CYP46A1 is a brain-specific enzyme responsible for cholesterol homeostasis. Inhibition of CYP46A1 activity serves as a therapeutic target for excitatory neurotoxicity. Sesame is a common medicine and food resource; its component lignans possess various pharmacological activities. In this study, the inhibitory effects of sesame lignans on CYP46A1 activity were investigated. Inhibition kinetics analyses revealed that sesamin and sesamolin produce mixed partial competitive inhibition of CYP46A1, while sesamol produces non-competitive inhibition. Notably, molecular simulations revealed that the sesame lignans have excellent orientations within the active cavity of CYP46A1. Importantly, the sesame lignans had high permeability coefficients and low efflux ratios. Furthermore, sesamin significantly reduced the levels of 24S-hydroxycholesterol in rat plasma and brain tissues, and down-regulated the protein expressions of CYP46A1, NMDAR2A, NMDAR2B, and HMGCR. Collectively, sesame lignans exhibit significant inhibitory effects on CYP46A1 activity, highlighting their potential therapeutic role in treating excitatory neurotoxicity. S-HC) and is the dominant cholesterol catabolic enzyme in the brain  is a brain-specific cytochrome P450 family enzyme encoded by CYP46A1. It converts cholesterol into 24S-hydroxycholesterol  and Huntington’s disease (HD) in animals . ConversSesame, also known as Sesamum indicum, is an annual plant that is a member of a widely cultivated genus. Sesame is an important oilseed with edible and potential nutritional and medical uses in most countries . Sesame in vivo.In our preliminary study, the inhibitory effects of sesamin, sesamolin, and sesamol on CYP46A1 were evaluated. Surprisingly, sesamin, sesamolin, and sesamol displayed significant inhibitory activity against CYP46A1. Therefore, we hypothesized that sesame has an effect on cholesterol metabolism in the brain by regulating the activity of CYP46A1, which, at least partly, explains its neuroprotective effect. Herein, we characterized the inhibitory kinetics of sesame lignans and explored the relationship between structure and inhibition. Moreover, the influence of sesamin on the activity and expression of CYP46A1 was investigated Testosterone and digoxin were obtained from Dalian Meilun Biotech Co., Ltd . 4-androsten-16β,17β-diol-3-one (16β-hydroxytestosterone) was obtained from Steraloids, Inc. . Sesamin (purity >98%) was obtained from Tokyo Chemical Industry Co., Ltd. Sesamolin (purity >98%) was obtained from Shanghai Aladdin Bio-Chem Technology Co., Ltd . Sesamol (purity >98%) was purchased from Sigma-Aldrich . Nicotinamide adenine dinucleotide phosphate (NADPH) and Carbamazepine were acquired from Shanghai Yuanye Bio-Technology Co., Ltd . The MDCK-MDR1 cell line was generously donated by Professor Su Zeng, Zhejiang University . Neuro-2a stable cell line overexpressed in human CYP46A1 (h-CYP46A1-Neuro-2a) was purchased from Hanbio Biotechnology Co., Ltd. The cDNA-expressed recombinant human cytochrome P450 (rhCYP) isoforms, including CYP46A1, derived from baculovirus-infected insect cells co-expressing NADPH-CYP reductase and cytochrome b5 were purchased from Cypex . A BCA Protein Assay Kit was purchased from Beijing Solarbio Science and Technology Co., Ltd. Anti-Cholesterol-24 Hydroxylase antibody was obtained from Sigma-Aldrich (Darmstadt Germany). NMDAR2A antibody and NMDAR2B antibody were purchased from Proteintech Group . HMGCR Rabbit mAb was obtained from ABclonal Technology Co., Ltd. Analytical reagent grade and high-performance liquid chromatography (HPLC) grade solvents were obtained from Tedia, Inc. .Km value), 100 mM potassium phosphate buffer (pH 7.4), 4 μL recombinant human CYP46A1 bactosomes , and 0.5 μL sesame lignans. After preliminary incubation at 37°C for 5 min, 10 μL NADPH  was added to initiate the reaction for 10 min. The reaction was stopped by adding the same volume of acetonitrile containing 1 μM of the internal standard. The mixture was left on ice until it was centrifuged at 20,000×g for 20 min at 4°C. Aliquots of the supernatant were stored at −20°C until analysis. The possible inhibitory intensities of the sesame lignans were evaluated by quantifying the 16-OH-TST generated.16β-hydroxytestosterone formation catalyzed by CYP46A1 was conducted at conditions linear for the duration of incubation and protein concentration. The incubation mixture (100 μL) consisted of 0.5 μL testosterone : 25 L/min; Ion Source Gas2 : 35 L/min; Curtain Gas (CUR): 30 psi; Collision Gas (CAD): 12 psi. MS/MS conditions for analyzed compounds are shown in Testosterone 16β-hydroxylation reaction samples were injected into an API 3200 triple quadrupole mass spectrometer  and an Agilent HP1200 LC system . The chromatographic separation was performed on a Hypersil ODS-BP column  at ambient temperature. IonSpray Voltage (IS): 4500 V; Temperature (TEM): 590°C; Ion Source Gas1  in the presence or absence of sesame lignans using Prism . Ki values were calculated using the equations for competitive inhibition  of the reaction. The α value determines the degree of change in enzyme affinity for the substrate following the binding of the inhibitor. When α is less than 1, binding of an inhibitor prevents substrate binding, and the mixed model is identical to non-competitive inhibition. When α is very large (α > 1), the binding of an inhibitor prevents substrate binding, and the mixed model depicts competitive inhibition. The type of inhibition was determined by fitting the data to enzyme inhibition models. The goodness-of-fit of kinetic and inhibition models was assessed using R2 values and parameter S.D. estimates. Dixon and Lineweaver-Burk plots were also used to characterize the inhibition kinetics. Kinetic constants are reported as the mean ± S.D. of the parameter estimate.Under the above incubation conditions, different concentrations of sesame lignans at the same concentration of testosterone (10 μM) were first used to measure the IChibition , non-comhibition , or mixehibition . The thrhibition .v=Vmax⋅To illustrate the potential interactions between the sesame lignans and CYP46A1, docking simulations were performed using the Sybyl/Surflex module. The structures of sesamin, sesamolin, and sesamol were downloaded from SciFinder. Based on the crystal structure of CYP46A1 (PDB ID: 2Q9F), the bioactive binding conformations of sesamin, sesamolin, and sesamol and soticlestat (a known specific CYP46A1 inhibitor) were evaluated using the empirical function Total Score. A 2D molecular descriptor  was used to show the interacting amino acid residue plot of the ligands located within the CYP46A1 active site. In addition, the docking results of sesamin, sesamolin, and sesamol were visualized using the PyMOL Molecular Graphics System, version 0.99 (DeLano Scientific LLC).®, PC membrane, 0.33 cm2 surface area, 0.4 μm pore size) in 24-well companion plates for 5 days. The culture medium was changed once every 2–3 days. To confirm the confluency of the cell monolayer, the trans-epithelial electrical resistance (TEER) of each well was measured before the transport assay using a resistance meter . The transport experiment was performed only on inserts with TEER values above 300 Ω cm2 for MDCK-MDR1 cells (Papp) and efflux ratio (ER) were calculated as follows , a membrane permeability assay was conducted with an MDCK-MDR1 monolayer. The transport assays were performed by seeding MDCK-MDR1 cells on a tissue culture plate insert  transport rate, and Papp, A→B is the apical to basal (A-B) transport rate. When the ER value is greater than or equal to 2, the investigated drug is considered to be a substrate of the efflux transporter  in cells that express P-gp, according to FDA guidelines .First, The Neuro-2a-CYP46A1 cells were seeded into 96-well plates at a density of 1 × 102 and 95% humidity. The Neuro-2a-CYP46A1 cells were seeded into six-well plates at a density of 1 × 105 cells per well and cultured for 24 h. Drug intervention experiments were conducted by replacing the medium with a serum-free medium containing sesame ligands (20 μM) for 24 h.MDCK-MDR1 cells and Neuro-2a-CYP46A1 cells were cultured in DMEM containing 10% (v/v) FBS, 1% nonessential amino acids, 100 U/mL penicillin G, and 100 μg/ml streptomycin, and incubated at 37°C in the presence of 5% COThe experiments were reviewed and approved by the Experimental Animal Centre of Dalian Medical University. All rats were obtained from Liaoning Changsheng Biotechnology co., Ltd. All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals . Wistar rats were randomly divided into the administration group (n = 5) and the control group (n = 5). The rats were provided with laboratory standard animal feed and kept under a 12-h light cycle. Sesamin (60 mg/kg) was given by intragastric administration daily for 4 weeks; the control group was given an equivalent volume of olive oil. The rats were anesthetized with pentobarbital sodium (40 mg/kg) and blood samples and brain tissue samples were collected for further assays.The cells and tissues were harvested with lysis buffer and cleaved at 4°C for 20 min. All samples were centrifuged at 12,000 × g for 15 min at 4°C. The protein concentrations were quantified by BCA regent kits. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins, and they were then transferred to PVDF membranes. After incubation of the membranes with 5% non-fat milk for 2 h, they were then incubated with the primary antibodies at 4°C overnight. After washing three times, the membranes were incubated with the secondary antibody for 2 h. The protein bands were detected by a Tanon-5200 imaging system and the densities were analyzed by ImageJ software. The GAPDH antibody served as the internal control for sample loading.S-hydroxycholesterol in rat plasma and brain tissues were measured by ELISA kits , according to the manufacturer’s instructions.The levels of 24The experimental procedure was performed as described previously . Total bp < 0.05 was considered statistically significant.The data were analyzed by one-way ANOVA and unpaired t-tests using GraphPad Prism software ; 50) of each compound on CYP46A1 enzyme activity was determined. As shown in Ki) values of 6.87 and 4.02 μM, respectively. Sesamol . The Papp values of sesamol, sesamin, and sesamolin were 35.32, 6.75, and 20.53 × 10–6 cm/s, respectively. Notably, their efflux ratios  were less than one and the ER values did not decrease to <50% of the ER in the absence of verapamil, which suggests that they are not substrates of efflux transporters, particularly P-gp, in MDCK-MDR1 cells. However, some uptake transporters might mediate their transmembrane transport. These results suggest that the sesame lignans are easily absorbed and distributed in brain tissue.Digoxin, a P-gp substrate, was first utilized as a probe drug to confirm the transmembrane transport mechanism. The transport of digoxin from the basal to the apical side was significantly greater than the opposite direction in MDR1-MDCK cells with ER values of 10.5, indicating a strong efflux transport mediated by P-gp. The result suggested that the transport model had been established effectively. The membrane permeabilities of sesamol, sesamin, and sesamolin across the MDCK-MDR1 monolayer compounds are shown in p < 0.05), 57.1% (p < 0.0001), and 26.1% (p < 0.05), respectively  once a day for 4 weeks. As shown in S-HC in the brain was significantly decreased by 29.3% (p < 0.01). Most of the circulating 24S-HC comes from the brain, so a reduction in brain synthesis reduces 24S-HC levels in plasma. The plasma concentration of 24S-HC in the control group was 103.69 ng/ml. In contrast, the plasma concentration of 24S-HC in rats treated with sesamin was 45.747 ng/ml (S-HC in plasma was significantly reduced by 55.88% (p < 0.01).To assess the effect of sesamin on the activity of CYP46A1 47 ng/ml . The levp < 0.05) and 31.6% (p < 0.05), respectively, compared with the control group (p < 0.05) and 43.5% (p < 0.001), respectively, in the sesamin-treated group compared with the control group  structures; MDP compounds interact with mammalian CYP enzymes in a variety of ways to inhibit or promote CYP enzyme activity . SesaminIn this study, sesamin inhibited the expression of the CYP46A1 protein in the rat brain but did not affect CYP46A1 mRNA. The promoter region of the human CYP46A1 gene, which has a high content of guanine and cytosine (GC), has no typical thymine-adenine-thymine-adenine (TATA) or cytosine-adenine-adenine-thymine (CAAT) boxes, according to transcriptional regulation studies. Aside from the increase in transcriptional activity produced by oxidative stress, researchers have not yet identified a treatment that can significantly alter the transcriptional activity of the CYP46A1 promoter structure . The find-aspartate receptors (NMDARs) are among the primary excitatory receptors on synapses, regulating the balance between neuronal excitation and inhibition (S-HC aggravates NMDAR-dependent excitatory toxicity in primary neuron culture after being subjected to oxygen-glucose deprivation (OGD), as well as reduced endogenous 24S-HC synthesis alleviates OGD-induced cell death (S-HC has also been identified as a powerful inducer of oxiapoptophagy, which involves simultaneous oxidative stress, apoptosis, and autophagy (in vitro and reduced the 24S-HC level in vivo, as well as down-regulated the expression levels of the NMDAR2A and NMDAR2B proteins in rat brain tissue. These findings strongly suggest that sesamin has therapeutic potential for neural hyperexcitation diseases.N-methyl-hibition . NMDARs hibition . Previouhibition . Notablyhibition . Exogenoll death . Besidesutophagy  (Gamba eutophagy . (Nury eutophagy . In thisin vitro and in vivo. Molecular simulations revealed that sesamin and sesamolin had excellent orientations within CYP46A1 by forming two water-hydrogen bonds, located near the heme of CYP46A1, which were consistent with their inhibitory intensities towards CYP46A1. Notably, the sesame lignans also had high permeability, implying that they may be easily absorbed and distributed in the brain. More importantly, sesamin dramatically lowered 24S-HC levels in vivo and lowered the protein expression levels of CYP46A1, NMDAR2A, NMDAR2B, and HMGCR in the brain tissues. Furthermore, These findings may help the development of effective therapies for neurological illnesses via modulation of CYP46A1 activity and provide a basis for further exploration of the therapeutic possibilities of targeting CYP46A1.In conclusion, naturally occurring sesame lignans exhibited remarkable inhibitory action against CYP46A1


Sepia officinalis) has represented an important resource for the fishery of the Adriatic Sea. In this study, for the first time, we assessed the presence of microplastics within cuttlefish eggs, more precisely in embryos and yolk. The presence of microplastics has already been described in common cuttlefish adult specimens. Our findings highlighted how this species is subjected to microplastic exposure starting from early-life stages. The possible effects of microplastic accumulation on embryo health and development should be investigated considering that the reproductive strategy of this species consists of adults laying eggs in separate batches during the single spawning season at the end of which both males and females die. Therefore, population stability primarily depends on the survival and correct development of embryos.Microplastic accumulation in aquatic environments could represent a potential risk to wildlife health and survival. Their transfer along the trophic web could threaten ecosystem stability. Over the years, the common cuttlefish  eggs and their association with embryonic development. Cuttlefish eggs were sampled from four different sites along the Marche region . Embryo and hatchling biometric parameters were evaluated and the internal structural integrity was examined through histological analysis. MPs were detected and characterized in embryos and yolk samples. MPs were identified in all sites (size < 5 µm), however, their presence has not been associated with an impairment of either embryo or hatchling internal structures. Noteworthy, the highest number of MPs (in both yolk and embryo samples) were found in Numana , where the lowest hatchling size was observed. On the other hand, the highest embryo mantle length was associated with the lowest number of MPs detected (9%) in Ancona. Overall, only MP fragments and sphere types  were observed, and the most frequent polymers were Polyvinyl chloride (52%), Polypropylene, and Cellulose acetate (15% both). Further studies are needed to assess the possible MP effects on the yolk quality and assimilation.Once they reach the aquatic environment, microplastics (MPs) are accidentally ingested by aquatic biota, thus entering the food chain with possible negative effects. The present study investigated, for the first time, MP presence in cuttlefish ( Danio rerio) under laboratory conditions highlighted several adverse effects associated with MP exposure and internalization such as larval hypoactivity, intestinal inflammation, oxidative stress, transcriptional changes in the immune response, metabolism disorders, and microbiome alteration [Over the last 50 years, plastic production has experienced steady and significant growth thanks to its involvement in several industry sectors  and consequently, also the number of microplastics (MPs), conventionally defined as small plastic particles (size comprise between 1 µm and 5 mm), has inevitably and rapidly increased . Existinteration ,12,13,14teration .Sepia officinalis) has always represented an important resource in the fishery history of the Mediterranean Sea [Dosidicus gigas), and two deep-sea squids , are the only four cephalopod species studied in terms of ingestion and accumulation of plastic fragments [The tendency of MPs to accumulate and move through the trophic web also implies a potential risk to human health. Many species involved in the bioaccumulation mechanism have a high commercial interest and are consumed daily by humans . Therefonean Sea ,18,19. Tnean Sea . Despiteragments ,10,21,22Evidence of MP presence in wild adult specimens of common cuttlefish was demonstrated by Abidli et al. (2019)  and by O2PO4–H2O + NaOH + Formaldehyde (36.5%) + Glutaraldehyde (25%) + H2O) at 4 °C for 24 h and successively washed in 70% ethanol three times (15 min each) and stored in the same ethanol solution at 4 °C to perform histological analysis.Cuttlefish eggs were collected, in collaboration with local fishermen, from the fishing gears where trapped females had released their eggs. Samplings were performed within June 2021 from 4 different areas/sites off the coast of the Marche region, site A, from an area off the coast of Senigallia (AN); site B, off the coast of Ancona (AN); site C, off the coast of Numana (AN); and site D, off the coast of San Benedetto del Tronto. Eggs were collected from each sampling site and successively embryos were sampled. Only the latest embryonic stages (28–29) were selected following the identification criteria suggested by Boletzky et al. (2016)  based onDue to the different times of spawning among the sampling sites, only in site B (Ancona) and site C (Numana), embryos ready to hatch were identified at sampling time. Therefore, spontaneous non-premature hatchlings from site B and site C were collected during the sampling day and their weight, and mantle length were carefully measured (n = 40 per site), 10 hatchlings per site were stored in formalin solution at 4 °C for 24 h and successively washed and stored in 70% ethanol solution at 4 °C to perform histological analysis.According to Italian legislation (D.L. 04/04/14 N.26 art1 a.1), ethical approval is not required for experiments carried out on vertebrates and cephalopods at embryonic or larval stages that are unable to feed themselves independentlyHistological analysis was performed following the method described by Chemello et al. . Samplesw/v) and incubated at 40 °C for 24 h. After 24 h, the digestates were filtered in fiber-glass filters  using a vacuum pump connected to a filter funnel. The filter membranes were dried at room temperature and placed into glass Petri dishes until the visual inspection for the identification and characterization of plastic particles.Microplastic extraction was performed at the laboratory of developmental and reproductive biology at the Department of Life and Environmental Science (DISVA) of Polytechnic University of Marche . Yolk and embryo pools were digested in a pre-filtered 10% KOH solution  made with deionized water and KOH tablets . MP extraction was performed following a modified version of the existing protocol described by Di Renzo et al. (2021)  and summ−1, 532 nm, or 785 nm laser diode). Raw Raman spectra were polynomial baseline-corrected, and vector-normalized . The chemical composition of the detected particles was identified by comparing the collected Raman spectra with several spectral libraries of polymers and pigments [Quantification and identification of microplastics were performed following the method described by Ragusa et al. (2022) . The inspigments ,28. SimiDuring sample collection, storage, processing, and analysis efforts were implemented to avoid microplastic contamination. For this purpose, a plastic-free protocol was adopted during all