Cinnamomum species, including true cinnamon (Cinnamomum verum) and less explored species . UPLC-MS led to the annotation of 74 secondary metabolites belonging to different classes, including phenolic acids, tannins, flavonoids, and lignans. A new proanthocyanidin was identified for the first time in C. tamala, along with several glycosylated flavonoid and dicarboxylic fatty acids reported for the first time in cinnamon. Multivariate data analyses revealed, for cinnamates, an abundance in C. verum versus procyandins, dihydro-coumaroylglycosides, and coumarin in C. cassia. A total of 51 primary metabolites were detected using GC-MS analysis encompassing different classes, viz. sugars, fatty acids, and sugar alcohols, with true cinnamon from Malaysia suggested as a good sugar source for diabetic patients. Glycerol in C. tamala, erythritol in C. iners, and glucose and fructose in C. verum from Malaysia were major metabolites contributing to the discrimination among species.The present study aimed to assess metabolites heterogeneity among four major  Cinnamomum − in the negative ion mode. Common fragmentation pathways for PAs include Retro Diels–Alder fission (RDA-F) [M − H-152]−, heterocyclic ring fission (HRF) [M − H-126]−, benzofuran-forming fission (BFF) [M − H-122]−, and quinine methide cleavage (QMC) that yielded characteristic ions for sequencing of PAs [Proanthocyanidins show UV-absorbance maxima at 235 and 280 nm corresponding to the absorbance spectrum of flavan-3-ols. UPLC-ESI-MSg of PAs .− at m/z 1151.2454 (C60H47O24)−] and [(M + H)+ at 1153.2626 (C60H49O24)+] respectively, indicate a procyanidin trimer with A-type interflavanyl bonds. It had main fragment ions at m/z 863 [−288 amu], attributed to the loss of the upper (epi)catechin unit, and m/z 575 [M − H-(2 × 288)]−, indicating the presence of the A-type bond between the third and the terminal flavan-3-ols units (C. cassia and C. iners while being absent in other specimens. Peak L10  showed MS2 fragments at m/z 863 [−288 amu] due to the loss of upper EC unit and m/z 573 due to the successive loss of terminal EC unit −] showed fragment ion peaks at m/z 711 [M − H-152] due to RDA, and m/z 573 [M − H-290] related to the loss of terminal (epi)catechin suggesting for the presence of A linkage between the top and middle units (− at 863.1853 (C45H35O18)−] were at m/z 577 [M − H-286] (loss of extension A-type (E)C unit), m/z 427 (loss of extension (E)C unit and RDA fragmentation) and m/z 289 related to the terminal (E)C unit (− at m/z 861.1699 (C45H33O18)−] and [(M + H)+ at m/z 863.1811 (C45H35O18)+], respectively, suggested for a procyanidin trimer with two A-type interflavonoid linkages. It was detected only in C. cassia and C. tamala. The MS2 fragment ions at m/z 575 [M − H-286]− are due to the loss of upper EC unit by QM cleavage and consequently annotated as (epi)catechin trimer with double A linkage. It was noticed that no PA trimers were detected in both C. verum accessions from either origin . This may account for its less astringent taste than C. cassia based on its tannins content  were at m/z 313 (due to RDA fragmentation) and m/z 161 G unit) annotated as epigallocatechin-O-caffeate. It was previously detected in tea extract, albeit it is the first time that it has been detected in cinnamon. The pyrogallol moieties of (epi)gallocatechins  are more reactive than the catechol moieties of (epi)catechins regarding their antioxidant activity [30H41O12)−] showed fragment ions at m/z 467 [M − H-126] due to HRF and m/z 305 due to QM cleavage between EC and EG units assigning this peak as gallo(epi)catechin-(epi)catechin.The main fragments detected in peak L51 [(M − H)activity . Peak L5− 995.2404 (C51 H47 O21)−] catechin units attached to chrysin and a hexose moiety. Further investigation is required to confirm this identification using other spectroscopic tools.A novel procyanidin was detected in peak L37 [(M − H)7 O21)−]  in C. ta− [m/z 147.0447 (C9H7O2)−] with the main fragment ion at m/z 103 [(M − H-44)−], identified as cinnamic acid, and more prominent in C. verum from Malaysia and Pakistan. Cinnamic acid exhibited potential antibacterial activity [− m/z 295.1155 (C15H19O6)−] was identified as cinnamyl-O-hexoside. Peak L24 [(M − H)− m/z 427.1673 (C20H27O10)−] was identified as cinnamyl-O-pentosylhexoside (rosavin), previously reported in C. cassia [m/z 293 [M − H-134]− due to the loss of cinnamyl alcohol, while the fragment ions at m/z 233 and 191 appeared due to sugar losses. Herein It was detected only in C. cassia; thus, it may be used as a marker for this species. Peak L27 [(M − H)−m/z 429.1762 (C20H29O10)−] with a mass difference of 2 amu compared to compound L24 was identified as dihydrocinnamyl-O-pentosylhexoside, which is the first time being reported in cinnamon  [(M − H)− m/z 395.2781 (C23H39O5)−], with its fragment ion at m/z 263 [M − H-132]−, was identified as cinnamoylcinnamate-O-pentoside detected only in C. iners species. Whether peaks L27 and L63 could serve as markers for that species has yet to be confirmed. Peak L39 [(M + H)+m/z 133.0652 (C9H9O)+] was identified as (E)-cinnamaldehyde that was reported to exhibit antibacterial, antifungal [C. verum and C. iners compared to other species and to likely account for their pungent taste. Peak L42 [(M + H)+m/z 163.076 (C10H11O2)+] was identified as methoxy-cinnamaldehyde, found most prominent in C. verum species. C. verum was reported to exhibit antitumor activity due to its richness in methoxy-cinnamaldehyde [+ m/z 135.081 (C9H11O)+] was identified as cinnamyl alcohol that was previously detected in bark and twigs of C. cassia −, followed by m/z 165 [M − H-162]−, suggestive for the presence of free carboxylic group and annotated as dihydrocoumaroyl-O-hexoside and was identified in C. cassia, C. tamala and C. verum from Pakistan, while peak L19 showed fragment ion at m/z 165 [M − H-162]−, followed by m/z 121 [M − H-162-44]−, assigning it as dihydrocinnacasside. Likewise, peaks L16 and L17 showed the same (M − H)− at m/z 459 with a mass difference of 132 amu than peaks L18 and L19, and annotated as dihydrocinnacasside-O-pentoside and dihydrocoumaroyl-O-pentosylhexoside, respectively. Dihydrocinnacasside-O-pentoside was identified in all Cinnamomum species except C. cassia. Peak L33 [(M + H)+m/z 147.0446 (C9H6O2)+] was identified as coumarin found most abundant in C. cassia, consistent with previous results ensuring its richness in coumarin with though health risks when consumed regularly. The lowest level of coumarin among the studied species was found in C. iners. This finding, together with the high level of cinnamaldehyde, poses its use as a flavoring agent instead of the more expensive true cinnamon.Compounds L16, L17, L18, L19, and L33 were coumaroyl derivatives, a precursor to cinnamates. Peaks L18 and L19 showed the same molecular ions at Cinnamomum species, including peak L28 [(M − H)−m/z 595.1699 (C27H29O17)−] and peak L29 [(M − H)−m/z 593.1848 (C28H33O14)−] assigned as naringenin di-O-hexoside in C. iners and C. tamala and isorhamnetin-O-pentosyldeoxyhexoside in C. cassia and C. tamala, respectively. A novel O- and C-glycosylated flavonoid (peak L30) was detected for the first time in C. cassia and C. tamala. Peak L30 [(M − H)−m/z 609.1998 (C27H29O16)−] showed main fragment ions at m/z 447 [M − H-162]− (due to loss of O-linked hexose sugar), and m/z 327 [M − H-162-120]−, indicating the presence of C-linked hexose moiety and annotated as luteolin-O-hexosyl-C- hexoside +] showed a fragment ion at m/z 297 [M + H-17]+ and was identified as norboldine. Antiplasmodial and antiviral activities of norboldine were reported  and a fragment ion peak at m/z 297 due to the opening of ring B and loss of methylene imine group as typical of aporphines and identified as corydine [+m/z 328.1528 (C19H22NO4)+] showed an ultraviolet absorption spectrum similar to that of peak L21 and was identified as norisocorydine. Peak L22 [(M + H)+ m/z 330.1680 (C19H20NO4)+] showed UV max typical of benzylisoquinolines annotated as reticuline, previously isolated from C. camphora ), cinnacassin L (peak L46 [(M − H)− at m/z 281.1168 (C17H18O3−)]), and one neolignan; and hydroxy-dimethoxyepoxy-neolignenediolethyl ether (peak L74 [(M − H)− at m/z 385.1657 (C22H26O6−)]), which were previously detected in the twigs of C. cassia  and cinncassiol A [(M − H)− at m/z 381.1724 (C20H29O7−)].Lignans from different plant sources were reported to show neuroprotective activities being useful in the treatment and prevention of neurodegenerative diseases . In the . cassia . The thrisorders . Herein 2O [M − H-18]− and/or loss of carboxylic moieties [M − H-44]−. Peaks L64, L66, and L69 with [M − H]− m/z 279.232 (C18H31O2)−, 255.2328 (C16H31O2)−, and m/z 281.2483 (C18H33O2)−, respectively, constituted the major peaks in all specimen, especially C. verum from both origins and C. iners. They were tentatively identified as linoleic acid, palmitic acid, and oleic acid, respectively. A mass difference of 16 amu between peaks L64 and L55 was indicative of an extra hydroxy group and assigning peak L55 as hydroxy linoleic acid identified in C. tamala and C. verum of both origins. Likewise, peak L60, a trihydroxylated fatty acid, was detected in cinnamon for the first time as trihydroxyoctadecaenoic acid  with the main fragment ion at m/z 283 due to the loss of carboxylic group. It was detected in C. iners and C. verum of both origins. Three dicarboxylic fatty acids were observed for the first time in cinnamon annotated as hexadecanedioic acid (L53)  in C. iners and C. verum of both origins, octadecenedioic acid (L54)  in all Cinnamomum species, and hexadecanedioic acid methyl ester (L57)  in all Cinnamomum species except C. verum  from different locations together in one quadrant, while C. iners (CI) and C. cassia (CC) were at another quadrant with negative PC1 values. On the other hand, C. tamala (CT) was separated from the rest of the samples with positive PC1 values. The loading plot  as first-class against all other species to identify the markers that are most distant among examined cinnamon specimens. OPLS-DA, as a supervised multivariate data analysis technique, has greater potential in the identification of markers by providing the most relevant variables for the differentiation between two class groups. OPLS results confirmed PCA regarding the richness of C. tamala in protocatechuic acid at a p-value less than 0.05. In the positive ionization mode, another OPLS-DA model  of (E)-Cinnamaldehyde and norboldine with the lowest level of coumarin. A significantly higher level (p < 0.05) of protocatechuic acid was observed in C. tamala, while true cinnamon showed its richness in cinnamate, including cinnamic acid, (E)-cinnamaldehyde, and methylcinnamic confirmed the results obtained from MVA.Lastly, to distinguish between true cinnamon of different origins that are CV and CVM, both specimens were modeled against each other using OPLS-DA  in negatCinnamomum species were enriched in sugars and esters, while amino acids were present at much lower levels.Primary metabolites of cinnamon were assessed post-silylation using GC-MS analysis  in orderCinnamomum species except for CV (C. verum from Pakistan). The highest relative levels of total sugars were detected in CVM  at ca. 64%, followed by CT at 53%. Monosaccharides were present at much higher levels compared to disaccharides represented by psicose , glucose , and fructose (G31). Glucose  was the most dominant monosaccharide amounting to 23% in CVM versus the lowest levels in CI (C. iners) at ca. 2%. Psicose , a low-calorie monosaccharide sugar that is 70% as sweet as sucrose with anti-obesity and antidiabetic effects [Sugars were the most abundant class in all  effects , was detC. verum from Malaysia). Generally recognized as safe food additives, sugar alcohols are low digestible carbohydrates [Cinnamomum species except for CI (C. iners), where meso-erythritol (G43) and arabitol  were the major sugar alcohols detected at 16 and 6%, respectively. Among