O-β-linked N-acetylglucosaminylation (O-GlcNAcylation) modulates Tau phosphorylation and aggregation. We here focus on the role of the PHF-1 phospho-epitope of Tau C-terminal domain that is hyperphosphorylated in AD (at pS396/pS404) and encompasses S400 as the major O-GlcNAc site of Tau while two additional O-GlcNAc sites were found in the extreme C-terminus at S412 and S413. Using high resolution NMR spectroscopy, we showed that the O-GlcNAc glycosylation reduces phosphorylation of PHF-1 epitope by GSK3β alone or after priming by CDK2/cyclin A. Furthermore, investigations of the impact of PTMs on local conformation performed in small peptides highlight the role of S404 phosphorylation in inducing helical propensity in the region downstream pS404 that is exacerbated by other phosphorylations of PHF-1 epitope at S396 and S400, or O-GlcNAcylation of S400. Finally, the role of phosphorylation and O-GlcNAcylation of PHF-1 epitope was probed in in-vitro fibrillization assays in which O-GlcNAcylation slows down the rate of fibrillar assembly while GSK3β phosphorylation stimulates aggregation counteracting the effect of glycosylation.Phosphorylation of the neuronal microtubule-associated Tau protein plays a critical role in the aggregation process leading to the formation of insoluble intraneuronal fibrils within Alzheimer’s disease (AD) brains. In recent years, other posttranslational modifications (PTMs) have been highlighted in the regulation of Tau (dys)functions. Among these PTMs, the  O-β-linked N-acetylglucosaminylation (O-GlcNAcylation) (Alzheimer’s disease (AD) is defined by two types of lesions, the extraneuronal senile plaques made of Aβ peptides and neurofibrillary degeneration constituted by intraneuronal inclusions of hyperphosphorylated Tau proteins. Tau is a microtubule (MT)-associated protein which is mainly expressed in neuronal axons under six alternatively spliced isoforms in adult brain. Although it has been shown that its primary function is promoting assembly and stability of MTs , other iylation) .Tau is an intrinsically disordered protein and its primary structure is divided into four regions: an N-terminal projection domain, a proline-rich domain (PRD), a microtubule-binding domain (MTBD), and a C-terminal region  Mandelk. In its O-GlcNAc glycosylation is another dynamic PTM involved in regulation of Tau, notably through impairment of its phosphorylation and toxicity  and O-GlcNAc hydrolase (OGA)  . It has ormation , 2014. Ihibitors .O-GlcNAcylation and S404 phosphorylation act as a unique motif regulated by a direct PTM crosstalk inducing each other chemical shift changes that suggest a spatial proximity between both sites in full-length Tau protein  in the C-terminal domain of Tau by disrupting the sequential phosphorylation by GSK3β  into NcoI/XhoI allowing removal of the sequence coding for the polyhistidine tag. TauPHF1 protein corresponds to a mutated form of Tau2N4R in which every Pro-directed Ser/Thr phosphorylation site as well as S262, S356 and S191 were mutated into Ala to prevent phosphorylation except those of the PHF-1 phospho-epitope (pS396/pS404).TauS262A mutant was obtained by site-directed mutagenesis of Tau2N4R (longest isoform of 441 residues) . TauPHF1E. coli strains transformed with Tau mutants were grown in M9 minimal medium enriched with 15N as nitrogen source for 15N-labeling , 1 mM MgSO4, 10 ml MEM vitamin cocktails (Sigma), 100 mg ampicillin per liter) at 37°C until OD at 600 nm reached 0.8–0.9. For 15N/13C-labeling, 0.5g 15N/13C-Isogro® (Sigma) and 2g 13C6-glucose instead of glucose were used. Then, cultures were induced by 0.5 mM isopropyl-thiogalactoside (IPTG) and cells were grown at 37°C for approximately 3 hours. The bacterial pellets harvested by centrifugation were resuspended in 40ml per liter of culture of cation exchange equilibrium buffer  supplemented with 0.5% Triton X100, 2 mM DTT and cOmpleteTM EDTA-free protease inhibitor cocktail (Roche). Cell lysis was performed by high pressure homogenization and the soluble extract was obtained by centrifugation at 30,000 × g for 30 min at 4°C. Then, heating of the soluble extract at 80°C for 15 minutes followed by centrifugation was performed as a first purification step before cation-exchange chromatography . Homogeneous fractions containing full-length Tau proteins as checked by SDS-PAGE and MALDI-TOF mass spectrometry were pooled and buffer-exchanged in 50 mM ammonium bicarbonate prior to lyophilization.TauS262A and TauPHF1 mutants were produced as described previously for wild-type Tau . BrieflyO-GlcNAc Tau  at 31°C for 2 days. O-GlcNAcylated Tau protein was enriched by reverse phase high-pressure liquid chromatography . To increase amounts of Tau-G for NMR analyses, a second round of O-GlcNAcylation/purification was performed with the non-O-GlcNAc enriched fraction. Both O-GlcNAc-enriched fractions were pooled together. Following this procedure, an average of 1.8 O-GlcNAc per Tau molecule were found by MALDI-TOF mass spectrometry considering a mass increment of +203 Da per GlcNAc group  and roup see .E. coli by coexpression of human GST–CDK2 and S. cerevisiae GST–Cak1 using a pGEX vector , as described  , concentrated at 46 μM (as determined by Bradford assay), frozen in liquid nitrogen and stored at −80°C until use.CDK2 phosphorylated at T160 was produced in escribed . Human clthcare) . The comTau proteins (Tau or Tau-G) were incubated at a final concentration of 0.1 mM with the recombinant CDK2/cyclinA3 complex and 5 mM ATP in phosphorylation buffer at 22°C overnight. After incubation, the kinase was removed by a heating step at 80°C for 15 min and subsequent centrifugation. Tau proteins were then buffer-exchanged in 50 mM ammonium bicarbonate prior to lyophilization. Overall phosphorylation levels were determined by MALDI-TOF mass spectrometry analyses considering a mass increment of +80 Da per phosphate group. Tau or Tau-G proteins phosphorylated by CDK2 were hereafter named Tau-P or Tau-G/P, respectively .E. coli strains transformed with a pGEX vector  carrying the human gsk3β were grown in 1L LB at 37°C for 3 hours until OD at 600 nm reached 0.8–0.9, then the culture was cooled down to 20°C and protein induction was performed at 20°C overnight upon addition of 0.2 mM IPTG. Harvested cells were resuspended in 40 ml extraction buffer  and cell lysis was performed by high pressure homogenization. Soluble proteins were isolated by centrifugation at 30,000 × g for 30 min at 4°C followed by purification on 1 ml of glutathione sepharose resin  per liter of culture. Resin beads were incubated with the soluble extract at 4°C for 3 hours and extensively washed with the extraction buffer, then with phosphorylation buffer supplemented with 50% glycerol. The GST-GSK3β fusion protein on resin beads was stored at −20°C until further use.BL21(DE3) Tau proteins  were incubated at final concentrations of 0.1 mM with the recombinant GSK3β kinase  and 5 mM ATP at 30°C overnight in phosphorylation buffer. After incubation, resin beads were removed by centrifugation and heating of the supernatant was performed at 80°C for 15 min followed by centrifugation. Tau proteins were then buffer-exchanged in 50 mM ammonium bicarbonate prior to lyophilization. Overall phosphorylation levels were determined by MALDI-TOF mass spectrometry analyses. Tau, Tau-G, Tau-P or Tau-G/P proteins phosphorylated by GSK3β were hereafter named Tau-P(GSK3), Tau-G/P(GSK3), Tau-PP or Tau-G/PP, respectively .3)-OH was carried out with 2 eq. of the amino acid, HBTU, HOBt and DIPEA in DMF over a time of 6 h (2O (95/2.5/2.5) for 3 hours followed by precipitation with cold diethyl ether.All reagents, amino acids, and solvents were purchased from commercial suppliers and used without further purification if not further mentioned. Solvents- acetonitrile, dimethylchloride, dimethylformamide (DMF)- were purchased from ACROS ORGANICS. Peptides were synthesized on SyroXP-I peptide synthesizer  according to standard Fmoc/tBu chemistry using O-(benzotriazol-1-yl)-N,N,NORGANICS. Peptides were synthesiz-phosphate (HBTU)/ hydroxybenzotriazole (HOBt) and preloaded resins (Novabiochem). Fmoc-Ser/Thr(PO(OBzl)OH)-OH (Bachem) was activated with N-pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU)/ diisopropylethylamine (DIPEA) and coupled manually to the resin. DIPEA was added in 3-fold excess with respect to the amino acid and HATU. The reaction time was extended to 6 hours. The coupling of Fmoc-Ser(β-D-GlcNAc(Ac)e of 6 h . A mixtuPurification was carried out by preparative reversed phase HPLC on a Knauer Smartline system  equipped with a Luna C8 column  running with acetonitrile/0.1% TFA and water/0.1% TFA gradient at 20 mL/min. Purified peptides were characterized by analytical HPLC and high resolution MS. The analytical HPLC was carried out with a VWR-Hitachi Elite LabChrom system  equipped with a Luna C8 column . Peptide mass to charge ratios were measured by using an Agilent 6210 ESI-TOF  .15N-labeled proteins and unlabeled peptides were dissolved at 0.2 mM and 2 mM, respectively, in a buffer containing 25 mM NaH2PO4/Na2HPO4 pH 6.6, 25 mM NaCl, 2.5 mM EDTA, 1 mM DTT and 5% D2O either in a volume of 200 μl (in 3 mm tubes) or in 300 μl (in Shigemi tubes). All 1H spectra were calibrated with 1 mM sodium 3-trimethylsilyl-3,3’,2,2’-d4-propionate as a reference. 1H spectra were acquired with 64 scans and 32 dummy scans, and a spectral windows of 14 ppm centered on 4.7 ppm sampled with 32k points. For 2D and 3D experiments, a spectral window of 14 ppm centered on 4.7 ppm was used for the proton dimension.NMR experiments were performed at 293K on a Bruker 600 MHz SB Avance III HD and 900 MHz Avance NEO spectrometer  equipped with 5-mm cryogenic triple resonance probe heads. For NMR experiments, 1H-15N HSQC spectra were recorded at nitrogen-15 natural abundance with 128 scans per increment, with 2048 and 128 points in the proton and nitrogen dimensions, respectively, and with a window of 25 ppm centered on 118 ppm for the nitrogen dimension. 