Convergent approach for direct cross-coupling enabled by flash irreversible generation of cationic and anionic species.

In biosynthesis multiple kinds of reactive intermediates are generated, transported, and reacted across different parts of organisms, enabling highly sophisticated synthetic reactions. Herein we report a convergent synthetic approach, which utilizes dual intermediates of cationic and carbanionic species in a single step, hinted at by the ideal reaction conditions. By reactions of unsaturated precursors, such as enamines, with a superacid in a flow microreactor, cationic species, such as iminium ions, are generated rapidly and irreversibly, and before decomposition, they are transported to react with rapidly and independently generated carbanions, enabling direct C-C bond formation. Taking advantage of the reactivity of these double reactive intermediates, the reaction take place within a few seconds, enabling synthetic reactions which are not applicable in conventional reactions.

Maillard-type glycated collagen with alginate oligosaccharide suppresses inflammation and oxidative stress by attenuating the expression of LPS receptors Tlr4 and Cd14 in macrophages.

Inflammation and oxidative stress contribute to noncommunicable diseases (NCDs), with macrophages playing pivotal roles. Glycated collagen through Maillard-type glycation holds promise for enhancing anti-inflammatory properties, but its mechanism remains unclear. This study investigates the cellular mechanism and aims to contribute to expanding collagen utilization. Collagen was glycated with alginate oligosaccharide (AO) and glucose (Glc: as a comparative case) at 60 °C and 35% relative humidity for up to 24 h (C-AO and C-Glc, respectively). The anti-inflammatory activities of both C-AO and C-Glc were evaluated using an LPS-stimulated macrophage model. 18 h AO-glycated collagen (C-AO18 h) was found to significantly reduce the production of nitric oxide and proinflammatory cytokines (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß). In contrast, C-Glc did not exhibit enhanced anti-inflammatory activity during any of the glycation periods. The enhanced anti-inflammatory activity of C-AO18 h was attributed to its downregulating effect on LPS receptors (toll-like receptor 4, Tlr4; cluster of differentiation 14, Cd14) and myeloid differentiation primary response 88 (Myd88) mRNA expression, with suppression in receptor expression resulting in decreased phagocytic ability of macrophages against E. coli. In addition, compared with intact collagen, C-AO18 h exhibited improved antioxidant activity in the LPS-stimulated macrophage model, as it significantly upregulated superoxide dismutase (SOD) and catalase (CAT) activities while reducing malondialdehyde (MDA) levels. Overall, this study contributes to the development of collagen-based functional foods for mitigating inflammation and oxidative stress in NCDs.

Analysis of the Medication Persistence Rate for and Adherence to Oral 5-Aminosalicylic Acid Preparations in Japanese Patients with Ulcerative Colitis: Study Using a Nationwide Claims Database.

INTRODUCTION: 5-aminosalicylic acid (5-ASA) is the first-line drug for the treatment of mild-to-moderate ulcerative colitis (UC). Three oral sustained-release formulations are often used. However, no unified view of their actual use in routine medical practice has been presented to date. METHODS: Using a health insurance claims database, we extracted patients with an initial diagnosis of mild-to-moderate UC during the period from December 1, 2017, to March 31, 2022. For the three types of oral 5-ASA formulation, we calculated and compared descriptive statistics of medication persistence rates (MPR), proportions of days covered (PDC), and adherence proportion (PDC ≥80%) in the extracted population. RESULTS: An oral 5-ASA formulation was used in combination with a topical preparation (cohort 1) in 899 patients, and oral 5-ASA was used alone (cohort 2) in 1,829 patients. In cohort 1, MPR at days 151-180 with concomitant use of topical formulation was significantly higher for the Multi Matrix System™ (MMX) formulation (65.2%) compared with that for pH-dependent formulation (51.7%, p < 0.025), while MPR tended to be higher for MMX than for the time-dependent formulation (56.4%, not significant). During days 151-180 after starting the oral formulation, MPR for MMX (66.7% and 65.8%) was higher than for pH-dependent (55.9% and 55.3%) and time-dependent (57.6% and 55.9%) formulations in cohorts 1 + 2 and 2, respectively. In cohort 1, there was a significant difference between MMX (68.3%) and pH-dependent (57.1%) formulations, but no significant difference was seen with time-dependent formulations (61.8%). In terms of the proportion of adherence until day 180, MMX was significantly better than the other formulations. CONCLUSION: The analyses of the three oral 5-ASA formulations suggested that both MPR and medication adherence were better for the MMX formulation than for time-dependent or pH-dependent formulations.