all sugar alcohols, meso-erythritol (43) and arabitol  provide the lowest calories  [C. verum from Malaysia (12%), while the lowest levels were detected in the same species from Pakistan (2%), suggesting geographical origin impact. However, such a hypothesis should be confirmed by analyzing true cinnamon samples from other origins. Major sugar acids detected in CVM included galactonic acid γ-lactone  at 12% of total metabolites composition.CI and CT contained the highest levels of sugar alcohols at 29 and 19%, respectively, versus the lowest levels (5%) in CVM  , posing  kcal/g) , sugar aCinnamomum species (16–34%), reaching the highest content in CV. Major fatty acid esters included glycerol monostearate (G4) and followed by 1-monopalmitin (G3) in all Cinnamomum species. Glycerol monostearate (G4) is broadly used in bakery products to enhance the taste and appearance of flour foods owing to its anti-staling properties that rationalize the incorporation of cinnamon in pastry aside from its role as a natural flavor [Cinnamomum species was affected by the levels of their corresponding fatty acids.Fatty acyl esters constituted the second major class in all l flavor . Monoglyl flavor . The abuCinnamomum species at considerable levels reaching 12% in CV and accounting for its fatty taste [C. verum from Malaysia) contained the least free fatty acids level [Fatty acids were present in all ty taste . Stearicds level .C. tamala), which has the highest levels (8%). Oxalic (G18), (E)-cinnamic (G23), and quinic (G25) acids were the major constituents of this class. Oxalic acid (G18) is considered an anti-nutrient, whereas quinic acid (G25) exhibits anti-inflammatory and immune-enhancing activities [E)-cinnamic acid (G23) has a honey-like odor with anti-obesity effects [E)-cinnamic acid (G23) in CVM  compared to CV (C. verum from Pakistan) were in agreement with UPLC-MS results in negative ion mode (E)-cinnamaldehyde and its precursor (E)-cinnamic acid, which is more enriched in CVM than CV.Another primary metabolite class posing quantitative differences among examined cinnamon specimens was organic acids to impart a slightly bitter taste, especially in CT (tivities . As it w effects . The eleion mode . MoreoveCinnamomum species. Catechin (G27), a predominant component in tea, exhibited a bitter taste [Phenolics were more abundant in CT (5%) and CV (2%) than in other cinnamon samples, and they are considered natural antioxidants . Major per taste , though er taste  contribuC. iners) was more enriched in sugar alcohols represented by meso-erythritol (G43), while CV (C. verum from Pakistan) encompassed more fatty acyl esters, i.e., glycerol monostearate (G4) and 1-monopalmitin (G3). On the right side of the loading plot along PC1, CVM  was characterized by higher levels of sugar acids viz. galactonic acid γ-lactone (G37) and its isomer (G38) in addition to sugars viz., fructopyranose (G31), psicofuranose (G29/G30), and glucopyranose (32/33). Sugars are of low taxonomic value and are thus not clear markers for distinguishing CV and CVM accessions, which are dependent on agricultural practices or growing conditions [The GC-MS data were likewise analyzed using PCA  to assesnditions .p-value less than 0.001. The OPLS-DA score plot  compared to true cinnamon that displayed no specific markers.Considering CT distant segregation in the PCA model, it was modeled as one class using OPLS-DA analysis  versus aore plot  displayeore plot  illustraore plot b in addiore plot  was emplore plot  also disore plot  revealedCinnamomum species viz., C. cassia, C. iners, C. tamala, and C. verum were obtained from different sources with sample information presented in Bark specimens of four different Formic acid and acetonitrile (HPLC grade) were provided by Baker (The Netherlands). All other solvents, standards, and chemicals were obtained from Sigma Aldrich .−1 umbelliferone as an internal standard sonicated for 20 min with frequent shaking, then centrifuged at 12,000× g for 10 min to remove debris. The filtered extract through a 0.22 μm filter was subjected to solid-phase extraction using a C18 cartridge  as previously described [2 in both ionization modes, RT data, and reference literature and searching the phytochemical dictionary of natural products [Dried finely pulverized cinnamon specimens (10 mg) were extracted by adding 2 mL 70% MeOH, containing 10 μg mL escribed . Cinnamoescribed . Charactproducts .g for 10 min to remove debris. Next, 100 µL of the methanol extract was transferred into screw-cap vials and evaporated under nitrogen gas until complete dryness. Then, 150 µL of MSTFA (N-methyl-N-(trimethylsilyl)-trifluoroacetamide), previously diluted 1:1 (v/v) with anhydrous pyridine, was added and incubated for 45 min at 60 °C for derivatization. Silylated products were separated by an Rtx-5MS column  [n-alkane series, as well as matching the mass spectra obtained with the NIST and WILEY libraries and with standards when available. Before mass spectral matching, peaks were first deconvoluted through AMDIS software , and their abundance data were extracted using the MET-IDEA tool [Dried finely pulverized cinnamon specimens (100 mg) were extracted by adding 5 mL 100% MeOH, sonicated for 30 min with frequent shaking, then centrifuged at 12,000× µm film) . For evaDEA tool .p-value less than 0.05 to indicate significance between groups. The data table of MS abundances generated from either UPLC-MS or GC-MS was subjected to modeling, i.e., PCA , HCA , and OPLS-DA  using SIMCA-P version 13.0 software package . Subsequently, markers were determined by analyzing the S-plot, which revealed covariance (p) and correlation (pcor). All variables were Pareto scaled and mean-centered. Validation of models was evaluated by computing the diagnostic indices, i.e., Q2 and R2 values, and permutation testing of iterations.Each cinnamon group’s data were represented as the mean ± standard deviation (SD) of three replicates. One-way analysis of variance (ANOVA) was employed through IBM SPSS Statistics, Version 28.0.  with a C. verum as a flavoring agent and in functional foods. UPLC-MS analysis allowed the identification of 74 metabolites, of which a new proanthocyanidin suggested to encompass catechin, chrysin, catechin, and hexose was detected for the first time, trihydroxylated fatty acid (trihydroxyoctadecaenoic acid) and three dicarboxylic fatty acids  were detected for the first time in cinnamon, albeit, though other spectroscopic analysis, i.e., NMR still required for complete elucidation of these metabolites. In addition, a number of newly identified flavonoid glycosides included naringenin di-O-hexoside, isorhamnetin-O-pentosyldeoxyhexoside, and luteolin-O-hexosyl-C-hexoside. It revealed the richness of Chinese cinnamon in coumarin, while C. verum and C. tamala were rich sources of cinnamates. Norboldine, an aporphine alkaloid of potential inhibitory activity against type I HIV, was detected at high levels in C. iners species, warranting further assays of its extract against different viruses. Despite the great proximity between C. verum of both origins, UPLC-MS allowed the detection of a number of compounds that accounted for differences between both origins, including dihydrocoumaroyl-O-hexoside and lignans. The palatability and agreeable taste of cinnamon spice pose it as an ingredient in nutraceuticals. According to the UPLC-MS profile, C. iners was the closest species to official C. verum concurrent with a low level of coumarin with a relatively high level of cinnamaldehyde, suggesting the former as a potential substitute for true cinnamon regarding minimal health hazards.This study provides the most holistic map of cinnamon spice primary and secondary metabolites composition using a multiplex approach of UPLC-MS and GC-MS techniques analyzed using chemometric tools. Such metabolite profiling justifies the premium value of Cinnamomum species, with true cinnamon encompassing the highest levels compared to other specimens. Whereas C. iners showed the healthiest low-calorie sugar profile with lower sugars and high sugar alcohol levels at 29%, viz., meso-erythritol (16%) and arabitol (6%) and thus posing it as a sugar source for diabetics.Primary metabolites analysis by GC-MS revealed true cinnamon richness in fatty acids and acyl esters, though with qualitative variation among different origins. Our findings also revealed that sugars were the most discriminatory metabolites among C. tamala was the most chemically distinctive species attributed to the elevated dihydrocinnacasside pentoside, protocatechuic acid, and glycerol. In contrast, positive ion UPLC-MS mode revealed that C. iners was the most distant species, as it is rich in catechins and alkaloids, i.e., norboldine and norisocorydine. Among GC-MS and UPLC-MS employed analytical platforms, UPLC-MS in negative ion mode provided the most rational classification, with close segregation of CV and CVM specimens, and not observed in other PCA models. Novel markers revealed from this study to identify adulteration of true cinnamon (CV) with Chinese cinnamon (CC) included dihydrocoumaroyl-O-hexoside and dihydrocoumaroyl-O-pentosylhexoside in addition to the well-recognized coumarin. On the other hand, cinnamates represented by methyl cinnamate, (E)-cinnamaldehyde, and cinnamoyl alcohol were enriched in true cinnamon. Such chemical marker should aid in the detection of adulteration in true cinnamon, especially when present in extract lacking the typical morphological features to distinguish it from its allied drugs, i.e., Chinese type.MVA of GC-MS and UPLC-MS detected in negative ion mode data revealed that Cinnamomum species do not represent all accessions of cinnamon worldwide, our approach is certainly feasible for analyzing other Cinnamomum species from such further sources to exploit factors that might impact the metabolic makeup, i.e., storage, seasonal variation and growth stage. Combining our variable metabolite profile data with gene expression can further assist in exploring involved genes, evaluating biosynthetic pathways, and ultimately enhancing breeding. The isolation and complete identification of the discriminative chemo-markers along with the newly highlighted metabolites should follow on as future work.Although the selected