1H-13C HSQC spectra were recorded with 32 scans per increment, with 1440 and 512 points in the proton and carbon dimensions, respectively, and with a window of 80 ppm centered on 45 ppm for the carbon dimension.For peptide assignment, standard NOESY and TOCSY experiments were recorded on peptides with 200 and 69 ms mixing times respectively, with 4096 and 512 points and using a DIPSI2 sequence for mixing. TOCSY and NOESY spectra were recorded with 32 scans and 64 scans per increment (and 128 dummy scans), respectively, with spectral windows of 13.98 and 10.0 ppm in each proton dimension centered on 4.69 ppm. 15N-labeled proteins, 1H-15N HSQC spectra were recorded with 32 scans per increment and 32 dummy scans with 3072 and 512 points in the proton and nitrogen dimensions, respectively, and with a window of 25 ppm centered on 118 ppm for the nitrogen dimension. Heteronuclear experiments were recorded with a WATERGATE sequence for water suppression and a double INEPT (INsensitive nuclei Enhanced by Polarization Transfer) for sensitivity improvement. All experiments were acquired with a recycle delay of 1s.For 15N/13C-labeled Tau sample at 293K. The HNCACB and HN(CO)CACB experiments were recorded with non-uniform sampling with 32 scans per increment and 16 dummy scans, with 1540, 90 and 180 points in the proton, nitrogen and carbon dimensions, respectively, and with a window of 25 ppm and 60 ppm centered on 118 ppm and 44 ppm for the nitrogen and carbon dimensions, respectively. The HN(CA)NNH experiment was recorded with non-uniform sampling with 24 scans per increment and 16 dummy scans, with 2048, 120 and 100 points in the proton and both nitrogen dimensions, respectively, and with a window of 25 ppm centered on 118 ppm for the nitrogen dimensions.Assignment of phosphorylation sites in CDK2/GSK3β phosphorylated TauS262A protein required the acquisition of three-dimensional NMR experiments on O-GlcNAc and phosphorylation levels were determined for individual Ser/Thr residues based on intensity of their respective amide correlations of modified and non-modified isoforms from the 1H-15N HSQC experiment. Resonance assignment, peak intensity and integration are given for phosphorylation and/or O-GlcNAcylation sites in TauPHF1 proteins (see I(p) on the sum of total peak intensities including non-modified I(np/ng) and O-GlcNAcylated I(g) isoforms as described in Equation (1). Percentage of O-GlcNAcylation is calculated similarly as described in Equation (2). Note that modification of a given residue could affect resonances of vicinal residues leading to resonance splitting, each corresponding to a specific isoform (as exemplified by S400 and S404). Therefore, I(p), I(g) or I(np/ng) corresponds to the sum of peak intensities of a given residue in its phosphorylated, O-GlcNAcylated or non-modified state, respectively, which could occur as several resonances depending on the modification state of its neighbors.eins see . PercentO-GlcNAcylation can either be determined using peak integration to take into account changes of relaxation of amide 1H and 15N upon modification. However, overlapping peak could lead to misinterpretation of modification levels. A comparison of values obtained from peak intensities and peak integrals gives similar results . For the calculation of overall phosphorylation and O-GlcNAcylation levels, m/z increments of +80 Da and +203 Da were used, respectively.Tau proteins were analyzed by MALDI-TOF MS  in a linear positive ion mode with sinapinic acid matrix after ZipTip2PO4/Na2HPO4 pH 6.6, 25 mM NaCl, 2.5 mM EDTA, 0.33 mM DTT, and 50 μM ThT. Kinetics of fibril formation were monitored by ThT fluorescence emission at 490 nm (with excitation at 440 nm). Aggregation reactions of each Tau PTM isoform were performed in duplicate or triplicate and kinetics curves represented as mean ± standard deviation were fitted with a model of single-phase association with the GraphPad Prism software. Different batches of kinase and phosphorylated Tau were prepared and involved in aggregation reactions with kinetics curves similar as those depicted in Aggregation reactions were performed into 96-well black plate (Greiner) in a plate reader  at 37°C without agitation. A 5 mM aqueous stock solution of ThT (Sigma) was filtered through a 0.22μm-filter. Tau aggregation was induced by heparin  and kinetics were measured for 5–7 days with 10 μM Tau and 2.5 μM heparin (ratio 4:1) in a buffer containing 25 mM NaHe in fresh monomeric Tau at 25 μM in 100 mM MES pH 6.9, 2mM EGTA, 1mM MgCl2, 20 mM NaF, 1 mM DTT, 100 μM ThT. Control of seeds without fresh monomeric Tau was performed in the same conditions. As a control of the absence of free heparin, TauS262A protein at 25 μM was incubated with 0.25 μM heparin (ratio 100:1) in the same conditions than the seeding reactions. Seeding reactions were incubated for a total of 5 to 7 days at 37°C. Negative-staining TEM imaging and SDS-PAGE measurement of decrease in soluble monomer concentration upon assembly were performed at the aggregation end-point as described for heparin-induced aggregation assays.For aggregation reactions induced by seeding, seeds were prepared by incubating TauS262A (in its non-modified form) at 10 μM and heparin at 2.5 μM (ratio 4:1) for 7 days at 37°C. After sonication, the aggregation reaction was diluted at 1:10O-GlcNAcylation modulates phosphorylation of Tau by GSK3β with or without priming by the proline-directed kinase CDK2/cyclin A3 (referred hereafter as CDK2). As major O-GlcNAc sites were all found in the C-terminus of Tau . As the quantitative pattern of O-GlcNAcylation is dependent on the conditions used for the O-GlcNAc transferase reaction, we achieve a high level of O-GlcNAc modification by reverse phase high-performance liquid chromatography enrichment of O-GlcNAcylated species in order to unambiguously detect perturbations of phosphorylation patterns induced by O-GlcNAcylation. This procedure leads to an overall O-GlcNAc level of 1.8 ± 0.3 GlcNAc per Tau molecule and site-specific O-GlcNAcylation of 61% ± 2%, 27% ± 1% and 47% ± 2% on S400, S412 and S413, respectively, determined by high resolution NMR spectroscopy on both TauS262A-G and TauPHF1-G . However, subsequent GSK3β phosphorylation is significantly reduced as compared to non-O-GlcNAcylated Tau to ca. 36% and 40% on both S400 and S396 sites, respectively , the overall S404 phosphorylation level of 38% is distributed between three isoforms: 14% for PHF1-3P, 21% for gS400/pS404 and 3% for S400/pS404, the last two being not subjected to further phosphorylation at S400 and S396 (O-GlcNAcylation of respective S404 and S400 sites are not exclusive but rather competitive. The non-phosphorylated S404 isoform contains a higher proportion of gS400 (75%) than the pS404 isoform (55%) indicating that O-GlcNAcylation is preferentially associated to the non-phosphorylated isoform.Hence, and S396 . These dO-GlcNAcylation level of 60%. Lower values were measured indicating that O-GlcNAcylation not only reduces S404 phosphorylation by GSK3β (by two-fold) and directly prevents formation of PHF1-3P epitope, but also reduces S404 phosphorylation in the S400 non-O-GlcNAc fraction indicating a potential role of O-GlcNAcylation at S412 and S413 sites in modulation of PHF-1 epitope phosphorylation.Furthermore, levels of 26% and 38% for pS400/pS404 and gS400/pS404 isoforms, respectively, were expected considering a S404 phosphorylation level of 65% (as PHF1-3P) and S400 O-GlcNAc modifications within the C-terminal domain. Therefore, O-GlcNAcylation neither modulates CDK2 priming nor has a long-range influence on phosphorylation sites localized in the proline-rich domain.In contrast, remote phospho-epitopes localized in the proline-rich domain of TauS262A were not regulated by O-GlcNAcylation and combination of both PTMs on Tau conformation was investigated by high-resolution NMR spectroscopy  calculation , TauPHF1-P nor TauPHF1-PP were able to form fibrils as monitored by Thioflavin T (ThT) fluorescence during several days at 37°C and negative-staining transmission electron microscopy (TEM). Hence, we first stimulated aggregation of the diverse phosphorylated/ regions . Hence,  regions . We have regions . However regions . As the O-GlcNAcylation reduces the elongation rate of TauPHF1 as previously shown , both providing high levels of phosphorylation, only marginally forms fibrillar aggregates using heparin as external inducer as compared to the non-phosphorylated isoform see . Phosphoters see . Howeverters see . In contters see .O-GlcNAcylation by OGT to probe modulation of GSK3β-mediated phosphorylation pattern and its role on Tau self-assembly into PHF-like filaments. The phosphorylation pattern provided by GSK3β in TauS262A is highly regulated by CDK2 priming. While only the triple phosphorylated state of PHF-1 epitope or PHF1-3P (pS396/pS400/pS404) was observed without priming as previously shown , acting by both decreasing phosphorylation on S404 and disrupting sequential phosphorylation of S400/S396 by GSK3β as previously described in peptides  induces the same effect on the C-terminus conformation as single phosphorylation of S396 (Tau[392-411]-2P) or double phosphorylation of S396/S400 (Tau[392-411]-3P)  . It was izing it . In a stcylation . Here, iO-GlcNAcylation in fibrillar aggregation was then investigated in vitro in the presence of heparin or seeds of Tau2N4R to induce aggregation. High level or site-specific phosphorylation are likely to compete with Tau binding to heparin due to charge repulsion, as illustrated by all phosphorylated isoforms of TauS262A and TauS262A-G (see O-GlcNAcylation by OGT (Tau-G) both reduce fibrillar aggregation rates with a greater effect for O-GlcNAcylation, which also affects the initial phase of fibrillization related to nucleation processes , the significant amount of gS400/pS404 isoform (21%) could also contribute to a large extent to acceleration of Tau fibrillar aggregation thereby counteracting the O-GlcNAc-mediated inhibition. This would suggest that a combination of phosphorylation and O-GlcNAcylation could be deleterious in the same way than PHF-1 phosphorylation in fibrillar assembly.In contrast, phosphorylation by GSK3β accelerates aggregation rates of Tau, Tau-P or Tau-G isoforms by 1. 5-, 3.3- and 7.1-fold, respectively . Each ofO-GlcNAcylation does not intrinsically inhibit the fibrillization process. Phosphorylation of both Tau and Tau-G, on the other hand, induce the formation of individual fibrils to a lesser extent with greater length than seeds and no fibrillar clusters were observed indicating a distinct mechanism of fibrillization as compared to the non-phosphorylated isoforms. This suggests that at least part of heparin within Tau seeds is still accessible as shown in We have shown here that seeds of Tau2N4R formed with heparin are able to induce fibrillar aggregation of monomeric TauS262A isoforms bearing various PTMs into PHF-like filaments as detected by TEM . DespiteO-GlcNAcylation has been shown in various cellular models of tauopathies or in vivo in transgenic mice. In most cases, decreasing protein O-GlcNAcylation through OGT knock-down or by mimicking impaired glucose metabolism and uptake as in AD brain leads to increased site-specific phosphorylation of Tau  is likely an indirect process involving the O-GlcNAc-mediated regulation of enzymes implicated in phosphorylation dynamics (A reciprocal regulation of Tau phosphorylation and n of Tau , 2009. Cathology . Howeverathology . The propeptides , but doedynamics  or otherdynamics .in vitro and significantly counteracts inhibition of fibrillization mediated by O-GlcNAc posttranslational modifications although O-GlcNAcylation severely reduces the PHF1-3P isoform. Conversely, priming by CDK2 increases GSK3β phosphorylation in both Tau and O-GlcNAcylated Tau isoforms by providing a number of phosphorylation sites in the proline-rich domain which otherwise is not targeted by GSK3β activity. A conformational study of the impact of PTMs in the C-terminal region around the PHF-1 epitope shows that phosphorylation of S404 triggers a dynamic conformational change in the region immediately downstream S404 site that is enhanced by other phosphorylations of the PHF-1 epitope or S400 O-GlcNAcylation. These data suggest that multiple phosphorylations or combination of phosphorylation and O-GlcNAcylation within the PHF-1 epitope have the same conformational effect while increasing the formation of PHF-like filaments.In this study, we have shown that GSK3β exacerbates fibrillar aggregation of Tau protein The original contributions presented in the study are included in the article/CS-N designed research. F-XC, AL, CS-N, and XT performed research. CD, OR, NG, CH, and CS-N contributed new materials/analytic tools. F-XC, AL, XT, and CS-N analyzed data. IL and CS-N wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