phases of the analysis, and a dedicated room was used for the digestion of samples, filtration, and Raman microspectroscopy analysis steps. Plastic tools were replaced with sterilized glass ones. Cotton laboratory coats and disposable latex gloves were worn during all phases of the sampling and the analysis. All liquids, including ethanol and deionized water for cleaning and preparation of all solutions, were filtered through 1.6 µm pore-size filter membranes (Whatman GF/A). Work surfaces were thoroughly washed with 70% ethanol before starting all procedures. Glassware and instruments, including scissors and tweezers, were washed using dishwashing liquid, triple rinsed with 70% ethanol, and finally rinsed with 1.6 µm filtered deionized water. Moreover, environmental and procedural blanks were prepared and thoroughly analyzed to detect microplastic contamination deriving from the laboratory environment and other external sources. Regarding environmental blanks, a filter membrane soaked with 1.6 µm filtered deionized water was placed into an uncovered Petri dish and positioned each day in the above-mentioned dedicated room. A procedural blank was also prepared together with every batch of samples following the same procedure as samples, without adding yolks or embryos. The filters deriving from environmental and procedural blanks also were first inspected with the optical microscopy of XploRA Nano Raman Microspectrometer (Horiba Scientific) at ×100 magnification.t-test (two-tailed) after the normal distribution was verified using the Kolmogorov–Smirnov test. For all the analyses the significance was set at p ≤ 0.05.The statistical analyses were conducted using the GraphPad Software Prism8 for Windows. Means of MPs in each yolk, MPs per g of yolk, MPs in each embryo, number of MPs per g of the embryo, embryos’ dorsal mantel length and weight were analyzed through one-way ANOVA followed by Tukey’s multiple comparisons test, whereas hatchling parameters were analyzed through an unpaired p ≤ 0.0001, p ≤ 0.0001, and p ≤ 0.01, respectively) than those from site B (p > 0.05) due to the high variability of samples analyzed  in animals from site C compared to hatchlings from site B  and Polyvinyl chloride (PVC) were the two most frequent polymers among sampling sites .The sites with the highest number of MPs were Site A and C which both represented 37% of the total amount . Only twThe most frequent polymer identified was PVC which accounts for more than half of MPs observed (52%) followed by PP and CA (both 15%) a. The siSepia officinalis eggs. Cuttlefish eggs are surrounded by the eggshell capsule, a multilayer envelope secreted in synergy by the female oviductal gland, nidamental gland, and ink sac which acts as a protective barrier with limited permeabilization to gases and other chemicals  [Sepia officinalis, during vitellogenesis, follicular cells are organized as strings that enter the oocytes and synthesize the vitellogenin that will enter the oocyte [Danio rerio) [As an intermittent terminal spawner, the common cuttlefish lays the eggs in separate batches during the single spawning season at the end of which both males and females die . Considelements) . During lements) . MPs suslements) . This prlements) . In Sepie oocyte . The pose oocyte . Therefoe oocyte ,10. Baseo rerio) . These ao rerio) . Althougo rerio) ,38,39. Mo rerio) ,41. Baseo rerio) ,43. The o rerio) . The unpo rerio) . The absFocusing on MP density analyses, worthy of note is that the highest number of MPs (in both yolk and embryo samples) was found in site C where the lowest hatchling size was observed. In site B, the highest embryo mantle length was associated with the lowest number of MPs detected. Beyond the variability of these parameters, embryonic development also depends to a large extent on the amount and quality of the yolk sac as it represents the only food source for the embryo, supplying energy and structural components required for optimal embryonic and post-embryonic development . The yolFinally, despite the occurrence of MPs in embryos from all the investigated sites and their association with embryonic growth, the histological investigation revealed that there are no alterations in the morphology and functionality of the embryonic internal organs and tissues. This result highlights the fact that although there are MPs accumulated inside the embryos, these do not interfere with organogenesis. Although MP presence was not associated with any impairment of internal structures in both embryos and hatchlings, their persistence could affect the life of juveniles and adult specimens.Sepia officinalis embryos from different sites of the central Adriatic Sea was observed for the first time. These preliminary results could suggest that the presence of MPs does not impact embryo development and organogenesis. However, regardless of the pathway through which MPs enter the egg, a better understanding of their relationship with the embryo and hatchling health and growth is required since these species will experience chronic exposure to MPs from the embryonic development to the adult phase. Further studies are in process to assess the possible MPs effects on the yolk quality and assimilation.In this study, the presence of MPs in


We investigated whether the binding site and a competitive or allosteric inhibition of TAS1R2/TAS1R3 influence the time-dependent sensory perception and in vitro TAS1R2/TAS1R3-activation profiles. We compared time-intensity ratings of cyclamate, NHDC, acesulfame K, and aspartame with and without lactisole with the corresponding TAS1R2/TAS1R3-activation in transfected HEK293 cells. In combination with lactisole, cyclamate and NHDC demonstrated a shift of the dose–response curve corresponding to a competitive inhibition by lactisole in the sensory and the cell experiments. Allosteric inhibition by lactisole for aspartame and acesulfame K was seen in the cell experiments, but not the sensory ratings. In conclusion, the data do not support a major impact of the binding site on the time-intensity profile of the tested sweeteners. Beside 50 value, but a similar Emax and hillslope value. In contrast, allosteric inhibition through binding to two different binding-sites, for example with aspartame and ace K, is characterized by a similar EC50 value in addition to a decreased Emax and hillslope value  measurement . To anald method . Thus, i22.1All compounds used for sensory evaluations were obtained in food grade (FG) quality. Citric acid, ethanol, monosodium glutamate, sodium chloride and sucrose were purchased from local supermarkets and pharmacies . Caffeine  and NHDC  was obtained from Merck KGaA . Acesulfame K (>  98 %), aspartame (> 99 %), cyclamate (>  99 %), lactisole (> 99 %), iron lactate-IIhydrate, and tannic acid (nat.) were kindly provided in FG quality by the Symrise AG . Compounds used in cell experiments were aspartame, cyclamate , neohesperidin dihydrochalcone (≥  95 %), acesulfame K (≥  99 %) from Sigma-Aldrich; lactisole  from Cayman Chemical.2.2For performing sensory analysis, a panel of 37 test persons  rated the test solutions. All panelists gave their written informed consent for participating in the panel and had to complete a sensory training. First, a training of the basic tastes sweet by 10.0 g/L sucrose, bitter by 0.3 g/L caffeine, salty by 2.0 g/L sodium chloride, sour by 0.3 g/L citric acid and umami by 0.6 g/L monosodium glutamate  was condsec) by moving the slider control in the software according to their perceived sweet intensity. The three minutes of evaluation were chosen to capture as much of the lingering as possible without wearing out the panelists during the evaluation. A total of 350 measuring time points was recorded within the pre-set time frame, especially to see differences in the first seconds of evaluation, which are important for the onset. The panelists were asked to take the sample (20 mL) into their mouth, while starting the timer and simultaneously the evaluation of sweet intensity over the time, and to spit out the sample after ten seconds while continuing the evaluation of the sweetness intensity until the end of the three minutes.For evaluation of the time-dependent parameters of the selected sweet test compounds, a TI-measurement was applied, using the software EyeQuestion® 4.11.74  online. The 7–8 ascending concentrations of the test compounds were pre-tested by selected panelists (n = 3 – 4) to be in a sensory consumable range, namely 0.01 – 50 mM ace K, 0.01 – 20 mM aspartame, 0.1 – 100 mM cyclamate and 0.001 – 1.0 mM NHDC. Each test compound and concentration was tested alone and in combination with 0.46 mM (100 ppm) and 0.92 mM (200 ppm) lactisole. The concentrations were chosen because lactisole (sodium 2-(4-methoxy phenoxy) propanoate) is commonly used up to 150 ppm in food  and typisec of evaluation as “max. intensity”, the time-point of the first maximum intensity as “onset” in sec, the intensity at 90 sec as “lingering” effect, and the area under the curve from the time-intensity plots as “AUC” as a marker for the overall intensity and duration.For comparison of the time-dependent properties, the following descriptors were used: the maximum intensity at the first 30 Calcium mobilization assayAll sensory experiments were conducted at the Christian Doppler Laboratory for Taste Research at the Institute for Physiological Chemistry, University of Vienna, Austria, at room temperature (21 – 23 °C).Gi3, and the two subunits of the human sweet taste receptor, TAS1R2 and TAS1R3 . Then, plates were placed in a fluorometric imaging plate reader  for automated compound application and measurement of fluorescence changes. Fluorescence changes of control wells not induced with tetracycline were subtracted from the data. Next, measurements were corrected for background fluorescence. Dose-response relationships of three independent experiments each performed in duplicates were calculated with SigmaPlot 14.0 software using the function f(x) = min+(max − min)/(1+(x/EC50)nH).For the functional characterization of the human sweet taste receptor, we used HEK293 Flp-In T-Rex cells stably transfected with the G protein chimera Gα15d TAS1R3 . The G pd TAS1R3 . Briefly2.4TI-ratings were recorded and analyzed using the software EyeQuestion® 4.11.74  online and MS Excel 16.0 . The statistical analysis and graphical representation were performed with GraphPad Prism 9.0 for sensory results and with SigmaPlot 14.0 for cell experiments. To evaluate the sensory dose–response effects of ascending sweetener concentration w/o lactisole, an asymmetrical five-parameter curve (Richard́s five-parameter dose–response curve) was fitted. The data are presented