Cyanidin-3-O-glucoside (C3G) is a natural colorant with anti-diabetic properties, while its underlying mechanisms remain far from clear. Here, we investigated the protective role of C3G on palmitic acid (PA)-induced pancreatic beta cell dysfunction and further decipher its possible molecular mechanisms. Both primary isolated mouse islets and the INS-1E cell were used, and treated with a mixture of PA (0.5 mM) and C3G  for different durations . We found that C3G could dose-dependently ameliorate beta cell secretory function and further alleviate cell apoptosis. Mechanistically, the primary role of the PKR-like ER kinase (PERK) endoplasmic reticulum (ER) stress pathway was detected by RNA sequencing, and the PERK-pathway-related protein expression, especially the pro-apoptotic marker C/EBP homologous protein (CHOP) expression, was significantly downregulated by C3G treatment. The critical role of CHOP in mediating the protective effect of C3G was further validated by small interfering RNA. Conclusively, C3G could ameliorate PA-induced pancreatic beta cell dysfunction targeting the CHOP-related ER stress pathway, which might be used as a nutritional intervention for the preservation of beta cell dysfunction in type 2 diabetes mellitus. The widespread epidemic of type 2 diabetes mellitus (T2DM), affecting over 10.5% of the global population, causes a huge burden for public health . Elevate2O2 exposure, glucotoxicity, and amylin or Ab1-42 treatment [Cyanidin-3-O-glucoside (C3G), the most abundant anthocyanin, derived from deep-colored fruits and vegetables, belongs to the polyphenol of the flavonoid group ,8. The mreatment ,15,16,17Cyanidin-3-O-glucoside was purchased from TAUTO BIOTECH . Palmitic acid (PA) and fatty acid-free-BSA were purchased from Sigma-Aldrich .The rat insulinoma INS-1E cell line was provided by FENGHUISHENGWU, Ltd. . INS-1E cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin , 1% 1 mM sodium pyruvate, 10 mM HEPES , and 0.05 mM 2-mercaptoethanol .2 and 37 °C, islets with intact borders were collected to use for glucose-stimulated insulin secretion assay (GSIS) experiments. Cell viability was measured by the CCK8 assay  and the process was performed according to the manufacturer’s instructions.Islets of Langerhans were isolated from C57BL/J6 male mice aged 8–12 weeks as previously described and were approved by the Animal Care and Utilization Committee of Sun Yat-sen University  . Briefly4·7H2O, 1.2 mM KH2PO4, 2.5 mM CaCl2, 5 mM NaHCO3, and 0.1% Fatty-acid-free BSA) and incubated for an hour at 37 °C. Then, the glucose-free KRBH was discarded and incubated with 3 mM and 25 mM glucose KRBH for one hour each in turn, and the supernatants were collected for insulin assays . The glucose concentrations were based on previous studies on pancreatic beta cell function [For GSIS assay, INS-1E cells were grown in 12-well plates for two days and primary islets were collected in individual tubes for instant use (10 islets/tube), after which cells/islets were treated with 0.5 mM PA prepared as previously described  with or function ,21. Cell5 cells) with 5 µL Annexin V-FITC and 5 µL PI added at the end of the intervention. After being gently vortexed, the mixture needed an incubation for 20 min at room temperature avoiding light. Finally, the cell apoptosis rate was determined by utilizing an EPICS XL-MCL flow cytometer  and analyzed by FlowJo™ software . Annexin V binds to the membrane of early apoptotic cells and PI stains the DNA of late apoptotic or necrotic cells; thus, the apoptotic cells include early apoptotic cells (Annexin V+/PI−) and late apoptotic cells (Annexin V−/PI+).Cell apoptosis was examined by flow cytometry using the Annexin V-FITC/propidium iodide (PI) apoptosis detection kit . INS-1E cells were seeded in 6-well plates treated with BSA, PA, or PA plus 50 µM C3G for 24 h. Then, cells were suspended with Annexin V Binding Buffer (500 µL/5 × 10Cell apoptosis rate was further confirmed by 4′-6-diamidino-2-phenylindol (DAPI)/PI immunofluorescent staining . INS-1E cells were grown on glass coverslips on 35 mm confocal dishes and after 36 h the cells were treated with BSA only, 0.5 mM PA, or PA containing 50 µM C3G. One hour prior to the end of treatment, 10 µg/mL of PI and 20 µg/mL of DAPI were added to the culture medium. At the end of incubation, cells were washed 3 times with clean PBS and fixed with 4% paraformaldehyde at 4 °C for 15 min. Cells were mounted with antifade mounting medium  and observed under a laser scanning confocal microscope . Random images were acquired from different sites within the confocal dish and image analysis was performed to calculate cell apoptosis rate based on the ratio of the fluorescence area of PI (staining late apoptotic and necrotic cells) to DAPI .p < 0.05) genes to make heatmap, volcano maps, and Gene Set Enrichment Analysis (GSEA) enrichment plots using the OmicStudio tools . The assigned GEO accession number is GSE196704.INS-1E cells were seeded in 6-well plates treated with BSA, PA, or PA plus 50 µM C3G for 24 h. At the end of treatment, total RNA was extracted utilizing Trizol reagent  with three replicate samples collected for each group and sent to LC Bio  for further processing of the samples and RNA sequencing (RNA-seq) analysis. Briefly, total RNA was quantified and the mRNA within was purified using Dynabeads Oligo (dT) . Then, mRNA was fragmented and reverse-transcribed to cDNA by SuperScript™ II Reverse Transcriptase . Finally, the processed products were amplified with PCR, and the 2 × 150 bp paired-end sequencing (PE150) was performed on an Illumina Novaseq™ 6000  following the vendor’s recommended protocol. The differentially expressed genes (DEGs) were selected with fold change >2 and statistically significant  was applied to perform qPCR on an Applied Biosystems Prism 7000 sequence detection system. The target primers were designed based on mRNA sequences given in NCBI Genebank and were verified on NCBI-primer blast.Quantitative real-time PCR (qPCR) was performed as previously described . BrieflyThe primers were: toll-like receptor 4 (TLR4) Sense: GATCTGAGCTTCAACCC, Anti-sense: TTGTCTCAATTTCACACCTGGA; G-protein-coupled receptor 40 (GPR40) Sense: GCTGCCTTCCCCTTTGGATA, Anti-sense: TGTTGATGCCCAGGGAACTG; C/EBP homologous protein (CHOP) Sense: CCCCAGGAAACGAAGAGGAAG, Anti-sense: AATCTGGAGAGCGAGGGCTT; glucose transporter 2 (GLUT2) Sense: AATTTGGACCGGCACATGCT, Anti-sense: CTGAGGCCAGCAATCTGACTA; PKR-like ER kinase (PERK) Sense: GCGGCAGTGAGAAGTGGAAT, Anti-sense: ACCTTCCAATCAGCAACGGA; β-actin Sense: ATATCGCTGCGCTCGTCGT, Anti-sense: CATACCCACCATCACACCCTGG.Whole-cell protein was extracted by RIPA Lysis Buffer with Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail, EDTA-Free (100×), and protein concentration was measured by Pierce BCA Protein Assay Kit . Total protein was separated by SDS-PAGE, transferred to PVDF membrane , and detected by SuperSignal™ West Femto Maximum Sensitivity Substrate . Primary and secondary antibodies used in the experiments were listed as follows: anti-PERK, anti-Phospho-PERK (Thr980), anti-glucose-regulated Protein 78 (GRP78), anti-CHOP, anti- eukaryotic initiation factor 2 alpha (eif2α), anti-elF2 alpha (phospho S52), anti-cleaved-caspase-3, anti- B cell lymphoma 2 (BCL2)-associated X protein (BAX), anti-BCL2 , mouse anti-rabbit IgG-HRP, and m-IgGκ BP-HRP .The supernatant of INS-1E cells was collected after the intervention mentioned in 2.3 to detect inflammatory factor  levels using the ELISA kit . The detailed procedures were followed in accordance with the manufacturer’s instructions.CHOP small interfering RNA (siRNA) oligos with control oligos, and RNAFit transfection reagent were provided by HANBIO . The transfection process was performed according to the manufacturer’s instructions. After 36 h of transfection, INS-1E cells were then exposed to PA or C3G for GSIS assay and were collected for WB as described above.p < 0.05 was taken to be statistically significant.Data were shown as mean ± SEM, and one-way ANOVA followed by LSD post hoc test was utilized for comparing differences between multiple groups. All experiments were performed at least in biological triplicate. SPSS 25.0  was used for all analyses. For WB and immunofluorescence data, representative images were shown in all figures. To determine whether C3G could protect the secretory function of the pancreatic beta cell, we first treated INS-1E cells with 0.5 mM PA in combination with a gradient concentration of C3G  for different time points  A–C. No cThen, we further confirmed the above observation in the primary isolated islets. Compared to the BSA group, GSIS was decreased by 52% due to PA exposure and that was significantly restored by C3G treatment, with GSIS increasing by 127% and stimulatory index increasing by 35%, respectively D.To explore whether C3G could have a protective effect on PA-induced apoptosis, we first checked the cell apoptosis rate by applying both Annexin V-FITC/PI double staining flow cytometry and DAPI/PI fluorescent staining. Compared with the control group, the apoptosis rate was dramatically augmented by 188% under PA exposure, while C3G treatment remarkably reduced the apoptosis rate by 47% A. FurtheCell-apoptosis-related proteins, including BAX, BCL2, and cleaved caspase-3, were further detected by WB C. ComparTo investigate the molecular mechanisms that mediated the protective effect of C3G on PA-induced beta cell dysfunction, we first used qPCR and WB to detect the expression of cell membrane receptors, including toll-like receptor 4 (TLR4) and G-protein-coupled receptor 40 (GPR40), which recent studies had reported to be involved with beta cell apoptosis and insulin secretory capacity ,24,25. NNext, RNA-seq was applied to screen the differentially expressed genes, and the results were analyzed using different methods. The pathway enrichment analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed to find the most significantly altered pathways, and GSEA was utilized to quantify the expression of gene sets under relevant pathways to see if the expression was up- or downregulated. As shown in The ER stress pathway is mainly mediated by three endoplasmic reticulum transmembrane proteins, including PERK, inositol-requiring enzyme 1 (IRE1), and transcription factor 6 (ATF6) ,27. BaseWe then further tested the secretion of several inflammatory factors and found that interleukin-1beta (IL-1β) secretion was decreased by 42% in the PA + C3G group compared to the PA group C.To verify the critical role of CHOP-medicated ER stress, we applied CHOP siRNA (siCHOP) to suppress the expression of CHOP; scrambled control RNA sicon was applied in the control group. siCHOP2 was selected in subsequent experiments, which induced CHOP reduction by more than 50% A. ComparAnthocyanin C3G has long been interesting as a plant-derived product with anti-diabetic properties, but the molecular mechanism is still not fully understood. In the present study, we demonstrated, for the first time, that C3G could potently restore the pancreatic beta cell’s secretory function and reduce apoptosis under PA exposure. Furthermore, we demonstrated that CHOP-mediated ER stress was the central mechanism of the PA-induced pancreatic beta cell dysfunction and could be effectively restored by C3G supplementation. Conclusively, the study provided a new angle for understanding the anti-diabetic beneficial effect of C3G via preserving pancreatic islets from lipotoxicity.2O2, amylin, or Ab-42, has been observed and reported by several studies [Previous studies reported that long-term high PA intake caused a significant decrease in serum insulin levels and islet morphology atrophy in wild-type mice and that exposure to a mixture of palmitic and oleic acids triggered an increased rate of apoptosis in INS-1E cells ,28. Here studies ,30,31,32A number of studies have suggested that TLR4 and GPR40, also known as free fatty acid receptor 1 (FFAR1), are expressed on beta cell membranes. It is reported that they could respond to medium- and long-chain saturated fatty acids, and mediate the downstream pathways that play a critical role in FFA-induced beta cell apoptosis and dysfunction ,33,34,35It has been reported by recent studies that ER stress has a great impact on pancreatic beta cells, and PERK, one of the three known ER stress transmembrane receptors, is involved with proinsulin folding and cell survival ,27,36. TNext, we used siRNA to further verify the critical role of CHOP in mediating the protective role of C3G in PA-induced pancreatic cell dysfunction. After knocking down the pro-apoptosis marker of ER stress, both WB and flow cytometry results supported that inhibition of ER stress pathway could suppress cell apoptosis. Interestingly, the combined effect of C3G and siCHOP2 presented the most significant decrease in cell apoptosis, which indicated that C3G did ameliorate beta cell ER stress to cut down the PA-induced damage. The ability of C3G to alleviate ER stress in hepatocytes, endothelial cells, retinal cells, and neuronal cells and, thereby exerting various health-prompting benefits, as reported previously ,43,44,45Although it is hard to expect the in vitro C3G dosage could be achieved from merely food or dietary supplements . With thIn summary, the data presented in our study demonstrated the protective role of C3G on PA-induced beta cell dysfunction through alleviation of the CHOP-mediated ER stress pathway. Our study provides direct evidence of the protective effect of C3G on pancreatic beta cell dysfunction upon fatty acid stress, which might help to fully understand the anti-diabetic properties of C3G and develop C3G as a nutritional intervention targeting beta cell lipotoxicity in obesity and its related diabetes.