An octa-screw extruder (OSE) is equipment for pelletizing, blending, and mixing polymers and composites. In this study, the degree of resin filling, residence time distribution (RTD) of molten resin, and temperature profile in the octa-screw extruder were evaluated both experimentally and numerically. An intermeshing corotating parallel octa-screw kneading extruder was used for the experiments. For the comparison study, the results obtained from this extruder were compared with the twin-screw extruder. High-density polyethylene was selected as the material for extrusion. Meanwhile, a numerical code, based on a 2.5 D finite element method derived from the Hele–Shaw flow model, was developed to simulate the octa-screw extrusion process. The empirical outcomes suggest that octa-screw extrusion exhibited a narrower RTD of the molten resin compared with the twin-screw extrusion, suggesting better extrudate quality. The octa-screw extrusion also showed a lower temperature profile than twin-screw extrusion. The results of the simulation were also found to be in good agreement with experimental measurements. Experimental and numerical investigations of an OSE enable detailed comprehension and visualization of resin distribution in the entire length of the OSE, thus providing advantages in terms of process optimization. The self-wiping corotating parallel octa-screw extruder (OSE) is a high-efficiency polymer-processing machine for the pelletizing, blending, alloying, compounding, devolatilizing, chemical reacting, and die forming of molten polymers . During The flow of material in OSEs is complex, and it can be difficult to mathematically predict flow patterns . SeveralThis work investigated the octa-screw extrusion process, both experimentally and numerically, and assessed the degree of fill, residence time distribution (RTD), and temperature profile in the extruder. An intermeshing corotating parallel octa-screw kneading extruder was used for the experiments. High-density polyethylene (HDPE) and a heat-resistant masterbatch were selected as the materials for experiments. Meanwhile, a numerical code, based on a reconstructed flow analysis network (FAN) scheme, the Hele–Shaw flow model, finite element method (FEM), and a downwind pressure updating technique , was devThe polymeric material used in this study was HDPE  with a molecular weight of 3.4 × 106 Da. The EX-3052J-OIIB-MB33 model heat-resistance masterbatch purchased from Nippon Pigment Company Ltd., Tokyo, Japan, was used for the experiments.An intermeshing corotating parallel octa-screw kneading extruder  with a length over diameter (L/D) of 45 and a diameter of 15 mm was used for the experiments. As shown in eFF) that describes the degree of fill for each screw element was determined using the following equation:emaxwt is the maximum weight of the polymer when the element is completely filled, and ewt is the weight of the residual polymer on the screw element.OSEs and TSEs were first operated, until they reached a steady state, to determine the degree of fill in the multiscrew extruders . The macThe heat-resistance masterbatch of 12 mg was first blended with HDPE of 20 g via a dry mixer for subsequent extrusions to identify the RTD. The extruded resins at the die exit were collected every 30 s during extrusion. The collected samples were then analyzed using a thermogravimetric analyzer . The temperature of the TGA was programmed to rise from 30 °C to 500 °C at a constant heating rate of 5 °C/min. TGA tests were performed under an airflow of 60 mL/min. After the complete pyrolysis of the polymers, the amount of the remaining masterbatch was weighted with a balance of ±0.1 mg accuracy. RTD was then defined as the variation in weights of the residual masterbatch with time.A numerical code, based on 2.5 D FEM derived from the Hele–Shaw flow model , was user, θ, z) system to simulate the flow pattern in an OSE  direction and axial (z) direction arep is the pressure and η is the viscosity of the molten fluid. In this model, the screw is assumed to remain stationary and the barrel is considered to rotate relative to the screw with an angular velocity of Ω. Assuming the pressure gradient in the calculation element remains constant, then the flow velocities become:sR is the screw radius. The strain rates with rθ and rz components can be, respectively, defined as:Meanwhile, the equations of motion along the circumferential ). Extrusion is one of the most used processes for producing polymeric materials, and the thermal homogeneity of the process has paramount importance for the manufacture of high-quality extruded products ,21. HencThe influence of extrusion speed on the temperature profiles was also numerically simulated. The simulated temperature was also compared with the experimental data to check the validity. The results in The comprehension of the RTD is critical for polymer extrusion because it is related to the level of polymer degradation and special processes such as reactive extrusion . The theFinally, it should be noted that the OSE is similar to the ring extruder (or planetary roller extruder), except that the screws are arranged on a flat plane. It has been reported that the planetary roller extruder is one of the most successful multi-screw extruders in pelleting and compounding powdered paint and recyclate . SchmidtWe experimentally and numerically investigated the octa-screw extrusion process and assessed the degree of fill, RTD, and temperature profile in the extruder. An intermeshing corotating parallel octa-screw kneading extruder was employed for the experiments. Meanwhile, a numerical code based on a reconstructed FAN scheme, the Hele–Shaw flow model, FEM, and a downwind pressure updating technique was developed to simulate the octa-screw and twin-screw extrusion processes. The empirical outcomes suggest that octa-screw extrusion exhibited a narrower resin distribution time than the twin-screw extrusion. The simulated results also showed that the OSE displays a lower temperature distribution than the TSE did during the extrusion process. This proves the advantages of the OSE in terms of superior mixing capability with minimized thermal degradation of polymeric materials in the extrusion process. The results of the simulation were also found to be in good agreement with experimental measurements. Experimental and numerical investigations of an OSE enable detailed comprehension and visualization of resin distribution over the length of the OSE, thus enhancing their capability in terms of process optimization.


Developing molecules that emulate the properties of naturally occurring ice-binding proteins (IBPs) is a daunting challenge. Rather than relying on the (limited) existing structure–property relationships that have been established for IBPs, here we report the use of phage display for the identification of short peptide mimics of IBPs. To this end, an ice-affinity selection protocol is developed, which enables the selection of a cyclic ice-binding peptide containing just 14 amino acids. Mutational analysis identifies three residues, Asp8, Thr10 and Thr14, which are found to be essential for ice binding. Molecular dynamics simulations reveal that the side chain of Thr10 hydrophobically binds to ice revealing a potential mechanism. To demonstrate the biotechnological potential of this peptide, it is expressed as a fusion (‘Ice-Tag’) with mCherry and used to purify proteins directly from cell lysate. Developing molecules that emulate the properties of naturally occurring ice-binding proteins (IBPs) is a daunting challenge. Here, the authors demonstrate the use of phage display for the identification of short peptide mimics of IBPs, which resulted in the identification of a cyclic ice-binding peptide containing just 14 amino acids. These proteins achieve the remarkable molecular recognition feat of selectively binding ice in the presence of a large excess of water inducing the macroscopic properties of thermal hysteresis (non-equilibrium depression of freezing point), ice recrystallization inhibition (IRI) and dynamic ice shaping, all characteristic of macromolecule–ice interactions. There is large structural diversity between IBPs from the relatively large and rigid Marinomonas primoryensis AFP stabilized by extensive calcium binding to the smaller and flexible antifreeze glycoproteins (AFGPs)1.Ice-binding proteins (IBP) ) are produced in psychrophilic species as an evolutionary mechanism to enable survival in sub-zero climates2. For example, the AFGPs are based on a glycosylated tripeptide motif Ala-Ala-Thr, with naturally occurring variants ranging from 4–55 repeating tripeptide units (2.4–34 kDa). These naturally occurring IBPs have provided structural information that has guided the design of a variety of synthetic peptide-based analogues over the past years. The synthesis of IBP analogues, however, is often very challenging, in particular on a large scale, and activities are variable4.There is a great interest to unravel and understand the interactions between IBPs and ice. This is not only of fundamental interest but can also help to identify design principles that facilitate the development of synthetic materials that emulate the ice-binding properties of IBPs. Analysis of the amino acid sequence and structure of IBPs has provided some insight into structure–function relationshipsEscherichiacoli cell lysate.In this work, we report a conceptually alternative approach to the development of synthetic IBP mimics. Rather than building upon existing structural know-how, this study explores the use of phage display, a biological, combinatorial discovery tool, for the identification of short peptide mimics of IBPs. Since the starting point of the phage display process is a randomized peptide library, this strategy vastly expands the diversity space that can be screened for potential ice-binding peptides. This may help to provide further structural insight into the key structural determinants that govern the properties of IBPs. Phage display, furthermore, generally leads to short peptides that are composed of naturally occurring amino acids, which may also enhance the synthetic accessibility of these compounds. Using phage display, a 14 amino acid, cyclic peptide ice recrystallization inhibitor is discovered. The solution structure of the peptide is characterized by NMR spectroscopy, and mutational analysis experiments reveal three residues, Asp8, Thr10 and Thr14, which are found to be essential for ice binding. Molecular dynamics (MD) simulations show that the peptide binds to ice adopting specific conformations through a combination of both hydrophobic interactions (Thr10 and Thr14) and hydrogen bonding (Asp8). Finally, to illustrate the potential use of short ice-binding peptides, the discovered sequence is used as a purification tag to extract overexpressed recombinant mCherry fluorescent protein from 7. Over the years, the scope of this technique has expanded and phage display has been successfully applied for the identification of short peptide ligands that can selectively bind to other targets including metals, semi-conductors, ceramics as well as a range of biological and synthetic polymers15. Ice, however, is an exceptionally challenging target substrate as it presents a dynamic interface. Ice is formed in water and is constantly growing/melting.Phage display allows the discovery of high-affinity, short peptide ligands against a broad range of protein substrates16. The phage library was designed to accommodate the formation of both mono- and bicyclic peptides17. The cyclic peptide library was generated by encoding four cysteines into each peptide while randomizing the remaining residues. Oxidation of all four cysteine residues can lead to the formation of two disulfide bridges and subsequently, a variety of bicyclic peptide