Plantainoside D is widely existed in the herbs and possesses various pharmacological activities, making it possible to co-administrate with other herbs. Its effect on cytochrome P450 enzymes (P450) is a risk factor for inducing adverse drug-drug interactions. To assess the effect of plantainoside D on the activity of major P450 isoenzymes in human liver microsomes.The Cocktail method was conducted in human liver microsomes in the presence of probe substrates. The activity of P450 isoenzymes was evaluated by the production of corresponding metabolites. The concentration-dependent and time-dependent inhibition assays were performed in the presence of 0, 2.5, 5, 10, 25, 50, and 100 μM plantainoside D to characterize the inhibitory effect of plantainoside D.50 values of 12.83, 8.39, and 14.66 μM, respectively. The non-competitive manner and competitive manner were observed in the CYP3A inhibition (Ki = 7.16 μM) and CYP1A2 (Ki = 6.26 μM) and 2D6 inhibition (Ki = 4.54 μM), respectively. Additionally, the inhibition of CYP3A was found to be time-dependent with the KI of 1.28 μM−1 and Kinact of 0.039 min−1.Significant inhibition was observed in the activity of CYP1A2, 2D6, and 3A, which was concentration-dependent with the ICin vitro, implying its potential of inducing interactions with CYP1A2-, 2D6-, and 3A-metabolized drugs. Although further in vivo validations are needed, the feasibility of the Cocktail method in evaluating P450 activity has been verified.Weak inhibitory effects of plantainoside D on the activity of CYP1A2, 2D6, and 3A were revealed The online version contains supplementary material available at 10.1186/s12906-022-03671-5. Plantago asiatica L. is a commonly used herb in the treatment of cardiovascular disease, which possesses the activity of preventing hyperlipidemia and atherosclerosis, two major risk factors of cardiovascular diseases [P. asiatica L., which is mainly responsible for its pharmacological activities. Previous studies have demonstrated the protective effect of plantainoside D on cardiac muscle cells from the damage of adriamycin by suppressing ROS generation and NF-kB activation [Due to the increasing incidences and mortality of cardiovascular disease, great efforts have been devoted to improving clinical therapeutic and precaution strategies, among which traditional Chinese medicine has gradually ranked a critical position –3. Plantdiseases –6. Planttivation . Plantaitivation .P. asiatica L. or other source herbs of plantainoside D, there would be a certain co-administration of other herbs with similar or auxiliary indications, which improves the potential of herb-herb interactions.The potential interaction between different active ingredients of co-administrated herbs would induce adverse therapeutic effects and even toxicity. The liver is the main site of xenogeneic metabolism, including drugs, herbs, and also some toxics. The biotransformation processes in livers are primarily mediated by various enzyme systems in the microsomes, where cytochrome P450 enzymes (P450s) play vital roles . AdditioThis study focused on the effect of plantainoside D on the activity of several P450 isoenzymes, including CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A in human liver microsomes. The kinetic models and inhibition characteristics of affected isoenzymes were also evaluated, aiming to provide theoretical reference for the clinical prescription of plantainoside D original herbs or other CYP-metabolized herbs applicable for cardiovascular diseases.Plantainoside D Figure S was obtaThe activity of P450s was evaluated by corresponding metabolites using HPLC with Agilent 1260 Series Rapid Resolution HPLC. The reaction system was prepared with the following compositions: HLMs with certain protein concentrations, specific substrates of different isoenzymes, PBS buffer solution, plantainoside D or specific inhibitors, and an NADPH generating system as previously reported , 17. Bes50 was evaluated. Furthermore, the inhibition model of P450 isoenzymes was estimated in the presence of various concentrations of corresponding substrates and fitting with Lineweaver–Burk and Dixon plots, and the inhibition constant was calculated by the following equations:The effect of plantainoside D was primarily evaluated with the concentration of 0, 2.5, 5, 10, 25, 50, and 100 μM and the values of ICI is the concentration of plantainoside D, Ki is the inhibition constant, S is the concentration of the substrate, and Km is the substrate concentration at half the maximum velocity (Vmax) of the reaction.where Km. Additionally, the values of KI and Kinact were evaluated in the presence of substrates approximate to fourfold Km according to the equation: A total of 1 mg/mL HLMs were firstly incubated with 20 μM plantainoside D and the NADPH-generating system for 30 min. Then, incubation with 20 μL aliquot was carried out for 0, 5, 10, 15, and 30 min in the presence of substrates concentration close to P < 0.05 indicates statistical significance. The correlated fitting analyses were performed with Graphpad Prism 7.0 software.The comparison was performed with the student’s t-test using SPSS 26.0 software. 50 value of 12.83 μM (Fig. 50 = 8.39 μM, Fig. 50 = 14.66 μM, Fig. In the presence of 0, 2.5, 5, 10, 25, 50, and 100 μM of plantainoside D, the activity CYP1A2 was suppressed, and the activity decreased with the concentration elevated with the ICKm (Fig. The result of the Lineweaver–Burk plot showed that the inhibition of CYP3A by plantainoside D was best fitted with the non-competitive inhibition model with the constant Vmax (Fig. Both the inhibition of CYP1A2 and 2D6 were demonstrated to be best fitted with a competitive inhibition model with a constant max Fig. B and C. KI and Kinact were calculated as 1.28 and 0.039 (Fig. The inhibitory effect of plantainoside D on CYP3A was enhanced with the increasing incubation time, while the inhibition of CYP1A2 and 2D6 showed no significant changes with time Fig. A. The ti039 Fig. C.Fig. 4TDue to the wide involvement of P450s in the metabolism processes of various drugs, herbs, and other kinds of xenobiotics, the changes in their activity would significantly affect the therapeutical efficiency or even induce toxicity. Because of the limited sources and the poor repeatability of liver cells and several factors that would complex the P450 enzyme kinetics, liver microsomes are usually used to assess the inhibition characteristics of P450 isoenzymes and drug metabolism . The pre50 values were obtained. IC50 is a critical reference that represents the inhibition degree and guides the prescription of plantainoside D. Previously, IC50 < 100 μg/mL was considered a strong inhibition, while IC50 > 100 μg/mL was a weak inhibitory effect [in vivo physiological situation. The hepatic concentration of plantainoside D is a critical factor determining whether the inhibition of P450s occurs and how the degree is. Therefore, the specific interaction needs further clinical validation.The inhibition of CYP1A2, 2D6, and 3A were all found to be concentration-dependent, which was enhanced by the increasing plantainoside D concentrations, and corresponding ICy effect . The inhThe non-competitive model was best fitted with the inhibition of CYP3A, while the competitive model was best fitted with CYP1A2 and 2D6 inhibition. Both non-competitive inhibition and competitive inhibition are reversible, which can be reversed or weakened by increasing the concentration of substrates . The inhin vivo investigations.Taken together, plantainoside D served as a non-competitive inhibitor of CYP3A and competitive inhibitor of CYP1A2 and 2D6. The inhibitory effect of plantainoside D was concentration-dependent and time-dependent. The potential of plantainoside D or its source herbs interacting with co-administrated herbs or drugs needs further Additional file 1:Fig S1. The chemical structure of plantainoside D.


Pseudomonas borealis INP by AlphaFold suggests that the central domain of 65 tandem sixteen-residue repeats forms a beta-solenoid with arrays of outward-pointing threonines and tyrosines, which may organize water molecules into an ice-like pattern. Here we report that mutating some of these residues in a central segment of P. borealis INP, expressed in Escherichia coli, decreases ice nucleation activity more than the section’s deletion. Insertion of a bulky domain has the same effect, indicating that the continuity of the water-organizing repeats is critical for optimal activity. The ~10 C-terminal coils differ from the other 55 coils in being more basic and lacking water-organizing motifs; deletion of this region eliminates INP activity. We show through sequence modifications how arrays of conserved motifs form the large ice-nucleating surface required for potency.Bacterial ice nucleation proteins (INPs) can cause frost damage to plants by nucleating ice formation at high sub-zero temperatures. Modeling of  Ice nucleation proteins have the same tandemly arrayed water-organizing motifs seen in some antifreeze proteins, but on a larger scale. The authors show that mutation, interruption, and truncation of these arrays reduce ice nucleation activity indicating that the two protein types share a common mechanism. Pseudomonas syringae1, P. fluorescens2, Erwinia herbicola3, and P. borealis4. Due to the broad distribution of ice nucleation-active bacteria on the surfaces of various plant species5 and their ability to initiate ice formation at temperatures as warm as −2 °C, INPs represent the primary source of frost damage in nature6. Once nucleated on frost-sensitive plants, ice grows both inter- and intracellularly, causing physical disruption of cell membranes7. Since increased INP expression can be triggered by nutrient starvation8, phytopathogenic strains of ice-nucleating bacteria may have evolved INPs to aid in plant tissue colonization and nutrient access9. Airborne INP-bearing bacteria may also have an important role in the hydrological cycle by initiating precipitation in clouds11. Despite the ubiquity of ice-nucleating bacteria and their importance in frost damage to crops and in precipitation, relatively little is known about the structure of INPs and the mechanism through which they trigger ice formation.Ice nucleation proteins (INPs) induce heterogeneous ice formation in supercooled water. They have been described in certain Gram-negative bacterial isolates, including those of 14. In 1993, Kajava and Lindow modeled the central domain of InaZ from P. syringae as a series of antiparallel β-sheet clusters in a planar zigzag arrangement that enabled interdigitation of monomers to form large aggregates14. Two later models were inspired by similarities in repeat sequence to the beta-solenoid fold15 used by some antifreeze proteins (AFPs) such as the spruce budworm and mealworm beetle AFPs18. The P. syringae INP model of Graether and Jia13 was built on a coil of UDP-acetylglucosamine acyltransferase (PDB 1LXA), shortened from 18 to 16 residues and repeated three times to model a 48-residue segment as a beta-solenoid. In contrast, the model of ref. 19, but it also produced a beta-solenoid structure for a section of eight 16-residue coils from P. borealis INP (PbINP). The repeating outward-projecting tyrosine and neighboring serine residues were proposed to form a dimerization surface in this latter model.The large size  and the binding of INPs to the outer bacterial membrane may have hindered their structural characterization. However, several models have been proposed for the central repetitive region comprised of ~50–80 tandem copies of a well-conserved 16-residue sequence18. In these AFPs, the threonyl side chains form two parallel arrays that constitute the ice-binding site, as demonstrated by site-directed mutagenesis20. The spacing of these threonine residues on a flat beta-sheet matches closely the spacing of oxygen atoms in the ice lattice on the basal and primary prism planes17. One proposal for the mechanism by which AFPs bind to ice is that these threonine residues organize water molecules in an ice-like arrangement so that they merge with the quasi-liquid layer on the ice lattice and then, as the system cools, serve to freeze the AFPs onto ice22. Ice growth is then inhibited by the Kelvin