Based on the type of spatial deviation, we also classified the PS trajectory into 16 pattern errors. Seven (19%) out of 37 implanted screws were considered unacceptable (deviation distances > 2.0 mm or angulation > 5°), and 14 (38%) were inaccurate (> 0.5 mm and ≤ 2.0 mm or > 2.5° and ≤ 5°). CT-to-fluoroscopy registration was superior to preopCT/C-arm , and percutaneous was slightly better than open but did not reach significance . Regarding pattern error, the tendency was to have more axial than sagittal shifts. Using a quantitative method to categorize the screw 3D position, only 10.8% of the screws were considered unacceptable. However, with a more rigorous concept of inaccuracy, almost half were non-optimal. We also identified that, unlike some previous results, the O-arm registration delivers more accurate implants than the preopCT/C-arm method.Robotics in medicine is associated with precision, accuracy, and replicability. Several robotic systems are used in spine surgery. They are all considered shared-control systems, providing "steady-hand" manipulation instruments. Although numerous studies have testified to the benefits of robotic instrumentations, they must address their true accuracy. Our study used the Mazor system under several situations and compared the spatial accuracy of the pedicle screw (PS) insertion and its planned trajectory. We used two cadaveric specimens with intact spinal structures from C7 to S1. PS planning was performed using the two registration methods (preopCT/C-arm or CT-to-fluoroscopy registration). After planning, the implant spatial orientation was defined based on six anatomic parameters using axial and sagittal CT images. Two surgical open and percutaneous access were used to insert the PS. After that, another CT acquisition was taken. Accuracy was classified into  Spine robotics is growing in importance and usage. Theoretically, they provide accuracy and replicability while accounting for patient-specific anatomical characteristics. Today’s systems are considered shared-control robots, providing "steady-hand" manipulation instruments while the surgeon controls the remainder of the procedure .Numerous studies have addressed the benefits of robotics over other insertion techniques by linking the extent and rates of screw breaching –4. StillOur study compared the spatial accuracy of PS insertion along a planned trajectory between different registration methods and surgical access. Specific distances and angles between the PS and surrounding anatomical landmarks were measured for both planned trajectories and inserted screws, using preoperative and postoperative CT images, respectively.The main objective of this cadaveric spine project was to define which registration method and surgical access showed the slightest deviation between the surgical plan and the final result. In addition, we investigated if there was a pattern of error for screw placement between the various robot-assisted methods, such as an average tendency to shift medially or superiorly.This project used two cadaveric human samples  with the entire spine from C7 to S1 (19 vertebrae each). A preoperative CT scan image  was taken in both spines to check the integrity of the bone and the posterior spine soft tissue. The specific image protocol used in the preoperative CT (zero-degree gantry angle and 0.625 mm slice thickness) was the same for the preopCT/C-arm registration method. Three screw dimensions were selected . This specific implant model can fit both open or percutaneous surgical methods. The robotic system used (Mazor X) was recently acquired, the first in Canada.preoperative CT scan is used to plan the procedure, but the tracking device is acquired through a separate intraoperative fluoroscopy. The two images are merged, allowing the robotic arm to move under continuous navigation. Ultimately, both methods can guide screw insertion for up to six vertebral levels, depending on the patient's anatomy and spine region.The setup process for Mazor X has been described elsewhere . BrieflyTwo human cadaveric specimens, "W" and "B," were used. The preopCT/C-arm method was tested under percutaneous and open surgical access, while the O-arm method was only tested using percutaneous access. Screws were inserted