Human adenoviruses (HAdVs) within species B, C, and E are responsible for highly contagious and potentially severe respiratory disease infections. The traditional method to type these pathogens was based on virus neutralization and hemagglutination assays, which are both time-consuming and difficult, particularly due to the nonavailability of reagents. Subsequent molecular typing based on the partial characterization of the hexon gene and/or the restriction enzyme analysis (REA) of the genomes is inadequate, particularly in identifying recombinants. Here, a rapid, simple, and cost-effective method for molecular typing HAdV respiratory pathogens is presented. This incorporates three pairs of universal PCR primers that target the variable regions of the three major capsid genes, i.e., hexon, penton base, and fiber genes, that span the genome. The protocol enables typing and characterization of genotypes within species B, C, and E, as well as of some genotypes within species D and F. To validate this method, we surveyed 100 children with HAdV-associated acute respiratory infections identified by direct immunofluorescence . Throat swab specimens were collected and analyzed by PCR amplification and sequencing; these sequences were characterized by BLAST. HAdVs were detected in 98 out of 100 (98%) samples, distributing as follows: 74 HAdV-B3 (74%); 10 HAdV-E4 (10%); 7 HAdV-C2 (7%); 2 HAdV-C6 (2%); 1 HAdV-B7 (1%); 1 HAdV-C1 (1%); 2 co-infection (2%); and 1 novel recombinant (1%). This study is the first detailed molecular epidemiological survey of HAdVs in Hong Kong. The developed method allows for the rapid identification of HAdV respiratory pathogens, including recombinants, and bypasses the need for whole genome sequencing for real-time surveillance of circulating adenovirus strains in outbreaks and populations by clinical virologists, public health officials, and epidemiologists. Adenoviridae family, which are nonenveloped double-stranded DNA viruses (Human adenoviruses (HAdVs) belong to the  viruses . As huma viruses . A measu viruses . These r viruses . Althoug viruses .To date, 113 HAdV genotypes have been identified, characterized, and reported using whole genome analysis, including the original 51 serotypesIn immunocompromised patients, particularly in the organ transplant setting, the most commonly reported adenovirus types include HAdV-C1, -C2, -C5, -A12, -A31, -B3, -B11, -B16, -B34, and -B35 . These tAs a result, it is important to type HAdVs accurately and rapidly for clinical diagnoses and epidemiological investigations in order to provide information on the pathogen, including the distribution of infections by individual and specific types, as well as to detect and characterize emergent strains in the context of outbreaks . As recoHAdVs were traditionally typed according to serum neutralization and hemagglutination-inhibition tests, which are time-consuming and reagent-limited .Molecular typing based on either partial sequence characterization of HAdV hexon gene or restriction enzyme analysis (REA) of the genomics DNA are improvements but still not effective, for example, in their difficulty to identify recombinants . HAdV isWhole genome sequencing is very useful for epidemiological surveys and understanding archived intriguing pathogens  but may At the same time, we characterized the molecular epidemiology of HAdVs circulating among inpatient and outpatient children during the ARD outbreaks in the late summer and early autumn of 2014 in Hong Kong, using our newly-developed HAdV molecular typing protocol. This is the first detailed molecular epidemiological survey of HAdVs circulating in Hong Kong.Human adenovirus genotypes HAdV-B3, -B7, -B11, -B14, -B21, -B55, -C5, -D19, -E4, and -F41 have been isolated, identified, studied, and archived in our laboratory . MoleculThis is a retrospective survey of an ARD outbreak in pediatric outpatients and inpatients with influenza-like symptoms in Queen Mary Hospital, Hong Kong from July 2014 through October 2014. For further details on the samples, please see The study protocol was approved by the Institutional Ethics Committee of Queen Mary Hospital in accordance with its guidelines for the protection of individual privacy, and adhering to the principles of the Declaration of Helsinki. Patient consent for using left-over specimens was waived.The HAdV-positive nasopharyngeal swab specimens collected from 100 patients were inoculated onto A549 cells. These were cultured in a maintenance medium  at 37°C in an atmosphere containing 5% (v/v) carbon dioxide. Cytopathic effect (CPE) was monitored for 5–7 days, following which, if no CPE was observed, the cells would be frozen and thawed three times and then passaged in A549 cells again to check for CPE.The penton base, hexon, and fiber gene sequences from HAdV-A18, -B3, -B7, -B11, -B14, -B16, -B21, -B34, -B35, -B50, -B55, -B66, -B68, -C1, -C2, -C5, -C6, -D19, -D37, -E4, -F41, and -G52 were extracted from genome data archived in GenBank. These were aligned using ClustalW to find regions with high sequence similarities. Primers targeting the three major capsid genes were designed based on these bracketing conserved regions, and these were subsequently obtained from Invitrogen .PCR conditions were determined, and reactions were conducted in a total volume of 20 μl comprising 1× Taq Master Mix (10 μl), primer F , primer R , DNA template (1 μl), and water (8 μl). Primers Penton-F and Penton-R were designed for both the PCR amplification and the subsequent DNA sequencing of the amplified penton base gene product. PCR conditions are as follows: 94°C for 1 min; 34 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 100 s; and a final extension of 72°C for 10 min. Similarly, Primers HVR-F and HVR-R were used for both the PCR amplification and subsequent DNA sequencing of the hexon gene product. Hexon PCR conditions are as follows: 94°C for 1 min; 34 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 100 s; followed by a final extension of 72°C for 10 min. Finally, Primers Fiber-F and Fiber-R were designed for both the PCR amplification and subsequent DNA sequencing of the fiber gene product. The PCR amplification conditions for this are as follows: 94°C for 1 min; 34 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 72 s; followed by a final extension of 72°C for 10 min.All three major capsid genes of the HAdV isolates were PCR-amplified using the corresponding pairs of universal primers noted above, respectively. These PCR products were purified and then DNA-sequenced directly with both PCR primers. The assembled DNA sequences were characterized by a BLAST survey of the NCBI GenBank database. Genotype identity of each clinical specimen was determined based on the penton base, hexon, and fiber sequences to which they showed the highest sequence identities.As noted, archived genome sequences from GenBank were used for the alignments and extraction of the penton base, hexon, and fiber gene sequences. Their accession numbers are as follows: HAdV-A12 (X73487), HAdV-B3 (DQ099432), HAdV-B3 (AY599834), HAdV-B7 (AY594255), HAdV-B7 (KC440171), HAdV-B11 (AY163756), HAdV-B14 (AY803294), HAdV-B16 (AY601636), HAdV-B21 (AY601633), HAdV-B34 (AY737797), HAdV-B35 (AY128640), HAdV-B50 (AY737798), HAdV-B55 (JX491639), HAdV-C1 (AC_000017), HAdV-C2 (J01917), HAdV-C5 (AC_000008), HAdV-C6 (KF268129), HAdV-D9 (AJ854486), HAdV-E4 (AY594253), HAdV-F40 (KU162869), and HAdV-G52 (DQ923122).Molecular Evolutionary Genetics Analysis (MEGA) software, version 7.0,Three pairs of universal primers were designed for PCR amplification and DNA sequencing of the HAdV penton base, hexon, and fiber genes. Primers Penton-F and Penton-R were designed based on the conserved regions of the penton base sequences. Within the alignment of sequences, primer sequences selected for Penton-F and Penton-R are highly conserved in the majority of penton base genes, which ensures that all of the analyzed adenovirus types within species A to G could be PCR-identified . The resFor the hexon, the universal amplification primers HVR-F and HVR-R were designed similarly. The primer sequences are conserved in HAdV genotypes across all of the species analyzed, yielding a PCR product of about 1,685 bp . This amPrimers Fiber-F and Fiber-R were designed for fiber gene. Point mutations of primer Fiber-F exist only in one or two nucleotides located in the middle of the primers; this ensures an effective PCR amplification of the different HAdV genotypes . These pGenomic DNA from reference samples of HAdV-B3, -B7, -B11, -B14, -B21, -B55, -C5, -D19, -E4, and -F41 was extracted and amplified by PCR amplification and identified by subsequent sequencing using these three pairs of universal primers . The PCRA total of 100 HAdV-positive samples were analyzed using this protocol . These iThe clinical diagnoses included one case of fatal pneumonia caused by HAdV-3; three cases of bronchitis; three cases of diarrhea; two cases of febrile seizure; two cases of rash; and 74 cases of upper respiratory tract infection URTI; .Hundred throat swabs specimens were successfully amplified by this PCR protocol using the three pairs of universal primers. These PCR products were sequenced using the same primers. BLAST analysis confirmed that 74 cases were HAdV-B3 (74%); 10 cases were HAdV-E4 (10%); seven cases were HAdV-C2 (7%); two cases were HAdV-C6 (2%); one case was HAdV-B7 (1%); one case was HAdV-C1 (1%); two cases were co-infected by different types of HAdVs  (2%); one case was putative recombinant (P1H2F2) (1%); the remaining two cases were PCR negative . The twon = 74) was the most prevalent type. The three capsid genes formed a subclade with another China HAdV-3 isolate Guangzhou01 circulating in 2004 (n = 10). This is unexpected as HAdV-E4 was formerly and apparently constrained to military populations  can be found in the article/supplementary material.The studies involving human participants were reviewed and approved by Institutional Ethics Committee of Queen Mary Hospital. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.DS and QZ contributed to the study design and manuscript writing. XW, JZ, WL, LQ, JO, PW, and QZ contributed to data analysis and data visualization. WZ, JW, DS, and QZ contributed to manuscript revision. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Key Research and Development Program of China (2018YFE0204503), the National Natural Science Foundation of China (32170139 and 81730061), the Natural Science Foundation of Guangdong Province , and the Fundamental Research Funds for the Central Universities (21622101).