structures. When only two cysteine residues are paired via a disulfide bridge, monocyclic peptides are obtained. Cyclic peptides have reduced conformational flexibility, which results in lower entropic penalties upon target binding, and increased diversity as compared to linear libraries of the same length18. To further increase diversity, a mixture of six different libraries varying in length from 9 to 14 amino acids was used. For the initial panning experiments, the mixture of phage libraries contained ~2 × 1010 unique peptides sequences17. For the affinity selection step, first an ice shell was formed inside a round bottom flask that was rotated in an ethylene glycol bath held at −0.5 °C. Then, a phage library solution was incubated with the ice for approximately 2 min to capture ice-binding phages. The incubation time was limited to just 2 min in order to prevent ice overgrowth and preserve phage function. After that, the remaining liquid, containing unbound phages, was decanted and the ice was washed with pre-cooled buffer solution. Next, the ice was melted, releasing bound phages that were subsequently used to infect E. coli to amplify phages available for iterative panning cycles. This strategy bears resemblance to the rotary ice-affinity purification method that has been used to isolate IBPs from organisms including bacteria, insects and plants21. By measuring the output phage titer after each round of selection, the enrichment of the phage library towards ice could be assessed. Repetition of the affinity selection cycle resulted in a gradual increase in the output phage titer. After five rounds, an ~15-fold increase in the output phage titer was determined  onto a glass coverslip, which is resting on an aluminium block cooled to −78 °C. This results in the instantaneous formation of a thin layer of ice, which is subsequently transferred to a cryostage and annealed at −8 °C for 30 min. After 30 min, the relative size of the ice crystals is evaluated and compared with those of a control buffer sample that does not contain a peptide. Figure peptide 8 showed significant IRI activity. While all peptides1–9 are based on sequences that have been identified via phage display, the absence of IRI activity for peptides1–7 and 9 indicates that phage affinity does not necessarily translate into peptide affinity. One possible reason for this can be that binding of a phage, which presents five copies of the peptide, to ice can proceed via multivalent interactions, whereas peptides 1–9 bind in a monovalent fashion. Figure peptide 3 and peptide 8, respectively, and illustrate the significant difference in ice grain size due to peptide 8.As a first screen to assess their activity, peptide 8 with ice, ice shaping experiments were performed. These experiments were carried out by sandwiching a droplet of a 1 mg/mL solution of peptide 8 in PBS containing 40% (w/v) sucrose between two glass coverslips. These are subsequently transferred to a cold stage, which is rapidly cooled to −50 °C, and held for 2 min. Then, the ‘sandwich’ is warmed to −8 °C and annealed for 30 min. The presence of sucrose reduces nucleation and crystal growth, allowing for the monitoring of the growth and development of individual ice grains. Figure peptide 3. As can be seen, the individual ice crystals have a disc-like shape, which is typical of uninhibited ice growth. This observation indicates that peptide 3 is unable to bind ice. The same disc-like shape present in Fig. peptide 8. In this case, the ice crystals are faceted. Ice crystals become faceted when ice growth is inhibited in some way, like by bound IBPs or in this case peptide 8. This observation is important because ice-binding and IRI activity are not linearly related. There are many examples of compounds, like small AF(G)P mimics, that have high IRI activity but are unable to bind to ice25.To probe the interactions of peptide 8 was examined by solution NMR spectroscopy. NMR experiments were performed with 6 mg/mL peptide 8 dissolved in 0.5× PBS at pH = 6.0 with 10% deuterium oxide (D2O). Such high concentration enables the acquisition of 1H, 15N and 1H, 13C heteronuclear single quantum coherence spectra at natural abundance, allowing for full characterization. Supplementary Figs. 1H–15N and 1H–13C correlation spectra that were recorded to assign the peptide backbone structure. Supplementary Table 1H Larmor frequency) and at different temperatures, revealed very few NOEs. All NOE crosspeaks were found to arise from consecutive residues with no long-range H,H correlations  as compared to the Cys2 and Cys7 Cβ shifts that are located at 28.13 and 28.12 ppm. The more downfield shifts of the Cys3 and Cys13 signals are indicative of the formation of a monocyclic peptide that contains a disulfide bridge between Cys3 and Cys13.The structure of m oxide DO. Such h8 is shown in Fig. 8, constrained CS-Rosetta27 was used. Based on the recorded chemical shifts, 1500 model structures were generated, which were first sorted based on their relative energy , and small-angle X-ray scattering (SAXS) analysis of 1 mM solutions of peptide 8 in 1× PBS pointed toward the existence of a single species and the absence of any higher order, aggregated assemblies under these conditions  and oxidizing (black square) conditions. As can be seen, the peptide is only active when it is oxidized, suggesting the cyclization is essential. The activity of peptide 8 is comparable to that of other IRI active compounds such as PVA56 (red circles) and truncated type I AFP (blue triangles)  Fig.  in spitepeptide 8, two mutants were prepared in which several cysteine residues were replaced by serine  as a starting point. Using CHARMM3633 and the TIP4P/Ice34 force fields for the peptide and water molecules, respectively, a total of 20 independent unbiased simulations were performed. The simulations were run at 265 K and for 200 ns each. Trajectory analyses plotting the number of ice-like and liquid water molecules around the methyl group of the Thr10 residue for each of the 20 independent MD simulations can be found in Supplementary Fig. peptide 8 was placed in a random orientation situated away from the growing ice front. Figure peptide 8 from two different perspectives upon binding to ice. The snapshots in Fig. peptide 8 starts to bind. Once peptide 8 has bound to ice, further ice growth is substantially hindered, as shown in Fig. peptide 8 to ice is predominantly entropically driven by the hydrophobic de-solvation of the methyl groups, which in turn is strongly influenced by the conformational space the peptide can occupy. Analysis of the C–S–S–C torsional angle, φ, around the disulfide bond  before the peptide binds with ice. This analysis reveals that in particular in the short time frame prior to ice binding there is an increase in the number of water molecules within the solvation shell of Thr10 methyl groups. This suggests that the ability of peptide 8 to interact with ice appears to be enhanced by specific conformations, which maximize the amount of water molecules within the solvation shell of Thr10. As a whole, these findings unequivocally underline the critical role of Thr10 in binding of peptide 8 to ice. In contrast, the contributions of Thr14 and Asp8, which were also identified via mutational analysis as essential residues, are less clear. Analysis of the hydrogen bonding interactions between peptide 8 and ice peptide 8. Supplementary Fig. peptide 8 to ice with the intermolecular distances during ice binding. Qualitatively, the distribution of distances that is observed corresponds fairly well with the distances that were obtained from the solution NMR analysis  at the C-terminal end of mCherry. The mCherry-peptide 8 fusion was isolated from the bacterial cell lysate via a single ice-affinity purification step. To this end, the crude bacterial cell lysate was added to a round bottom flask that was covered with a preformed, thin layer of ice. After 15 min, the remaining liquid was decanted. This resulted in the formation of a scarlet coloured ice shell within the flask (Fig. peptide 8 as an ice-affinity tag, rotary ice-purification of E. coli cell lysate expressing mCherry modified with a C-terminal peptide 8 tag was compared with that of E. coli cell lysate containing overexpressed GFP, which shares high sequence and size similarity to mCherry, without an ice-tag. The total protein content in the cell lysate, as well as in the ice phase and in the liquid phase was assessed by the BCA assay as well as via fluorescence spectroscopy. Under UV-irradiation, the ice shell of the flask used for the GFP “purification” showed very little fluorescence as compared to the ice shell that was used to capture the peptide8-tagged mCherry (Supplementary Fig. peptide 8-tagged mCherry lysate resulted in isolation of ~50% of the total protein content, as compared to just ~20% in case of the GFP containing control lysate (Supplementary Fig. peptide 8-modified mCherry was accompanied by an increase in protein purity, whereas the control experiment with the GFP lysate showed no improvement in protein purity (Supplementary Fig. The ice-binding properties of ins Fig. . In a fiask Fig. . SDS-PAGask Fig. . The ideask Fig. . To furtPhage display was successfully used to identify a 14 amino acid, cyclic ice-binding peptide. By combining ice-binding activity studies with NMR and modelling tools, it was established that the peptide has a cyclic structure and features three residues essential for ice binding. The strategy presented here complements traditional de novo design approaches to ice-binding peptides. Since the phage display ice-affinity selection process starts from a randomized peptide library, a vast diversity space can be screened for potential ice-binding peptides. The short peptides that can be identified with the help of phage display may help to provide further structural insight into the key structural determinants that govern the properties of IBPs. Furthermore, since phage display generally leads to short peptides that are composed of naturally occurring amino acids, the strategy reported here may also enhance the synthetic accessibility of novel ice-binding peptides.Further information on research design is available in the Supplementary InformationReporting Summary