as means ± SEM from at least 16 single evaluations. Statistical significance between the different test compounds and concentration-dependent effects was assessed by two-way ANOVAs with Tukey post hoc test using Graph Pad Prism. The normal distribution of the data sets was checked by evaluation of the kurtosis (between −7 to + 7) and the skewness (between −3 to + 3). Equal variance was tested using Levenés test. In case the assumptions of a normal distribution and equal variance were not met, the non-parametric ANOVA on ranks  with Dunn's post-hoc test without the calculation of interaction was applied. A Pearson product moment correlation was conducted to investigate relationships between sensory results and cell responses of the tested sweeteners.3In the present study, the impact of selected sweet tasting compounds, namely acesulfame K (ace K), aspartame, cyclamate and NHDC on the sweet taste receptor TAS1R2/TAS1R3 was evaluated. First, TI-measurements for sweetness of the test compounds were conducted in order to obtain time-dependent parameters in dependence of concentration with and without the combined application of the sweet taste inhibitor lactisole. As lactisole acts either as a competitive or allosteric inhibitor for sweeteners based on their binding site at the sweet taste receptor, the aim of this experiment was to detect whether a different binding site at the sweet taste receptor also translates into differences on the temporal sensory profile.A broad, for panelists acceptable concentration range of each test compound was used, in absence or presence of 0.46 mM and 0.92 mM lactisole (100 ppm and 200 ppm). sec, which is similar to the temporal sweetness recordings of lactisole alone , respectively, for 0.46 and 0.92 mM lactisole .As a second parameter, the AUC of the TI-curves in the tested range of concentrations was compared, to assess the summated overall sweetness impression exerted by the different sweeteners w/o lactisole as a function of duration and intensity. The concentration dependency of the AUC for the four sweeteners w/o lactisole is displayed in sec , Ace K (sec (27 sec). Aspartame responses (sec (23 sec) fell between these extremes. Thus, the activation of the sweet taste receptor via the orthosteric (TAS1R2-VFD) or allosteric (TAS1R3-TMD) binding site seems to have no consistent effect on the speed of signal onset. Also signal decays showed considerable differences. The only sweetener-induced receptor activation showing a signal decay back to baseline or even below was documented for Ace K  and the slowest for NHDC . Aspartame and cyclamate exhibited identical signal decay times with 93 sec (73 sec). Again, signal decay seems to be independent on the interaction site.The results of the sensorial time-intensity measurements were compared to activation of the sweet taste receptor in transfected HEK293 cells. The functional analyses of a sweet taste receptor expressing mammalian cell line confirmed previous results on the differential lactisole sensitivities of the sweet taste receptor responses to sweeteners . Sweetenyclamate  G reache), Ace K  E and NH), Ace K  H stimulesponses  F with 4or Ace K  E after or Ace K  F, cyclaor Ace K  G and NHor Ace K  H stimul50 values for the sensory data and derived from the transfected HEK293 cells revealed a strong correlation  or competitive  inhibition with typical shift in the dose–response relationships.sec. This sweet aftertaste of lactisole was dose-dependent and also present at lower sweetener concentrations at which lactisole inhibited the sweetness of the test compounds completely. A certain sweet aftertaste of lactisole and other sweet taste inhibiting compounds is well-known and was previously described as “sweet water taste”, as it can be induced by rinsing with water after the inhibitor stimulus (sec, also saliva secretion, without the necessity of rinsing with water, can induce a sweet aftertaste impression, which supports the idea of the dissociation from the receptor to be important for the sweet water taste of lactisole. We propose that not only water can induce the sweet lactisole effect, but also salivary secretion during tasting. Because of the clear noticeable sweet-water effect of lactisole, we only used the values up to 30 sec to identify the maximum sweetness for the sweeteners.As markers for the temporal characteristics of the sweetness perception, the maximum intensity, the AUC from the respective TI-plots, as well as onset and lingering were investigated. As expected, with rising concentration of the sweeteners, also the sweetness rating, represented by the maximum intensities as well as the AUC of the corresponding TI- plots were increased. Two, respectively three for the cell experiments, different concentrations of lactisole were included in the present study which are in a relevant range for food applications and cell experiments . A sweetstimulus . To elucstimulus . The resin vitro (Focusing on the TI-parameters and the effect of lactisole on the different sweeteners, the shift of the dose–response relationships of the maximum intensity for cyclamate and NHDC fit into the model of competitive inhibition in combination with lactisole. Similarly, the shifts in the dose–response relationships of the AUC can be best explained by a competitive inhibition model. As the binding site of cyclamate, NHDC, and lactisole has been previously shown to be located at the TMD of TAS1R3 , these rin vitro  and addiin vitro . This himax in the sensory experiments was less reduced than expected. This was not the case in the in vitro experiments, where pronounced reductions in the maximal signal amplitudes already at 100 µM of lactisole were evident. A reason for the discrepancy between the results obtained from the cell model and the sensory study regarding the Emax might be that some of the test compounds such as ace K are also activating other taste receptors, e.g. bitter taste receptors (The inhibitory effect of lactisole on ace K and aspartame was expected to reflect an allosteric inhibition mode, as these two sweeteners have been described to bind with high affinity to the VFD of TAS1R2 . The doseceptors , that mieceptors . The intmax was shown, although the increased EC50 values in combination with lactisole do not fit into an allosteric inhibition model. For typical allosteric inhibition induced by lactisole, a similar EC50 and reduced hillslope would have been expected (2+-release of heterologous transfected cells and point mutations, that the binding sites for ace K and aspartame, although located both at the VFD of TAS1R2, have strikingly different and specific binding residues (For ace K, a significant reduction of the Eexpected  and was expected , which cexpected , althougresidues . Since tresidues . Supportresidues , indicatresidues . Also, aresidues . The recresidues . Howeverresidues . HoweverTo summarize, in combination with lactisole, cyclamate and NHDC demonstrate a shift of the dose–response curve corresponding to a competitive inhibition by lactisole in the sensory and the single receptor cell experiments. In contrast to the expectations, aspartame was able to overrule the lactisole inhibition in higher concentrations in the sensory experiments, which could argue for a second, low-affinity binding site at the TAS1R3-TMD. Moreover, the effect of lactisole on the temporal markers of the sensory profile AUC, onset, and lingering for the sweeteners was independent of the major binding site of the sweeteners. In conclusion, the data do not support a major impact of the binding site on the time-intensity profile of the tested sweeteners. Future studies are needed to assess the effect of lactisole and further compounds to confirm their intensity and lingering effects in cell experiments related to sensory properties.Corinna M. Deck: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization. Maik Behrens: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation, Writing – original draft, Writing – review & editing, Visualization. Martin Wendelin: Conceptualization, Methodology, Writing – review & editing. Jakob P. Ley: Conceptualization, Methodology, Resources, Writing – review & editing, Supervision, Project administration, Funding acquisition. Gerhard E. Krammer: Conceptualization, Resources, Writing – review & editing, Supervision, Funding acquisition. Barbara Lieder: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Data curation, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors M. Wendelin, J. P. Ley, and G. E. Krammer are employees of the Symrise Distribution GmbH or Symrise AG, respectively.


Hyperpolarization activated cyclic nucleotide-gated channel 1 (HCN1) is expressed throughout the nervous system and is critical for regulating neuronal excitability, with mutations being associated with multiple forms of epilepsy. Adaptive modulation of HCN1 has been observed, as has pathogenic dysregulation. While the mechanisms underlying this modulation remain incompletely understood, regulation of HCN1 has been shown to include phosphorylation. A candidate phosphorylation-dependent regulator of HCN1 channels is 14-3-3. We used bioinformatics to identify three potential 14-3-3 binding sites in HCN1. We confirmed that 14-3-3 could pull down HCN1 from multiple tissue sources and used HEK293 cells to detail the interaction. Two sites in the intrinsically disordered C-terminus of HCN1 were necessary and sufficient for a phosphorylation-dependent interaction with 14-3-3. The same region of HCN1 containing the 14-3-3 binding peptides is required for phosphorylation-independent protein degradation. We propose a model in which phosphorylation of mouse S810 and S867 (human S789 and S846) recruits 14-3-3 to inhibit a yet unidentified factor signaling for protein degradation, thus increasing the half-life of HCN1. Ih) carried by HCN1 channels play essential roles throughout the nervous system in tempering excitability, setting pacemaking activity, and coordinating signal integration [Hyperpolarization activated currents  and the terminal tripeptide Ser-Asn-Leu, thus bridging a 300 amino acid long intrinsically disordered region . Differe14-3-3 proteins are a conserved family of phosphoserine/phosphothreonine binding proteins ubiquitously expressed in eukaryotes and particularly enriched in the brain. There are over a hundred proteins that have been thoroughly validated as 14-3-3 client proteins. These include proteins participating in cellular functions as diverse as fatty acid metabolism and apoptosis , 39. BioHere we demonstrate that HCN1 is a novel 14-3-3 client protein and this interaction requires phosphorylation of two serines in the intrinsically disordered C-terminal