Development of a liquid chromatography-based versatile bioanalysis for bevacizumab based on pretreatment combining aptamer affinity purification and centrifugal ultrafiltration concentration.

We report on the development of a versatile and accurate bioanalytical method for bevacizumab using a pretreatment method combining affinity purification with anti-idiotypic DNA aptamers and centrifugal ultrafiltration concentration, followed by liquid chromatography (LC)-fluorescence analysis. An affinity purification method using Sepharose beads as an affinity support removed immunoglobulin G and a large amount of coexisting substances in the serum sample. Purified bevacizumab was separated as a single peak by conventional LC and detected fluorometrically, showing good linearity (R2 = 0.999) in the range of 5-200 µg/mL, sufficient to analyze bevacizumab concentrations in the blood of bevacizumab-treated patients. By combining this purification method with a concentration method using a centrifugal filtration device that inhibits non-specific adsorption of bevacizumab, the quantitative range was reduced by a factor of 10 while showing good linearity (R2 = 0.999) in the 0.5-20 µg/mL range. The developed analytical method is expected to be used not only for general bioanalysis of therapeutic mAbs in clinical settings, but also for next-generation antibody drugs that show drug efficacy at low concentrations and for analysis of trace samples.

Variation in shrimp tropomyosin allergenicity during the production of Terasi, an Indonesian fermented shrimp paste.

Terasi is a fermented shrimp paste in Indonesia. We examined the effect of the Terasi manufacturing process on the abundance of the allergen tropomyosin (TM) and its IgG/IgE-binding ability. Terasi was produced from three shrimps, Akiami (Acetes japonicus), Okiami (Euphausia pacifica), and Isazaami (Neomysis awatchensis). Protein degradation and TM IgE-binding activity were examined by immunoblotting using anti-TM rabbit IgG and competitive enzyme-linked immunosorbent assays using shrimp-allergic patients' sera. The processing caused TM degradation, and the IgG-specific response in Akiami meat disappeared at the second fermentation step but remained in both Okiami and Isazaami Terasi. In contrast, TM IgE-binding in all meats decreased gradually during manufacturing and nearly completely disappeared in Akiami Terasi. Conclusively, Terasi production is an effective manufacturing process to reduce the IgE-binding ability of TM, and Terasi can be recognized as a low allergenic seafood when produced under an appropriate manufacturing condition.

Glycation with uronic acid-type reducing sugar enhances the anti-inflammatory activity of fish myofibrillar protein via the Maillard reaction.

The role of carboxyl group in uronic acid in enhancing the anti-inflammatory activity of fish myofibrillar protein (Mf) was investigated, when lyophilized Mf was reacted with various reducing sugars at 60 °C and 35% relative humidity through the Maillard reaction. After pepsin and trypsin digestion, the anti-inflammatory activity was evaluated by measuring the secretions of tumor necrosis factor-α, interleukin-6, interleukin-1ß, and nitric oxide in lipopolysaccharide-stimulated RAW 264.7 macrophage. The anti-inflammatory activity of Mf was not affected by glycation with glucose or galactose, whereas strongly enhanced by glycation with uronic acid-type reducing sugars: glucuronic acid, galacturonic acid, and alginate oligosaccharide. These results indicate that the presence of carboxyl group in reducing sugar is important for enhancing the anti-inflammatory activity of Mf. This study also shows that the enhanced effect could depend upon the number of carboxyl group in bound reducing sugar.

Efficacy of Low-Dose Estrogen-Progestins and Progestins in Japanese Women with Dysmenorrhea: A Systematic Review and Network Meta-analysis.