effect23. However, this hypothesis was not supported by vibrational sum-frequency generation spectroscopy24 or by a modeling study25, which suggested that water ordering is not essential for AFP activity. For the much larger INPs that are thought to self-associate on the outer bacterial membrane26, it has been suggested that the organization of many ice-like water molecules on one surface may trigger ice nucleation30. Thus, there may be a common theme to the mechanisms of action of AFPs and INPs: the ordering of ice-like water molecules by a regular array of residues on a flat protein surface. AFPs would only need a small number of ice-like water molecules to adsorb to ice and stop its growth by the Kelvin effect31. These small proteins can only slightly raise the temperature for the nucleation of new ice crystals32. However, large INPs and their aggregates order enough ice-like water molecules to initiate nucleation at high sub-zero temperatures33. The importance of the organized water molecules in INP activity has even been questioned34. Notably, these authors, using sum-frequency generation spectroscopy, found that both active and heat-inactivated INPs showed no difference in the ordering of interfacial water molecules upon cooling. However, another study using sum-frequency generation and 2D infrared spectroscopy did provide evidence for a structural ordering of water by INPs30.An exciting discovery of the solenoid models was the regular placement of a TxT motif where the two threonine residues on the beta-strand project outwards as they do in several insect AFPsPbINP truncations, mutations, and central domain interruptions on ice nucleation temperatures. We show that E. coli expressing recombinant PbINP with fewer repeats containing putative water-organizing residues reproducibly freeze at lower temperatures, where a minimum central domain length of 13–15 repeats is required for activity. Conservative mutations of threonine and tyrosine in the water-organizing motifs reduce freezing temperatures in both full-length and shortened PbINP central domains. A similar decrease in ice nucleation efficiency from the insertion of a functional bulky domain into the central domain indicates the importance of an uninterrupted water-organizing surface. Based on the PbINP AlphaFold model and deletions from different regions of the central domain, we propose that the ~10 repeats adjacent to the C-terminal domain contribute to the formation of INP aggregates required for efficient ice nucleation.Clearly, more experiments are needed to clarify the INP mechanism of action. Here we tested the water ordering hypothesis by examining the impact of PbINP begins with a 110-residue globular domain followed by a long flexible linker  expressed in E. coli have shown that deletions of 1–34 repeats from the central repetitive region reduced the ice nucleation temperature by up to 8.5 °C, in a manner generally consistent with deletion size35. These analyses were limited because the apparatuses used could only measure droplet freezing at temperatures far above the homogeneous ice nucleation temperature. Here, we have been able to measure the lower limits of ice nucleation activity of recombinant E. coli cultures bearing PbINP constructs by using two small volume freezing assays described in Methods: WISDOM, wherein monodisperse nanoliter-sized droplets are suspended in an oil mixture, and BINARY, involving microliter-sized droplets sealed in individual compartments. Ice nucleation for the various cultures was compared by determining the fraction of frozen droplets (fice) as a function of temperature in both systems  for each repeat deletion culture against the number of repeats (nrep) remaining in its central domain , i.e., those that froze heterogeneously, contain only one nucleator  to −6 °C (at 109 Nm g−1).Previously, it has been reported that wild-type ure Fig. . Data frOne of our initial assumptions was that all INP repeats would be roughly equivalent in their contribution to heterogeneous ice nucleation. We investigated this supposition by examining the match of each 16-residue repeat to the consensus sequence AGYGSTQTAGEESSLT Fig. . Extensi12. In the WebLogo plot of the 11 C-terminal repeats, the putative water-organizing residues and the Y at position 3 are largely absent and a well-conserved R appears at position 12. The difference in solenoid coils between the first 54 that are water-organizing and the last 11 that generally lack water-organizing motifs is illustrated in Fig. The degree of conservation within the more consistent 54 N-terminal repeats and the more variable 11 C-terminal repeats is depicted using WebLogo plots, illustrating sequence conservation through bit scores as well as the frequency of alternative residues at each position Fig. . Apart fPbINP, pairs of restriction sites were exploited to generate deletions of the central domain’s N-terminal (SacII), middle (EagI), and C-terminal (PstI) portions in each case deleting 32 repeats. Using the restriction nuclease EcoRI, it was possible to delete the 41-residue C-terminal domain. The four corresponding ice nucleation curves from BINARY for cultures bearing these deletions were compared to controls with full-length PbINP  is facing outward from the solenoid core , into the repetitive region of PbINP, which has an estimated height of 2.2 nm and width of 1.0 nm. An advantage of using mRuby2 for this purpose is that its fluorescence indicates the functional production of the construct by the bacteria. Initially, the mRuby2 DNA was inserted into a 46-repeat construct rather than the 65-repeat construct to help accentuate any effects of the steric disruption that might have been muted in a full-length construct background. The insertion then replaced repeats 9 to 28, and a second construct re-positioned mRuby2 towards the C terminus by twelve INP repeats to replace repeats 21 to 40 . This observation suggested that ice nucleation activity might be correlated with the longest uninterrupted water-organizing surface in the central domain, which would be 37 repeats. This hypothesis was tested with the second mRuby2 construct (R21-mRuby2-PbINP), where the bulky fluorescent protein was shifted towards the C terminus by twelve repeats, such that the largest continuous surface spanned 25 repeats. The WISDOM and BINARY T50 values for R21-mRuby2-PbINP were −11.5 and −11.4 °C, respectively, which are 2.3 and 2.8 °C lower than those of the initial R9 construct. This shift to lower ice nucleation temperatures shows that the continuity of the water-organizing surface is important for potent ice nucleation activity.Fluorescence microscopy images after growth and induction of els Fig. . Cultureely Fig. . These vPbINP domains for which we were unable to find structural homologs in the database. The sequence for the globular fold at the front half of the N-terminal domain is conserved in INPs from other species and strains of ice-nucleating bacteria. The putative unstructured region that follows is also a common feature of these proteins, although differing in length and sequence. It is tempting to speculate that this long linker region might offer flexibility during the assembly of an INP superstructure or aggregate that is thought to be needed for ice nucleation at sub-zero temperatures as high as −2 °C40.AlphaFold predicted structures of the N- and C-terminal PbINP suggests that it forms a cap over the C-terminal end of the central solenoid repeats that covers the hydrophobic core of the solenoid and engages the beta strands of the last coil. Capping structures are essential for the stability of beta-solenoids41, and without them, the solenoid tends to unravel or form amyloid fibrils42. The complete loss of ice nucleation activity seen with the deletion of the PbINP C-terminal domain is consistent with previous findings in the literature on inaZ35 and Erwinia ananas INP43. Indeed, AlphaFold predicted an almost identical structure for the C-terminal domain of InaZ to that of PbINP . We were then able to interrogate this differential by insertion of oligonucleotides that encoded three, four or five repeats. The restoration of three repeats to a total of 15 boosted the ice nucleation temperature to −23 °C. Between 15 and 18 repeats, a single repeat addition was associated with a 2–3 °C increase in TPbINP using AlphaFold in combination with the WEBLOGO plots indicate that the C-terminal 11 coils of the central repeat region appear to diverge in sequence and presumably also diverge from an ice nucleation function. Notably, the previously well-conserved water-organizing motifs are largely absent from this portion of the solenoid. Instead, the appearance of a regular array of R at position 12 in the coil suggests another function for these repeats (R-coils), which we speculate could be associated with the formation of a patch of INPs. This would appear to be critical because deletion of the C-terminal 33 repeats from the central core eliminated all ice nucleation activity, whereas the loss of the same number of repeats from the center or the N-terminal end of the central domain resulted in only a minor reduction in ice nucleation temperature. In one model  can be predetermined in a rational manner with high accuracy.The consistency in our in vivo expression protocols, combined with “blind” measurements employing two independently established ice nucleation apparatuses, add rigor to our results and enable reliable comparisons between gth Fig. , which sPbINP. For example, the TxT motif appears in the same location in all coils of the INP solenoid model and would form a tandem parallel array indistinguishable from the ice-binding sites of insect AFPs like those from Tenebrio molitor and Dendroides canadensis beetles18 and spruce budworm16 except in its much greater length. To evaluate the role of the TxT motifs in ice nucleation, we used the same mutagenesis strategy that established their ice-binding role in AFPs. Threonine residues at intervals along the tandem parallel array were replaced by Y, S, and K to disrupt the flatness and water-organizing ability of this surface while being compatible with the underlying beta-sheet structure of the solenoid. T to Y mutations were previously effective in delineating the ice-binding site of T. molitor AFP20 and the bacterium, Marinomonas primoryensis, ice-binding protein50. T to S mutations, which remove the crucial hydrophobic methyl group, were effective in knocking out the ice-binding activity of type I AFP53. Mutations to K also successfully knocked down the activity of another type I AFP54.In designing the mutagenesis constructs, we were guided by similarities between recurrent motifs from some structurally characterized AFPs and conserved residues in the central domain of 12, where the second INP lies parallel but is 180° rotated, this motif can be viewed as a lateral extension of the TxT motif to produce a wider water-organizing surface. This resembles the ice-binding sites seen in some beetle and moth AFPs that have four56 or five57 parallel ranks of T. The first and third positions of this SLT motif are less well-conserved than those in the TxT sequence, which is also observed in the ice-binding site of the grass Lolium perenne AFP58. That the T50 values of the TxT and SLT mutants in full-length constructs are comparable in activity to the deletion of the corresponding region suggests that their ability to serve as templates for ordered arrays of water molecules has been completely disrupted  maintained by AlphaFold’s designers; the translated PbINP gene was used as the input sequence. To compare the β-solenoid coils, a 16-residue section matching the consensus sequence for repeats 1–54 (AGYGSTQTAGEESSLT) was isolated from both the AlphaFold model and the originally proposed PbINP β-solenoid model12. This comparison and the predicted C-terminal region fold were visualized using PyMOL v2.5.0 .The structure of P. borealis INP (GenBank accession number: EU573998) was altered to introduce silent mutations that generated useful restriction sites. To produce internally truncated PbINP constructs, we confirmed that pairs of introduced restriction sites were unique, with the sites appearing only twice in the synthetic PbINP and not elsewhere in