tail of HCN1. Using cultured cells, we observed that the 14-3-3 interaction sites overlapped with a phosphorylation-independent site involved in degradation of HCN1.The protocol for maintaining mice and obtaining tissue for experiments was approved by the University of Iowa IACUC.http://n2t.net/addgene:79888; RRID:Addgene_79888) [http://n2t.net/addgene:74255; RRID:Addgene_74255) [eSpCas9(1.1)_No_FLAG_AAVS1_T2 was a gift from Yannick Doyon  . The pSNe_79888)  to make e_74255)  and the http://n2t.net/addgene:68375; RRID:Addgene_68375) [http://n2t.net/addgene:14757; RRID:Addgene_14757) [Δ3, 14-3-3 and SNAP-HCN1Δ804–814; Δ861–871. The cloning of serine to alanine substitutions into AAVS1-CAG-SNAP-HCN1 was performed by Genscript Cloning Services.Homology directed repair templates for targeted knock in to the AAVS1 site were generated using AAVS1_Puro_PGK1_3xFLAG_Twin_Strep, a gift from Yannick Doyon  , as the e_14757) , to makehttp://n2t.net/addgene:20861; RRID:Addgene_20861) [CT (CT598-910) reporter from which truncations were made up to the transmembrane domain (control reporter).pDisplay-AP-CFP-TM was a gift from Alice Ting  . The C-tAll sequences were validated by Sanger sequencing . Novel plasmids are available upon request–AAVS1-CAG-mRuby3-HCN1 (wild-type and mutants), AAVS1-CAG-SANP-HCN1 (wild-type and mutants), and SNAP-TM-HCN1 (various truncations and targeted mutations).2 in a humidifying incubator. Cells were passaged every two to three days and all experiments were performed prior to passage 25. Knock in cell lines were generated by co-transfecting cells 80% confluent in 6-well plates with 2 μg of AAVS1 homology directed repair template plasmid (wild-type and mutant versions of AAVS1-CAG-SNAP-HCN1 and AAVS1-CAG-mRuby3-HCN1) and 0.5 μg of AAVS1 targeted Cas9 plasmid (eSpCas9(1.1)_No_FLAG_AAVS1_T2). 24 hours after transfection, cells were treated with a high dose of puromycin, 5 μg/mL. After 48 hours, cells were switched to a maintenance dose of 2 μg/mL Puromycin which was maintained throughout. For transient transfections, 8 μg plasmid/10 cm plate or 2 μg plasmid/well of 6-well plate was used and cells harvested 24 hours post-transfection. All transfections were performed using Lipofectamine 2000 (ThermoFischer Scientific cat. # 11668027) at a 2:1 Lipofectamine (μL) to plasmid DNA (μg) ratio as described by the manufacturer.HEK293 (ATCC CRL-3216) were cultured in DMEM (Gibco cat. # 11965092) supplemented with 10% fetal bovine serum (Gibco cat. # 26140079), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco cat. # 15140122), and 0.5 μg/mL amphotericin B (Gibco cat. # 15290018). Cells were maintained at 37°C with 5% CO6His14-3-3ζ was a gift from Dr. Christian Ottmann [E. coli and protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at mid-log phase. Induced cells were lysed using B-PER Bacterial Protein Extraction Reagent (ThermoFischer Scientific cat. # 90084) and purified using HisPur Ni-NTA resin (ThermoFisher Scientific cat. # 88221) or a HisTrap HP column . Purified 6His14-3-3ζ was then dialyzed into PBS. Quality of the purification was assessed by SDS-PAGE and Coomassie staining, and protein concentration was measured by absorbance at 280nm.pPROEX-HTb vector for expression of human  Ottmann . This plΔ3, 14-3-3 were performed by Eurofins Discovery . Recordings were made using the Qpatch automated patch clamp system. Recording conditions: Intracellular Solution (mM): 70 KF, 60 KCl, 15 NaCl, 5 HEPES, 5 EGTA, 4 MgATP, 0.01 cAMP (pH 7.3 by KOH). Extracellular Solution (mM): 110 NaCl, 30 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, 5 Glucose (pH 7.35 by NaOH). Current recordings at tests pulses from –30 to –130 mV were provided, as was the capacitance of each cell. From the data provided, the current density and percentage of maximal current were obtained and plotted against the voltage. Plots were fit to a Boltzman sigmoidal. All electrophysiology data are presented as mean ± standard error. For comparisons between cell lines, the maximal current density was compared using t-test.Current recordings from HEK293 cell lines expressing mRuby3-HCN1 or mRuby3-HCN1All antibodies used are detailed in 2PO4, 1.8 mM KH2PO4, pH 7.4) supplemented with cOmplete Mini, EDTA free protease inhibitor cocktail and PhosSTOP phosphatase inhibitor cocktail (Millipore Sigma cat. # 11836170001 and 4906845001). For tissue preparation, wildtype (C57Bl6/J) or HCN1 knockout mice as described in [All samples were homogenized on ice in lysis buffer  was added to antibody-bound resin overnight at 4°C and washed with lysis buffer. Protein was eluted with 2X reducing sample buffer.6His14-3-3ζ was incubated with Ni-NTA resin in 5% BSA dissolved in lysis buffer supplemented with 25 mM imidazole for 3 hours at 4°C; 5 mg of 6His14-3-3ζ was used for pulldown from brain, 0.5 mg for pulldown from retina and HEK293 cells expressing full-length HCN1, 0.1 mg for pulldown of SNAP-TM-HCN1CT reporters. For the control, Ni-NTA resin was treated identically without the addition of 6His14-3-3ζ. 1.5 mL of whole brain lysate , 400 μL of retinal lysate , 400 μL of HCN1 expressing HEK cell lysate (50% of lysate from 10 cm dish– 300–500 μg), or 300 μL of reporter expressing HEK293 cell lysate (95% of lysate from well of 6-well plate– 300–400 μg) was added to control or 6His14-3-3ζ conjugated resin and incubated overnight at 4°C. Resin was then washed with lysis buffer supplemented with 25 mM imidazole and protein was eluted with 250 mM imidazole in 8% SDS in 0.1 M Tris pH 7.8.For 14-3-3 pulldowns, 598-910 or CT731-910). All pulldown data presented as mean ± standard deviation.For pulldowns using the SNAP-TM reporters, the amount of each reporter pulled down was compared using a t-test or 1-way ANOVA followed by Dunnett’s multiple comparison to compare the truncated or mutant reporters to control  in cold PBS for thirty minutes on ice. The reaction was quenched by the addition 50 mM Tris-HCl, 150 mM NaCl, pH 7.5). Cells were briefly washed with PBS prior to addition of lysis buffer. For each replicate, equal amounts of protein (200–300 μg) for each experimental sample was added to 75 μL of High capacity Neutravidin agarose (ThermoFischer Scientific cat. # 29202) and incubated at 4°C overnight. Resin was washed with lysis buffer supplemented with 0.5% SDS. Protein was eluted by incubation for 5 minutes at 75°C in 2X reducing sample buffer prepared from 4X LDS sample buffer and 10X reducing agent. Eluted protein was then examined by western blot. T-test was used to compare mRuby3-HCN1 and mRuby3-HCN1Stable HEK293 cell lines were grown to confluency on a 35mm dish. The day before the pulse-chase experiment, cells were trypsinzied, resuspended in 10 mL of media, and 0.5 mL of the cell suspension was plated in each well of a 24-well plate. The following day cells were treated with 1 μM SNAP-cell TMR-Star (New England BioLabs cat. # S9105S) in complete culture media for 45 minutes at 37°C; for the chase, media was replaced with media containing 3 μM SNAP-Cell block (New England BioLabs cat. # S9106S) and cells maintained at 37°C. Aliquots of cells were harvested immediately at the end of the pulse or at 4, 8, 12, and 24 hours into the chase. Lysates were immediately frozen and processed once the final timepoint was collected. Lysates were quantified by BCA assay and equal total protein concentrations were analyzed by western blotting against the SNAP-tag with membranes imaged for SNAP-HCN1 conjugated SNAP-Cell TMR-star (600 nm channel on LI-COR Odyssey FC) prior to blocking.Δ804–814; Δ861–871 and SNAP-HCN1S810A; S867A to SNAP-HCN1. Data presented as mean ± standard deviation.The labeled SNAP-HCN1 fluorescent signal was normalized to SNAP-HCN1 western blot intensity. This value was then normalized to the 0-hour chase timepoint to give the % labeled SNAP-HCN1. The half-life was calculated by fitting the data to a one-phase decay curve. 2-way ANOVA followed by Dunnett’s multiple comparison was used to compare SNAP-HCN1in vivo [653SRMRTQSPPVY, B = 804VRPLSASQPSL, C = 861TLFRQMSSGAI, the serine marked in bold is the predicted phosphorylated residue), are in the long, intrinsically disordered portion of the HCN1 C-terminal tail which contains 37 serines and 30 threonines . Intriguingly, one of the predicted peptides (peptide A) overlaps the ER retention motif in HCN1.We analyzed the sequence of HCN1 using 14-3-3Pred, a prediction tool for the identification of phospho-peptides likely to serve as 14-3-3 ligands . Severalin vivo . The thr6His14-3-3ζ (hereafter referred to simply as 14-3-3). 14-3-3 immobilized on Ni-NTA beads, but not the unconjugated control beads, pulled down HCN1 from whole brain lysate, retinal lysate, and lysate from HCN1 expressing HEK293 cells  were deleted while targeted alanine substitution was performed at the site corresponding to peptide A to prevent phosphorylation while preserving the di-arginine ER retention motif (647SRMRTQSPPVY to 647SRMRAQAPPVY) [1/2: -118.6 ± 0.1 mV and mRuby3-HCN1Δ3, 14-3-3 V1/2: -116.1 ± 0.1 mV  . Whole-c± 0.1 mV . These Vlls i.e. , 54. The neurons , 56. Giv6His14-3-3ζ. We first tested if the C-terminus of mouse HCN1 was sufficient for 14-3-3 association. Amino acids 598–910 of HCN1 (CT598-910) was fused to a transmembrane domain (from the PDGF-receptor) with an extracellular SNAP-tag as shown in 598-910, but not the control base reporter, was pulled down by 14-3-3 . Further deletion that left peptide A intact (CT598-698) resulted in complete loss of interaction   . The com 0.0001) .598-910; p = 0.0359), supporting our earlier decision to disregard the prediction of a fourth putative 14-3-3 binding site. Second, HCN1 fragments lacking the region containing peptide C (amino acids 850–880) resulted in loss of half the pull down efficiency . Third, another drop in pulldown efficiency was observed when the serial truncation also resulted in loss of the region containing peptide B .Serial deletions of 30 amino acid blocks from the most distal portion of the C-terminal tail of HCN1 generated three findings . First, 598-910; p = 0.0227). Finally, site-specific mutations were used to delete just the 10 amino acid blocks corresponding to peptides B and C from the distal C-terminus  or to mutate the phosphorylation sites of those peptides with serine to alanine replacements . Both sets of mutations reduced 14-3-3 interaction significantly . This data indicates that in unstimulated HEK293 cells, 14-3-3 associates with two sites in the disordered tail of HCN1 centered around S810 and S897.To determine whether the distalmost C-terminus of HCN1 was sufficient to mediate the interaction with 14-3-3, the proximal region of the C-terminus including peptide A was removed leaving