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ramulus Mori (Sangzhi) alkaloids (SZ-A) from Morus alba L. were approved in 2020 for the treatment of T2DM. In this study, we examined the therapeutic effects and mechanism of SZ-A on obesity and NAFLD in mice. Mice (C57BL/6J) fed a high-fat diet (HFD) for 14 weeks were treated with SZ-A for another 6 weeks. HFD-induced weight gain was reduced by SZ-A in a dose-dependent manner. SZ-A treatment significantly stimulated adiponectin expression and secretion in adipose tissue and 3T3-L1 adipocytes. Additionally, SZ-A markedly reduced hepatic steatosis  and expression of pro-inflammatory and pro-fibrotic genes. SZ-A regulated lipid metabolism and oxidative stress , superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione (GSH)) in the liver. Palmitic acid-induced insulin resistance and lipid accumulation in HepG2 cells were also repressed by SZ-A. Collectively, SZ-A protected mice from HFD-induced NAFLD through an indirect effect of improved systemic metabolism reducing bodyweight, and a direct effect by enhancing the lipid metabolism of HepG2 cells. The weight-loss effect of SZ-A in mice was partly due to improved fatty oxidation instead of influencing food consumption.Nonalcoholic fatty liver disease (NAFLD), obesity, and type 2 diabetes mellitus (T2DM) have highly related mechanisms.  Non-alcoholic fatty liver disease (NAFLD) has emerged as the most common cause of chronic liver disease worldwide, affecting approximately 25% of the population . The preDisturbed lipid homeostasis and metabolic stress-induced damage are the key pathogenetic features of NAFLD ,9. AMP-aTo date, no pharmacological therapies have been approved for NAFLD treatment. Given the complexity of NAFLD pathophysiology, agents targeting different molecular processes of hepatic metabolism, oxidative stress-induced cell death, inflammation, and fibrosis have been investigated in preclinical models and clinical trials .These agRamulus Mori (Sangzhi) alkaloid (SZ-A) tablets, extracted from Morus alba L. (mulberry twig), were approved by the China National Medical Products Administration in 2020 for T2DM treatment in China . The main components of SZ-A powder  are alkaloids (50% or more by weight), including 1-deoxynojirimycin (DNJ), 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), and fagomine (FA). Though the prophylactic effects of DNJ have been reported to protect against high fat diet-induced liver steatosis through the regulation of the gut microbiota [crobiota  and mitocrobiota , the appcrobiota . Chroniccrobiota . In addicrobiota . SZ-A excrobiota . Tissue crobiota , which rIn the present study, we investigated whether SZ-A mitigates obesity and NAFLD in HFD-fed mice by directly acting on the liver and adipose tissues. The direct effects and mechanisms of action of SZ-A and its major components on hepatocytes and adipocytes were studied in vitro. Our results demonstrate the potential of SZ-A as a natural anti-diabetic agent for the treatment of obesity and NAFLD in mice.Ramulus Mori (Sangzhi) alkaloids (SZ-A) powder , an HFD control group by intragastrical administration of saline , the SZ-A treatment groups by intragastrical administration , an HFD control group by peritoneal injection (HFD i.p. n = 10), and an SZ-A treatment group by peritoneal injection . The mice in the NC group were given a standard diet, and the mice in other groups were fed with HFD  for 14 weeks. Then the mice were given saline i.p., saline i.g., SZ-A 100 mg/kg/d i.g., SZ-A 300 mg/kg/d i.g., SZ-A 400 mg/kg/d i.g., and SZ-A 200 mg/kg/d i.p. for 6 weeks, respectively. Mice in the HFD and treatment groups were fed with HFD during the treatment. Bodyweights were measured weekly during the experimental period. Food intake was measured per cage. At the end of these experiments, mice were fasted overnight before being euthanized. Blood samples were collected and then kept at room temperature for half an hour before centrifugation at 3000 rpm for 15 min to obtain the serum samples. Tissues including liver, abdominal adipose tissue, perirenal fat, and subcutaneous fat were carefully collected, kept in liquid nitrogen, and then stored at −80 °C until analysis. Total body fat was measured using a Pharma Scan 70/16 US small animal MRI . All animal care and experimental procedures were approved by Beijing Laboratory Animal Research Center . Six-week-old male C57BL/6J mice were housed at 24 °C with a 12 h light/dark cycle. After 1 week of acclimatization to the environment, the mice were randomly divided into eight groups: a normal control group , low-density lipoprotein cholesterol (LDL-C), C-reactive protein (CRP), high-density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were measured using automatic biochemistry analyzer  with appropriate commercial assay kits from Biosino Biotechnology and Science Inc. . Serum leptin level was determined using a mouse leptin ELISA kit  according to the manufacturer’s instruction. g for 5 min at 4 °C, and the supernatant was collected for the assay. Hepatic total cholesterol (TC) and triglyceride (TG) were determined by COD-PAP and GPO-PAP with the commercial kits . To measure the hepatic lipid peroxidation, the liver malondialdehyde (MDA) concentrations were monitored using a thiobarbituric acid reactive substances (TBARS) assay kit , and calculated using a standard curve. The reduced glutathione (GSH) content was determined using a thiol-specific reagent, dithionitrobenzoic acid, according to the manufacturer’s protocol , and the adduct was measured at 420 nm. Glutathione peroxidase (GPx) activity was determined using a glutathione peroxidase assay kit . In brief, the activity of GPx was measured using the assay kit based on the principle that oxidation of glutathione and H2O2 could be catalyzed by GPx to produce oxidized glutathione (GSSG) and H2O. According to the xanthine oxidase method, the activity of superoxide dismutase (SOD) was determined by superoxide dismutase activity colorimetric assay kit using water-soluble tetrazolium salt . The concentration of proteins was estimated using a bicinchoninic acid (BCA) assay kit .The frozen liver tissues were homogenized in cold PBS (pH 7.4). The suspension was centrifuged at 3000× 3 were excised and placed in a tissue container, which was then filled with Tissue-Tek OCT compound gel  and frozen in liquid nitrogen. The frozen samples were cut into 7 μm slices and stained with Oil Red O solution  for evaluation of fat accumulation. After removing the staining solution with 70% ethanol and distilled water, the sections were counterstained by hematoxylin for 2 min, washed in tap water for another 5 min, and mounted with glycerin. Images of stained liver sections were obtained with an Olympus microscope–camera system. In order to observe the ultrastructure of liver, a small part of liver tissue was fixed in glutaraldehyde solution (2.5%) for 4 h at 4 °C and post-fixed in 1% osmic acid with 0.1 M phosphate buffer for 1 h at 4 °C. After dehydration, infiltration, and embedding, liver tissue samples were cut and stained with uranyl acetate. Images were taken under a transmission electron microscope  at 80 kV. Fresh liver tissue, inguinal adipose tissue (iWAT), epididymal adipose tissue (eWAT), and brown adipose tissue (BAT) were fixed in 10% paraformaldehyde solution, embedded in paraffin wax, and cut at 5 μm for hematoxylin–eosin (H&E) staining. Liver sections of each animal with volumes of approximately 1 cm2 at 37 °C.Human hepatocellular carcinoma cell line (HepG2 cells) and 3T3-L1 cell line were obtained from the Cell Resource Center, Peking Union Medical College . The HepG2 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, and maintained in 100 mm dishes in the presence of 5% COThe PA-induced medium was prepared with serum-free DMEM using the methods described by Arwa et al. . A 100 mv/v) fetal bovine serum, 1 μmol/L DEX, 10 μg/mL insulin, and 0.5 mmol/L IBMX every 2 days for an additional 4 days. Cells were incubated with fresh DMEM containing 10% fetal bovine serum and 10 mg/mL insulin for another 2 days. Finally, the media were replaced with DMEM containing only 10% FBS and 1% penicillin–streptomycin for 2 days, after which the cells were used.3T3-L1 pre-adipocytes were grown to confluence in DMEM containing 10% calf serum and 1% penicillin–streptomycin for 4 days. Then the cells were induced to differentiate with DMEM containing 10% , DNJ (40 µg/mL), FA (10 µg/mL), DAB (10 µg/mL), and 0.125 mmol/L PA were added in the culture medium at the same time for 24 h. The cells were washed with PBS and lysed in lysis buffer (1% Triton X-100 and 1% PMSF in PBS) for 4 h at 4 °C. The TG level of the cells was measured by GPO-PAP . The protein concentrations were measured using BCA, and results were expressed as a fold change of intracellular TG level compared to the non-treated control.HepG2 cells were plated in 6-well plates at 4 × 10Glucose consumption and cellular glycogen concentration in HepG2 cells was assessed according to the previous report with a few modifications . In shor4 cells/mL and induced as described in cell culture and differentiation protocol. The differentiated 3T3-L1 preadipocytes were treated with different concentrations of SZ-A , DNJ , FA , and DAB  during differentiation. On the last day of the differentiation process, the media were collected, and concentrations of adiponectin were measured by ELISA .The 3T3-L1 cells were seeded into 6-well plates at 2 × 10® . RNA was quantified by a Nano-300 Micro-Spectrophotometer , and 1 µg RNA was reverse transcribed into cDNA using a reverse transcription system  according to the manufacturer’s protocol. The PCR amplification protocol consisted of 30 s at 95 °C, 10 s at 95 °C, and 30 s at 60 °C for 40 cycles. The purity of the PCR products was determined by melting curve analysis. The relative amount of each gene was calculated using 2−ΔΔCT. The level of transcripts was normalized, using β-actin as an internal standard. The primers used are provided in Total RNA was isolated from liver tissues using TRIzolg for 30 min at 4 °C. The protein concentration was detected by BCA assay. Equal amounts of protein samples were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with blocking buffer (10% blocker milk in PBS) for 1 h and incubated with first antibodies including β-actin (1:1000), p-AMPK (1:1000), AMPK (1:1000), PPARα (1:1000), PPARγ (1:1000), PGC1α (1:1000), adiponectin (1:1000), and UCP2 (1:1000) overnight at 