Adamussium colbecki appears to remain ice-free in shallow Antarctic marine environments where underwater ice growth is prevalent. In contrast, scallops colonized by bush sponges in the same microhabitat grow ice and are removed from the population. Characterization of the Antarctic scallop shells revealed a hierarchical micro-ridge structure with sub-micron nano-ridges which promotes directed icing. This concentrates the formation of ice on the growth rings while leaving the regions in between free of ice, and appears to reduce ice-to-shell adhesion when compared to temperate species that do not possess highly ordered surface structures. The ability to control the formation of ice may enable passive underwater anti-icing protection, with the removal of ice possibly facilitated by ocean currents or scallop movements. We term this behavior cryofouling avoidance. We posit that the evolution of natural anti-icing structures is a key trait for the survival of Antarctic scallops in anchor ice zones.The presence of supercooled water in polar regions causes anchor ice to grow on submerged objects, generating costly problems for engineered materials and life-endangering risks for benthic communities. The factors driving underwater ice accretion are poorly understood, and passive prevention mechanisms remain unknown. Here we report that the Antarctic scallop  Adamussium colbecki appears to remain ice-free in shallow Antarctic marine environments where underwater ice growth is prevalent. They find that micro-ridge structures in the shell promote directed icing that may prevent the dangerous accumulation and firm attachment of buoyant ice.Wong et al report that the Antarctic scallop  Currently, growing efforts have focused on anti-icing surfaces developed for in-air environments7 to avoid, repel or retard ice formation. Promising strategies employ either micro-/nano-textured or very smooth liquid interfaces10 to encourage the shedding of accreted ice. Specific topological modifications have also been fabricated that redirect ice growth to only a subset of the surface, potentially reducing overall adhesive forces11.The unwanted interaction with ice in our natural environments presents challenges with undesirable consequences: expansion or growth of frost/ice can impact power generation , damage infrastructure , or be lethal to organisms14 are not understood, and mechanisms for its prevention remain unknown. Presently lacking a suitable terminology, we define the term “cryofouling” to describe the unwanted accumulation of ice on submerged surfaces. Naturally-occurring surfaces that avoid cryofouling may already exist, given that various cold-blooded organisms survive16 in the shallow marine environments surrounding Antarctica where cryofouling is prevalent and often hazardous to aquatic life18.In contrast to in-air environments, the driving factors of ice accretion on underwater surfaces19 below its expected salinity- and pressure-dependent equilibrium freezing point. In coastal Antarctica, supercooled waters are produced by the interaction of coastal waters with massive, thick floating ice shelves producing small, freely floating ice crystals in the water column21. In some high-Antarctic environments, near-surface ocean waters are regularly supercooled by ~0.01 °C for up to half of each year22. Seawater supercooling in Antarctica, albeit minimal in magnitude, may drive substantial volumes of ice accretion on submerged surfaces18. For example, in McMurdo Sound (78 °S latitude), the supercooled conditions , presumably having evolved or serendipitously possessing characteristics that permit them to avoid cryofouling, survive and thrive in the shallow anchor-ice zone.In shallower areas where anchor ice is common, the cryofouling of organisms can interfere with behavioral or biological processes, or remove individuals from the seabed given the buoyancy of accreted iceAdamussium colbecki, the Antarctic scallop, avoids cryofouling of the exterior surfaces of its mineral shells. Using a comparative approach, we compare the results of ice growth and adhesion experiments on Antarctic scallop shells to those performed on two temperate Atlantic scallop species that do not encounter ice in their habitats. Based on results of molecular phylogenetic analyses, Placopecten magellanicus  belongs to the same, small subclade as the Antarctic scallop, while the Argopecten irradians  is more divergent  revealed that the prevalence and thickness of the anchor-ice formations are highest in the shallowest locales and decreased with increasing depth of the seabed. From the underside of the surface sea ice (~2–3 m) to about 6 m depth, anchor ice formed a 60-cm-thick mat, covering greater than 90% of the seabed , nor did any scallops appear to be frozen to the actively growing anchor-ice matrix on which they sat.Similar to the well-studied locales in southeastern McMurdo SoundHomaxinella balfourensis) that had colonized the external shell surfaces of Antarctic scallops. In these observations, the growth of ice24 on the epizoic sponges caused involuntary flotation of the host scallops due to the buoyancy28 of the ice attached to the sponge  laminated with proteinaceous periostracum. Surface composition was found to be identical at different locations of the analyses, including along concentric growth rings, micro-ridges, or the micro-valleys in between. The surface roughness was determined using atomic force microscopy (AFM) and revealed that the peaks of the concentric growth rings were the roughest (RMS roughness of 135 ± 57 nm), followed by the radial micro-ridges (RMS roughness of 96 ± 3 nm). In contrast, valley floors between these micro-ridges were relatively smooth (RMS roughness of 38 ± 9 nm), interrupted only by small protuberances  that were irregularly dispersed throughout the radial micro-valleys.Next, we determined the elemental composition and roughness at different positions on the shell surface. Energy-dispersive X-ray spectroscopy revealed that the shell surface predominately consists of oxygen, carbon, calcium, traces of silicon, sodium, aluminum, and magnesium  chamber at controlled humidity (60%). Compared to the temperate control species, ice nucleation on the Antarctic scallop shell occurred later and subsequent ice growth was directed toward specific surface features  on a surfaceres Fig. . These r32. Since more liquid water resides at the growth rings, here the nucleation of microscopic ice is more likely33. (2) Upon the formation of these microscopic ice domains as ice stripes (on growth rings), the vapor field around the ridges becomes compressed. The compressed vapor field generates locally steeper vapor gradients and thus stronger vapor fluxes towards the ice on the ridges  initial condensation-frosting on growth rings, followed by (2) continuous frost growth. (1) The presence of sharp/rough edges (such as those on the growth rings), is known to enhance liquid nucleation by reducing the energy barrierges Fig. . As a coA caveat remains, as the shell surface temperature during frosting assays (in-air) is significantly lower (c. −10 to −15 °C) than in nature. In its natural habitat, the lowest temperatures experienced by surfaces immersed in seawater Fig.  are rare37) and/or environmentally induced forces , or a combination of the above. This is particularly relevant once a sufficiently high ratio of ice volume to attachment surface area has been achieved. To provide complementary insight, both in-air and underwater ice-adhesion measurements were performed.We performed ice-adhesion measurements to test whether the apparent cryofouling avoidance of the Antarctic scallop’s shell in nature could arise from reduced adhesion forces between adhered ice and the shell’s surface. If adhesion is sufficiently low, ice could detach and float away under behavioral  to shells underwater was determined using a custom-built apparatus. The apparatus determined the forces required for ice detachment when adhered ice was pulled perpendicular to and away from the shell surface. Underwater ice-adhesion experiments were performed in saltwater (35 g/L NaCl), simulating the seawater conditions in Antarctica. A cold source beneath a small section of the shell induced ice growth up to 6 mm in height above the shell surface, , ≥3.7 cm3 of ice would need to be attached per mm2 of ice-shell contact area for the passive removal of ice under its own buoyant forces. Therefore, the “passive” removal of ice from Antarctic scallops must result from a combination of surface-to-environment factors. That is, the removal of ice crystals with small contact areas, such as platelet ice with its fine dendritic structure, may be facilitated by drag forces arising in natural water currents or induced by the rapid opening and closing of the scallop’s valves during swimming behaviors37. In these cases, adherent ice on the microstructures of the shell would likely experience the initiation of cracks that would subsequently facilitate ice removal given water movements or other physical disturbances.The strength of ice adhesion 39. This time period roughly coincides with the onset of the major glaciation of Antarctica (c. 35 mya). The Antarctic scallop, Adamussium colbecki, appears to have arisen more recently, in the late Pliocene (<5 mya), and remains the only extant scallop species in Antarctica39. Given that fossils of other, earlier members of the genus also appear to display similar surface microstructure39, this functional micro-ornamentation could conceivably be a lineage-specific adaptive trait that evolved in response to the threat posed by the presence of underwater ice formation in Antarctica. In the alternative, the cryofouling avoidance observed for the extant Adamussium colbecki may simply be a serendipitous outcome of pre-existing shell microstructure that evolved under unrelated developmental and environmental selection pressures. Regardless of the origin of the trait, the anti-cryofouling characteristics are clearly important for the survival of the Antarctic scallop today, at least within the shallow anchor-ice zone of coastal Antarctica. Establishing the biological relevance, ecological impact and the evolutionary timeline of the Antarctic scallop’s anti-cryofouling capabilities merit additional investigations, as they are beyond the scope of this work.Whether the cryofouling-avoidant surfaces of the Antarctic scallop’s shell arose under evolutionary selection pressure remains unclear. Based on fossil records, the exclusively Antarctic scallop genus For decades, ecologists have considered anchor ice to be an important agent of disturbance in the shallow-water benthic communities of McMurdo Sound, Antarctica. However, the mechanisms underlying the differential effects of underwater ice growth among species remain unexplained. Our observations of Antarctic scallops in their natural environment suggest that their exposed mineral shells passively avoid cryofouling, even in benthic habitats impacted by sustained underwater ice production. Characterization of these scallops’ shells revealed the presence of a unique distribution of growth rings and micro-ridges, with icing experiments suggesting that these structures modify the growth and/or adhesion of ice to the shell surface. In this model, the concentric growth rings promote directed icing events, leaving micro-ridges and valleys  ice-free. This directed ice growth behavior would result in the formation of fragmented ice crystals having large grain boundaries, thus helping to prevent the dangerous accumulation and firm attachment of buoyant ice that could lead to death or otherwise impact normal scallop behaviors. This specialized shell surface phenotype was not observed in two scallop species from temperate climates, in which ice-adhesion strength was determined to be markedly higher. Together, our observations support the inference that the Antarctic scallop’s possession of a cryofouling-avoidant shell surface, together with environmental and/or behavioral factors, this could be a key trait contributing to the species’ success in icy, shallow nearshore habitats of Antarctica. These findings provide a tentative step towards a better understanding of the complex interaction of polar benthic invertebrates with the intriguing phenomenon of underwater ice growth in supercooled water.Adamussium colecki  were collected from New Harbor, Antarctica by SCUBA divers in November 2015. Temperate control species, including Agropecten irradians  and the Placopecten magellanicus  were obtained from the Marine Biological Laboratory, collected in the Atlantic Ocean in the vicinity of Woods Hole, MA, USA. Shells of all species were opened, cleaned of adherent body tissues, thoroughly rinsed in fresh deionized water, and then allowed to dry at room temperature prior to their use in experiments or analyses.Live 2 scan area) on the Antarctic scallop. The surface elemental composition and its variation across the exterior valve surface were determined using energy-dispersive X-ray spectroscopy (EDS) for Ca, C, O, F, Na, Mg, Al, Si, and Ca. This was performed on the growth rings, micro-/nano-ridges, and micro-valleys.The exterior surfaces of Antarctic scallop valves were analyzed using scanning electron microscopy . Atomic force microscopy (AFM) was used to provide nanometer-resolution mapping  of surface geometries and surface roughness . A perforated aluminum plate  was mounted on the end of a force probe. The ice-adhesion strength (N/m2 or Pa) of the adhered ice was determined by retracting the force probe vertically, normal to and away from the valve surface (30 µm/s) using a motorized stage until the ice detached from the shell surface.In-air frosting was performed using a custom apparatus in an air-filled climate-controlled (20 °C) chamber at a controlled humidity (about 60% relative humidity; Supplementary Fig. All experiments were performed at least three times and the reported error bars are standard errors.Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Reporting Summary