only a 180 amino acid long fragment of the distal C-terminus that retains the disordered polyQ stretch as a spatial linker (residues 731–910). This construct was pulled down slightly more efficiently than the construct containing the full-length C-terminus . The need for kinase activity is consistent with the canonical mechanism of 14-3-3 recruitment.We next tested the requirement for kinase activity. HEK293 cells transfected with the reporter CTled down . Only trΔ804–814; Δ861–871) and the other with just the central serine residues replaced with alanine, aka ‘phosphonull’ mutants . HCN1 expression was measured in surface biotinylation assays. We expected both mutant versions of HCN1 would be expressed at ~2 fold higher levels as we saw previously for HCN1Δ3, 14-3-3. However, SNAP-HCN1Δ804–814; Δ861–871 expressed at roughly six-times the level of SNAP-HCN1 both in total  and at the surface . To our surprise, SNAP-HCN1S810A; S867A did not exhibit a change in total expression and the change in surface expression was small   .S810A; S867A was slightly reduced to 3.0 hours and the relative amount of labeled SNAP-HCN1S810A; S867A was decreased slightly compared to SNAP-HCN1 at 8 hours . However, the half-life of SNAP-HCN1Δ804–814; Δ861–871 was significantly increased to 5.4 hours with the relative amount of labeled SNAP-HCN1Δ804–814; Δ861–871 being elevated compared to SNAP-HCN1 at 4 hours  and 8 hours . A longer half-life indicates increased stability of the protein and would lead to the increased static expression levels we observed.To investigate the dynamics of SNAP-HCN1 expression we used pulse-chase assays exploiting the ability of the SNAP-tag to covalently bind substrate. Cells were incubated with a fluorescent SNAP-substrate for 45 min which was then washed out and replaced with a non-fluorescent SNAP-substrate so that any SNAP-HCN1 synthesized after the pulse could not be fluorescently labeled. Cells were harvested at several time points for the next 24 hours. Following SDS-PAGE and transfer onto PVDF membrane, the amount of labeled SNAP-HCN1 was measured by direct fluorescence detection and the amount of total SNAP-HCN1 was measured by western blotting against the SNAP-tag . The halThe key finding of this study is the identification of an interaction between HCN1 and the signaling adaptor 14-3-3. 14-3-3 can interact with two sites centered on S810 and S867 in the intrinsically disordered cytoplasmic C-terminus of mouse HCN1. These 14-3-3 interaction sites also overlap with an independent HCN1 degradation signal.in vivo. In that study, peptides containing the second 14-3-3 binding site (S810) were not resolved, leaving it untested whether this site is phosphorylated in vivo [HCN1 phosphorylation has been implicated in either modulating channel activity or expression, likely depending on which sites are phosphorylated. Yet, the previously characterized HCN1 regulatory proteins are not known to require HCN1 phosphorylation for binding. In a recent phosphorylation site mapping study, one of the residues (S867) we identified as necessary for 14-3-3 association with HCN1 was shown to be phosphorylated  in vivo . While S in vivo . Thus, wWe focused on the C-terminus of HCN1 as it contains the most evolutionarily conserved predicted 14-3-3 bindings sites, but it is possible that 14-3-3 may interact with other regions of HCN1. The cytoplasmic N-terminus of HCN is intrinsically disordered, like the distal C-terminal tail, and contains several serine and threonine residues. This includes T39 which was shown to be phosphorylated  and liesin vivo. The choice of model system likely explains the ambiguous results we obtained testing the effect of individual kinase inhibitors. That experiment would be better carried out in vivo, under physiologically relevant conditions, ideally where phosphorylation can be induced rather than relying on manipulation of any basal phosphorylation of HCN1. That said, it is intriguing that we obtained the strongest effects in HEK293 cells by inhibiting PKC and p38MAPK which have both been implicated in neuronal regulation of HCN1 [One caveat of this study is the use of HEK293 cells as our primary model system. While a useful system to express the various constructs used in this study and examine the function of HCN1 mutants, we recognize that these cells do not adequately reflect neurons endogenously expressing HCN1. Thus, while the findings presented here implicate S810 and S867 as necessary for the 14-3-3 interaction, additional residues may be phosphorylated and accessible to 14-3-3  of HCN1 , 60. Ano of HCN1 –64.The major conceptual challenge presented by our data was the observation that 10-amino acid deletions centered on S810 and S867 both prevented 14-3-3 association and decreased the turnover rate of HCN1 leading to increased surface expression and higher current density. While site-specific serine-to-alanine (phosphonull) mutations of S810 and S867 prevented 14-3-3 association but did not significantly affect the static expression levels of HCN1. This indicates that a strong degradation signal is overlapping but distinct from the 14-3-3 interaction with the C-terminus of HCN1. One possibility is that 14-3-3 is a non-essential component of a complex that promotes HCN1 channel degradation. This hypothesis is unsatisfying and suggests that this interaction has no functional role despite S810 and S867 being highly conserved. Instead, we propose that the simplest model involves competition between the degradation signal (working via a yet uncharacterized Factor X) and phosphorylation-mediated recruitment of 14-3-3 . In HEK2in vivo systems that represent normal neuronal homeostasis and responsiveness to neuro-modulatory signaling.The region of HCN1 that associates with 14-3-3 is intrinsically disordered, so it is better to think of it as existing in a population of different conformations rather than in just one fixed state. That means binding by multiple proteins may not be competitive when HCN1 channels are considered en masse. Thus, we must consider how the various factors acting on the C-terminus of HCN1 act in coordination, whether they function in competitive or cooperative terms. This is especially important when we consider TRIP8b, an endogenous HCN channel accessory protein absent from HEK293 cells. TRIP8b binds to both the extreme C-terminal Ser-Asn-Leu tripeptide of HCN1 and the cyclic nucleotide binding domain, spanning the intrinsically disordered region containing the 14-3-3 binding sites –32. AnotTaken together, this work demonstrates that 14-3-3 interacts with the C-terminus of HCN1 in a phosphorylation dependent manner. Phosphorylation has been implicated as a means to regulate HCN1 channel function and, to our knowledge, this is the first HCN1 interacting partner identified whose binding depends on phosphorylation of the channel. While we are unable to definitively identify the function of this interaction, our work demonstrates that 14-3-3 association occurs at sites involved in regulating HCN1 turnover, identifying this region as an area of interest for further studies into regulation of HCN1 channels. Uncovering all the mechanisms regulating HCN1 is important as this channel is an attractive drug target for amelioration of various neurological and neuropsychiatric conditions.S1 Fig731-910 were treated with kinase inhibitors (detailed in A) representative pulldown B) comparison of all experimental replicates, dark grey bar is the untreated control, purple bars highlight the treatments that generated a statically significant reduction in pulldown efficiency with adjusted p-value shown.HEK293 cells expressing reporter-HCN1 CT(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Raw images(TIF)Click here for additional data file.


Phaseolus lunatus (lima beans), compared to the commonly consumed P. vulgaris (red kidney bean) and beans in the Glycine and Vigna genera. The results indicated that soybeans in the Glycine genus contained the highest isoflavone contents, especially glycitein (1825–2633 mg/100 g bean) and daidzein (1153–6471 mg/100 g bean), leading to potentially higher enzyme inhibitory activities  than those from other genera. Interestingly, lima beans with low isoflavone content (up to 2 mg/100 g bean) still possessed high inhibitory activities against lipase (12–21% inhibition) and β-secretase (50–58% inhibition), suggesting that bioactive compounds other than the isoflavones might be responsible for these activities. Isoflavone contents and enzyme inhibitory activities in Vigna beans were diverse, depending on the particular cultivars. The information gained from this study can be used for further investigation of bioactive components and in-depth health properties, as well as for future breeding of a new lineage of bean with specific health potentials.Legumes and pulses are important food components with various phytochemicals and health benefits. However, the health-related bioactivities of some underutilized species remain uninvestigated. To breed a new bean lineage with particular health-related properties, this study investigated phenolics  and the in vitro inhibitory activities of the enzyme relevant to some non-communicable diseases in underutilized cultivars of  Phaseolus vulgaris L.) cultivated in Spain, utilizing high-performance liquid chromatography coupled with electrospray time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) [p-coumaric acid, ferulic acid and syringic acid) and two flavonoids (vitexin and isovitexin) were dominantly found in mung beans (Vigna radiate L.) harvested from different regions in China [Beans in the Leguminosae family are one of the most widely eaten global economic plants; they are inexpensive, nutrient-dense, genome-safe  and can -TOF-MS) . These w-TOF-MS) . Four phin China . Furtherin China . Worldwiin China , suggestin China . From thPhaseolus vulgaris L.), which exhibited an α-amylase inhibitor, along with starch to adult rats resulted in significantly lower blood sugar levels compared to the control group, indicating the anti-diabetic effects of beans via the inhibition of the starch digesting enzyme, α-amylase [Phenolics are non-nutritive compounds of great interest for clinical nutrition due to their wide range of positive health effects, such as antioxidant, anti-cancer and anti-Alzheimer’s disease (AD). The World Health Organization (WHO) (2021) stated that non-communicable diseases (NCDs) accounted for 71% of all fatalities worldwide, or 41 million deaths annually . Interes-amylase . A daidz-amylase . The inhBioactive compounds and health-related information have been widely reported in the most commonly consumed beans but some underutilized beans