the pET-24a vector backbone. Furthermore, restriction sites were introduced at equivalent points in the repeats such that they maintained the periodicity of the central repetitive domain. For example, an internal truncation would join position four of a 16-residue repeat to position five of some later repeat. The DNA sequence for enhanced green fluorescent protein  was ligated to the 3′-end of the PbINP sequence via a hexanucleotide encoding two linker residues (Asn-Ser). The entire DNA sequence was codon-optimized for expression in Escherichia coli without disrupting any planned silent mutations or restriction sites and synthesized by GenScript . This construct was sub-cloned into pET-24a and sequenced in both directions. As with all constructs produced by GenScript, a certificate of analysis confirmed that the correct DNA sequence had been synthesized and successfully cloned into the expression plasmid. Biological materials produced and used in this study can be obtained from the corresponding author.The gene encoding the E. coli Top10 cells (Invitrogen) according to the manufacturer’s instructions. The production of PbINP deletion constructs followed identical steps, differing only in the restriction endonuclease used for plasmid digestion. Plasmid DNA, encoding full-length PbINP, was purified from overnight culture using the GeneJET Plasmid Miniprep Kit (Thermo Scientific). Eluted full-length PbINP plasmid DNA was mixed with the applicable restriction enzyme (New England Biolabs) and buffer for digestion, then subjected to agarose gel electrophoresis. The larger PbINP vector fragment (~8500–6500 bp) was excised from the DNA agarose gel and purified using the QIAquick Gel Extraction Kit (Qiagen) before overnight incubation with T4 DNA ligase. E. coli Top10 cells were transformed with 1–2 µL of ligation product to produce individual colonies that were grown overnight in Luria broth (LB) and kanamycin. Deletion constructs were then purified using the miniprep kit and analyzed by restriction digest and gel electrophoresis. Sanger sequencing  confirmed deletions.The plasmid was transformed into chemically competent PbINP deletion construct (GenScript). Internal deletions to generate 15-, 16-, and 17-repeat constructs were done as described above.To generate constructs with 15, 16, and 17 repeats, a 288-bp synthesized DNA , thereby splitting the 46 repeats in the central domain into two uninterrupted sections with 9 and 37 repeats. Similarly, the R21-mRuby2-PbINP construct was of the same length but had mRuby2 inserted in the place of bases 1475 and 2387 (repeats 21–40), which splits the central domain into 21 and 25 repeats. This latter construct was synthesized by GenScript.The R9-mRuby2-PbINP construct was measured in the Arctic Express (DE3) strain of E. coli  since its constitutive expression of two cold-adapted chaperones promotes correct folding of proteins at low temperatures68. Electrocompetent cells were mixed and incubated on ice with constructed PbINP vector, then the solution was pipetted into a chilled cuvette and subjected to electroporation according to the manufacturer (Bio-Rad). After the addition of 500 µL LB, cells were allowed to recover at 37 °C for 1 h and were then spread onto LB-agar plates.The activity of each E. coli cultures were grown in LB supplemented with 25 μg/mL of gentamicin and 100 μg/mL of kanamycin at 37 °C with shaking at 235 rpm until the culture reached an OD600 of ~0.6. Expression of PbINP variants and the full-length construct was induced using 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) overnight at 10 °C with shaking at 235 rpm. Bacterial samples were stored at 4 °C for 2 days prior to ice nucleation measurements to potentially encourage the formation of higher-order INP structures69. All samples were analyzed for ice-nucleating activity in a “blind” manner: bacteria were shipped in tubes marked only with a sample number, and the genetic identity of each PbINP construct was revealed after ice nucleation measurements had been performed.All 600 of ~0.6 and stored in 1.5-mL vials at −80 °C. When these samples were subsequently assayed, the cultures were thawed at 37 °C in a water bath for 4 min, followed by three wash steps with LB broth and centrifugation to remove the glycerol before being restored to their original volume in LB. There were no statistically significant differences in the ice nucleation temperatures of fresh versus previously frozen cultures.Those samples not assayed on the day of preparation were cryopreserved in 15% glycerol after reaching an ODPbINP were achieved using identical protocols. After centrifugation and determination of the mass of the resulting cell pellet, bacteria were resuspended in sterile phosphate-buffered saline  to 1 mg/mL. An aliquot (100 µL) of this suspension was diluted in 9.9 mL of PBS and thoroughly mixed, yielding a 10−2 cell dilution, with subsequent dilutions of 10−4, 10−6, and 10−8 prepared. A 100-µL sample from each of these dilutions was spread onto separate solid media plates, which were incubated at 10 °C. The number of colony-forming units (CFU) was determined after overnight growth.Serial dilutions of cultures expressing 71. Briefly, ~120 nanoliter-sized droplets (80–100 µm diameter) containing bacteria at a concentration of 1 mg/mL were generated in a microfluidic device in a narrow junction pressed by an oil phase and subsequently trapped in an array of chambers. The microfluidic device was then placed on a commercial temperature-controlled stage , with the freezing of the individual droplets tracked by their brightness using an optical microscope  equipped with a charged-coupled device (CCD) camera. Homogeneous ice nucleation of pure water droplets was observed below −35 °C, at a cooling rate of 1 °C per min, in agreement with theoretical predictions. The temperature uncertainty of the apparatus was calculated at ±0.3 °C. The frozen fraction fice, representing the accumulated fraction of frozen droplets at each temperature, was derived for each sample. T50, the temperature at which 50% of the droplets are frozen , was used to compare different samples. Ice nucleation experiments were repeated twice using different microfluidic chips. The period between each repetition was up to 40 days, and the samples were cryopreserved during the time gap between the measurements as described above in subsection “Culturing of bacteria”.Freezing experiments on suspensions containing live bacteria were performed using the Weizmann Supercooled Droplets Observation on a Microarray (WISDOM) apparatus72 apparatus was used for ice nucleation experiments with freeze-dried PbINP-expressing bacterial cultures that were reconstituted with double-distilled H2O. For each sample, three separate replicates were performed using ~64 droplets per trial. Droplets (0.6 µL) were placed on a hydrophobized glass plate and separated by a polydimethylsiloxane spacer, then they were cooled at a rate of 1 °C per min using a commercial cooling stage . The droplets’ freezing temperatures were detected by a change in the average gray value in the image of each droplet, observed with a CCD-Camera . Any droplets with direct contact with the device’s polydimethylsiloxane spacer were not considered because of the potential for heterogeneous ice nucleation of this surface, and hence an effect on the freezing temperature of the droplets cannot be excluded. The apparatus was calibrated in terms of the absolute temperature and the rate of cooling. The overall uncertainty in the reported freezing temperature is ±0.3 °C.The Bielefeld Ice Nucleation ARraY (BINARY)E. coli cultures were filled to their original volume by adding double-distilled water, and the resuspended samples were tested directly on the BINARY apparatus as well as samples diluted 100-fold in phosphate-buffered saline (PBS). As references for the undiluted and diluted experiments, respectively, we prepared an aqueous sucrose solution with a concentration of 10% weight/volume and diluted a portion of this solution with PBS buffer by a factor of 100.The freeze-dried ice and T50 as those described for the WISDOM experiments above. The T50 value obtained for pure double-distilled water on BINARY is −29.5 °C. This temperature can be regarded as the lower limit for heterogeneous freezing experiments of samples in pure water measurable in this apparatus (without the colligative effects of solutes).For the evaluation of the freezing events, we used the same definitions of fThe number of consensus-matching residues in each repeat was determined to assess sequence conservation across the central domain. A plot of the number of consensus-matching residues (out of a maximum of 16) for each repeat was produced using the sequence comparison output from Molecular Evolutionary Genetics Analysis (MEGA) software. The amino acid sequence for the last 48 residues of the N-terminal domain, the 65 sixteen-residue central domain repeats, and the first 32 residues of the C-terminal domain were compared to the consensus sequence AGYGSTQTAGEESSLT. The “Distance” analytical tool was used to “compute pairwise distances” using the “No. of differences” model to output the number of divergences per 16 residues. This number, subtracted from 16, is equivalent to the number of consensus-matching residues. Using a data analysis program, these values were plotted against the repeat position, where the N-terminal domain residues correspond to repeats −3 to −1, the central domain is represented by repeat positions 1–65, and the first 32 C-terminal domain residues are designated “repeats” 66 and 67.PbINP constructs were obtained using an inverted microscope  equipped with a high-speed CMOS camera . Post-induction E. coli cultures were imaged at 100x magnification in both the green and red fluorescence channels to detect GFP and mRuby2 using emission ranges of 495–540 nm and 570–625 nm, respectively. Overlaid images were produced using the IX83 microscope’s associated software.Fluorescence microscopy images of fluorescently tagged Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Earth is warming and sea levels are rising as land‐based ice is lost to melt, and oceans expand due to accumulation of heat. The pace of ice loss and steric expansion is linked to the intensity of warming. How much faster sea level will rise as climate warms is, however, highly uncertain and difficult to model. Here, we quantify the transient sea level sensitivity of the sea level budget in both models and observations. Models show little change in sensitivity to warming between the first and second half of the twenty‐first century for most contributors. The exception is glaciers and ice caps (GIC) that have a greater sensitivity pre‐2050 (2.8 ± 0.4 mm/yr/K) compared to later (0.7 ± 0.1 mm/yr/K). We attribute this change to the short response time of glaciers and their changing area over time. Model sensitivities of steric expansion (1.5 ± 0.2 mm/yr/K), and Greenland Ice Sheet mass loss (0.8 ± 0.2 mm/yr/K) are greater than, but still compatible with, corresponding estimates from historical data (1.4 ± 0.5 and 0.4 ± 0.2 mm/yr/K). Antarctic Ice Sheet (AIS) models tends to show lower rates of sea level rise (SLR) with warming (−0.0 ± 0.3 mm/yr/K) in contrast to historical estimates (0.4 ± 0.2 mm/yr/K). This apparent low bias in AIS sensitivity is only partly able to account for a similar low bias identified in the sensitivity of global mean sea level excluding GIC (3.1 ± 0.4 vs. 2.3 ± 0.4 mm/yr/K). The balance temperature, where SLR is zero, lies close to the pre‐industrial value, implying that SLR can only be mitigated by substantial global cooling. The rate of ice sheet mass loss and ocean expansion has a near linear relationship to global mean surface warming in both models and dataThe modeled transient sensitivity of the sea level budget to warming is compatible with historical behavior except for Antarctic mass lossModels of Antarctic mass loss may be biased as data shows increasing losses with warming in contrast to models In the case of SLR, however, the initial transient response to warming is a change in the sea level rate due to the slow response of ice sheets and oceans. The TSLS is therefore defined as the instantaneous change in rate of