INTRODUCTION: Although several studies suggest beneficial effects of low-dose estrogen-progestins (LEPs) and progestins on dysmenorrhea in Japanese women, the difference in efficacy between drugs remains unknown. METHODS: We identified studies by searching the MEDLINE, Cochrane Library, and ICHUSHI databases and included randomized controlled trials (RCTs) that used total dysmenorrhea score and visual analogue scale (VAS) as outcome measures to evaluate LEPs and progestins for primary and secondary dysmenorrhea. We analyzed results by meta-analysis and network meta-analysis (NMA). RESULTS: We identified 10 articles on eight RCTs and included seven drugs (six LEPs and one progestin, i.e., dienogest) and placebo in the analysis. Meta-analysis showed improvements in total dysmenorrhea score and VAS for almost all drugs compared with placebo. In NMA, VAS in secondary dysmenorrhea improved more with dienogest than with norethisterone/ethinylestradiol (mean difference - 25.84 [95% CrI - 44.46 to - 7.15]). In the comparison of administration regimens, VAS improved more with progestin-continuous than LEP-cyclic and the surface under the cumulative ranking (SUCRA) of LEP-extended and progestin-continuous appeared to be higher than that of LEP-cyclic. CONCLUSIONS: We confirmed that LEPs and dienogest are effective for primary and secondary dysmenorrhea and suggest that continuous regimens may be more effective than cyclic regimens in improving outcomes.

Comparison of Maillard-Type Glycated Collagen with Alginate Oligosaccharide and Glucose: Its Characterization, Antioxidant Activity, and Cytoprotective Activity on H2O2-Induced Cell Oxidative Damage.

To improve the antioxidant activity of collagen molecules using Maillard-type glycation, the relation between antioxidant activity and progress indexes for the Maillard reaction must be understood. In this study, lyophilized tilapia scale collagen was mixed with a half weight of alginate oligosaccharide (AO) or glucose and incubated at 60 °C and 35% relative humidity for up to 18 h to produce the Maillard-type glycated collagen (C-AO and C-Glu, respectively). As glycation progressed, the amount of conjugated sugar coupled with UV-vis absorbance at 294 nm and 420 nm increased more rapidly in C-Glu than in C-AO, and the available lysine decreased rapidly in C-Glu compared with C-AO. The early-to-middle- and late-stage products of the Maillard reaction were involved in enhanced antioxidant activity of digested C-AO and digested C-Glu, respectively. Additionally, C-AO acquired the antioxidant activity without marked available lysine loss. The cytoprotective effect of collagen in H2O2-induced damage was enhanced by glycation, achieved by reducing malondialdehyde content and increasing superoxide dismutase and catalase activities. These results indicate that AO is an excellent reducing sugar that enhances the health benefits of collagen without excessive loss of lysine, which is a nutritional problem of the Maillard-type glycation.

Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation.

Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) are a promising cell source to treat age-related macular degeneration (AMD). Despite several ongoing clinical studies, a detailed mapping of transient cellular states during in vitro differentiation has not been performed. Here, we conduct single-cell transcriptomic profiling of an hESC-RPE differentiation protocol that has been developed for clinical use. Differentiation progressed through a culture diversification recapitulating early embryonic development, whereby cells rapidly acquired a rostral embryo patterning signature before converging toward the RPE lineage. At intermediate steps, we identified and examined the potency of an NCAM1+ retinal progenitor population and showed the ability of the protocol to suppress non-RPE fates. We demonstrated that the method produces a pure RPE pool capable of maturing further after subretinal transplantation in a large-eyed animal model. Our evaluation of hESC-RPE differentiation supports the development of safe and efficient pluripotent stem cell-based therapies for AMD.

Development of a DNA aptamer that binds to the complementarity-determining region of therapeutic monoclonal antibody and affinity improvement induced by pH-change for sensitive detection.

Therapeutic monoclonal antibodies (mAbs) are successful biomedicines; however, evaluation of their pharmacokinetics and pharmacodynamics demands highly specific discrimination from human immunoglobulin G naturally present in the blood. Here, we developed a novel anti-idiotype aptamer (termed A14#1) with extraordinary specificity against the anti-vascular endothelial growth factor therapeutic mAb, bevacizumab. Structural analysis of the antibody-aptamer complex showed that several bases of A14#1 recognized only the complementarity determining region (CDR) of bevacizumab, thereby contributing to its extraordinary specificity. As the CDR of bevacizumab is predicted to be highly positively charged under mildly acidic conditions and that DNA is negatively charged, the affinity of A14#1 to bevacizumab markedly increased at pH 4.7 (KD = 44 pM) than at pH 7.4 (KD = 12 nM). A14#1-based electrochemical detection method capable of detecting 31 pM of bevacizumab at pH 4.7 was thus developed. A14#1 could be potentially useful for therapeutic drug measurement as a novel ligand of bevacizumab.