4 °C, then washed three times with Tris-buffered saline with Tween 20 (TBST) and incubated with the secondary antibody conjugated to anti-mouse (1:5000) or anti-rabbit (1:5000) HRP-conjugated secondary antibodies for 1 h. Bands were detected using enhanced chemiluminescence (ECL) and visualized by Tanon-4600SF chemiluminescence imager . HepG2 cells, liver tissue, and iWAT were lysed in ice-cold lysis buffer containing protease inhibitor cocktail  and 1 mmol/L phenylmethanesulfonyl fluoride (PMSF) for 30 min, and were subjected to centrifugation at 10,000× ® Ultra™ RNA Library Prep Kit for Illumina® according to the manufacturer’s instructions. Library quality was checked using the Agilent Bioanalyzer 2100 system. After cluster generation, the library was sequenced using an Illumina Novaseq platform, and 150 bp paired-end reads were generated. RNA sequencing data have been submitted to the Gene Expression Omnibus (GEO) under accession number (GSE199105). RNA extraction and RNA-seq analysis were carried out by Novogene . Briefly, total RNA from liver tissues was isolated using TRIzol reagent according to the manufacturer’s instructions, and RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer2100 system . The mRNA was purified using poly-T oligo-attached magnetic beads. After fragmentation, libraries were constructed using NEBNextHigh quality reads were aligned to the Ensemble mouse (mm10/GRCm38) reference genomes with HISAT2 (version 2.0.5) software. Differential expression analysis was performed using the DESeq2 R package (1.20.0). The resulting p-values were adjusted using Benjamini and Hochberg’s approach for controlling the false discovery rate. Transcripts with a Padj <0.05 and a fold change >1.2 were assigned as differential expression genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs were implemented by Novogene .t-test. Multiple groups were compared using one-way or two-way analysis of variance (ANOVA) followed by the Tukey’s test depending on the experiments. Differences where p < 0.05 were considered statistically significant. Image J software  was used to quantify the relative expression of proteins and adipocyte area. Graphs were produced using Prism 8 software . Numeric results are expressed as the mean ± SEM. Two experimental groups were analyzed using unpaired two-tailed Student’s To examine the therapeutic effects of SZ-A on obesity and NAFLD, 6-week-old C57 mice were fed an HFD for 14 weeks and administered SZ-A (i.g.) for an additional 6 weeks with continuous HFD feeding A. After Next, we examined the direct effect of SZ-A on adipose tissue and adipocytes. Fewer immune cells infiltrated the eWAT of the SZ-A-treated mice A. BAT frNAFLD is characterized by lipid overload, oxidative stress-induced cellular damage, inflammation, and fibrosis. Liver weight and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and C-reactive protein (CRP) levels were significantly elevated in mice administered an HFD diet. Here, we found that the serum levels of ALT, AST, and CRP were lower in SZ-A-treated mice than in control HFD mice A–C. LiveNext, we examined the effects of SZ-A on inflammation and fibrosis. Macrophage markers (F4/80 and CD68) and pro-inflammatory cytokines  and monocyte chemoattractant protein-1 (MCP1)) were significantly upregulated in the livers of HFD-fed mice. However, mRNA expression levels of macrophage markers and cytokines were attenuated in the SZ-A group H. LectinTo test whether SZ-A exhibited therapeutic effects outside the intestinal tract, HFD-fed mice were administered SZ-A (200 mg/kg) daily via intraperitoneal injection for 6 weeks. Bodyweight was significantly reduced by SZ-A at 200 mg/kg A, which n = 6 per group). A total of 436 genes were differentially expressed  were significantly diminished after SZ-A treatment. Moreover, the mRNA levels of lipid β-oxidation genes (proliferator-activated receptor-α (PPARα) and PGC1α) were increased in SZ-A-treated mice A. SZ-A iTo investigate the direct effects of SZ-A on hepatocytes, HepG2 cells stimulated with PA were used as models of insulin resistance and lipid accumulation. PA-stimulated HepG2 cells developed insulin resistance. Compared to the PA group, SZ-A significantly increased glycogen content and glucose consumption in HepG2 cells A,B. We aMice develop obesity and NAFLD when fed with HFD. Excessive lipids accumulate in the adipose tissue, which leads to obesity. Ectopic lipid accumulation in the liver induces oxidative stress and liver injury. Chronic inflammation and fibrosis occur because of metabolic stress. SZ-A is a group of alkaloids that undergo rapid distribution in liver and adipose tissues after administration. The weight-loss effect of SZ-A was partly mediated by activation of PPARα. Reduced fat mass is beneficial for systemic improvement of metabolism. SZ-A stimulates adiponectin expression and secretion, which mediates the crosstalk between liver and adipose tissue. AMPK is activated in the liver and HepG2 cells by direct and indirect effects of SZ-A. The phosphorylation of ACC by p-AMPK inhibits the function of ACC to inhibit lipogenesis and enhance lipid β-oxidation. PPARα and PGC1α are also increased in the liver. In conclusion, SZ-A protects mice from NAFLD through direct effect on hepatocytes and indirect effect mediated by weight loss. AMPK and PPAR signaling are involved in the protective effects of SZ-A .Morus alba L., has been shown to reduce hyperglycemia in patients [NAFLD, obesity, and T2DM are common manifestations of metabolic syndrome that shares the same mechanisms, such as hyperlipidemia, insulin resistance, hyperglycemia, and inflammation. Increased hepatic gluconeogenesis promotes insulin resistance and T2DM, further aggravating NAFLD. Excessive fat leads to hypertrophy of adipocyte cells, necrosis, inflammation of adipose tissue, and increased FFA in the liver, which are risk factors for NAFLD . SZ-A, tpatients  and was In the present study, we used mice induced by HFD for 14 weeks as a model, followed by administration of SZ-A for another 6 weeks to study the therapeutic effect of SZ-A. We found that SZ-A ameliorated HFD-induced obesity and inhibited the increase in serum total cholesterol levels. Liver FFAs are derived from adipose tissue lipolysis, de novo lipogenesis, and diet. The fates of FFAs are β-oxidation and triglycerides formation. Excessive FFAs in the liver induce lipotoxicity when their supply and disposal are disturbed [To further investigate the target tissues that mediate the protective effect of SZ-A on obesity and NAFLD, SZ-A was intraperitoneally administered to HFD-induced mice for 6 weeks. We found that the bodyweight of mice treated with SZ-A was significantly lower than that of mice in the control group. Serum ALT and AST levels, hepatic steatosis, inflammation, and fibrosis were also reduced. These results imply that SZ-A may exert a protective effect on organs other than the intestinal tract. It has been reported that the major distribution tissues of DNJ, FA, and DAB are the gastrointestinal tract, liver, and kidney, which provide a rational basis for the direct effect in these tissues .Here, we focused on the direct effects of SZ-A and its major constituents, DNJ, FA, and DAB, on hepatocytes and adipocytes. PA induces lipid accumulation and insulin resistance in hepatocytes. In vitro studies revealed that SZ-A alleviated PA-induced lipid accumulation in HepG2 cells, indicating a direct effect of SZ-A on hepatocytes. It is mainly DNJ and DAB that play a role in activating the p-AMPK/p-ACC pathway and lowering liver triglycerides and cholesterol. SZ-A and its different constituents were added during the differentiation of 3T3-L1 adipocytes. We found that SZ-A promoted adiponectin secretion in 3T3-L1 adipocytes. DNJ and DAB are major constituents that stimulate adiponectin secretion. In terms of its anti-inflammatory effects, it has been reported that DNJ and FA are effective in inhibiting supernatant IL-6, whereas DAB is more effective in suppressing TNFα . These rObesity is an important pathogenic factor for NAFLD and T2DM. Agents with mechanisms of insulin sensitization and bodyweight loss have been studied for NAFLD treatment. It is reported that treatment resulting in a 5% weight loss decreases hepatic fat content . The effAdipose tissue plays an important role in systemic nutrient and energy homeostasis. Intercellular and interorgan communication is mediated by adipokines secreted by adipocytes. Adiponectin has been shown to inhibit inflammation, suppress oxidative stress, and accelerate fatty acid oxidation by binding to adiponectin receptors . The syn2+/calmodulin-dependent protein kinase regulates PGC1α expression [Lipid overload and oxidative stress-induced cellular damage are important factors in NAFLD pathogenesis. Hepatic lipid accumulation is the result of a disequilibrium between lipid production and consumption. AMPK is a master regulator of cellular energy homeostasis . Phosphopression . PGC1α ipression . We founpression . We founOxidative stress reflects the imbalance between ROS production and scavenging . ROS cauOur study has some limitations. The protective effect of SZ-A on NAFLD cannot be separated from its weight-loss effect in this study. More studies are needed to compare the effect of SZ-A to food restriction resulting in the same weight loss on HFD-induced NAFLD. The mechanism of bodyweight loss is not clear, though we found that oxidation of fatty acid was influenced by SZ-A administration in mice. Mouse models such as methionine-choline deficient (MCD) diet-induced NASH should also be tested for effects of SZ-A in mice. We have tested the effect of SZ-A on HepG2 cells in vitro. However, the effect of SZ-A on hepatic stellate cells requires further investigation. Besides, the direct target and mechanism of individual components of SZ-A remains unknown, though the effects of SZ-A on adipocyte adiponectin expression and hepatocyte triglyceride metabolism were reported. In this study, HFD-induced obesity, hepatic steatosis, oxidative stress, inflammation, and fibrosis in mice were ameliorated by SZ-A administration. We speculate that SZ-A may act through multiple pathways. PPARα contributes partly to reduced bodyweight. SZ-A attenuates NAFLD by both weight loss and direct effects on HepG2 hepatocytes. AMPK and PPAR signaling pathways play important roles in the protective effects of SZ-A. In summary, the present study in mice revealed that SZ-A is a promising agent for the treatment of complex diseases, such as obesity and NAFLD.