SLR associated with a change in temperature  projections remain highly uncertain due, in large part, to inadequate understanding of how the ice sheets covering Antarctica and Greenland will respond to climate forcing. Various approaches have been developed to attempt to address this uncertainty including community‐based model intercomparison projects  of the IPCC and their Special Report on Oceans Cryosphere and Climate (SROCC) had weaker sensitivities than indicated by the observational and proxy sea level record  brings together an advanced set of participating climate models compared to CMIP5. CMIP6 models were forced by an updated set of emissions scenarios, utilizing the Shared Socioeconomic Pathways (SSPs), creating a broader selection of possible futures. For the first time, CMIP6 included an ice sheet modeling intercomparison  or calculated based on potential temperature and salinity in CMIP6 output. For historical outputs, the thermosteric sea‐level change time series are divided into four periods which are: 1850–1900, 1900–1950, 1950–2000, and 1992–2014. For the future climate scenario, we have investigated SSP1‐2.6, SSP2‐4.5, and SSP5‐8.5, for two time periods: 2016–2050, and 2051–2100. Table 2.4The ice sheet and GIC contributions to SLR have been the focus of two model intercomparison projects—ISMIP6 and GlacierMIP. Edwards et al.  emulatedMass loss from the ice sheets can be partitioned into ice dynamic and surface mass balance (SMB) components. To make an assessment as to which of these components is driving the ice sheet's TSLS, we used the output from the ISMIP6 ice sheet models that were forced by CMIP6 models, reported in Payne et al. . The CMI3The major contributors to SLR can be viewed as large reservoirs. The ice sheets and GIC are reservoirs of freshwater, and the ocean is a reservoir of heat. Any change in the stock of these reservoirs will result in a change in sea level. A steady state is characterized by a balance in the fluxes to and from these reservoirs, for example, gains from snow fall must be balanced by losses from melt and discharge. The initial impact of a change in climate will be a shift in the flux balance of every reservoir, and thus a change in the corresponding sea level rates. However, the fluxes to and from reservoirs are not only influenced by external forcing but will also depend on the stock in the reservoir. The long wave radiation losses from the ocean surface depend on sea surface temperature and are thus connected to ocean heat, and total melt losses from GIC depend on the remaining glaciated area. This leads to a feedback between the stock in the reservoir and the net fluxes. The reservoir leaks or gains until it finds a new equilibrium with the imposed climate, and the equilibration process can be characterized by an e‐folding timescale. The ocean and ice sheets are giant reservoirs that change size slowly and have multi‐centennial equilibrium response times. GIC, however, come in many sizes—every glacier or ice cap with its own response time. Often glacier response times are measured in decades.In this paper, we focus on the century scale response of the primary contributors to the sea level budget. The chosen time frames are relatively short compared to the response times we expect from most contributors. Thus we will be focusing on the initial “transient” response. We use GMST as an indicator for the intensity of the climate forcing, and investigate how sensitive the transient response is to a change in forcing intensity, that is, mean temperature. As we are primarily concerned with changes in the response rather than absolute values, we disregard small quasi‐constant components of the sea level budget such as the effect of ocean bottom deformation (order 0.1 mm/yr)  is defined as the initial increase in the rate of SLR to an increase in global mean surface air temperature  and two projection periods . The steric contribution is based on CMIP6, and thus covers the entire set of target periods. However, the ice emulator only provides estimates for the future contribution. This is a limitation inherited from the ISMIP6 protocol. We require at least three points in every regression, and reject all poorly constrained TSLS estimates with a standard error greater than 3 mm/yr/K. This quality filter is particularly useful for the 2016–2050 period where the GMST for the different scenarios has not yet deviated by much. We use a different set of periods when we estimate the historical TSLS from observations as we are limited by data availability.In order to estimate the TSLS we regress GMST against the rate in the sea level contribution. We simply use linear least squares regression for model data. However, for observational estimates we use weighted least squares regression as not all data are equally certain. We weight every data point by the inverse of the estimated standard error in the sea level rate. Confidence intervals in the historical TSLS estimates are determined using a Monte Carlo approach where we perturb the estimated rate and temperature according to the reported standard errors. These perturbations are assumed to be independent, and thus we are assuming no error covariance between the estimated rates in different periods. Fully covariant errors in the sea level rates would only affect the estimated intercept but not the slope. We therefore argue that this assumption has minimal impact on the estimated TSLS confidence intervals.The spread between CMIP6 runs should not be interpreted as representative of the uncertainty distribution. Some earth system models have been run multiple times, and this can lead to a bias if all CMIP6 runs are treated as equally probable samples from an uncertainty distribution. We therefore average all the model runs from an individual Earth System Model (ESM), with the only exception being those models with perturbed physics which are treated as if it was a different ESM. In this study, CanESM5 is the only model with two different perturbed physics members. Given that there exists substantial difference between different perturbed physics members of CanESM5 in volume‐averaged ocean temperature  with the steric estimate from a random sample from the CMIP6 ensemble where each run (j) has been assigned a probability weight. The weight, w, is designed to account for how well temperatures match. We writem) based on these weights therefore has a small temperature misfit. We make a first order adjustment to the steric rate (S˙m) to account for this misfit as followsm is the sensitivity estimated for mth CMIP6 model. This is a small adjustment as the model weights ensure that the temperature misfit is small. This combination strategy ensures that the CMIP6 ensemble is weighed such that it is consistent with future temperatures used by the ice emulator and consequently AR6 as the emulator was designed to be consistent with the AR6.There is not a simple one‐to‐one relationship between the ESMs used for the steric model, and the model samples from the ice emulator. It is therefore not trivial to produce a fully consistent model estimate of GMSL. However, it is clear that at the very least we must ensure that only models with similar climate sensitivity are paired. We therefore pair each ice emulator sample  are consistent (1.7 ± 0.5 and 2.1 ± 0.8 mm/yr/K) but show substantially more scatter as the data span a smaller temperature range and, consequently, is less well constrained. The observations indicate a sensitivity of 1.4 ± 0.5 mm/yr/K.The observational data imply a balance temperature of −0.9 ± 0.2 K which is close to the pre‐industrial value. This suggests that to mitigate SLR in the future would require a substantial reduction in present‐day GMST, noting that internal variability has been responsible for about 5 cm of sea level change over the pre‐industrial Common Era  and ocean thermal forcing  with increasing global average temperature throughout Antarctica, which agrees with previous independent modeling studies even going back as far as the fourth IPCC assessment report in 2007 , indicating that increases in accumulation under warmer conditions outweigh any losses from other processes. In fact, as was the case for the WAIS, ice dynamics shows no obvious trend with temperature Figure  suggestiThe spread in dynamic response to global mean temperature in the EAIS is greater than in the WAIS from 2051 to 2100, especially for the highest global mean temperatures Figure . HoweverFor both EAIS and WAIS, the early twenty‐first century time period tends to produce lower rates of sea level contribution, with a smaller spread, than the late twenty‐first century time period for a comparable temperature change. This is indicative of the diverging responses of the ice sheet model ensemble members over time, as some continue with a linear trend in the sea level contribution for the duration of the century, whereas others, particularly at the high tail end of the distribution have a super‐linear response , but smaller sensitivity post 2050 (3.0 ± 0.4 mm/yr/K). This is in line with the central TSLS estimate derived from expert assessments for the entire twenty‐first century (+4.2 mm/yr/K [90%: 2.6–11 mm/y/K])  to warming which is marginally greater but compatible with the historical estimate . This could indicate a bias in either the model or the observational estimates, or that the transient sensitivity is increasing over time. Results from a structured expert judgment  and argued that this raises concerns regarding model skill , and change due to dynamical sea level is then accounted for. Often it is assumed that the dynamical sea level scales with global mean steric expansion. In practice, this means that the local sea level is a weighted sum of all the individual contributors. If we explicitly account for GIC, local vertical land motion, weather and tidal variability (e.g., following Frederikse et al., Beyond the year 2100, we expect feedbacks to play an increasing role in the ocean heat uptake and ice sheet mass loss. We therefore expect the TSLS of these contributors to start deviating from historical values and from a linear trend. Eventually the response can no longer meaningfully be considered transient, and it will be more useful to consider the equilibrium sensitivity to warming and sea level commitment (Clark et al., Supporting Information S1Click here for additional data file.


Streptococcus pneumoniae: quercetin.The effectiveness of current antimicrobial methods for addressing for food-borne Gram-positive pathogens has dropped with the emergence of resistant strains. Consequently, new methods for addressing Gram-positive strains have to be developed continuously. This includes establishing novel targets for antimicrobial discovery efforts. Eukaryotic choline kinases have been highly developed as drug targets for the treatment of cancer, rheumatoid arthritis, malaria and many other conditions and diseases. Recently, choline kinase (ChoK) has been proposed as a drug target for Gram-positive species generally. The aim of this work was to discover novel, natural sources of inhibitors for bacterial ChoK from tea extracts. We report the first natural bacterial ChoK inhibitor with antimicrobial activity against  Streptoccus pneumoniae (sChoK) and blocking the growth of S. pneumoniae cells was discovered [The effectiveness of current antimicrobial methods for addressing for food-borne Gram-positive pathogens has dropped with the emergence of resistant strains. Consequently, new methods for addressing Gram-positive strains have to be developed continuously. This includes establishing novel targets for antimicrobial discovery efforts. Eukaryotic choline kinases have been highly developed as drug targets for the treatment of cancer, rheumatoid arthritis, malaria and many other conditions and diseases. Recently, choline kinase (ChoK) has been proposed as a drug target for Gram-positive species generally  after thscovered . Later, scovered . ChoK inscovered , but so scovered .S. pneumoniae, PCho is incorporated into two types of teichoic acids: lipoteichoic acid (LTA) and cell wall teichoic acid (CTA) [All ChoK isoforms phosphorylate choline (Cho) into phosphocholine (PCho). In the case of id (CTA) . LTA andid (CTA) . LTA is id (CTA) , and theid (CTA) .lic gene locus. The LicB gene expresses a Cho transporter which collects Cho from the external environment. LicA codes for sChoK, which phosphorylates Cho into PCho. LicC is a gene coding for cytidylyl transferase, which converts PCho into CDP–choline. The LicD1 and LicD2 PCho transferases remove PCho from CDP–choline and attach them to N-Acetylgalactosamine  residues located on pre-teichoic acid glycan subunits [The sChoK enzyme is an element of the pathway that mediates the decoration of teichoic acids with PCho via the intermediary CDP–choline. Genes that are part of this pathway are expressed via the subunits . These rsubunits . The Tacsubunits . Meanwhisubunits .S. pneumoniae and a colorimetric method for detecting sChoK as models, we screened eight different soluble tea extracts for anti-ChoK and antimicrobial activity with the ultimate aim of discovering novel ChoK inhibitors capable of preventing the growth of Gram-positive bacteria. These teas are Espinheira Santa, Peppermint, Lemon Broth, Wormwood, Rose Hips, Rooibos, Hibiscus, and Saint-Johns’ Wort. These teas have been previously been identified as having antimicrobial activity, however their drug targets have not been described  of 3.86 +/− 1.25 corresponded well with the measured IC50 of quercetin. The α-value is an index competitivity: the higher α, the stronger the competitivity of an inhibitor with the substrate being assayed, with an α reaching infinity being indicative of a purely competitive mode of inhibition; an α = 1 reveals a non-competitive mode of action, and an α-value between 1 and infinity being indicative of a somewhat competitive (mixed) mode of inhibition. With respect to choline, an α-value > 1 was measured, which was indicative of a mixed mode of inhibition, which was consistent with a Kminh/Kmfree of >1 and a Vmaxinh/Vmax free of <1. Meanwhile, an α-value of 1 was measured in the case of ATP, which was indicative of a non-competitive mode of action, a conclusion supported by a Kminh/Kmfree of ~1 and a Vmaxinh/Vmax free of <1.In order to gain some insight into the binding model of quercetin to sChoK, kinetic assays performed on the purified recombinant sChoK using the LDH/PK method to determine the mechanism of action of quercetin. The two natural substrates of sChoK are choline and ATP. The concentration of quercetin was fixed at its IC choline A or ATP  choline B were ad choline A. On the choline C: KI, α,Taken together, these results suggest that quercetin physically enters the choline binding site and displaces the choline substrate, while leaving the ATP binding site in the catalytic site untouched. Using this information, we defined the binding site of querecetin as those residues which interact with choline in a docking study of quercetin into the sChoK binding site. As shown in the most energetically favorable structure that emerged from this study, quercetin is modelled to have a binding mode that overlaps in part with the choline binding site. Therefore, quercetin can be considered a classic competititve inhibitor. Moreover, the binding mode of quercetin could be used as a scaffold to rationally design inhibitors that more effectively interact with the choline binding site.We report here an initial screening of 8 tea extracts. This screening revealed that all 8 teas contained inibitors of sChoK, and that each had some antimicrobial activity that correlated well with their inhibitory activity. The flavonoid quercetin was found to be a competitive inhibitor of sChoK. Importantly, while querecetin has been identified as a promising natural inhibitor, the full library of potential sChoK inhibitors has not yet been determined. Therefore, further studies that make use of alternative extraction and chromatagraphic methods are warranted to fully mine the inhibitory potential of these teas. In addition, this research has implications for many diseases linked to the activity of eukaryotic ChoKs including human isoforms. These diseases include cancer, rheumatoid arthritis, and malaria. Indeed, querecetin could function as a potential natural therapeutic against these diseases via inhibition of their respective ChoKs. This study represents an important initial step towards establishing a discovery pipeline for natural ChoK inhibitors with a wide range of therapeutic applications.All reagents were purchased from Sigma-Aldrich except for brain–heart infusion (BHI) broth, which was purchased from BD-Biosciences., concentrated in an Eppendorf Vacufuge Concentrator at room temperature for 24 h and freeze-dried for 2 days (Labconco). Samples were stored at 4 degrees and then dissolved in deionized water and filtered with a 0.22 µM before use.Three grams of each type of tea leaf were steeped individually in 150 mL of boiling deionized water and was infused overnight for 16 h. The extracts were then filtrated with a 0.22 µM MCE syringe filters The ability of the tea extracts to inhibit sChoK enzymatic activity was assayed in mixtures containing 8 mg/mL tea extracts and BL21 (DE3) cell extracts containing sChoK. A colorimetric method was used to quantify Cho consumption and PCho production as described previously .Inhibitory concentrations of both tea extracts and quercetin were determined as previously described .One hundred µL volume holes were then made into the Mueller–Hinton Plates agar plates inoculated with 5 mg of each tea extract was dissolved in 100 µL of deionized water, these mixtures were used to fill the holes made in the plates. Plates were then incubated overnight at 37 °C. A qualitative assessment was made the next day of the strength of inhibition, according to the relative diameters of the inhibition zones.sChoK was expressed and purified as described previously .The ability of the tea extracts to inhibit choline kinase enzymatic activity was assayed in the mixture’s tea extracts and BL21 (DE3) cell extracts containing either human or bacterial choline kinase. A colorimetric was used to quantify choline consumption and phosphocholine production as described previously .m and IC50 and initial velocities were measured using serial dilutions of the second substrate. The (%I) was calculated by dividing each initial velocity in the absence of compound with its corresponding velocity in the absence of compound. %I versus concentration and fitted into the following equation The mechanism of action of the inhibitors with respect to each substrate was determined as previously described using the PK/LDH method as the method of detection . BrieflyUsing the information from the kinetic data indicating the competitive nature of quercetin with respect to choline, quercetin was docked into choline binding site of apo-sChoK structure as a model (RCSB accession #4R77) using the Patchdock server. The quercetin .SDF structure file was downloaded from the PubChem database  and conv


Canine histiocytic sarcoma cells (DH82 cells) were persistently infected with CDV-Ond, CDV-Ondneon, CDV-Ondneon-vasostatin and CDV-Ondneon-GM-CSF and characterized on a molecular and protein level regarding their vasostatin and GM-CSF production. Interestingly, DH82 cells persistently infected with CDV-Ondneon-vasostatin showed a significantly increased number of vasostatin mRNA transcripts. Similarly, DH82 cells persistently infected with CDV-Ondneon-GM-CSF displayed an increased number of GM-CSF mRNA transcripts mirrored on the protein level as confirmed by immunofluorescence and Western blot. In summary, modified CDV-Ond strains expressed GM-CSF and vasostatin, rendering them promising candidates for the improvement of oncolytic virotherapies, which should be further detailed in future in vivo studies. Canine histiocytic sarcoma (HS) represents a neoplasia with poor prognosis. Due to the high metastatic rate of HS, there is urgency to improve treatment options and to prevent tumor metastases. Canine distemper virus (CDV) is a single-stranded negative-sense RNA (ssRNA (-)) virus with potentially oncolytic properties. Moreover, vasostatin and granulocyte-macrophage colony-stimulating factor (GM-CSF) are attractive molecules in cancer therapy research because of their anti-angiogenetic properties and potential modulation of the tumor microenvironment. In the present study, an  Cancer is one of the leading causes of death in dogs  and caniViral oncolysis is based on the premise that virus infection, amplification and spread within a tumor leads to its eradication . In genein vitro [in vitro [Due to its close relationship to the measles virus, which has already been tested as an oncolytic agent in clinical studies in human medicine , canine in vitro  and in min vitro . Furtherin vitro ,12,13. Sin vitro  and an ain vitro .in vivo [The inhibition of tumor angiogenesis is one of the most important starting points for anti-tumor therapies, as the continuous growth of tumors is dependent on a sufficient blood supply . Vasostain vivo . Interesin vivo . The modulation of the tumor microenvironment represents another promising approach for anti-tumor therapy. Classically activated macrophages (M1) are known to be pro-inflammatory and anti-tumoral, while alternatively activated macrophages (M2) behave contrarily ,21. The Nowadays, genetic engineering can further enhance the anti-tumoral properties of viruses . Indeed,neon-vasostatin) or GM-CSF (CDV-Ondneon-GM-CSF). Based on these previous findings and taking into consideration the results obtained in the canine histiocytic sarcoma model using DH82 cells, CDV-Ond represents a very promising candidate for oncolytic virotherapy. Therefore, CDV-Ond strains were engineered in order to express vasostatin or GM-CSF in infected cells to potentiate the therapeutic effect. The aim of this study was to characterize DH82 cells persistently infected with engineered CDV-Ond strains expressing either vasostatin (CDV-OndThe current experiments were performed with early passages (passage 4 to 6) of all persistently infected cell lines.neon-GM-CSF and CDV-Ondneon-vasostatin were compared to non-infected DH82 cells and DH82 cells persistently infected with CDV-Ond and CDV-Ondneon, respectively. The infection status of the aforementioned persistently infected cultures was assessed by immunofluorescence (IF) staining and reverse transcription quantitative PCR (RT-qPCR) for CDV-nucleoprotein (CDV-NP). CDV-NP was detected in more than 95% of the cells in infected cultures regardless of the strain, whereas non-infected controls were negative (p > 0.05), as shown in DH82 cells persistently infected with CDV-Ondnegative . Moreoveneon pi, CDV-Ondneon-GM-CSF pi, CDV-Ondneon-vasostatin pi) displayed more than 80% mNeonGreen-positive cells, whereas non-infected cultures and cells infected with DH82 CDV-Ond were negative , DH82 CDV-Ond pi cells (p = 0.0029) and DH82 CDV-Ondneon pi cells (p = 0.0029), as shown in The number of GM-CSF mRNA transcripts was significantly higher in DH82 CDV-Ondneon-GM-CSF pi cells revealed a significantly higher number of GM-CSF immunolabeled cells compared to non-infected DH82 cells (p < 0.0001), DH82 CDV-Ond pi cells (p = 0.0009) and DH82 CDV-Ondneon pi cells (p = 0.0001), respectively, as shown in In order to verify the qPCR data on a protein level, fluorescent immunolabeling was performed. DH82 CDV-Ondneon-GM-CSF pi revealed a significantly increased amount of intracellular GM-CSF (p = 0.0187) compared to all other groups that did not show any band, as shown in neon-GM-CSF pi cells showed an additional band at 14 kDa. DH82 CDV-Ondneon-GM-CSF pi cells revealed significantly more 14 kDa GM-CSF (p = 0.0052) compared to all other groups that did not show any band at 14 kDa , DH82 CDV-Ond pi (p = 0.0286) and DH82 CDV-Ondneon pi cells (p = 0.0286), as shown in The number of N-terminal calreticulin (vasostatin) mRNA transcripts was significantly higher in DH82 CDV-OndImmunohistochemical staining for N-terminal calreticulin did not display statistically significant differences in the number of immunolabeled cells in all investigated cultures independent of the infection state . Immunolneon-vasostatin pi cells  compared to DH82 CDV-Ond pi cells , as shown in neon revealed no significant changes compared to DH82 cells persistently infected with CDV-Ondneon-vasostatin. Furthermore, the immunoblotting