in sequence from cranial to caudal vertebral levels. The robotic technique and implant dimensions are described in Table DICOM formatted preoperative and postoperative images were analyzed on a personal computer using the Mazor system software . For each screw trajectory, specific distances and angles were calculated concerning anatomical landmarks on axial and sagittal planes. Each plane had two distances  and one angular measurement.S—Cont Ped). For the distal axial measurement, we first needed to identify an axial midline, which can be challenging given the natural asymmetry of vertebral processes. We, therefore, opted to use the center point of the spinal canal and vertebral body, through which we defined a central vertebral line. The distance from the tip of the screw to the central vertebral line was then measured (S—Midline). Finally, we calculated the angle subtending the screw’s axial trajectory with the central vertebral line (Ax Angle).Considering the axial plane Fig. , the proS—Inf Ped). The distal measure contemplated a line from the tip of the screw to the inferior endplate of the vertebral body (S—Inf EP). And the angular measurement subtended the angle between the screw trajectory and a line tangent to the inferior endplate (Sag Ang).On the sagittal plan Fig. , the proFor each implanted screw, the arithmetic difference between the preoperative plan and the final screw position was calculated from the six above measures (four distances and two angles). This permitted us to identify superior-inferior and medial–lateral shifts between planned and actual screw trajectories. For example, in the axial plane, a positive value indicates a medial deviation and a negative value indicates a lateral deviation. While in the sagittal plane, a positive value is characterized by inferior variation and a negative value by superior deviation.optimal, inaccurate and unacceptable. Optimal was defined for the screws with planned and postop differences ≤ 0.5 mm for direct distance and ≤ 2.5° for angle. Differences of > 2.0 mm and > 5° would be deemed unacceptable. The inaccurate group involved screws where the final trajectory deviated beyond the limits of measurement but remained within the acceptable range .Accuracy was classified into three groups: inaccurate and unacceptable groups) were classified according to their deviation pattern error into eight suboptimal trajectories. The patterns are illustrated in Fig. All suboptimally placed screws  was conducted to identify which measures contributed to the maximal variation in screw placement error.Statistical analyses were completed in R (2022.07.2 + 576). Contingency tables were analyzed using Fisher’s exact test. To compare the performance of different pedicle screw insertion methods, the maximal distance deviation for each screw was compared across groups using the Kruskal–Wallis rank sum test. Post hoc pairwise comparisons were conducted using the Mann–Whitney Thirty-seven screws were inserted on the right side of the two cadaveric specimens. We could not insert the right S1 implant on cadaver W due to poor bone quality (low density) on that specific vertebra. Otherwise, all PS were inserted as planned (Table unacceptable (distance > 2.0 mm or angulation > 5°). On cadaver W, they were in the lumbar region (L3 and L4), while on cadaver B, they were in the lower thoracic  and lumbosacral (L5 and S1) regions. Comparing the three insertion methods, the percutaneous O-arm performed best with no unacceptable implants. The percutaneous preopCT/C-arm method had the next best performance with only two unacceptable screw placements (15%), and the open preopCT/C-arm technique had the poorest performance with five unacceptable screws (38%). All seven unacceptable screws had significant deviations in the axial plane and predominantly in the lateral direction. At the same time, only 3/7 were deviated in the sagittal plane and predominantly in the inferior direction.From all implanted screws, 7/37 (19%) were considered Inaccuracy was identified in 14/37 (38%) of the implants, where at least one of the measures deviated from the planned trajectory by > 0.5 mm and ≤ 2.0 mm for distances or > 2.5° and ≤ 5° for angulation. Sixteen screws had optimal results (43%). Both