Insight into the Ferrier Rearrangement by Combining Flash Chemistry and Superacids.

The transformation of glycals into 2,3-unsaturated glycosyl derivatives, reported by Ferrier in 1962, is supposed to involve an α,ß unsaturated glycosyl cation, an elusive ionic species that has still to be observed experimentally. Herein, while combination of TfOH and flow conditions failed to observe this ionic species, its extended lifetime in superacid solutions allowed its characterization by NMR-based structural analysis supported by DFT calculations. This allyloxycarbenium ion was further exploited in the Ferrier rearrangement to afford unsaturated nitrogen-containing C-aryl glycosides and C-alkyl glycosides under superacid and flow conditions, respectively.

Digestibility in the gastrointestinal tract and migration to blood of ß'-component (Onk k 5), a major salmon roe IgE-binding protein.

The major allergen of chum salmon (Oncorhynchus keta) roe is the ß'-component (Onc k 5, ß'-c), which is a yolk protein and a fragment of vitellogenin. When yolk content containing ß'-c was orally administered to mice, ß'-c passed through the gastrointestinal tract and was excreted in feces without marked degradation. The direct administration of ß'-c to ligated jejunal and ileal loops showed that ß'-c was absorbed through the small intestine and transferred into the blood. Immunohistochemical staining showed that orally administered ß'-c was distributed from the apical side to the basal side of intestinal epithelial cells, suggesting that endocytosis may be involved in the intestinal absorption of ß'-c. In conclusion, ß'-c is absorbed along a large portion of the small intestine and circulates in the blood stream without significant digestion. The resistance of ß'-c to gastrointestinal digestion seems to contribute to its strong allergenicity.

Anti-Idiotype DNA Aptamer Affinity Purification⁻High-Temperature Reversed-Phase Liquid Chromatography: A Simple, Accurate, and Selective Bioanalysis of Bevacizumab.

This study presents a simple, accurate, and selective bioanalytical method of bevacizumab detection from plasma samples based on aptamer affinity purification⁻high-temperature reversed-phased liquid chromatography (HT-RPLC) with fluorescence detection. Bevacizumab in plasma samples was purified using magnetic beads immobilized with an anti-idiotype DNA aptamer for bevacizumab. The purified bevacizumab was separated with HT-RPLC and detected with its native fluorescence. Using aptamer affinity beads, bevacizumab was selectively purified and detected as a single peak in the chromatogram. HT-RPLC achieved good separation for bevacizumab with a sharp peak within 10 min. The calibration curves of the two monoclonal antibodies ranged from 1 to 50 µg/mL and showed good correlation coefficients (r² > 0.999). The limit of detection (LOD) and lower limit of quantification (LLOQ) values for bevacizumab were 0.15 and 0.51 µg/mL, respectively. The proposed method was successfully applied to the bioanalysis of the plasma samples obtained from the patients with lung cancer and may be extended to plan optimal therapeutic programs and for the evaluation of biological equivalencies in the development of biosimilars.

High-Throughput Bioanalysis of Bevacizumab in Human Plasma Based on Enzyme-Linked Aptamer Assay Using Anti-Idiotype DNA Aptamer.

We propose a highly selective, sensitive, accurate, and high-throughput bioanalysis method for bevacizumab utilizing an anti-idiotype DNA aptamer. With this method, bevacizumab in a plasma sample was reacted in a 96-well plate immobilized with the aptamer and further reacted with a protein A-HRP conjugate. The resulting HRP activity was colorimetrically detected using a microplate reader. The calibration curve of bevacizumab ranged from 0.05 to 5.0 µg/mL, and showed a good correlation coefficient ( r2 = 1.000). The limit of detection was 2.09 ng/mL. We also demonstrated both the possibility of highly sensitive detection using luminol chemiluminescence and the repeated use of affinity plates. The proposed method is applicable for planning optimal therapeutic programs and for an evaluation of the biological equivalencies in the development of biosimilars.

LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis.

The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR1 1 . Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death2-8. In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype9-11. Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1-/- (also known as Rbck1-/-) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3-/-Casp8-/-Hoil-1-/- embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.