are missing these data, leading to low market value and reduced use in food applications. Scant information is also available on breeding new lineages within the same genera with particular properties. Previously, we reported the nutritive values and antioxidant activities of ten original lineage beans collected by the Genebank Research and Development Group, Biotechnology Research and Development Office, Ministry of Agriculture and Cooperatives, Thailand, in underutilized lima beans and commonly consumed beans including red kidney bean, red bean, azuki bean, black gram, mung bean and soybean . To furtPhaseolus lunatus L. cultivar ‘38’ (BK083065), P. lunatus L. cultivar ‘47’ (BK083066), P. lunatus L. cultivar ‘59’ (BK083064), P. vulgaris L. cultivar ‘112’ (BK083063), Vigna umbellata (Thunb.) Ohwi and H. Ohashi cultivar ‘107’ (BK083067), V. angularis (Wild.) Ohwi and Ohashi cultivar ‘108’ (BK083062), V. mungo (L.) Hepper cultivar ‘CN4’ (BK083061), V. radiata (L.) Wilczek cultivar ‘CN84-1’ (BK083072), Glycine max (L.) Merrill cultivar ‘SJ5’ (BK083060) and G. max (L.) Merrill cultivar ‘CM60’ (BK083057). All beans were ground into fine powder using a grinder . The powdery samples were kept at −20 °C until analysis.Ten bean cultivars collected in the Genebank Research and Development Group, Biotechnology Research and Development Office, Department of Agriculture (DOA), Thailand, were planted in the research field of Chiang Mai Field Crops Research Center, Department of Agriculture, Chiang Mai, Thailand. Bean seeds were collected in April 2020 with physical appearances shown in v/v) ethanol (20 mL) before shaking in a 30 °C water bath shaker  for 2 h. The mixture was then centrifuged at 3800× g using a refrigerated centrifuge  for 15 min. The supernatant was collected and filtered through 0.45 µM polyethersulfone membrane syringe filter for further analysis.The extraction of bean samples followed previous research . Brieflyv/v) methanol containing 0.5 g tert-butylhydroquinone) and shaken in an 80 °C water bath shaker  for 2 h. To stop the reaction, the mixture was incubated on ice for 5 min before adding 1% (v/v) ascorbic acid (100 µL). Sonication utilizing an ultrasonic bath  was performed for 5 min. Then, the volume of the mixture was adjusted to 50 mL using 62.5% (v/v) methanol containing 0.5 g tert-butylhydroquinone and filtered through a 0.22 µM polytetrafluoroethylene membrane syringe filter. The filtrate was loaded onto a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) system as described below.The supernatant collected in The LC-ESI-MS/MS system was used to identify phenolics in the lineage beans. The system was equipped with a Dionex Ultimate 3000 series ultrahigh-performance liquid chromatographer (UHPLC), a TSQ Quantis Triple Quadrupole mass spectrometer (MS) and a diode array detector with a Chromeleon 7 chromatography data system (version 7.2.9.11323) from Thermo Fisher Scientific .p-coumaric acid , luteolin (>98.0% HPLC), kaempferol (>97.0% HPLC), genistein (>98.0% HPLC), myricetin (>97.0% HPLC), syringic acid (>97.0% T), ferulic acid , cinnamic acid (>98.0% HPLC), naringenin , quercetin  and sinapic acid  from Tokyo Chemical Industry ; rutin (≥94% HPLC), gallic acid (97.5–102.5% T), vanillic acid (≥97% HPLC), rosmarinic acid (≥98% HPLC) and cinnamic acid (≥99% HPLC) from Sigma-Aldrich ; galangin (≥98.0% HPLC) from Wuhan ChemFaces Biochemical Co., Ltd. ; isorhamnetin (≥99.0% HPLC) from Extrasynthese ; daidzein (≥98.0% HPLC), daidzin (≥98.0% HPLC), glycitin (≥98.0% HPLC), glycitein (≥98.0% HPLC) and genistin (≥98.0% HPLC) from Tokyo Chemical Industry .The peak retentions and mass chromatograms of the bean samples were compared with commercial standards including twelve phenolic acids and eighteen flavonoids (including six isoflavones). These standards were apigenin (>98.0% HPLC), (−)-epigallocatechin gallate (>98.0% HPLC), 3,4-dihydroxybenzoic acid (≥97% T), 4-hydroxybenzoic acid , hesperidin , chlorogenic acid , caffeic acid , m/z mass range, 3500 V positive ion, 30 Arb sheath gas (N2), 15 Arb auxiliary gas (N2), 325 °C ion transfer tube temperature and 350 °C vaporizer temperature. Full scanning (FS) and selective reaction monitoring (SRM) modes were selected for qualitative analysis. The MS/MS ion fragments (parent ions and SRM transitions) were achieved using specific collision energies and radio frequencies (RF-lens), as shown in The reaction monitoring transitions were optimized for commercial standards. Each standard (10 µL of 20 µg/mL) was loaded onto the LC-ESI-MS/MS system using a flow rate of 0.5 µL/min with 10 min run time for phenolics and 20 min run time for isoflavones. Positive fragment ions were generated utilizing a heated electrospray ion source (HESI) and determined using a TSQ Quantis Triple Quadrupole MS with parameters as follows: 50–1000 v/v) formic acid in Milli-Q water  at a flow rate of 0.5 mL/min as shown in y = ax ± c) and correlation coefficient of linear regression (R2) of each standard were obtained using the plot between concentrations (x) and ion peak areas (y).To create the calibration curves, a mixture of all commercial standards was prepared in methanol and diluted to different levels to obtain the linear range. The standard mixture (10 µL) was chromatographically separated using a 2.1 mm × 100 mm, 2.6 μm Accucore RP-MS column  and a gradient mobile phase consisting of acetonitrile (solvent A) and 0.1%  and Gen 5 data analysis software. Commercially available enzyme inhibitors, including orlistat (lipase), acarbose (α-amylase) and saxagliptin (DPP-IV), were performed as positive controls. All chemicals and reagents were received from Sigma-Aldrich .In vitro bioactivities of ten bean extracts were performed as inhibition of the key enzymes relevant to obesity (lipase), type II diabetes ) and AD (acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and β-secretase (BACE-1)) using the well-established protocols previously reported ,15,16,17A is the initial velocity (V0) of the reaction with enzyme but without the bean extract (control), a is the V0 of the reaction without enzyme and the bean extract (control blank), B is the V0 of the reaction with enzyme and the bean extract (sample), and b is the V0 of the reaction with the bean extract but without the enzyme (sample blank).The inhibitory activity was calculated as a decline in enzyme kinetics using the following equation.V0 to fluorescence absorbance at specific wavelength.The inhibitory activity of BACE-1 was determined using a fluorescence detection as an end-point assay; thus, the percentage of inhibition was determined utilizing Equation (1) but changing n = 3). The results were represented as mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) and a Duncan’s multiple comparison test were performed with significant differences at p < 0.05. The statistical analysis was performed using the Statistical Package for the Social Sciences . Principal component analysis (PCA) and hierarchical cluster analysis (HCA) of phenolics and in vitro health-related properties were analyzed using XLSTAT® .All experiments were carried out in triplicate on three independent sets of samples  and daidzein (352–1334-fold higher) than genistein. The content of aglycone isoflavones were also higher than their glycosylated forms . However, genistein presented a lower amount than its glycoside isoflavone, genistin (3–10-fold lower).Among the phenolics, beans were previously reported to contain a high content of flavonoids, especially isoflavones ,19. IsofVigna genus exhibited lower content of isoflavones than beans in the Glycine genus. Among the beans in the Vigna genus, the red rice bean ‘107’ and azuki bean ‘108’ exhibited all of the investigated isoflavones with the exception of glycitein, which was absent in both cultivars. These two cultivars also possessed higher content of daidzein than genistein (344–378-fold higher), while the content of daidzin was higher than glycitin (3–9-fold higher) and genistin (7–9-fold higher). The mung bean ‘CN84-1’ only exhibited daidzein and glycitein and their glycosides, while genistein and genistin were undetected in this cultivar. The glycitein content was higher than daidzein (1.5-fold higher). A similar result was observed with their glycosylated forms (3.1-fold higher in glycitin). Furthermore, the content of aglycones was higher than their corresponded glycosylated forms (26-fold higher in daidzein and 12.3-fold higher in glycitein). The last cultivar in this genus, black gram ‘CN4’, only contained daidzein and its glycoside, with a daidzein content 36.3-fold higher than diadzin.Beans in the Phaseolus genus possessed the lowest content of isoflavones. The lima bean ‘38’ exhibited only genistein and diadzin, while daidzein was lower than the limit of detection (LOD) (4.15 µg/mL). The lima bean ‘59’ exhibited only glycitein, while the only isoflavone detected in lima bean ‘47’ was diadzin, with a content lower than the LOD (0.04 µg/mL). The red kidney bean ‘112’ exhibited similar isoflavones as the lima bean ‘38’, with detected the glycitein also lower than the LOD (0.14 µg/mL).Beans in the Glycine genus, ‘SJ5’ and ‘CM60’, exhibited high isoflavone content; however, both cultivars possessed low amounts of naringenin, while the soybean ‘CM60’ also exhibited kaempferol at levels lower than the LOD (0.122 µg/mL) [In addition to isoflavones, LC-ESI-MS/MS analysis using twenty-four commercial phenolic standards (twelve flavonoids and twelve phenolic acids) indicated that all of the bean types did not possess phenolic acid, while some flavonoids (other than isoflavones) were detected in low amounts . Soybean2 µg/mL) .Vigna genus possessed various types of flavonoids but at a low content. Interestingly, the red rice bean ‘107’ exhibited a moderate amount of kaempferol and trace amounts of quercetin at levels lower than the LOD (0.05 µg/mL) [Beans in the 5 µg/mL) . The azu5 µg/mL) , naringe5 µg/mL)  and kaemPhaseolus genus also exhibited various types of flavonoids but in low amounts. The lima bean ‘38’ exhibited low isorhamnetin content and lower than the LOD contents of quercetin, naringenin and kaempferol. The lima bean ‘47’ only exhibited trace amounts of isorhamnetin, while the lima bean ‘59’ contained lower than the LOD contents of naringenin and kaempferol. The red kidney bean ‘112’ also exhibited low quercetin content and lower than the LOD content of kaempferol and isorhamnetin (0.016 µg/mL) [Likewise, beans in the 6 µg/mL) .The enzymes investigated in this study are the key targets for medicinal-based drug design to prevent and treat NCDs. All bean extracts were investigated regarding their inhibitions against these key enzymes compared