of culture mediums with an anti-N-terminal calreticulin antibody displayed protein bands at 27 kDa in all groups of persistently CDV-infected DH82 cells and non-infected controls. Densitometrical analysis showed that DH82 cells persistently infected with CDV-Ondneon-vasostatin  expressed significantly more 27 kDa N-terminal calreticulin than cultures persistently infected with CDV-Ondneon , as shown in neon-vasostatin.The immunoblotting of intracellular proteins with an anti-N-terminal calreticulin antibody revealed detectable bands at 27 kDa, as expected in all investigated cultures. However, densitometrical analysis showed significant differences in sizes and intensities of bands at 27 kDa in DH82 CDV-Ondin vitro characterization of canine histiocytic sarcoma (DH82) cells persistently infected with canine distemper virus strain Onderstepoort genetically modified to express either vasostatin or granulocyte and macrophage colony-stimulating factor (GM-CSF). Viruses with inserted immunomodulatory or anti-angiogenetic genes broaden the spectrum of mechanisms leading to tumor regression and the prevention of tumor spread and metastases, leading to enhanced oncolytic properties of the armed viruses [The present study represents an  viruses ,29,30,31neon CDV-Ondneon-vasostatin and CDV-Ondneon-GM-CSF) revealed a high percentage of infected cells ranging from 86% to 94%. These data show that the genetically engineered viruses behaved similarly to the non-modified CDV-Ond strain regarding the percentage of infected cells. Furthermore, the cumulative population doubling assay revealed that a persistent infection of DH82 cells with the different viruses  had no effect on the growth rate that did not differ significantly over time during the experiment. The present results are in agreement with previously published cumulative population doubling assays of DH82 cells and DH82 cells persistently infected with CDV-Ond [The immunolabeling for CDV-nucleoprotein (NP) revealed that up to 98% of DH82 cells were successfully infected with CDV-Ond. Similarly, DH82 cells infected with the genetically engineered viruses , closely related to CDV, has already been reported to lead to tumor regression in a murine model for human Burkitt’s lymphoma . Interesro study .neon-GM-CSF revealed a significantly higher number of GM-CSF mRNA transcripts compared to all other groups. This result suggests that the inserted GM-CSF gene in CDV-Ondneon-GM-CSF is a stable construct in early cell passages. The higher number of GM-CSF transcripts corresponded on the protein level with an increased number of GM-CSF-immunolabeled cells and high amounts of protein, suggesting a CDV-Ondneon-GM-CSF infection-induced effect on DH82 cells. However, the relatively low number of GM-CSF-expressing cells compared to the very high amount of intra- and extra-cellular protein as determined by immunoblotting might originate from single DH82 cells producing very high amounts of this protein after infection with CDV-Ondneon-GM-CSF. In the present study, DH82 cells persistently infected with CDV-Ondin vitro in a tumor neutralization assay via stimulating peripheral blood mononuclear cells [However, Western blot results demonstrated two unexpected protein bands with molecular weights higher than 14 kDa, most likely representing various glycosylated forms of GM-CSF . The lowar cells .In vitro, CDV-Ondneon-GM-CSF behaves similarly to the parenteral CDV-Ond strain while producing increased amounts of GM-CSF, rendering the engineered strain highly attractive to enhance the aforementioned mechanism of regression via TAMs. This might be attributed to a higher number of TAMs and additionally by a re-polarization of TAMs into the anti-tumoral M1 phenotype. However, the functionality for an in vivo treatment has to be evaluated in further studies. Interestingly, other viruses have also been successfully employed for the same purpose, such as Herpes simplex virus type I with a GM-CSF insertion and deletion of γ34.5 and ICP47 genes, which has been approved by the Food and Drug Administration for advanced melanoma treatment since 2015 ,38. One neon-vasostatin, a higher number of vasostatin mRNA transcripts were found. However, the increased level of vasostatin mRNA transcripts was not mirrored on the protein level. Indeed, the number of cells with membranous immunolabeling for vasostatin showed no significant differences between all groups. Unfortunately, the vasostatin was not tagged in the modified CDV-Ondneon-vasostatin strain, rendering a discrimination between virus-induced and regularly cell-produced vasostatin impossible. Moreover, the intracellular and extracellular (culture medium) levels of vasostatin, detected by immunoblotting, further corroborate the fact that there might be a post-translational interference after the vasostatin mRNA translation that prevents the final protein formation. One possible explanation might be the involvement of non-membranous organelles that assemble in response to acute cellular stress, known as stress granules [neon-vasostatin, despite the high number of vasostatin mRNA transcripts.In DH82 cells infected with CDV-Ondgranules  and procgranules . They argranules . One of granules . Among cgranules . This mi2+ levels under stress conditions [neon-vasostatin cells and to verify its functionality. The downregulation of calreticulin mRNA levels and expression on the protein level was observed in stress conditions . One of nditions . Cellulanditions . Moreovenditions . These pin vitro characterization on a transcriptional and protein level of two CDV-Ond strains engineered for expressing vasostatin and GM-CSF, respectively, revealed that CDV-Ond, as a ssRNA(-) virus [neon-vasostatin a good candidate for future functional studies in vitro and in vivo. The production and secretion of GM-CSF by DH82 cells persistently infected with CDV-Ondneon-GM-CSF confirmed this candidate as a promising tool for future functional in vitro studies and in vivo murine xenograft investigations in a model for HS using DH82 cells. In conclusion, the -) virus , is a goThe current study focused on early passages of persistently CDV-infected DH82 cells to maximize potential effects due to very high percentages of CDV-positive cells. Future studies should also focus on higher passages in order to demonstrate that the virus is able to constantly infect the cell population and to see if this will affect the production of vasostatin or GM-CSF.neon, CDV-Ondneon-vasostatin or CDV-Ondneon-GM-SCF with 100 µL of the stock solution. After inoculation for 2 h in the incubator, the inoculum was replaced by culture medium. After reaching a persistent infection state of DH82 cells infected with CDV-Ondneon, CDV-Ondneon-vasostatin and CDV-Ondneon-GM-CSF, cells were passaged every 7–10 days when the cultures were nearly 90% confluent. All cells were cultured in T75  and T25 flasks  in an incubator at standard conditions . The medium was changed every 2–3 days. In the present study, experiments were performed using passage 47 of the non-infected DH82 cells and passage 172 of the persistently CDV-Ond-infected DH82 cells. Moreover, due to a decreased number of cells expressing mNeonGreen in the later passages, passages 4–6 of DH82 cells persistently infected with CDV-Ondneon, CDV-Ondneon-GM-CSF or CDV-Ondneon-vasostatin were used. Non-infected canine histiocytic sarcoma (DH82) cells obtained from the European Collection of Authenticated Cell Cultures (ECACC No. 94062922) and persistently canine distemper virus (CDV) strain Onderstepoort (CDV-Ond)-infected DH82 cells (DH82 CDV-Ond pi) were cultured as previously described ,12,13,14neon pi, DH82 CDV-Ondneon-vasostatin pi and DH82 CDV-Ondneon-GM-CSF pi cells were seeded into T75 flasks and cultured over 3 weeks. The medium was changed every 2–3 days and the cells were counted at every cell passage performed in weekly intervals. The population doubling (PD) was calculated as previously described [10 /log2 [To investigate cell growth rate, a cumulative population doubling assay was performed. Non-infected DH82 cells and DH82 CDV-Ond pi, DH82 CDV-Ondescribed ,13 accorer)/log2 . The cumneon, as previously described [Recombinant viruses used in this study were derived from CDV-Ondescribed ,47. Subsneon, CDV-Ondneon-vasostatin and CDV-Ondneon-GM-CSF were centrifuged  and diluted logarithmically from 100 to 10−10 in Dulbecco’s Modified Eagle’s Medium  containing 10% fetal bovine serum , 1% penicillin/streptomycin  and zeocin  and titrated in quadruplicates in 96-well microtiter plates  containing Vero.DogSLAM cells (1.5 × 104/well). Following the 7-day incubation period under standard conditions, the cells were examined for cytopathogenic effect. In addition, the 50% log10 tissue culture infectious dose per milliliter (TCID50/mL) was calculated. Analysis was performed in three independent samples per group.Virus titration was performed as previously described . Brieflyneon pi, DH82 CDV-Ondneon-vasostatin pi and DH82 CDV-Ondneon-GM-CSF pi cells was extracted using TRIzol  and purified with RNeasy Mini Kit  and RNase free DNase Kit  according to the manufacturer’s protocols. The RNA concentration was spectroscopically measured at 260 nm using the GeneQuant pro . Transcription to cDNA was performed using Omniscript , RNAse OUT  and Random Hexamers  according to the manufacturer’s protocols. Reverse transcription was performed using a Biometra Thermocycler T-Gradient ThermoBlock  under the following conditions: 25 °C for 10 min, 37 °C for 1 h and 93 °C for 5 min. The total RNA of non-infected DH82 cells and DH82 CDV-Ond pi, DH82 CDV-OndPrimers were designed using Primer3 web version 4.1.0 or taken from the literature ,49. All 2, 0.2 mM dNTPs Mix  and primers at a concentration of 300 nM each. Gel electrophoresis for PCR amplicons was run in 2% agarose gel  over 45 min at 90 V, 100 W and 150 mA. Qualitative PCR was performed using a Biometra Thermocycler T-Gradient ThermoBlock  under the following conditions: denaturation at 94 °C for 1 min followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C (vasostatin), 58 °C  or 59 °C (CDV) for 2 min, extension at 72 °C for 1 min and a final extension at 72 °C for 5 min using the Taq DNA Polymerase Kit  with 1.25 mM MgCl2 to 108 copies per sample. Reverse transcription quantitative PCRs were performed in quadruplicates with negative controls as previously described [® Gel and PCR Clean-up Kit  was used for isolation of PCR amplicons out of the agarose gel according to the manufacturer’s protocol. Standard curves were prepared using serial-diluted PCR amplicons in a concentration ranging from 102 to 108 copies/µL. Quantitative PCR was performed using the Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix . Primers were diluted to a final concentration of 150 nM each. The reaction was performed using the AriaMx Real-time PCR System  at the following conditions: denaturation at 95° for 3 min and 40 cycles at 95 °C for 5 s, 57 °C (CDV), 58 °C (GM-CSF), 60 °C (vasostatin) or 64 °C (GAPDH) for 10 s, followed by one cycle at 95 °C for 30 s, 65 °C for 30 s and 95 °C for 30 s. The melting curve was established by initial denaturation at 95 °C for 1 min, followed by 40 cycles, starting with 55 °C and increasing the temperature by 1 °C per cycle. GAPDH was used as a housekeeping gene for standardization of relative gene expression. The relative gene expression was calculated with the formula X/Y × 100, where X = gene expression of the target and Y = expression level of the housekeeping gene (GAPDH). Standard curves for estimation of copy numbers were generated using RT-PCR amplicons from 10escribed . The Nucneon pi, DH82 CDV-Ondneon-vasostatin pi and DH82 CDV-Ondneon-GM-CSF pi was carried out in three independent samples from each cell type as formerly described [TM BCA Protein Assay Kit . For protein normalization, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping protein. The same amount of proteins were separated on 15% SDS-PAGE gels. Subsequently, proteins were transferred to a nitrocellulose membrane  blocked with 5% milk  in 0.1% Tween 20 in phosphate-buffered saline (PBS) for 1 h. Immunoblotting was performed using a primary antibody directed against canine GM-CSF , N-terminal calreticulin  and GAPDH . Horseradish peroxidase (HRP)-conjugated rabbit anti goat , goat anti rabbit  and rabbit anti mouse  antibodies were used as secondary antibodies. Protein bands were visualized using chemiluminescent substrate  and a ChemiDoc MP Imaging System . Densitometric analysis was performed to quantify band sizes and intensities using ImageJ version 1.51.0 (https://imagej.nih.gov/ij/). Subsequently, intracellular GM-CSF and N-terminal calreticulin amounts were normalized against the amount of GAPDH.Immunoblotting of non-infected DH82 and DH82 CDV-Ond pi, DH82 CDV-Ondescribed ,12,50. Fneon pi and DH82 CDV-Ondneon-vasostatin pi cell pellets were obtained. Two to three-micrometer-thick slides were obtained from each cell pellet. Immunolabeling was performed in triplicates with negative controls as previously described [® Elite® ABC Kit, Vector Laboratories, Burlingame, CA, USA) were applied for 30 min and 20 min, respectively. A 3′3′-diaminobenzidine (DAB) system  was used for the detection of positive reactions. Nuclei were counterstained with Mayer’s hemalum . Information regarding antibody details, antigen retrieval and blocking serum are reported in neon pi, DH82 CDV-Ondneon-vasostatin pi and DH82 CDV-Ondneon-GM-CSF pi. Therefore, pictures were taken in 5 evenly distributed fields per cell pellet at a 200× magnification using a microscope  equipped with an Olympus D72 camera . For analysis, the relative number of cells with membranous staining was counted and divided by the total number of cells.To investigate the cellular localization of vasostatin, immunohistochemistry for N-terminal calreticulin was performed. Formalin-fixed paraffin-embedded (FFPE) non-infected DH82, DH82 CDV-Ond pi, DH82 CDV-Ondescribed ,51,52. Bneon pi, DH82 CDV-Ondneon-vasostatin pi and DH82 CDV-Ondneon-GM-CSF pi in 5 randomly selected fields per well. Therefore, pictures were taken at a 200× magnification using a microscope  equipped with an Olympus DP-72 camera and using Olympus CellSense standard software version 2.3. For analysis, the number of positive cells was counted and divided by the total number of cells.In order to verify the GM-CSF production within the investigated cells and the infection status with CDV-Ond, the cells were immunolabeled for anti-canine GM-CSF and CDV-nucleoprotein (NP), respectively. The immunostaining of DH82 cells was performed in triplicates with negative controls in duplicates in 96-well microtiter plates , as previously described . Brieflywww.sas.com). The level of significance was set at p ≤ 0.05. Graph creation was carried out using GraphPadPrism version 8.0.1 for Windows . For descriptive statistics, median and range were calculated. For the analysis of data obtained from the cell population doubling assay, immunofluorescence, immunoblotting, virus titration, immunohistochemistry and RT-qPCR, the non-parametric Mann–Whitney U test was used. Statistical analysis of mNeonGreen and CDV-NP expression was performed using the Wilcoxon signed-rank test. Statistical analysis was performed with SAS software version 7.1.5.0 (SAS Institute, Cary, NC, USA,


Science, Bridget S. Gosis et al. demonstrate that selective inhibition of the mammalian target of rapamycin complex 1 (mTORC1) signaling through deletion of the RagC/D guanosine triphosphatase-activating protein folliculin (FLCN) in mice enhances activation of transcription factor E3 (TFE3) in the liver and protects against nonalcoholic fatty liver disease (NAFLD).1In a recent study published in 2 In the clinic, there are currently no FDA-approved medicines for the targeted therapy of NAFLD. Intriguingly, targeting FLCN to inhibit mTORC1 for the treatment of NAFLD does not affect other substrates of mTORC1 and is, therefore, able to avoid unanticipated feedback loops and undesired side effects.1NAFLD is the primary cause of chronic liver disease and affects one-fourth of the adult population worldwide.4 thus collaborating the reaction of cellular metabolism to external cues , growth factors , and metabolic intermediates ,4 RagA/B must be in the GTP-bound state for mTORC1 to be recruited to the lysosomal surface. RagC/D contributes to dimer formation, although its GTP/GDP-binding state is less important for mTORC1 lysosomal recruitment. The sophisticated machinery of trimeric GATOR1 (GTPase-activating protein activity toward Rags 1), which functions as a GAP (GTPase-activating protein), precisely modulates the nutrient-dependent transition of RagA/B activity. The GATOR2 complex counteracts the activities of GATOR1. Several cytoplasmic amino acid sensors, including Sestrin2, CASTOR1, and SAMTOR, sense intracellular amino-acid levels and transmit their information to mTORC1 via GATOR complexes. The solute carrier SLC38A9 facilitates intralysosomal amino acid level sensing, and the lysosomal v-ATPase complex is necessary for the preservation of the lysosomal proton gradient4  and the pentameric lysosomal complex Ragulator.4 Briefly, in response to upstream stimulations, activation of mTORC1 and canonical substrate phosphorylation need nutrient-stimulated Rag activity and GF-dependent Rheb activation . Growth factors (GFs) binding to their receptors can activate PI3K-Akt signal and subsequently block the GAP activity of the Rheb inhibitor TSC (trimeric tuberous sclerosis complex), resulting in Rheb conversion to the functional GTP-bound state. Rheb binding to mTORC1 induces profound conformational changes that result in the activation of the canonical mTORC1 substrates S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1).4 This selective phosphorylation of mTORC1 has been clarified by recent studies in which researchers discovered that S6K and 4E-BP1 comprise a TOR signaling (TOS) motif, a five amino acid region directly recognized and linked by mTORC1 through its subunit Raptor, thereby enabling substrate recruitment and phosphorylation. Instead of the TOS motif, TFE3/B possess a Rag-binding region (RBR) in their N-terminal regions, allowing them to engage with Rags in this way. TFE3/B interaction is only possible when RagA/B and RagC/D are connected to GTP and GDP, respectively. Upon that basis, TFE3/B phosphorylation is acutely susceptible to upstream inputs that affect Rag activity, such as nutrients, but is insensitive to other mTORC1-activating stimulations, such as growth factors. Among these regulatory mechanisms, RagA/B activity is essential for mTORC1 lysosomal localization and, as a result, for its action on all substrates. Furthermore, RagC/D activity is strictly required for TFE3/B recruitment and phosphorylation, and it serves a secondary role in mTORC1 lysosomal recruitment and phosphorylation of the canonical substrates S6K and 4E-BP1.4 Because of this, it is possible to separate canonical and noncanonical signaling downstream of mTORC1 by modulating FLCN  separation from the lysosomal surface and boosts its GAP activity toward RagC/D, which transforms GTP-RagC/D into GDP-RagC/D and facilitates the activation of mTORC1. Excitingly, FLCN clearance in a variety of cells suppresses mTORC1-mediated phosphorylation and inactivation of the transcription factor E3/B (TFE3/B) family without altering mTORC1-driven phosphorylation of its canonical substrates S6K and 4E-BP1.1 Hepatocyte-specific FLCN deletion in mice preferentially inhibits mTORC1-mediated phosphorylation of TFE3 and promotes its entrance into the nucleus, but has a modest effect on the canonical substrates S6K and 4E-BP1. Targeting this FLCN:mTORC1:TFE3 arm appears to simultaneously induce lipid consumption programs and suppress de novo lipogenesis. When TFE3 is released into the nucleus, Insig2 is activated to prevent the proteolytic processing of SREBP-1c, enhance the interaction of TFE3 with chromatin close to SREBP-1c, and further inhibit adipogenesis-related genes (Fig.mTORC1 has the ability to trigger selective responses to a wide variety of stimuli, which has been demonstrated to be related to some physiological and pathological conditions. Suppression of FLCN in the liver by Bridget S. Gosis et al. has been shown to protect mice against NAFLD and partially reverse these processes, indicating that FLCN could be a therapeutic target for NAFLD.5 Approximately 500 different functions are attributed to the liver when working with other systems and organs. Therefore, how to properly target NAFLD without disrupting other liver processes remains a key difficulty. mTORC1 governs hepatic lipid production and catabolism, but it also regulates numerous other hepatocyte pathways; hence, inhibiting mTORC1 could result in unwanted feedback loops. This study1 permitted selective control of liver cell metabolism by the FLCN:mTORC1:TFE3 arm and exhibited very few undesired effects in the treatment of NAFLD, thus opening up a new therapy option for NAFLD.As a multi-pathogenic disease, NAFLD affects a wide range of intrahepatic cell types and involves an intricate web of signal transduction pathways.


Then, ACT and FCT were subjected to peptide sequencing using LC-MS-QTOF to identify the potential AG and AA inhibitors. Molecular docking was performed on peptides with the highest number of hotspots and PeptideRanker score to study their interactions with AG and AA enzymes. Among the peptides identified, SPRRF, FE, and RR were predicted to be the most active peptides against AG, while AA inhibitors were predicted to be RPR, PPGIP, and LRP. Overall, hydrolysates prepared from lupin proteins using alcalase and flavourzyme may be useful in formulating functional food for managing diabetics.The use of natural ingredients for managing diabetes is becoming more popular in recent times due to the several adverse effects associated with synthetic antidiabetic medications. In this study, we investigated the in vitro antidiabetic potential (through inhibition of α-glucosidase (AG) and α-amylase (AA)) of hydrolysates from lupin proteins pretreated with ultrasound and hydrolyzed using alcalase (ACT) and flavourzyme (FCT). We further fractionated ACT and FCT into three molecular weight fractions. Unfractionated ACT and FCT showed significantly ( Diabetes, a chronic metabolic disease, is one of the leading causes of premature mortality in the world today. According to the International Diabetes Federation (IDF), one in ten adults has diabetes globally, implying that over 537 million people live with this chronic disease. This number is projected to increase dramatically to over 643 million by 2030 and 783 million by 2045 . In 2021Several antidiabetic agents, including miglitol, emiglitate, acarbose, and voglibose, have been developed over the years, are commercially available, and are effective as antidiabetic drugs . HoweverLupinus angustifolius L.) is a highly sustainable leguminous bean with a protein profile comparable to soybean. It has a very high protein content (33.9–43.4%) and less competition for direct consumption like soybean. Enzymatic hydrolysis of lupin proteins has been reported to yield hydrolysates with antihypertensive, antidiabetic, antioxidant, and antimicrobial activities [Lupin , alcalase , α-amylase , α-glucosidase , 4-Nitrophenyl-β-d-glucopyranoside (ρNPG), 3,5-dintrosalicylic acid (DNSA), and starch (EC 232-679-6) were procured from Sigma Aldrich . Membrane sheets used for ultrafiltration  were procured from Sterlitech Corporation . Precast gel, precision plus standard, Coomassie blue, 2-mercaptoethanol, Tris/Glycine/SDS buffer, and Laemmli sample buffer were purchased from Bio-Rad Laboratories . Lupin protein isolate (LPI) was purchased from Prolupin GmbH  and kept at 4 °C prior to analysis.Flavourzyme  LPI solution was made in a beaker using MilliQ water, with continuous stirring for approximately 30 min at ambient temperature. Prior to enzymatic hydrolysis, the solution was ultrasonicated using Bandelin Sonoplus Ultrasonic Homogenizer HD3400  with 400 W fixed power, 20 kHz frequency, and a 25 mm diameter probe at 60% amplitude. The sonication process was performed for 5 and 10 min at an ultrasonic intensity of 52.24 W/cm2 and 104.5 W/cm2, respectively. To maintain an ambient temperature, the beaker was placed in an icebox. The solution was then adjusted to the optimum temperature  and pH  of the proteolytic enzymes and inoculated with the enzymes using a 3% enzyme/substrate