cadavers had a similar number of inaccurate implants , although there was a tendency for more errors to be in the axial plane. When combined with Unacceptable screws, this difference became significant, with more errors appearing in the axial plane  compared to the sagittal plane.Using the screw error pattern classification Table , we founN = 11, median and IQR = 0.9  mm) and preopCT/C-arm  image registration methods were compared to one another. The former was significantly more accurate . When only the surgical access method was considered, the percutaneous approach had slightly better outcomes than the open approach , but results did not reach significance .The two registration methods (O-arm and preopCT/C-arm) and two surgical approaches (open and percutaneous) were analyzed separately and in combination. Using the maximum deviation in distance measurements between the preoperative plan and the final trajectory for each screw, the O-arm , followed by the percutaneous preopCT/C-arm , and finally, open preopCT/C-arm . Groupwise analysis with the Kruskal–Wallis rank sum test found these differences significant (Χ2(2) = 9.18, p = 0.01). A post hoc pairwise study using the Mann–Whitney U test and Benjamini–Hochberg correction for multiple comparisons found the percutaneous O-arm method to perform significantly better than the percutaneous and open preopCT/C-arm methods (p = 0.02). The two preopCT/C-arm methods performed similarly (p = 0.61).Looking at the performance of registration methods and surgical approaches in combination Fig. , the perScrew—Pedicle distance in the sagittal plane, with a further 51% from the two axial measures. These results reinforce the prior findings that the most significant errors in screw placement are in the axial plane. By grouping screws according to the insertion method and using a Gaussian approximation for the error distribution along principal components, Fig. Considering all four distance deviations for each screw, we conducted a principal component analysis (PCA), identifying linear combinations of error that most contributed to inaccurate screw placement. The two main components plotted in Fig. Although robotic spine systems can offer many advantages, including reduced radiation exposure and decreased invasiveness, the increment in precision remains essential in favor of their use.Gertzbein–Robbins scale) to check accuracy when the implant is inserted in a broad pedicle (> 7 mm). In this context, for a 5-mm-diameter PS to still be considered acceptable (< 2 mm breach), it is necessary to have no more than a 3 mm shift from the original trajectory. However, in a narrow pedicle (< 4 mm), the same 3 mm shift is not an appropriate measure. This qualitative method is vague and cannot be applied to all patients and spinal levels. In discordance with this method, we used a spatial orientation or quantitative approach to compare PS accuracy in the current study.In the spine, there are several methods to measure implant accuracy. Some use the criteria of "in" or "out" , others Postoperative Screw Error Patterns. This involved using anatomically derived measures of screw position, which described 3D screw trajectories, and analyzing how final screw placements deviated from the planned trajectories. By classifying trajectory errors, it allows robot calibration, avoiding future deviances. Another benefit of our measurement methodology is that we used the contralateral pedicle as one of the measures in the axial plane, as opposed to the more commonly used ipsilateral pedicle [Our experiment measured preoperative and postoperative screw distances and angles in two anatomic planes. A variation of this method has been used before , 12, but pedicle –9. Sever pedicle . None ofUnacceptable (> 2.0 mm for distance and > 5° for angulation) and Inaccurate . Ideally, a robotic system should produce no noticeable screw trajectory errors, but technology has yet to mature. In addition, it is challenging to measure screw trajectories from CT images without a repeatability error of less than 0.5 mm or 2.5°. To minimize this error, we measured preoperative and postoperative images using the same planning software. A third-party application was considered less consistent.The discussion of how to define