Taurochenodeoxycholic acid (TCDCA) is one of the main components of bile acids (BAs). TCDCA has been reported as a signaling molecule, exerting anti-inflammatory and immunomodulatory functions. However, it is not well known whether those effects are mediated by TGR5. This study aimed to elucidate the interaction between TCDCA and TGR5. To achieve this aim, first, the TGR5 eukaryotic vector was constructed. The expression level of TGR5 in 293T cells was determined by immunofluorescence, real-time quantitative PCR , and Western blot. The luciferase assay, fluorescence microscopy, and enzyme-linked immunosorbent assay (ELISA) were recruited to check the interaction of TCDCA with TGR5. TCDCA treatment in 293T cells resulted in TGR5 internalization coupled with a significant increase in cAMP luciferase expression. Our results demonstrated that TCDCA was able to bind to the TGR5 receptor and activate it. These results provide an excellent potential therapeutic target for TCDCA research. Moreover, these findings also provide theoretical evidence for further TCDCA research. BAs are major components of bile and can be divided into free bile acids and conjugated bile acids based on their chemical structure. Free bile acids include cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), and lithocholic acid (LCA). Conjugated bile acids include glycocholic acid (GCA), taurocholic acids (TCA), glycochenodeoxycholic acid (GCDCA), and TCDCA . The maiS)-methyl-cholic acid , which has a similar structure to TCDCA, activates TGR5 and stimulates adenylate cyclase (AC), intracellular cAMP production, and protein kinase A (PKA) activation . All of the data were calculated based on the comparative Ct formula, and each sample was normalized to the GAPDH expression level. Relative mRNA expressions were analyzed according to the Ct values, which were based on the equation: 2The 293T cells were cultured in 24-well plates with starvation treatment. The pCMV-EGFP-TGR5 plasmid was transiently transfected into the 293T cells using lipofectamine 2000. The culture medium was replaced with 10% DMED after 6 h. The expression of TGR5 was observed with an inverted fluorescence microscope after 24 h.In order to observe the TGR5 receptor located in the membrane of the cells. The 293T cells were transfected with pCMV-EGFP-TGR5 for 24 h and were rinsed with phosphate-buffered saline (PBS) after the medium was discarded. The 293T cells were fixed in an immunostaining fixative for 15 min at room temperature and were washed with PBS three times (5 min each time). Then, these cells were permeabilized with 0.5% Triton X-100 for 10 min, washed with PBS three times (5 min each time), and blocked at room temperature for 1 h. Primary TGR5 antibodies at a 1:50 dilution were added and were incubated overnight at 4 °C. Three post-primary antibody washes were conducted, followed by the incubation of the 1:100 diluted secondary rhodamine-labeled fluorescence antibodies for 1 h at room temperature. Next, the 293T cells were washed and were subsequently stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature. DAPI was washed with PBS, and TGR5 expression was visualized with a confocal microscope .g and 4 °C for 5 min. After supernatant was discarded, 300 μL of complete lysis buffer was added into the samples and was incubated at 150 rpm for 20 min. The samples were vortexed and centrifuged at 14,000× g and 4 °C for 20 min. The supernatant was collected and stored at −80 °C.The whole-cell protein was extracted using a Nuclear Extract kit following the manufacturer’s protocols. In brief, the cells were washed with pre-cooled PBS/phosphatase inhibitor three times. Then, the supernatant was removed, and the pellet was re-suspended with 1 mL of PBS/phosphatase inhibitor and was centrifuged at 200× The 293T cells were seeded in 6-well plates when cell fusion reached 70%, and the pCMV-TGR plasmid was transferred by means of transient transfection for 48 h. Total protein and concentration quantification were conducted according to the bicinchoninic acid assay (BCA) kit protocol. Equal amounts of protein (40–60 μg) were loaded into 8–12% SDS-PAGE gels and were transferred to polyvinylidene difluoride (PVDF) membranes. The TGR5 protein expression was determined using the primary TGR5 and GAPDH antibodies at 1:1000 and 1:3000 dilution. Horseradish peroxidase (HRP) conjugated secondary antibodies were used for band detection, and the bands were visualized using maximum sensitivity substrate. Chemiluminescence signals were measured using the enhanced chemiluminescence (ECL) system.−5 M of TCDCA was added for 1 h. Then, the internalization of the TGR5 receptor was observed under an inverted fluorescence microscope.The 293T cells were transiently transfected into the pCMV-EGFP-TGR5 cells. After 24 h, 10TM Luciferase Assay System.The 293T cells were cultured in 24-well plates with DMEM medium containing 10% FBS. When cell fusion reached 70%, the culture medium was changed to DMEM. pGL4.29 and pCMV-TGR5 plasmids were co-transferred into the 293T cells by Lip2000. After 24 h of transfection, the cells were treated with TCDCA  and 100 μM of TLCA for 5 h, respectively. Finally, luciferase intensity was measured using a Bright-GloThe pCMV-TGR5 plasmid was transfected into the 293T cells and was incubated for 24 h. Then, 10–5 M of INT-777 treatment was implemented for 1 h. The rest of the 293T cell groups were treated with different concentrations of TCDCA and TLCA for 5 min, respectively. The cAMP ELISA Kit determined the intracellular cAMP.p < 0.05.Each experiment was performed at least three times. All of the values are reported as mean ± S.D. between experimental groups and controls were assessed by one-way ANOVA with Dunnett’s test using SigmaStat . The level of statistical significance was set at The TGR5 fragment was amplified from the human placenta tissues and was purified by PCR A,B. ThenThe mRNA expression of TGR5 in the 293T cells that had been transfected with the pCMV-TGR5 plasmid was determined by qPCR. Meanwhile, TGR5 protein expression was analyzed with the TGR5 polyclonal antibody by means of Western blot. We found that TGR5 was significantly increased in 293T cells with the pCMV-TGR5 plasmid compared to the control. The gene and protein expression of TGR5 were consistent .To observe the expression and localization of TGR5 in the 293T cells that had been transfected by the pCMV-EGFP-TGR5 plasmid, the 293T cells were first incubated with the anti-TGR5 antibody and the secondary antibody labeled with rhodamine and DAPI. We found that the pCMV-EGFP-TGR5 plasmid was expressed in the 293T cells compared to in the negative control cells of the pCMV-EGFP plasmid using fluorescence confocal microscopy, confirming that TGR5 was expressed in the 293T cells A. MeanwhTo directly observe the interaction between TCDCA and the TGR5 receptor, the pCMV-TGR5 and pCMV plasmids were transfected into 293T cells and with 100 μM of TCDCA treatment for 15 min, respectively. The invagination of the TGR5 receptor was observed using the inverted fluorescence microscope. The results indicated that the membrane fluorescence took place invagination in the 293T cells that had been treated with TCDCA for 15 min compared to the control group. The phenomenon also revealed that TCDCA interacted with the TGR5 receptor .To further explore whether TCDCA interacts with the TGR5 receptor, 293T cells that were overexpressing TGR5 were treated with different concentrations of TCDCA  and 100 μM of TLCA for 1 h, with TLCA being used as a positive control. Meanwhile, IBMX acted as a TGR5-specific agonist, and DMEM acted as a negative control. A multi-function microplate reader determined that the TGR5 luciferase was a reporter gene. As a result, TCDCA and TLCA enhanced TGR5 luciferase reporter gene expression remarkably compared to the negative control. Moreover, the expression of the TGR5 luciferase reporter gene significantly increased in in the 293T cells that were overexpressing TGR5 and that had been treated with 100 μM of TCDCA than it was the 1 μM and 10 μM concentrations of TCDCA. TCDCA could significantly enhance the TGR5 luciferase reporter gene expression because TCDCA interacted with the TGR5 receptor .The 293T cells were treated with different concentrations of TCDCA , 0.05 μM of INT-777, and 100 μM of TLCA for 1 h. The cAMP content was determined using an ELISA kit. It was determined that TCDCA and TLCA resulted in a remarkable increase in the cAMP content. Surprisingly, we also found that the cAMP content was augmented in the 293T cells that had been treated with 1 μM TCDCA, and it continued to increase in the 100 μM of TCDCA. Thus, these findings demonstrate that TCDCA enhances cAMP content in a concentration-dependent manner, indicating that TCDCA increases cAMP content through the TGR5 receptor .In this study, we investigated the interaction between TCDCA and TGR5. Our results indicated that TCDCA could activate the TGR5 receptor in 293T cells. Our previous research showed that TCDCA increased the apoptosis rate of NR8383 cells through the PKC-JNK signaling pathway ; we alsoMany methods have been applied to aid in research focusing on the interaction between ligands and receptors in the biological field, such as radioactive labeling, fluorescent labeling, scintillation proximity assay (SPA), and fluorescence resonance energy transfer (FRET). However, it is challenging to label TCDCA using isotopes and fluorescence due to the fact that it is a particular structure ,17,18,19+ macrophages by increasing the cAMP content to regulate NF-κB-p65 activation in the macrophages of liver cancer cells [The TGR5 receptor mediates a variety of signal pathways that are related to cancer, inflammation, diabetes, and obesity. Additionally, TGR5 receptor agonists significantly increase the cAMP content while also inhibiting downstream extracellular-signal-regulated kinase (ERK) signaling, inducing proliferation and inflammation in non-ciliated cholangiocytes . Moreoveer cells . Glucagoer cells . Our resTCDCA acts on 293T cells, leading to TGR5 internalization as well as increases in cAMP and luciferase reporter. These results show that TCDCA can bind with the TGR5 receptor and can activate it, providing the experimental basis for the molecular mechanism of anti-inflammatory, immune regulation by TCDCA as well as a novel theoretical basis on which to seek a new target TCDCA.


