PB T or OA SF T cells. In contrast, PD-1+ cells made up 20.9 +/- 8.6% (mean +/- SD) of RA SF T cells. In RA SF, PD-1 was expressed more predominantly on CD4+ T cells than on CD8+ T cells. As well as expressing CD45RO and CXCR3, CD4+ PD-1+ T cells were mostly CTLA-4 positive and CD26 negative, and were enriched in CD45RB(low) cells. Intracellular cytokine staining revealed that CD4+ PD-1+, but not CD4+ PD-1-, T cells produced interleukin 10 (IL-10), and that CD4+ PD-1+ T cells produced less IL-2 than CD4+ PD-1- T cells. CONCLUSION: PD-1+ T cells in RA SF are enriched, and phenotypic analysis suggests that these cells constitute a unique anergic T cell subset in RA SF.

Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4.

Signal transduction through Toll-like receptors (TLRs) originates from their intracellular Toll/interleukin-1 receptor (TIR) domain, which binds to MyD88, a common adaptor protein containing a TIR domain. Although cytokine production is completely abolished in MyD88-deficient mice, some responses to lipopolysaccharide (LPS), including the induction of interferon-inducible genes and the maturation of dendritic cells, are still observed. Another adaptor, TIRAP (also known as Mal), has been cloned as a molecule that specifically associates with TLR4 and thus may be responsible for the MyD88-independent response. Here we report that LPS-induced splenocyte proliferation and cytokine production are abolished in mice lacking TIRAP. As in MyD88-deficient mice, LPS activation of the nuclear factor NF-kappaB and mitogen-activated protein kinases is induced with delayed kinetics in TIRAP-deficient mice. Expression of interferon-inducible genes and the maturation of dendritic cells is observed in these mice; they also show defective response to TLR2 ligands, but not to stimuli that activate TLR3, TLR7 or TLR9. In contrast to previous suggestions, our results show that TIRAP is not specific to TLR4 signalling and does not participate in the MyD88-independent pathway. Instead, TIRAP has a crucial role in the MyD88-dependent signalling pathway shared by TLR2 and TLR4.