pedicle screw accuracy and its importance is sparse in the literature. Rampersaud et al. are among the few groups addressing this question . In theiOrtel et al. quantified only the axial angle using a "midsagittal line" passing in the middle of the vertebra, taking the spinous process as a reference [Kleck and al. also measured the axial angle but included a direct distance from the entry point to the tip of the screw [Guha et al. measured both axial and sagittal angles [A few others have used the quantitative concept to measure their implants utilizing direct distances and angles to compare planned and postoperative images , 12, 15.he screw . AlthougGertzbein–Robbins scale for measuring accuracy [In our study, the incidence of unacceptable screws in our sample 10.8%) was consistent with other studies , 16–19, % was conaccuracy . In addiGertzbein–Robbins scale and found no difference between the O-arm and preopCT/C-arm registration techniques for unacceptable screws (> grade I) [In numerous articles, o-arm accuracy has been compared against fluoroscopy; overall, it performs better , 20–23. grade I) , 24. Ourgrade I) . In our Kantelhardt et al. [In terms of surgical access, apart from the advantages or disadvantages of the two techniques, some scientific studies claim that open exposure can tamper with screw accuracy when soft tissue retraction is not optimal or if soft tissues encumber the surgical robotic arm , 3, 25. Our study has several limitations. Despite what some consider a positive factor  lacks inOur robotic cadaveric study used a quantitative method to categorize the screw 3D position. With that, the preoperatively planned and postoperative screw locations could be compared. Our methodology was designed to help elaborate a screw error pattern classification. Moreover, it allowed grouping.In our casuistic, only 10.8% of the screws were considered unacceptable; conversely, if the new, most rigorous concept of inaccuracy was added, almost half were non-optimal. We also identified that, as opposed to some previous results, the O-arm registration delivers more accurate implants than the preopCT/C-arm method.Although our sample is restricted to only 37 screws, the results may suggest changes in the next generation of robotic systems. The future designs compel new methods to improve accuracy and must offer tools to correct repeated erratic patterns.


In white adipose tissue, phosphorylation of residues Ser296/Ser297 was enhanced upon agonist activation whilst Thr306/Thr310 did not become phosphorylated. By contrast, in immune cells from Peyer’s patches Thr306/Thr310 become phosphorylated in a strictly agonist-dependent fashion whilst in enteroendocrine cells of the colon both Ser296/Ser297 and Thr306/Thr310 were poorly phosphorylated. The concept of phosphorylation bar-coding has centred to date on the potential for different agonists to promote distinct receptor phosphorylation patterns. Here, we demonstrate that this occurs for the same agonist-receptor pairing in different patho-physiologically relevant target tissues. This may underpin why a single G protein-coupled receptor can generate different functional outcomes in a tissue-specific manner.Free fatty acid receptor 2 (FFAR2) is activated by short-chain fatty acids and expressed widely, including in white adipocytes and various immune and enteroendocrine cells. Using both wild-type human FFAR2 and a designer receptor exclusively activated by designer drug (DREADD) variant we explored the activation and phosphorylation profile of the receptor, both in heterologous cell lines and in tissues from transgenic knock-in mouse lines expressing either human FFAR2 or the FFAR2-DREADD. FFAR2 phospho-site-specific antisera targeting either pSer G protein-coupled receptors (GPCRs) routinely are constitutively phosphorylated or become phosphorylated on serine and threonine residues, located either within the third intracellular loop or the C-terminal tail, following exposure to activating agonist ligands . This isi, Gq, and G12/G13 families (i proteins and reduction in cellular cAMP levels (2+ and Gq-mediated manner (A number of GPCRs are able to bind and respond to short chain fatty acids (SCFAs) that are generated in prodigious quantities by fermentation