to well-known enzyme inhibitors.Phaseolus genus exhibited higher inhibitory activities than soybeans in the Glycine genus and the red rice bean ‘107’ in the Vigna genus. However, no clear trend in lipase inhibition was observed in the Vigna genus that contained both the highest inhibitory provider, azuki bean ‘108’ and the lowest red rice bean ‘107’.One medicinal targeted pathway to control obesity is through the inhibition of the lipid-degrading enzyme, lipase. The inhibition of this enzyme leads to slow digestion of lipids, resulting in a lower rate of fat accumulation within the body . Using aPhaseolus genus, ‘38’, ‘47’ and ‘59’ exhibited high inhibitory activities (25.04–31.06%), while the red kidney bean ‘112’ in the same genus exhibited lower inhibition (14.15%). Both soybeans in the Glycine genus exhibited similar lipase inhibitory activities (24.77–26.33%), compatible with the lima beans ‘47’ and ‘59’. Likewise, beans in the Vigna genus exhibited distinctive degrees of inhibition. The red rice bean ‘107’ exhibited the highest inhibition, similar to the lima bean ‘38’, while the azuki bean ‘108’ and black gram ‘CN4’ possessed the lowest inhibitory activities, similar to the red kidney bean ‘112’ in the Phaseolus genus. For α-glucosidase inhibitory activities, all 10 bean cultivars exhibited 6.43–59.83% inhibition using an extract concentration of 12.5 mg/mL (Glycine genus exhibited higher α-glucosidase inhibitory activities than beans in the Vigna (1.1–2.2-fold higher) and Phaseolus (1.2–9.3-fold higher) genera. Other than the carbohydrate degrading enzymes, all 10 bean cultivars also exhibited DPP-IV inhibitory activities ranging from 11.95 to 51.62% inhibition using extract concentration of 12.5 mg/mL , while beans in the Vigna and Phaseolus genera exhibited both high and low inhibitory activities. In the Vigna genus, the black gram ‘CN4’ provided high inhibitory activities (51.62%), while lower inhibitions were observed in the azuki bean ‘108’ (4.3-fold lower). Similarly, among the Phaseolus beans, the lima bean ‘59’ exhibited higher inhibitory activity (47.33%) than lima beans ‘38’ and ‘47’ (1.2–1.3-fold higher) and the red kidney bean ‘112’ (3.6-fold higher).For control of type II diabetes, inhibition of carbohydrate-hydrolyzing enzymes, α-amylase and α-glucosidase, leads to low glucose production and absorption into the body, ,24. Anot.5 mg/mL . Among t.5 mg/mL . Among tGlycine genus exhibited AChE inhibitory activities with 11.76 and 18.76% inhibitions, respectively  and Vigna (1.7–2.8-fold higher) genera. Likewise, using an extraction concentration of 2 mg/mL, all 10 bean cultivars exhibited BACE-1 inhibitory activities ranging from 16.38 to 58.23% (Phaseolus genus showed high inhibitory activities (>50%), while the beans in the Vigna genus exhibited different degrees of inhibition. In this genus, the black gram ‘CN4’ and mung bean ‘CN84-1’ exhibited high inhibitory activities (>50%), while less than half of these inhibitory activities were observed in the red rice bean ‘107’ and azuki bean ‘108’. Soybeans in the Glycine genus exhibited low to moderate inhibitory activities (22.95–29.83%).The main hypotheses for medicinal targeted AD treatment are through increased cholinergic neurotransmitters and lower β-amyloid formation. In the cholinergic hypothesis, the inhibition of AChE and BChE increases the neurotransmitter concentrations , while tectively . No AChEe lowest . Soybeano 58.23% . The limGlycine genus, ‘SJ5’ and ‘CM60’, were projected together with all tested isoflavones, enzyme inhibitory activities against α-glucosidase, α-amylase, DPP-IV, AChE and BChE as a cluster. This result suggested that the soybeans, ‘SJ5’ and ‘CM60’, were rich in isoflavones with high α-glucosidase, α-amylase, DPP-IV, AChE and BChE inhibitory activities compared to the other bean cultivars, and also implied that the high amounts of isoflavones contributed to high enzyme inhibitory activities because other cultivars located far away from the tested isoflavones possessed low amounts of isoflavones. Interestingly, the red kidney bean ‘112’ and azuki bean ‘108’ were projected close to the lipase inhibitory activities, while the lima beans, ‘38’, ‘47’ and ‘59’, mung bean ‘CN84-1’ and black gram ‘CN4’ were projected close to BACE-1 inhibitory activities, suggesting particularly high enzyme inhibitory activities in these bean cultivars. However, all of these bean cultivars possessed minor amounts of isoflavones, implying that other phytochemicals in the beans may play a role in suppressing the activities of these two enzymes. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were conducted to investigate the correlations between the ten bean cultivars and their properties. The mean data of the identified isoflavones and inhibitory activities against lipase, α-glucosidase, α-amylase, DPP-IV, BChE, AChE and BACE-1 were used in the PCA and HCA analyses. A biplot between the observations (ten bean cultivars) and variables (isoflavones and enzyme inhibitory activities) was generated, as shown in A HCA was further used to determine clusters in all of the tested bean cultivars and beans with similar properties were grouped together. The dendrogram B shows tPhaseolus beans , (2) Vigna beans (V. umbellata (Thunb.) Ohwi and H. Ohashi or red bean cultivar ‘107’, V. angularis (Wild.) Ohwi and Ohashi or azuki bean cultivar ‘108’, V. mungo (L.) Hepper or black gram cultivar ‘CN4’, and V. radiata (L.) Wilczek or mung bean cultivar ‘CN84-1’) and (3) Glycine beans (G. max (L.) Merrill or soybean cultivars ‘SJ5’ and ‘CM60’) were investigated regarding their bioactive compounds  and in vitro inhibitory activities against the key enzymes relevant to some NCDs. The results demonstrated that soybeans in the Glycine genus, ‘SJ5’ and ‘CM60’, contained the highest contents of isoflavones, while traces of other phenolics were detected in all genera. With different amounts of isoflavones, the soybeans also exhibited high inhibitory activities against α-amylase, α-glucosidase, DPP-IV, AChE and BChE. By contrast, the Phaseolus beans exhibited high inhibitory activities against lipase and BACE-1, while no clear trends in the isoflavone contents and enzyme inhibitory activities were observed in the Vigna beans.Original lineage beans in Thailand were reported as a rich source of nutrients, phenolics and antioxidant activities that varied according to genera . These bSoybeans are good sources of isoflavones. Our results indicated that two soybeans, ‘SJ5’ and ‘CM60’, contained exceptionally high content of isoflavones, especially daidzein and glycitein, and concurred with previous literature, suggesting that soybeans are excellent isoflavone providers ,19. In o50 values of daidzein against α-amylase and α-glucosidase were 301 and 48 µM, respectively, and stronger than acarbose, a competitive inhibitor of α-amylase and α-glucosidase [2 who had a high soy product intake and lower risk of type II diabetes development [Due to the distinguished amounts of isoflavones, soybeans exhibited high inhibitory activities against α-glucosidase, α-amylase, DPP-IV, AChE and BChE. The inhibition of carbohydrate-degrading enzymes, α-glucosidase and α-amylase, slows down the rate of glucose absorption into blood vessels, thus maintaining circulating glucose levels . Phenolicosidase . Howevercosidase . Interescosidase , while icosidase . Similarelopment . Adults elopment . Thus, tOne hypothesis of AD occurrence is cholinergic deficiency, involving over-activity of two cholinesterase enzymes, AChE and BChE, which are neurotransmitter, acetylcholine, degrading enzymes. Thus, over-active cholinesterases can lead to insufficient neurotransmission, deterioration of cholinergic neurons and finally cognitive decline . No infoEven though the lima bean ‘38’, ‘47’ and ‘59’ contained trace amount of isoflavones, these beans exhibited high inhibitory activities against lipase and BACE-1. Compared to the other 12 bean cultivars, low TPC in the lima bean was also previously reported as 1.17 mg GAE/g bean, leading to trace lipase inhibitory activity . While oA similar result was observed in BACE-1 inhibitory activity of our lima beans. Strong BACE-1 inhibitions were detected, while these beans exhibited low isoflavone content. Other bioactive components such as active proteins/peptides might be responsible for these inhibitory activities. BACE-1 inhibitors can be divided into peptidic and non-peptidic groups ,44. SeveGlycine genus that were rich in isoflavones exhibited high α-glucosidase, α-amylase, DPP-IV, AChE and BChE inhibitory activities compared to other bean cultivars. These results implied that isoflavones might potentially act as enzyme inhibitors. However, lima beans in the Phaseolus genus exhibited high lipase and BACE-1 inhibitory activities even though they contained trace amounts of isoflavones, suggesting that other bioactive compounds such as active peptide/protein might be responsible for these activities.This study demonstrated that soybeans in the Vigna beans, on the other hand, exhibited diverse enzyme inhibitory activities and isoflavone contents, depending on the particular cultivars.Beans have been widely reported for their bioactive compounds and health-promoting properties but information is lacking, especially on the underutilized lima beans. This study investigated phenolics, especially isoflavones, and inhibitory activities against the key enzymes relevant to the control of obesity (lipase), diabetes  and AD . The soybean ‘SJ5’ and ‘CM60’ exhibited relatively high enzyme inhibitory activities against α-glucosidase, α-amylase, DPP-IV, AChE and BChE, possibly due to their exceptionally high isoflavone contents, especially daidzein and glycitein. Interestingly, lima beans exhibited high lipase and BACE-1 inhibitory activities even though they contained low isoflavone contents, suggesting that other bioactive compounds might be responsible for these activities. Our results provide a valuable reference for further investigation of bioactive components (other than phenolics) and in-depth health properties of underutilized lima beans. The information from this study can also be used to benefit other bean cultivars and promote the fulfillment of missing health information by further comprehensive investigation of cell culture and in vivo or clinical studies. The investigation of the bioaccessibility of these beans and the bioactivities of the digested beans would be an interesting direction for future work. This information can support future applications in functional food development and breeding strategy within the same genus to produce new progeny with both high bioactive agents and advantageous health properties.