Here, using extensive all-atomfree energy simulations, we show how the motor’s directionalitynaturally arises from the interplay between intraprotein interactionsand energetics of protonation of the c-ring. Notably, our calculationsreveal that the strictly conserved arginine in the a-subunit (R176)serves as a jack-of-all-trades: it dictates the direction of rotation,controls the protonation state of the proton-release site, and separatesthe two proton-access half-channels. Therefore, arginine is necessaryto avoid slippage between the proton flux and the mechanical outputand guarantees highly efficient energy conversion. We also providemechanistic explanations for the reported defective mutations of R176,reconciling the structural information on the Fo motorwith previous functional and single-molecule data.F Because energetics of the c-ring rotation has been found to stronglydepend on the protonation state of the B and R carboxylates,30 we first calculated their pKa’s in the Fo initial state (0°)using the alchemical approach . We found (inset in Ka ofthe B site (8.4 ± 0.3) is markedly higher and that of the R site(1.9 ± 0.2) is markedly lower compared to pH of their respectivecompartments , even if the predictedshifts are somewhat overestimated. This finding suggests that protonaffinities for the B and R sites are fine-tuned to the proton gradientdirection, promoting the rate of the proton transfer via Fo under physiological conditions. The observed difference in pKa between the B and R sites seems to resultmostly from a different degree of hydration of both carboxylates . The pKa valuesfor the remaining eight proton-carrying c-subunits that are exposedmostly to lipid environment  were computed to be at least 11.To understandthe mechanism of action of Finset in 2 that pK–B0; blue curves in 30As can be seen, the free energyprofiles determined for the dominantprotonation state  but alsothe rate of (de)protonation might play an important role in the directionalrotary mechanism.30 In particular, protonationof the B site could even be expected to be a rate limiting step, giventhat a single file of water molecules confined between the helicesof the a-subunit connects this site to the rest of the half-channel.32 Therefore, we examined the effect of the B site deprotonation onthe rotation energetics by recomputing the free energy of the rotationin the synthesis direction with both R and B sites deprotonated ,reflecting the change of environment around the B site carboxylatefrom polar and partially hydrated (at 0°) to fully hydrophobic(at 36°). This increase effectively inhibits the full rotarystep in this protonation state ensuring that the c-ring waits, ifnecessary, for the protonation of the B site to occur. Surprisingly,however, in the range up to 10°, the R–B– protonation state seems to kinetically favor the synthesisdirection even more than the R–B0 protonationstate, as the free energy barrier for the former is ∼3 kcal/mollower. Since the two curves change their slope around 10°, withR–B– becoming notably steeperand R–B0 flatter, it may suggest thatthe c-ring, starting from0°, first tends to rotate with the B site carboxylate deprotonatedup to 10°, where it stalls waiting for the carboxylate to getprotonated, to progress further governed by the less steep R–B0 profile. This hypothesis would agree with previousstructural studies and the recent single-molecule study that revealedthe existence of a 11° substep in the rotary mechanism of the E. coli Fo.34 To test whetherat 10° the B carboxylate is still connected to water in the proton-bindinghalf-channel, we calculated the carboxylate–water radial distributionfunction  and found that upon10° rotation the degree of hydration increases substantially,possibly making the proton binding from the half-channel even moreefficient than at 0°. Increasing hydration of the B carboxylatemay also account for an unexpectedly flat rotation free energy inthe R–B– protonation state inthe 0–10° range. Consistently, a doubly deprotonated stateof the c-ring was also found to contribute to the proton transferpathway in the recent coarse-grained simulations.13As can be seen, beyond10° the RFigures S1, S3, S7, and S8), which corresponds to a drastic decrease in thepKa of E59, early deprotonation of thepre-R carboxylate can also be expected to affect the rotation kinetics.To test this possibility, we determined the effect of deprotonationof the pre-R carboxylate on the synthesis free energy profile [(pre-R)−R–; green curve in −R– state, as opposed to the two otherexamined protonation states, the profile remains virtually flat upto 20°, which shows that early proton release might in fact promotefaster progression of the c-ring in the synthesis direction, furtherhighlighting the coupling between rotation and (de)protonation events.Because during the rotation in the synthesis directionthe c-subunitentering the R site (pre-R subunit) moves from hydrophobic to polarand well-hydrated environment (Supporting Information Methods for details). In –) and the central R176, stably oriented towards theR site , isa key differentiating factor largely responsible for the directionalpreference in the dominant R–B0 protonationstate. This finding supports the electrostatic picture of the Fo unidirectionality resulting from the semiquantitative andcoarse-grained models.30 Indeed, the intraprotein interactions involvingR– and R176 are 4.25 and 3 times, respectively,more effective in promoting the synthesis direction than those involvingthe third most contributing residue, i.e., also highly conserved R169,whose interaction with R– seems to favor the synthesistoo.Next, to gain atomic-level understanding of the directionalitymechanism, we examined residue–residue and residue–environmententhalpiccontributions to the interaction free energies along the mechanicalcoordinate . In all cases, interactions with lipid moleculeshave mostly negligible effect on directionality.Since strengthening of the electrostatic attraction between pairsof oppositely charged residues over the course of the rotation isaccompanied by their dehydration, the interactions of R35 and could be directly testedin single-molecule imaging experiments. Importantly, the removal ofthe second strongest interaction partner of the R–, R169, retains the asymmetry of the profile around 0° and therebythe wild-type kinetic preference for synthesis, only reducing theheight of the barriers in both directions .To directlytest the pivotal role of the central arginine (hereR176) in differentiating the rotation energetics in the synthesisvs hydrolysis direction, we further determined how its mutation toalanine (R176A) affects the free energy landscape governing the rotarymotion of the c-ring around 0° 4A. As seFigure S10) further substantiates the critical role of electrostatic attractionbetween the central arginine and the deprotonated R-site carboxylatein promoting the unidirectional rotation in the synthesis direction.This simple mechanism implies that any pair of oppositely chargedresidues could work in a similar manner. Indeed, the c-ring glutamate(here E59) can occasionally be replaced by aspartate without abolishingthe directionality, e.g., in some Gram-negative bacteria, including E. coli. In contrast, arginine cannot be replaced by anyother residue, including lysine, i.e., the only other residue positivelycharged at physiological conditions. To explore why this substitutionmight be detrimental, we first determined and compared the hydrolysisand synthesis free energy profiles for the lysine mutation (R176K)that was also previously shown to inhibit the ATP-driven proton pumpingactivity.24 It is clear from Figure S11) preserves a relatively high  barrier to therotation in the hydrolysis direction , it could be expected to interact with the carboxylatein the release site less favorably and thus lead to an undesired increaseof its pKa. To test this, we computedpKa of the R-site carboxylate in the R176Kmutant and, as a control, in the R176A mutant  and 2.2± 0.4 (R176K). Since the effect on pKa of the R-sitecarboxylate does not depend on the exact nature of a positively chargedresidue, we conclude that it alone cannot explain strict conservationof the central arginine.Because the side chain of K176 is muchmore flexible than thatof R176 . An unexpectedlyhigh increase in the flux observed for R176K seems to arise from afact that much more conformationally flexible lysine cannot act asan efficient water plug . One could expect that the connected channel can also allow protonsto leak across the membrane via Grotthuss mechanism downhill the gradientin a manner uncoupled from the c-ring rotation. Such rotation-uncoupledproton leakage has indeed been observed for the alanine mutant byMitome et al.,24 who showed that even whenthe c-ring is fused with the a-subunit and thus unable to rotate,the Fo can still act as a proton channel when the centralarginine is substituted by alanine. Although the continuous waterchannel is also predicted to be present in the R176 K mutant , and thus rotation in the synthesis directionis strongly preferred, consistent with the motor’s directionalityand high efficiency of energy conversion. Importantly, this kineticpreference arises from the interplay between the intraprotein interactionsand the energetics of protonation of carboxylates in the binding (B)and release (R) half-channels, consistent with recent coarse-grainedsimulations.30 Specifically, we predicted thatat 0° the B site is predominantly protonated and the R site deprotonated,and hence their pKa’s seem to befine-tuned to the direction of the proton gradient, accelerating theproton flow through Fo under physiological conditions.In agreement with simple early models and coarse-grained simulations,30 our energetic analysis demonstrated that in this dominant protonationstate the main contribution accounting for the much lower activationbarrier in the synthesis direction is the attraction between the strictlyconserved arginine in the a-subunit (R176 in the yeast Fo) and the deprotonated carboxylate at the R site. By recomputingthe free energies for the R176A mutant, we further confirmed thisfinding, showing that in the absence of R176 the directionality ofFo is largely abolished and the pKa of the R site unfavorably increases by5 units. Furthermore,the alanine mutation causes the B and R aqueoushalf-channels, which are well-separated by R176 in the wild type,to join in a single continuous pore through which water can readilymove across the membrane. Along with the abolished directionality,the existence of this shortcut provides mechanistic explanation forthe previously reported uncoupled proton transport caused by removalof the arginine side chain. Finally, we found that a positively chargedresidue, lysine, substituted for R176 facilitates deprotonation ofthe R site and preserves a high barrier to rotation in the hydrolysisdirection similar to the arginine. However, it also markedly increasesthe barrier in the synthesis direction which slows down the rotationby several orders of magnitude, further explaining why arginine hasto be conserved to ensure efficient coupling between proton translocationand rotary motion in the Fo motor.To sum up, we found that the free energy landscape governing theF