of dietary fibre by the gut microbiota . The mosfamilies . DespiteP levels , whilst d manner .A challenge in studying the molecular basis of effects of SCFAs in native cells and tissues is that the pharmacology of the various GPCRs that respond to these ligands is very limited  and ther297 in both the basal state and after addition of the orthosteric hFFAR2-DREADD agonists -hexa-2,4-dienoic acid (sorbic acid) .We performed mass spectrometry on a form of hFFAR2-DREADD with C-terminally linked enhanced yellow fluorescent protein (hFFAR2-DREADD-eYFP) following its doxycycline-induced expression in Flp-In T-REx 293 cells. These studies identified phosphorylation at residue Seric acid)  or 4-metic acid) . In addireatment . Based oorylated . Ser296 receptor  and withd Ser325 . Althoug2-DREADD  or pSer3296/pSer297 antiserum was able to identify a diffuse set of polypeptides centred at 70 kDa in preparations generated from vehicle-treated cells (296 and Ser297 were converted to Ala (hFFAR2-DREADD-PD1) , was induced . As such, this was not explored further. However, as we also generated both a hFFAR2-DREADD-PD3 variant, in which both Ser324 and Ser325 were altered to alanine  variants of hFFAR2-DREADD-eYFP with arrestin-3. MOMBA promoted such interactions with the intact DREADD receptor construct in a concentration-dependent manner with pEC50=4.62±0.13 M . The maximal effect, but not measured potency, of MOMBA was reduced for all the PD variants except hFFAR2-DREADD-PD2   the poteto MOMBA . Similared cells , howevered cells . Such st induced . To ensu induced . Althoug alanine , and an  alanine  in whichEADD-PD2 , but in EADD-PD2 . It is lEADD-PD2 . To extentiserum . This su296/Ser297 and in allowing phosphorylation of Thr306/Thr310 was further substantiated because both these effects of MOMBA were re-capitulated by the second hFFAR2-DREADD-specific agonist, sorbic acid -3-(2-(3-chlorophenyl)acetamido)-4-(4-(trifluoromethyl)phenyl)butanoic acid)   would beantisera . Additiontiserum . As CATPntiserum  this may samples . To conciminated , as was /pSer297 .306/pThr310 antiserum was also effective in detecting MOMBA-induced post-activation states of hFFAR2-DREADD-eYFP in immunocytochemistry studies. When induced in Flp-In T-REx 293 cells the presence of the receptor construct could be observed via the eYFP tag with or without exposure to MOMBA  rather than by MOMBA. Immunoblots using lysates from these cells with either of these antisera showed predominant identification of a group of polypeptides migrating with apparent mass in the region of 60 kDa after treatment of cells induced to express hFFAR2-eYFP with C3   . Recognion of C3 . Paralleon of C3  and confon of C3 . As withst CATPB . In addintiserum , confirm306/pThr310 antiserum was only able to identify hFFAR2-eYFP after exposure to C3 in this context  or vehicle. Imaging the presence of eYFP confirmed receptor expression in each case  and, in  context , whereased cells .To expand such comparisons into native tissues we generated a further transgenic knock-in mouse line. Here, we replaced mFFAR2 with hFFAR2-HA, again with expression of the transgene being controlled in a CRE-recombinase-dependent manner. To assess the relative expression of hFFAR2-HA in this line compared to hFFAR2-DREADD-HA in equivalent tissues of the hFFAR2-DREADD-HA knock-in mice, we isolated both white adipocytes and colonic epithelia from homozygous animals of each line and immunoprecipitated the corresponding receptors using anti-HA. Immunoblotting of such HA immunoprecipitates showed similar levels of the corresponding receptor in each and that the molecular mass of hFFAR2-HA was equivalent to hFFAR2-DREADD-HA .306/pThr310 antiserum compared to vehicle-treated samples  produced a marked increase in detection of hFFAR2-HA by the anti-pThr samples . By cont samples . Basal inificant  and littnificant . Such baNUS mice . To exteNUS mice . HoweverNUS mice . Immunobse to C3  that incse to C3 . Like cese to C3 . The antth CATPB  but this324/pSer325 hFFAR2-DREADD, we had no direct evidence from mass spectrometry performed on the receptor enriched from an HEK293-derived cell line expressing hFFAR2-DREADD-eYFP of phosphorylation of these sites. Moreover, we failed to find evidence to support such