Anti-inflammatory effects of dulse (Palmaria palmata) resulting from the simultaneous water-extraction of phycobiliproteins and chlorophyll a.

The use of dulse (Palmaria palmata) as a source of edible anti-inflammatory products was evaluated in this study. Phycobiliproteins and chlorophyll a were simultaneously extracted from lyophilized dulse leaves via water-extraction, and subjected to thermolysin digestion to produce thermolysin-digested water-extract (d-DWE). d-DWE significantly reduced tumor necrosis factor-α, interleukin-6, and nitric oxide in LPS-stimulated murine macrophages (RAW 264.7 cells), and orally administered d-DWE mitigated acute inflammation in carrageenan-induced paw edema of mice. Mass spectrometry revealed d-DWE contained peptide LRDGEIILRY (derived from phycoerythrin ß-chain) and chlorophyll a decomposition products, and they individually reduced the secretion of the proinflammatory mediators in LPS-stimulated RAW 264.7 cells. These results indicate the anti-inflammatory activity could be from a combined effect of phycobiliprotein and chlorophyll a decomposition products prepared from the water-extract of dulse. Thus, inexpensive and safe water-extraction method is effective for the extraction of anti-inflammatory components from dulse.

A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development.

Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.

Dienogest inhibits C-C motif chemokine ligand 20 expression in human endometriotic epithelial cells.

OBJECTIVE: C-C motif chemokine ligand 20 is thought to contribute to the development of endometriosis by recruiting Th17 lymphocytes into endometriotic foci. The present study investigated the effects of dienogest, a progesterone receptor agonist used to treat endometriosis, on C-C motif chemokine ligand 20 expression by endometriotic cells. STUDY DESIGN: Effects of dienogest on mRNA expression and protein secretion of C-C motif chemokine ligand 20 induced by interleukin 1ß were assessed in three immortalized endometriotic epithelial cell lines, parental cells (EMosis-CC/TERT1), and stably expressing human progesterone receptor isoform A (EMosis-CC/TERT1/PRA+) or isoform B (EMosis-CC/TERT1/PRA-/PRB+). RESULTS: Dienogest markedly inhibited interleukin 1ß-stimulated C-C motif chemokine ligand 20 mRNA expression and protein secretion in EMosis-CC/TERT1/PRA-/PRB+, which was abrogated by the progesterone receptor antagonist RU486. In EMosis-CC/TERT1/PRA+, dienogest slightly inhibited C-C motif chemokine ligand 20 mRNA and protein. In EMosis-CC/TERT1, dienogest slightly inhibited C-C motif chemokine ligand 20 mRNA, but had no effect on C-C motif chemokine ligand 20 protein. CONCLUSION: Dienogest inhibited interleukin 1ß-induced up-regulation of C-C motif chemokine ligand 20 in endometriotic epithelial cells, mainly mediated by progesterone receptor B.

The linear ubiquitin chain assembly complex regulates TRAIL-induced gene activation and cell death.

The linear ubiquitin chain assembly complex (LUBAC) is the only known E3 ubiquitin ligase which catalyses the generation of linear ubiquitin linkages de novo LUBAC is a crucial component of various immune receptor signalling pathways. Here, we show that LUBAC forms part of the TRAIL-R-associated complex I as well as of the cytoplasmic TRAIL-induced complex II In both of these complexes, HOIP limits caspase-8 activity and, consequently, apoptosis whilst being itself cleaved in a caspase-8-dependent manner. Yet, by limiting the formation of a RIPK1/RIPK3/MLKL-containing complex, LUBAC also restricts TRAIL-induced necroptosis. We identify RIPK1 and caspase-8 as linearly ubiquitinated targets of LUBAC following TRAIL stimulation. Contrary to its role in preventing TRAIL-induced RIPK1-independent apoptosis, HOIP presence, but not its activity, is required for preventing necroptosis. By promoting recruitment of the IKK complex to complex I, LUBAC also promotes TRAIL-induced activation of NF-κB and, consequently, the production of cytokines, downstream of FADD, caspase-8 and cIAP1/2. Hence, LUBAC controls the TRAIL signalling outcome from complex I and II, two platforms which both trigger cell death and gene activation.