Gastric cancer is one of the most common malignancies of the digestive system with a high lethal rate. Studies have shown that inherited and acquired mutations in pyruvate metabolism and citric acid cycle (P-CA) enzymes are involved in tumorigenesis and tumor development. However, it is unclear how different P-CA patterns affect the tumor microenvironment (TME), which is critical for cancer progression.This study mainly concentrated on investigating the role of the P-CA patterns in multicellular immune cell infiltration of GC TME. First, the expression levels of P-CA regulators were profiled in GC samples from The Cancer Genome Atlas and Gene Expression Omnibus cohorts to construct a consensus clustering analysis and identify three distinct P-CA clusters. GSVA was conducted to reveal the different biological processes in three P-CA clusters. Subsequently, 1127 cluster-related differentially expressed genes were identified, and prognostic-related genes were screened using univariate Cox regression analysis. A scoring system was then set up to quantify the P-CA gene signature and further evaluate the response of the patients to the immunotherapy.We found that GC patients in the high P-CA score group had a higher tumor mutational burden, higher microsatellite instability, and better prognosis. The opposite was observed in the low P-CA score group. Interestingly, we demonstrated P-CA gene cluster could predict the sensitivity to immunotherapy and ferroptosis-induced therapy.Collectively, the P-CA gene signature in this study exhibits potential roles in the tumor microenvironment and predicts the response to immunotherapeutic. The identification of these P-CA patterns may significantly accelerate the strategic development of immunotherapy for GC.The online version contains supplementary material available at 10.1186/s12935-022-02739-z. Helicobacter pylori, the incidence of GC is increasing and has shown a younger trend in recent years  dominant), C3 (inflammatory), and C4 (lymphocyte depleted), and the immune subtypes C2 had a high proliferation rate and is highly correlated with mutated gastric cancer . In the p = 0.154)  Fig. C. A defi54) Fig. E and F, Ferroptosis represents a new programmed cell death that is distinct from apoptosis and necrosis , 37. OurLDHA, CS, IDH3G were significantly higher in cancer tissues than those in normal tissues  The cumulative distribution function (CDF) curves in consensus cluster analysis for cluster numbers k = 2–9. (B) Relative change in the area under the CDF curve from k = 2 to k = 9. (C) The tracking plot for k = 2–9; The vertical axis shows consensus matrix k-value, the horizontal axis represents the GC samples. (D-L) Consensus matrix heat map when k = 2-9. DEGs differentially expressed genes; CDF, cumulative distribution function.Additional file 3: Figure S3. The correlation of the ferroptosis with P-CA. Heatmap of ferroptosis genes expression among P-CA clusters (A), and gene clusters (B). (C) Differences in the expression of genes related to ferroptosis among the three gene clusters. .Additional file 4: Figure S4. Verification of gene expression levels. Three upregulated genes including LDHA (A), CS (B), IDH3G (C). Immunohistochemical staining of IDH3G (D)


Human InsR, IGF1R, and IRR receptor tyrosine kinases (RTK) of the insulin receptor subfamily play an important role in signaling pathways for a wide range of physiological processes and are directly associated with many pathologies, including neurodegenerative diseases. The disulfide-linked dimeric structure of these receptors is unique among RTKs. Sharing high sequence and structure homology, the receptors differ dramatically in their localization, expression, and functions. In this work, using high-resolution NMR spectroscopy supported by atomistic computer modeling, conformational variability of the transmembrane domains and their interactions with surrounding lipids were found to differ significantly between representatives of the subfamily. Therefore, we suggest that the heterogeneous and highly dynamic membrane environment should be taken into account in the observed diversity of the structural/dynamic organization and mechanisms of activation of InsR, IGF1R, and IRR receptors. This membrane-mediated control of receptor signaling offers an attractive prospect for the development of new targeted therapies for diseases associated with dysfunction of insulin subfamily receptors. Receptor tyrosine kinases (RTKs) are ubiquitous receptors in the human organism. The human RTKs family contains 58 receptors divided into 20 subfamilies. The insulin receptor subfamily contains three highly homologous receptors: the insulin receptor (InsR), the insulin-like growth factor-1 receptor (IGF1R), and an orphan insulin receptor-related receptor (IRR) ,4. Membe2 [The insulin receptor subfamily has a unique structure among RTKs. Each receptor monomer consists of two disulfide-bonded polypeptide chains, α and β. On the cell surface, the receptors exclusively exist as preformed covalently bound dimers (αβ)2 . Molecul2 . Binding2 ,16,17,182 ,14,16 wi2 . Upon li2 ,18,20,212 . An addi2 .To date, the structural–dynamic information concerning TM segments of the insulin receptor subfamily remains scarce. Functionally, TMD anchors the receptors in the plasma membrane and participates in proper transmission of the ligand-binding signal. Moreover, substitution of InsR-TMD by TMD of glycophorin A (GpA) markedly suppresses insulin signaling . In addi952 and Pro941, respectively. Moreover, IGF1R-TMD has a higher flexibility in comparison with InsR-TMD. A distinctive feature of IRR-TMD is two short α-helices connected through a flexible linker Thr925AlaThrPro928. Thus, the conformational variability of TMDs and their interactions with surrounding lipids were found to differ significantly between these three representatives, suggesting the existence of membrane-mediated control of the receptor activation and functioning within the subfamily. Moreover, the observed TMD flexibly of all three proteins suggests a unique molecular mechanism of the receptor activation distinct in some details from other RTKs.In this work, we aim to compare the structures and dynamics of TMDs of all three representatives—InsR, IGF1R, and IRR—from the subfamily. In particular, we conducted structural studies of their TMDs in membrane-mimicking micelles using NMR spectroscopy. Furthermore, we probed conformational variability of the three TMDs using molecular dynamics (MD) simulations of the NMR-derived structures in different lipid bilayers. Each TMD model has some distinguishing features. Both InsR and IGF1R TMD structures form α-helices with a kink at residues Pro13C,15N-labeled peptides InsRtm, IGF1Rtm, and IRRtm, containing the hydrophobic TM segments flanked by short polar N- and C-terminal regions corresponding receptor fragments InsR951–982, IGF1R931–962, and IRR917–948, respectively. The peptides were solubilized in the aqueous suspension of dodecylphosphocholine (DPC) micelles at a detergent/peptide (D/P) molar ratio of 160 to shift the predominant dynamic equilibrium state of the peptides into monomer form [1H/13C/15N-heteronuclear NMR technique at 313 K, pH 6.7 [In order to characterize and compare the TMD structural–dynamic properties within the insulin receptor subfamily, we obtained the mer form . The pre, pH 6.7 ,31.1H/15N-HSQC spectra  7PHT, 7PH8, and 7PL4, respectively.According to the characteristic chemical shift distribution observed in the heteronuclear NMR  spectra A, the ov spectra . The str954–R980 and F933–R960, respectively, with a slight bend near the intramembrane proline residues P961 and P941. In contrast, IRRtm includes two helical regions: G919–T925 and V929–K946  bends the TMD helix with the angle of ~26° between n- and c-term helical part A955–G960 and P961–L979 and the distance of ~40 Å between A955 and R980. IGF1Rtm helix has two irregularities at P941 and G949G950. The bending angle of ~30° is observed between the n-term and the central helical part F933–L940 and P941–V948 followed by a ~10° kink at G949G950. Nevertheless, the distance between n-term F933 and c-term R960 of IGF1Rtm is the same 40 Å as for the other TMD structures, with the exception of the bent state of IRRtm.In all cases, the intramembrane prolines destabilize the TMD helices, causing a kink that structurally separates the helices into the N- (n-term) and C-terminal (c-term) parts. Pwo parts G, yellow. InsRtm G, blue a. InsRtm G, green 2 derived from 1H, 13C, and 15N chemical shifts revealed similar differences between the TMDs. While the internal parts of InsRtm and IGF1Rtm have strong helical conformation with slight weakening of helicity before the intramembrane proline residue  estimated from 15N CSA/dipolar cross-correlated transverse relaxation of the TMD amide groups are in agreement with this assumption  and local order parameters S residue C,D, IRRtsumption B. Moreovs, Δδ1HN E, which flexible B and accflexible F.In order to study conformational behavior in different membrane environments, the obtained NMR-derived structures of the TMDs from the insulin receptor subfamily were relaxed by MD in two explicit lipid bilayers consisting of POPC and DMPC surrounded by water molecules. POPC and DMPC membranes have different properties, such as acyl chain ordering and membrane thickness; POPC forms a bilayer with hydrophobic thickness of 2.9 nm while DMPC is thinner by approximately 0.14 nm . StartinAccording to RMFS values averaged over MD trajectories for each TMD B, the n-As shown in 936IAL940 before intramembrane proline P941  mainly lays parallel to the membrane surface in both lipid bilayers. The longer c-term helix (V929–K946) crosses the POPC and DMPC bilayers, having the tilt angle of ~15° and ~30°, respectively . This leads to increased mobility of the N-terminal part of TMD helices in the micellar medium mimicking a membrane. Moreover, IRR TMD undergoes a conformational transition between the two main I- and Γ-states corresponding to the straight and bent structures of the TMD helix divided by the P-hinge into mutually mobile N- and C-term parts. MD simulations of the NMR-derived structures in two explicit lipid bilayers composed of POPC and DMPC revealed that InsR TMD embedded into thin DMPC membrane can also switch between two main states I and Γ due to unfolding of the helix turn (forming P-hinge) adjacent to the intramembrane proline. At the same time, TMD of IGF1R compensates for the decrease in membrane thickness by tilting—this is consistent with previous MD simulations [940–988 fragments elongated by JM regions [In the present NMR studies, we found that all TMDs of InsR, IGF1R, and IRR have bends before the intramembrane proline residues Pulations . Neverthulations  have sho regions . This suAs noted above, the relatively hydrophilic surface regions A, which Although transmembrane signaling by type I receptors such as RTKs has not yet been fully elucidated, some molecular details of the process regulated by TMDs have been uncovered based on numerous structural, biophysical, and biochemical studies. Recently, we proposed a lipid-mediated mechanism implying that signal transduction through the membrane and allosteric regulation of type I receptors are inclusively mediated by coupled protein–protein and protein–lipid interactions ,46. To probe protein–lipid interactions, we used MD-averaged distributions of lipid density around TMDs. These data were visualized in terms of 2D density maps—projections on the surface of a cylinder built around the helix axis . IntriguBased on the totality of experimental and calculated data obtained in this work as well as published data, we came up with the following sequences of key events leading to RTKs of the insulin receptor subfamily activation and signal transduction schematically summarized in 932FIHLIIALPVAVLLIVGGLVIMLYVFHRKR962. InsRtm and IRRtm gene constructions contained N-terminal His-tag connected with target proteins by flexible GS linker MHHHHHHG-S952NIAKIIIGPLIFVFLFSVVIGSIYLFLRKR982 and MHHHHHHGS-A917GGLHVLLTATPVGLTLLIVLAALGFFYGKKR948, respectively. We use continuous-exchange cell-free expression (CECF) system based on E. coli S30 extract. The plasmids were used as DNA templates in the reactions [13C,15N-labeled algal amino acid mixture was used for production of isotopic labeled proteins. The reactions were incubated with gentle shaking overnight at 34 °C. The peptide precipitate was separated from the reaction mixture by centrifugation. The pellet was solubilized in a buffer containing 0.5% lauroyl sarcosine, 50 mM Tris/HCl, pH 8.0, 50 mM NaCl, and size exclusion chromatography was carried out using the Tricorn 10/300 Superdex 200 Increase column at 0.5 mL/min flow rate. Fractions containing the peptide were combined, then the peptide was precipitated by addition of TCA and washed with cold acetone three times. The pellet was solubilized in 1:1 (v/v) trifluoroethanol–water mixture and lyophilized. In order to incorporate TMDs into membrane-mimicking micelles or bicelles, the peptide powder was dissolved in 1:1 (v/v) trifluoroethanol–water mixture with the addition of n-dodecylphosphocholine-d38  at the peptide:detergent (P:D) molar ratio of 1:160 [3, and 5% D2O (v/v), pH 6.7.The DNA sequences encoding human TMD fragments of IGF1R , InsR , and IRR  were synthesized from six overlapping synthetic oligonucleotides by PCR and then cloned into pGEMEX-1 vector. The genes were under the control of the T7 promoter. The final construct of IGF1Rtm was with the leading methionine: M-Neactions  to produeactions ,52. The of 1:160 . The mix1H/13C/15N backbone resonances of TMDs were assigned with the CARA software [1H/15N-HSQC, 1H/13C-HSQC, 1H/13C-HSQC-CT (constant time version with evolution period of 28.6 ms), 1H/15N-TROSY, 1H/13C/15N-HNCO, 1H/13C/15N-HNCA, 1H/13C/15N-HN(CO)CA, 1H/13C-HCCH-TOCSY (with the mixing time of 17 ms), 1H/13C/15N-HNCACB,15N-edited TOCSY-, and NOESY-HSQC with the mixing time of 80 ms. The BEST-TROSY version [1H–1H NOE connectivities and torsion angle restraints. Torsion angle restraints, the helical secondary structure probabilities (HSP), and local order parameters S2 were obtained from the secondary structure probabilities estimated from the 1H, 13C, and 15N chemical shift values using the web-based program TALOS-N [1H–1H NOE connectivities were obtained with the CARA software through the analysis of the three-dimensional 15N-edited NOESY-HSQC spectrum. In order to characterize the intramolecular dynamics of the TMD fragments, the effective rotation correlation times τR were estimated for individual amide groups of the fragments based on 15N CSA/dipolar cross-correlated transverse relaxation experiment acquired in interleaved fashion for the reference and attenuated spectra using a 2D 1H/15N-ct-TROSY-HSQC-based pulse sequence [R was calculated as the ratio of peak intensities of the reference and attenuated spectra; the uncertainties were estimated from the noise level. The water accessibility of the TMDs residues was analyzed by chemical exchange of the amide protons with water directly detected by CLEANEX [1HN secondary chemical shifts, Δδ1HN, of the TMDs residues was calculated as the difference between actual chemical shift and typical random-coil chemical shift for a given residue [NMR spectra were acquired at 313 K on 600 and 800 MHz Bruker AVANCE III spectrometers equipped with triple-resonance with H/C/N triple resonance Z-gradient cryoprobe. The software  using thsoftware : 1H/15N- version  of the t version . The spa version  based on TALOS-N . Intramosequence  with the CLEANEX  experime residue .MD simulations and data processing were carried out using the GROMACS package version 2019.4 or higher. All-atom Amber14SB protein parameters with S-lipids and TIP3P water were used to describe intermolecular interactions together with PME electrostatics and Van der Waals cut-off at 1.5 nm. To estimate stability of all the systems, they were subjected to energy minimization, MD equilibration with restrained protein, and unrestrained 200-ns MD simulations. Each TMD fragment was inserted into a pre-equilibrated hydrated palmitoyloleoylphosphatidylcholine (POPC) or dimyristoylphosphatidylcholine (DMPC) bilayer with a conformation derived from NMR data and perpendicular orientation of the helix axis with respect to the membrane plane. Relaxation was carried out by 9-ns MD simulation started from fully “frozen” protein with stepwise unfreezing of terminal amino acids as follows: First, we used constraints imposed on all protein atoms and equilibrated the hydrated lipid bilayer for 3 ns at the constant temperature of 315 K. Then, we allowed sidechains and terminal protein residues to fit to the environment during 6-ns MD. Finally, production MD runs were performed without restraints. The structure stability was estimated in terms of the root-mean-square deviation (RMSD) of coordinates of all (or only backbone) protein atoms from the starting NMR structure, protein secondary structure change, and the tilt angle between the helix axis and the membrane normal. Molecular flexibility was evaluated as per-residue root-mean-square fluctuation (RMSF) averaged over the trajectory. Molecular hydrophobic–hydrophilic properties on the helix surface were estimated using the molecular hydrophobicity potential (MHP) approach, as described previously ,42. ProtIn summary, we can conclude that the TMD sequences of all three representatives of the insulin receptor subfamily encode information about their conformational lability, which is largely determined by the intramembrane proline residue and its neighboring residues. Under the conditions of a real lipid membrane, this is expressed in the distinct features of the structural–dynamic behavior of these TMDs. It is important to note that the extent of the conformational changes undergone differs for the three proteins and, in addition, it depends on the properties of the surrounding water–lipid media. Variation of these parameters allows each of the representatives of the signaling receptor subfamily to respond sensitively to activating stimuli  and the current state of the local membrane environment. This, in turn, can provide the receptors with a certain “margin of safety” necessary for the stable and robust operation of these important cell signaling systems. On the other hand, it gives them a certain flexibility, helping to synchronize with their neighbors in the “signal orchestra”—the surrounding membrane receptors. This membrane-mediated control of signaling of the insulin family receptors would also influence receptor dysfunction upon pathological conditions, which should be taken into account when developing new targeted therapies for human diseases, including neurodegenerative ones.