phosphorylation, using the generated antiserum, in either the cell line or the mouse tissues we studied. As we will discuss for the other antisera, this does not however explicitly exclude that these sites might be phosphorylated in other tissues or settings. For the anti-pThr306/pThr310 hFFAR2-DREADD antiserum we also did not find direct mass spectrometry support for phosphorylation of these residues in the HEK293-derived cell line. However, in both immunoblotting studies performed on lysates of these cells and in immunocytochemical studies, this antiserum was able to identify hFFAR2-DREADD-eYFP in a sensitive and agonist activation-dependent manner. Moreover, experiments in which we denuded the receptor of phosphorylation by treatment with LPP demonstrated that the antiserum was only able to identify the phosphorylated receptor and mutation to Ala of both Thr306 and Thr310 also eliminated detection of the receptor, confirming the sites of modification.The ability to detect activated GPCRs in native tissues can provide insights into how such receptors are regulated and, in drug-discovery and target validation efforts, provide valuable information on target engagement. For most GPCRs, as well as interaction with members of the family of heterotrimeric G proteins, upon agonist occupancy this also results in rapid phosphorylation of various serine and threonine residues, usually within the third intracellular loop and/or the C-terminal tail. Often mediated by members of the GRK family of kinases such phosphorylation allows higher affinity interactions with arrestin proteins. Classically this is linked to desensitisation of receptor-mediated second messenger regulation, as binding of an arrestin usually occludes the G protein binding site. However, now and for a considerable period, it has been established that arrestin interactions can promote distinct signalling functions and that these might be dependent on the detailed architecture of the GPCR-arrestin complex . This ca297 and, in addition, agonist-dependent phosphorylation of Ser296. In the same way as discussed for the anti-pThr306/pThr310 antiserum we confirmed the specificity of this antiserum and showed in both immunoblotting studies and immunocytochemical studies that there was increased immunoreactivity of this antisera after cells expressing hFFAR2-DREADD were exposed to an appropriate agonist.To assess aspects of potential differential bar-coding we required a second antiserum able to identify a different site(s) of phosphorylation in hFFAR2-DREADD. Here, we did have direct mass spectrometry-derived evidence for basal phosphorylation of Ser306 and Thr310. To do so we took advantage of a knock-in transgenic mouse line that we have characterised extensively  was markedly different in these various tissues. As we were able to use the HA tag to immunoprecipitate the receptor from different tissues, SDS-PAGE and immunoblotting studies showed that the predominant form of the receptor in colonic epithelium migrated as a substantially larger species than that isolated from white adipocytes, and this was also the case when the receptor was immune-enriched from Peyer’s patches and associated mesenteric lymph nodes . De-glyc296/Ser297 and Thr306/Thr310 is mediated by distinct GRKs and that the expression patterns or levels of the GRKs vary between these cell types , anti-HA affinity matrix , cOmplete ULTRA Tablets, Mini, EASYpack Protease Inhibitor Cocktail , PhosSTOP , NuPAGE MOPS SDS Running Buffer , NuPAGE 4–12%, Bis-Tris, 1.0–1.5 mm, Mini Protein Gels , Tris-Glycine Transfer Buffer , Laemmli buffer , Nitrocellulose membrane , TSA tetramethylrhodamine (TMR) detection kit (AKOYA Biosciences), SKU NEL702001KT, Tissue culture reagents (Thermo Fisher Scientific). Molecular biology enzymes and the nano-luciferase substrate NanoGlo were from Promega. Polyethylenimine, Linear, MW 25000, Transfection Grade (PEI 25K) obtained from Polysciences. LPP was from New England BioLabs. MOMBA was from Flourochem (018789), whilst CATPB and compound 101 was from Tocris Bioscience.The generation and characterisation of transgenic hFFAR2-DREADD-HA-expressing and CRE-MINUS mouse lines are described in detail in Site-specific mutagenesis on the hFFAR2 receptor-DREADD-eYFP