Neuropeltis racemosa Wall. (Convolvulaceae) is wildly distributed in Asia. Its stem is used as the component in traditional Thai recipes for treatments of muscle rigidity, skin disorder, dysentery, and hypoglycemia. However, the chemical constituents and biological activities of N. racemosa have not been reported. From a screening assay, N. racemosa stem crude extract showed the potent effect on alpha-glucosidase inhibition at 2 mg/mL as 96.09%. The bioassay-guiding isolation led to 5 compounds that were identified by spectroscopic techniques as scopoletin (1), syringic acid (2), methyl 3-methyl-2-butenoate (3), N-trans-feruloyltyramine (4), and N-trans- coumaroyltyramine (5). Compounds 1, 4, and 5 exhibited an IC50 of 110.97, 29.87, and 0.92 µg/mL, respectively, while the IC50 of positive standard, acarbose was 272.72 µg/mL. Kinetic study showed that compound 1 performed as the mixed-type inhibition mechanism, whereas compounds 4 and 5 displayed the uncompetitive inhibition mechanism. The docking study provided the molecular understanding of isolated aromatic compounds  to alpha-glucosidase. Hence, this study would be the first report of isolated compounds and their anti-alpha-glucosidase activity with the mechanism of action from N. racemosa. Thus, these active compounds will be further studied to be the lead compounds among natural antidiabetic drugs. Diabetes mellitus (DM) is metabolic syndrome related to a hyperglycemia condition. In 2021, The International Diabetes Federation reported number of diabetes adults is 537 million and the trend will rise to 783 million in 2045 − ion peak at m/z 197.0456, and correlated with a molecular formula of C9H10O5. The structure of 2 was determined to be syringic acid.Compound 3 was obtained as a brown amorphous solid and dissolved in chloroform. The UV spectrum in chloroform demonstrated an absorption maximum (λmax) at 290 nm. The IR spectrum exhibited band at 2922, 1698-1595, 1457, 1260–1220, 1110, and 874–800 cm−1. The 1H-NMR spectrum showed three singlet proton signals at δH 1.2 , 3.8 , and 5.8 . The 13C-NMR spectrum showed six carbon signals that were observed: one quaternary carbon signal at δC 157.30 (C-3); one olefinic methine signal at δC 107.42 (C-2); one carbonyl signal at δC 186.84 (C-1); one methoxyl signal at δC 56.48 (OCH3-1), and two methyl signals at δC 29.69 (C-4) and at δC 29.35 (CH3-3). The HMBC correlations (3. The structure of 3 was elucidated as methyl 3-methyl-2-butenonoate.Compound elations  suggeste4 was obtained as a white amorphous solid and dissolved in methanol. The UV spectrum in methanol showed an absorption maximum (λmax) at 318 nm. The IR spectrum demonstrated band at 3434, 1652, 1542, and 978 cm−1. The 1H-NMR spectrum exhibited the methoxyl proton signal at δH 3.78 . The two doublets at δH 6.39  and 7.42  indicated the presence of two trans protons. The two doublet of doublet signals at δH 3.45  and 2.75  were determined as methylene protons which conjugated to secondary amine and aromatic parts, respectively. The residue five proton signals at δH 6.72 , 6.78 , 7.01 , 7.05 , and 7.11  were indicated as seven aromatic protons of two aromatic rings. The 13C NMR showed sixteen carbon signals; however, they were indicated as eighteen carbons: carbonyl carbon at δC 169.19 (C-1); the methoxyl carbon δC 56.42 (OCH3-6); two methylene carbons at δC 35.81 (C-2′) and 42.53 (C-1′); two olefinic methine carbon at δC 118.79 (C-2) and 142.01 (C-3); two quaternary aromatic carbon at δC 128.31 (C-4) and 131.22 (C-3′); three oxygenated aromatic carbons at δC 149.32 (C-6), 149.88 (C-7) and 156.94 (C-6′); five tertiary aromatic carbons at δC 111.63 (C-5), 116.42 (C-8), 123.22 (C-9) 130.72 (C-4′ and C-8′), and 116.28 (C-5′ and C-7′). The HR-ESIMS showed an [M + Na]+ ion peak at m/z 336.1206 , correlated with a molecular formula of C18H19NO4. The structure of 4 was determined to be N-trans-feruloyltyramine.Compound 5 was obtained as a white amorphous solid and dissolved in methanol. The UV spectrum in methanol showed an absorption maximum (λmax) at 295 nm. The IR spectrum demonstrated a band at 3434, 1637, 1541, and 980 cm−1. The 1H spectrum of 5 showed doublet signals at δH 6.38  and 7.31  which were assigned as two trans protons. The doublet of doublet signals at δH 3.45  and 2.75  were determined as two methylene protons. Other four proton signals, at δH 6.70 , 6.78 , 7.05 , and 7.38 , were assigned as protons on two symmetric aromatic rings. The 13C NMR showed thirteen carbon signals; however, they were indicated as seventeen carbons: one carbonyl carbon at δC 169.25 (C-1); two methylene signals at δC 35.82 (C-2′) and 42.53 (C-1′); two olefinic methine carbon signals at δC 118.49 (C-2) and 141.76 (C-3); two quaternary carbon signals at δC 127.77 (C-4) and 131.34 (C-3′); two oxygenated aromatic carbons at δC 156.92 (C-6′) and 160.50 (C-7); eight tertiary aromatic carbons at δC 130.3 (C-5 and C-9), 116.72 (C-5′ and C-7′), 116.28 (C-6 and C-8), and 130.72 (C-4′ and C-8′). The HR-ESIMS showed an [M + Na]+ ion at m/z 306.1101 , and correlated with a molecular formula of C17H17NO3. The structure of 5 was determined to be N-trans-coumaroyltyramine.Compound 50) showed the inhibitory ability of compounds (1) and two tyramine-derived amides (4 and 5) exhibited stronger alpha-glucosidase inhibition than the positive control, acarbose.To determined alpha-glucosidase inhibition, the percentage inhibition values at 2 mg/mL were used for the guided isolation, while the half maximal inhibitory concentration  were determined by secondary plot as shown in 4 and 5 indicated them to be typical of uncompetitive inhibition with Ki′ values were 51.81 and 1.99 µM, respectively, whereas the plot of 1 displayed mixed-type inhibition. The acarbose standard showed competitive inhibition with Ki values 264.46 µM. Therefore, these could be explained that compounds 4 and 5 were bound with alpha-glucosidase in separated site, compound 1 might both have interacted with the active site and separated site, while acarbose prohibited at the active site of enzyme. For a better understanding of compounds from N. racemosa, the molecular docking was performed.Compounds 1, 2, 4, and 5 that have aromatic part in their structures were evaluated for the protein-ligand complexes. The results indicated that these compounds were bound with the same residues at the entrance area of the active site of alpha-glucosidase, as shown in 1 blocked the alpha-glucosidase entrance area as the line in 4 and 5) were aligned at the exit part of the enzyme. This alignment would prohibit the release of the product from the alpha-glucosidase. These findings also agreed with previous studies of these two derivatives − .Orange amorphous solid; C6H10O2; UV (CHCl3) λmax 290 nm; IR νmax cm−1: 2922, 1698, 1645,1595, 1457, 1322, 1260, 1220, 1110, 874, 800; 1H NMR (CDCl3) δ: 5.80 , 3.8 , 1.20 ; 13C NMR (CDCl3) δ: 186.84 (C-1), 157.30 (C-3), 107.42 (C-2), 56.4 (OCH3-1), 29.69 C-4), 29.35 (CH3-3).Brown amorphous solid; Cmax 318 nm; IR νmax cm−1: 3434, 1652, 1542, 1515, 1457, 1269, 1125, 1032, 978, 819, 670; 1H (DMSO-d6) δ: 7.42 , 7.11 , 7.05 , 7.01 , 6.78 , 6.72 , 6.39 , 3.87 , 3.45 , 2.75 ; 13C NMR (DMSO-d6) δ: 169.19 (C-1), 156.94 (C-6′), 149.88 (C-7), 149.32 (C-6), 142.01 (C-3), 131.33 (C-3′), 130.72 , 128.31 (C-4), 123.22 (C-9),118.79 (C-2), 116.42 (C-8), 116.28 , 111.63 (C-5), 56.42 (OCH3-6), 42.53 (C-1′), 35.81 (C-2′); HR-ESIMS (positive-ion mode) m/z 336.1206 [M + Na]+ .White amorphous solid; UV (MeOH) λmax 295 nm; IR νmax cm−1: 3434, 1637, 1541, 1456, 1245, 1174, 1105, 980, 830; 1H (DMSO-d6) δ: 7.44 , 7.38 , 7.05 , 6.78 , 6.70 , 6.38 , 3.45 , 2.75 ; 13C NMR (DMSO-d6) δ: 169.25 (C-1), 160.50 (C-7), 156.92 (C-6′), 141.76 (C-3), 131.34 (C-3′), 130.72 , 130.53 , 127.77 (C-4), 118.49 (C-2), 116.72 , 116.28 , 42.53 (C-1′), 35.82 (C-2′); HR-ESIMS (positive-ion mode) m/z 306.1101 [M + Na]+ .White amorphous solid; UV (MeOH) λp-nitrophenol (pNP), and detected by visible light at 405 nm with a SPECTROstar Nano spectrophotrometer . The UV absorbance of the final product, pNP, was converted to velocity (V) by the following equation: The alpha-glucosidase inhibitory assay was slightly modified from Dej-adisai and Pitakbut, 2015 . This as50 value that performed the inhibition ability of samples was received from the calibration curve plotted between percentages of inhibition and five concentrations of samples.The ICpNPG (5–0.1 mM), the fixed enzyme concentration (1 unit/mL), and the 3 concentrations of each effective sample were evaluated for the manner of inhibition. The double reciprocal Lineweaver–Burk plot was used to determine the mode of inhibition, while the secondary plots conducted the inhibition constants. The inhibition constant was showed as Ki values when the inhibitor binds with free enzyme for competitive inhibition, but it was showed as Ki′ values when the inhibitor binds with the enzyme–substrate complex for uncompetitive inhibition. Briefly, the enzyme inhibition procedure was performed as mention above. The 6 concentrations of https://www.rcsb.org/, accessed on 1 May 2021), while the compounds of interest were downloaded from Pubchem . Before performing the experiment, both the alpha-glucosidase and the compounds of interest needed to be prepared properly. Firstly, Autodock Tool version 1.5.6 from the same institute as Autodock Vina was used to prepare the alpha-glucosidase and to identify the active site of this enzyme by using the native ligand, the glucose molecule, as a guideline [Autodock Vina version 1.1.2 from the Scripps Research Institute, San Diego, California, USA was used to investigate the molecular interaction in this study . The taruideline . The cenuideline . To perfuideline . The triuideline  was useduideline ,27.Neuropeltis racemosa which is used as the component of many traditional Thai recipes. The bioassay-guided fractionation on alpha-glucosidase inhibition revealed that the best active extract is the ethanolic extract. The extracts were separated by using classical column chromatographic techniques and five compounds were obtained. The spectroscopic techniques including UV-Vis, FTIR, HRMS, and NMR  analyses revealed the chemical structure of the isolated compounds. Then, the compounds were estimated for their bioactive potential of alpha-glucosidase inhibition. Moreover, three compounds  which have the better activity than the standard acarbose were chosen for the mechanism of action analysis. Some of them exhibited the significant alpha-glucosidase inhibition with different mechanism of action. Besides this, an in silico study exposed the alpha-glucosidase inhibition of selected compounds  at the molecular level. The parameters of molecular docking such as binding energy, desolvation, and torsion energy were used for the explanation of the molecular interaction. Based on the results, these findings justify the value of N. racemosa as a component of traditional medicine and resource of lead antidiabetic compounds for further study.This study would be the first report on the phytochemical investigation of