E-selective catalyst, with a readily available photocatalyst, enables the exclusive formation of the contra-thermodynamic Z-isomer. The scope and limitations of this unprecedented approach are discussed based on both computational and experimental mechanistic data.Olefin cross-metathesis is a cornerstone reaction in organic synthesis where stereoselectivity is typically governed by the structure of the catalyst. In this work, we show that merging Grubbs second generation catalyst, a classical  Light is magic! The combination of Grubbs second generation catalyst, a well-known catalyst for E-selective olefin metathesis, with a photosensitizer enables efficient access to the contra-thermodynamic Z-isomers. However, the feasibility of this dual catalytic (Ru/PC) system depends on the compatibility of the metathesis catalysts as well as the different intermediates generated during the metathesis process with the photocatalytic conditions , we used conditions previously reported by Grubbs Z : E ratios (80 : 20 to 90 : 10).7 These results are in good accordance with Zhang's report that cyanoarenes are competent catalysts for stilbene photoisomerization.14 The low Z : E ratio obtained when riboflavin  is used as a photocatalyst is obviously due to its low solubility in dichloromethane.As shown in E isomer was obtained exclusively  in the absence of photocatalyst, the reaction didn't proceed without Grubbs second-generation catalyst . Finally, isomerization didn't proceed in the dark (entry 11), and yield and stereoselectivity dropped when the reaction was carried out at room temperature (entry 12).Based on these results, photocatalyst PCf was selected for further optimization evaluations. Unsurprisingly, while the Z-stilbenes were obtained in good to excellent conversions (66 to 99) and high selectivity (Z : E ranging from 87 : 13 (8e and 8f) to 91 : 9 (8a)) except in the case of naphthyl stilbene 8i (Z : E = 75 : 25).With the optimized conditions in hand, we next evaluated the scope of the reaction by testing different styrenes bearing electron-donating or accepting groups at the para or the meta positions of the aromatic ring . Homodim13 the reaction is amenable to olefin cross-metathesis with aliphatic olefins 8l–8q. In these cases, improved yields and selectivities were observed when PCa was used instead of PCf. As depicted in ortho position of the aromatic ring. Interestingly the reaction works smoothly with 4-cyanostyrene, leading to the desired adduct 8o in good yield and Z : E = 74 : 26. Moreover, the reaction proceeds well with acrylates and good to excellent conversions were obtained (8r–8zf). The stereoselectivity observed in these cases was fair to excellent and the process shows good compatibility with various functionalities (ester (8t), chloro (8u), fluoro (8v), trifluoromethyl (8w), cyano (8za), and bromo (8zb)), confirming the broad scope of this novel tandem experimental protocol.Interestingly, in accordance with Grubbs' general empirical model for olefin reactivity,In order to gain further insights into the reaction mechanism, experimental and computational experiments were conducted.3. Excitations towards LUMO+7 are indeed the major components of singlet states S5 (3.25 eV/381 nm) and S6 (3.41 eV/363 nm) at the ground state geometry. Their oscillator strength is small with respect to that of state S7 (3.56 eV/348 nm) but not neglectable, thus dissociative 1MC states can be directly populated upon 355 nm irradiation, or more likely upon excitation to S7 followed by internal conversion to S6 or S5. The corresponding dissociative 3MC states (T6 at 2.69 eV and T7 at 2.93 eV) are expected to be similarly populated through additional intersystem crossing facilitated by spin–orbit coupling due to the metal center. These computational results are fully consistent with previous experimental results showing the efficiency of near visible light irradiation to generate 14-electron complexes from 2 or related compounds.12,15We first examined the interaction of blue light with Grubbs second generation catalyst to confirm that light was not deleterious to the metathesis process. In this context, several DFT calculations were performed, starting with inspecting the optimized ground state molecular orbitals ˙−. This efficient single electron transfer matches the redox potentials PCf* (E° = 1.41 V/SCE) and 2 (E° = 0.25 V/SCE) of both components. Additionally, the Stern–Volmer luminescence quenching experiment evidenced linear correlation with respect to the metathesis catalyst 2. This shows that 2 is an effective reductive quencher of PCf* (Ksv = 2.34 × 104 M−1) .17 This efficient process results in the generation of the 14-electron complex 9 through a classical dissociative pathway from neutral 2 rather from its radical cation (2)˙+ as in the case of bis-NHC ligated Ru complexes.18 Oxidation of 2 by *PC is thus not problematic since BET is fast  was observed by transient spectroscopy and measured to be diffusion-limited . Marked shortenings of the Ru–C(car) (car = N-heterocyclic carbene) and Ru–Cl distances are also noteworthy between 2 and 9  to occupy the volume set free by releasing the bulky PCyThe most remarkable ground state Kohn–Sham orbitals of 9 are the dσ* antibonding LUMO+1 (towards car) and LUMO+2 (towards ben), both displaying small contributions towards the bound C atom and large contributions in the vacant space opposite , and even lower between *9-3MLCT and 9   distance at 2.096 Å . This triplet state is nearly degenerate with its associated minimum energy crossing point , therefore very efficient nonradiative deactivation processes can be expected in the 14-e complex. The existence of this low-lying 3MC state is not relevant to metathesis itself but could be involved in catalyst photodegradation mechanisms.In the case of complex 9, a 3 loss or nonradiative deactivation. If light is absorbed by complex 9, efficient nonradiative deactivation channels are also available.To sum up, the minor fraction of light absorbed by complex 2 is not deleterious in that it could lead to PCyE-stilbene, which eventually gets isomerized into its Z form. The energy content of the triplet state PCf* is 51.3 kcal mol−1, i.e. larger than that of E-stilbene   processes, we have already established that the triplet states of 2 and 9 are too low lying to allow energy transfer to the organic compounds present in the reaction vessel . We then Table S4. This de− and (2)˙+. Due to a diffusion-limited BET, phosphine loss occurs from 2 rather than from its oxidized form. Then, the classical metathesis mechanism takes place to yield the E-alkene. A subsequent photoisomerization of the latter by PC* leads to the formation of the desired Z-isomer 8. The strong point is that for simplicity, the reaction is performed one-pot under continuous irradiation.Based on these mechanistic data, we propose the reaction mechanism shown in Z-olefins from the standard E-selective Grubbs second-generation catalyst. Joint experimental and computational investigations showed that the feasibility of this reaction results from a combination of (i) light being mostly absorbed by the photocatalyst PC, (ii) diffusion-controlled back electron transfer restoring Grubbs catalyst following its oxidation by PC* (iii) formation of the classical metathesis product E-alkene, and (iv) E–Z photoisomerization by a final energy transfer event from PC*.We have developed an orthogonal tandem catalytic (metathesis/photoisomerization) transformation enabling facile access to E-selective) geometric isomerization due to the formation of ruthenium hydride complexes.19 However, our results show that a high level of Z-selectivity can be achieved without any additives. This indicates that competitive ground state metal hydride isomerization processes can be avoided under our conditions.Finally, it is worth mentioning that metathesis reactions can often lead to positional isomerization or thermodynamic (Z-olefins.This work opens new avenues in the exploration of photometathesis that should lead to the development of practically simple approaches for the synthesis of Experimental and computational data have been provided in the ESI.‡S. E. C. and A. G. performed the synthetic experiments; S. C. performed the laser flash photolysis and fluorescence experiments; I. M. D. performed the computational studies; and S. L. designed and supervised the project. All authors analysed the results.There are no conflicts to declare.SC-013-D2SC03961C-s001SC-013-D2SC03961C-s002SC-013-D2SC03961C-s003SC-013-D2SC03961C-s004SC-013-D2SC03961C-s005SC-013-D2SC03961C-s006SC-013-D2SC03961C-s007SC-013-D2SC03961C-s008SC-013-D2SC03961C-s009SC-013-D2SC03961C-s010SC-013-D2SC03961C-s011SC-013-D2SC03961C-s012SC-013-D2SC03961C-s013SC-013-D2SC03961C-s014SC-013-D2SC03961C-s015SC-013-D2SC03961C-s016SC-013-D2SC03961C-s017SC-013-D2SC03961C-s018