ratio. Hydrolysis was allowed to proceed for 4 h and was terminated after 4 h by placing the solution in hot water at 95 °C for 15 min. The hydrolysate was then centrifuged, freeze-dried, and stored at −20 °C for further studies. This process yielded two hydrolysate samples: alcalase hydrolysate (ACT) and flavourzyme hydrolysate (FCT). Three batches of the hydrolysates were prepared, and experiments were performed in triplicates.Preparation of LPH was performed using the method of Fadimu et al. . A 10% (Protein hydrolysate prepared from the steps above (ACT and FCT) were fractionated into 3 molecular weight fractions (molecular weight cut-off (MWCO) of 1, 5, and 10 kDa) using the SEPA CF Cell membrane Filtration System . During the ultrafiltration process, the pressure of 25 bar and 3.5 LPM was maintained at a temperature of 15 °C. Six ultrafiltrated fractions were obtained for ACT  and FCT  corresponding to the MWCO used. All fractionated samples were freeze-dried and kept at −20 °C for analysis. SDS-PAGE analysis was carried out using precast polyacrylamide gel (4–20%) using the method of Laemmli  as descrw/v) and equilibrated for 3 min. Afterward, 200 µL of Dinitrosalicylic acid (DNSA) reagent was added to the mixture and then boiled for 5 min. Finally, the reaction mixture was cooled to room temperature, followed by dilution with Milli-Q water (5 mL). The absorbance of the final reaction mixture was measured using a plate reader  at 540 nm. α-amylase inhibitory activity was calculated using the equation below:The method of Wickramaratne et al. , as prev50 (concentration of peptides that inhibited 50% of α-amylase enzyme) value was estimated by plotting α-amylase inhibition (%) against sample concentration (mg/mL).ICd-glucopyranoside (5 mM) solution to the wells containing the enzyme and samples. The mixture was then incubated at 37 °C for 20 min. Afterward, the absorbance of the mixture was measured at 405 nm using a plate reader . The inhibitory activity was estimated using the equation below: Alpha-Glucosidase inhibitory activity was measured using the method of Lankatillake et al.  as descr50 (concentration of peptides that inhibited 50% of α-glucosidase enzyme) value was estimated by plotting α-glucosidase inhibition (%) against sample concentration (mg/mL).ICm/z. The mass spectrometric data obtained were then analysed using Peaks studio version 6.0 having average local confidence (ALC) exceeding 80%. Analysis of the bioactivity of the identified peptides was performed using the Peptide Ranker server at http://distilldeep.ucd.ie/PeptideRanker/ (accessed on 15 April 2022). Peptides with Ranker scores above 0.5 were selected for further analysis. The molecular mechanism of α-amylase and α-glucosidase inhibition by the selected peptides was established using PepSite 2, a web-based (http://pepsite2.russelllab.org/ (accessed on 15 April 2022)) platform for checking in silico molecular interaction between enzymes and peptides. Then, the lysosomal α-glucosidase (PDB ID: 5NN3) and α-amylase (PDB ID: 1SMD) crystal structures were downloaded from the PepSite 2 web-based program. A significant level below 5% (p < 0.05) was used to select the most potent peptides and their potential binding sites.LC-MS-QTOF was employed for the possible identification of peptides in samples with the highest inhibitory activities (ACT and FCT) using the method of Sarah et al. . Firstlyhttp://www.rcsb.org (accessed on 16 April 2022)). De novo peptide structure was obtained from the Pep-Fold 3 server (https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/#overview (accessed on 16 April 2022)) [http://wenmr.science.un.nl/haddock2.4/ (accessed on 17 April 2022)) as described by Honorato et al. [The 3D structures of lysosomal α-glucosidase (PDB ID: 5NN3) and α-amylase (PDB ID: 1SMD) were downloaded from the Ressource Parisienne en Bioinformatique Structurale (RPBS) website l 2022)) . Pre-pro22) . Preo et al. . The intp < 0.05.All measurements were conducted in triplicate, and data were reported as mean ± standard deviation. Analysis of variance (ANOVA) was performed using SPSS statistical software . Mean separation was done using Duncan’s Multiple Range Test (DMRT), and significant differences were defined as Short-chain proteins or peptides resulting from proteolysis reactions have different molecular weights. The degradation pattern of lupin protein hydrolysates generated using alcalase (ACT) and flavourzyme (FCT) and their various ultrafiltrated fractions  are presented in 50 values of lupin protein hydrolysates and their ultrafiltrated fractions , followed by unfractionated hydrolysates from flavourzyme (FCT) . This result is in accordance with previous studies which reported that hydrolysates generated using alcalase had significantly higher α-amylase inhibitory  ability than those prepared using flavourzyme, based on IC50 values [Diabetes is a chronic condition characterized by high glucose levels in the blood due to the inability of the pancreas to produce enough insulin needed to facilitate the transportation of glucose into the cells. Glucose is the final product of carbohydrate metabolism in the body. Delaying carbohydrate hydrolysis into glucose by inhibiting the critical enzyme required for the reaction has been an effective approach for managing blood glucose levels. Alpha-amylase is one of the enzymes that hydrolyze starch into glucose, and blood glucose levels may be effectively managed through its inhibition. The result of ICractions  showed t0 values ,10.50 value from 1.66 mg/mL to 4.87, 3.58, and 3.85 mg/mL, respectively. A similar trend was observed in the α-amylase inhibitory property of hydrolysates generated using flavourzyme. A significant increase in the IC50 value of FCT (1.98 mg/mL) to 3.52, 4.38, and 3.19 mg/mL was observed after ultrafiltration into 1 kDa, 5 kDa, and 10 kDa molecular weight fractions, respectively. The variations observed in the potency of the samples against α-amylase could be associated with the differences in their amino acid and peptide composition, as reported in the previous study [50 values upon fractionation. Fractionation of protein hydrolysates into various molecular weight fractions is not a guarantee for improved activity [In this study, ultrafiltration of ACT into 1 kDa, 5 kDa, and 10 kDa molecular weight fractions increased the ICus study , who repof FCT 1.8 mg/mL tactivity , but in 50 values for α-glucosidase inhibitory activity of hydrolysates and their ultrafiltrated fractions prepared from lupin proteins using alcalase and flavourzyme are presented in 50 value of 1.65 mg/mL) had higher α-glucosidase inhibitory activity than flavourzyme hydrolysate (FCT) . Upon fractionation into 1, 5, and 10 kDa molecular weight fractions, a general reduction in α-glucosidase inhibitory activity was observed. Among the fractions, the highest α-glucosidase inhibitory activity was recorded for the 10 kDa fraction of alcalase hydrolysate , closely followed by the 10 kDa fraction of flavourzyme hydrolysate . The lowest inhibitory activity was recorded in the 1 kDa fraction of ACT and FCT  and 5 kDa fraction of FCT . The low inhibitory activity observed in the ultrafiltrated fractions could be linked to a reduction in the composition of peptides in the various fractions since FCT and ACT contained all the peptides generated during the hydrolysis. In contrast, their ultrafiltrated counterparts possessed only a fraction of the peptides. These results indicate that the fractionation process decreased the concentration of peptides, thereby causing a reduction in their IC50 values. A similar observation has been reported for ultrafiltrated fractions of protein hydrolysates [Inhibition of α-glucosidase is one of several approaches for managing blood glucose levels in diabetic patients. This inhibition often disrupts the catalytic activity of the enzyme, and the overall effect is the reduction in postprandial blood glucose level due to a delay in glucose absorption. The antidiabetic effects of lupin protein hydrolysates have been studied but the peptides responsible and the mechanisms behind the impact remain unknown . The IC5olysates ,25. Over50 values compared with their ultrafiltrated fractions (http://distilldeep.ucd.ie/PeptideRanker/ (accessed on 15 April 2022))  are more likely to bind with the active site of α-amylase and α-glucosidase, causing them to lose their activity through the blockage of the substrate and catalytic sites. In total, 16 peptides were found to be active inhibitors of α-glucosidase  and flavourzyme (FCT) were selected for further studies due to their relatively higher ICractions . Peptideractions . In bothl 2022)) . In addipeptides . Peptidecosidase  and 14 fcosidase  based onp < 0.05). According to Hermans et al. [According to s et al. , the thrs et al. . Interess et al.  and Roigs et al.  reportedSimilarly, 14 peptide sequences from the flavourzyme hydrolysate (FCT) were predicted to have the capability to hinder the catalytic activity of α-glucosidase . In thisThe potential molecular interaction between the α-amylase enzyme and peptides from alcalase and flavourzyme hydrolysates are presented in p-value and number of hotspots using statistical significance , MLLL , and AIPPGIPY  could bind 6 sites or residues on α-amylase. Other peptides such as RW, PMLL, ML, AIPINNPGKL, HSDADFIL, RLL, LR, LRL, and LLPH also showed high α-amylase inhibitory potential by binding 5 active site residues. However, peptide FP could only bind to 3 sites  of the catalytic triad. This agrees with the study by Siow and Gan [To predict the way peptides bind onto the active site of the α-amylase enzyme, we used the Pepsite2 computational approach. To achieve this, we calculated the ificance . In this and Gan , as theyAmong peptides identified in the flavourzyme hydrolysate, ML, LP, and RPR demonstrated high α-amylase inhibitory capacity as they bind 5 hotspots on the triad . This coMolecular docking is the most effective approach for identifying the binding patterns or forces behind protein-ligand complexes and is widely used in drug research . Alpha-aAs indicated by molecular docking, all the peptides could easily bind on the active site of α-glucosidase with high binding energy . The tri50 value reported in the ACT (1.66 mg/mL) in comparison to FCT (1.98 mg/mL). Other α-amylase inhibitory peptides such as LP, MLLL, AIPPGIPY, and RPR also showed strong α-amylase inhibitory potential as they formed hydrogen bonds with at least one residue and interacted well with the substrate-binding pockets of α-amylase  and flavourzyme (FCT) and fractionated them into different molecular weight fractions. The IC50 values obtained showed that the original hydrolysates from alcalase and flavourzyme displayed higher inhibitory potentials towards α-glucosidase and α-amylase than their ultrafiltrated fractions. Lupin protein hydrolysates effectively inhibited the two enzymes involved in the management of diabetes (α-glucosidase and α-amylase) through disruption of enzyme-substrate interactions at the active site of the enzyme and delayed postprandial hyperglycaemia. Using an in-silico approach, peptides SPRRF, FE, RR from ACT and PPGIP, RPR, and LRP from FCT were predicted to be the most potent inhibitors of α-glucosidase. In contrast, MLLL, AIPPGIPY, and SPRRF from ACT and ML, LP, and RPR from FCT were predicted as the most active inhibitors of α-amylase, due to their abilities to bind several hotspots on carbohydrases. The mode of interaction between these peptides and the carbohydrases was explored using molecular docking, and the results indicated that the peptides have a very strong potential to bind to the catalytic site of the enzymes and inhibit them. Additionally, this study has established that peptides derived from lupin protein hydrolysates could bind to a higher number of bound residues on the active site of α-glucosidase in comparison to α-amylase. In summary, hydrolysates from lupin proteins generated using either alcalase or flavourzyme could be used as a functional ingredient in the development of nutraceuticals for diabetic patients. However, the potential activities of the identified peptides in their pure form should be validated in future studies by using in vivo, in vitro, and cell line assays.