construct was performed using the Stratagene QuikChange method  and as described in Cell culture, transfection, and generation of stable cell lines were carried on as described previously . HEK293 Cell lysates were generated from either the various Flp-In T-REx 293 cells following 100 ng/ml doxycycline treatment or from HEK293T cells following transient transfection, to express C-terminally eYFP-tagged hFFAR2receptor-DREADD receptor constructs (hFFAR2-DREADDreceptor-eYFP) and prepared as described previously .To remove phosphate groups, immunocomplexes were treated with LPP at a final concentration of 10 units/μl for 90 min at 30°C before elution with 2× SDS-PAGE sample buffer.eYFP-linked receptor constructs were immunoprecipitated from prepared cell lysates using a GFP-Trap kit (Chromotek) and immunoblotted as described previously . MembranFlp-In T-REx 293 cells expressing hFFAR2-DREADD-eYFP or hFFAR2-eYFP were seeded on poly-D-lysine-coated 13 mm round coverslips in 24-well plates and performed as described previously .BRET-based arrestin-3 recruitment assay was performed as described in 3 24.9, CaCl2 1.9, Mg2SO4 1.2, KCl 4.7, KH2PO4 1.2, glucose 11.7, pH 7.0) supplemented with protease and phosphatase inhibitors. To obtain colonic epithelium preparations, the colon was cut longitudinally and pinned flat on a sylgard-coated Petri dish (serosa down) containing ice-cold sterile oxygenated Krebs solution. The epithelium was gently removed using fine forceps.To establish the phosphorylation status of hFFAR2-DREADD-HA or hFFAR2-HA different tissues from hFFAR2-HA, hFFAR2-DREADD-HA, and the corresponding CRE-MINUS mice were dissected. Following cervical dislocation, the entire colon, Peyer’s patches, mesenteric lymph nodes, and adipose tissues were quickly removed and placed in ice-cold Krebs-bicarbonate solution  (for hFFAR2-DREADD-HA) or C3 (2–10 mM) (for hFFAR2-HA) for 20–30 min. For antagonist treatment, tissue samples were incubated with CATPB (10 µM) for 30 min prior to treatment with MOMBA/C3. Samples intended for western blot analysis were immediately removed and frozen at –80°C. For immunohistochemical analysis tissues were fixed using 4% paraformaldehyde (containing phosphatase inhibitors) for 30 min to 2 hr at room temperature.Dissected tissue samples were transferred to warm (30–35°C) Krebs-bicarbonate solution perfused with 95% Og (4°C). Protein quantification was performed using a BCA protein assay kit (Thermo Fisher Scientific).Frozen samples were homogenised in RIPA buffer (composition in mmol/l: Tris (base) 50 mM, NaCl 150 mM, sodium deoxycholate 0.5%, Igepal 1%, SDS 0.1%, supplemented with protease phosphatase inhibitor tablets). The samples were further passed through a fine needle (25G) and centrifuged for 15 min at 20,000 × 1 (Roche). HA-tagged receptor complexes were centrifuged (2000 × g for 1 min) and washed three times in RIPA buffer. Immune complexes were resuspended in 2× Laemmli sample buffer at 60°C for 5 min. Following centrifugation at 20,000 × g for 2 min, 25 μl of sample was loaded onto an SDS-PAGE on 4–12% Bis-Tris gel. The proteins were separated and transferred onto a nitrocellulose membrane. Non-specific binding was blocked using 5% bovine serum albumin (BSA) in Tris-buffered saline  supplemented with 0.1% Tween20 for 1 hr at RTP. The membrane was then incubated with appropriate primary antibodies in 5% BSA in TBS-Tween20 overnight at 4°C. Subsequently, the membrane was washed and incubated for 2 hr with secondary antibodies. After washing (3×10 min with TBS-Tween), proteins were visualised using Odyssey imaging system.HA-tagged receptors were immunoprecipitated overnight at 4°C from the tissue lysates using anti-HA Affinity Matrix beads from rat IgGIHC was performed as previously described by Signal amplification was performed as previously described by 296/pSer297-hFFAR2 and pThr306/pThr310-hFFAR2 were developed in collaboration with 7TM Antibodies GmbH.The rabbit phospho-site-specific antisera pSer2 beads packed into a StageTip column, and phosphorylated peptides were then eluted, dried, and then injected on an Acclaim PepMap 100 C18 trap and an Acclaim PepMap RSLC C18 column (Thermo Fisher Scientific), using a nanoLC Ultra 2D plus loading pump and nanoLC as-2 autosampler (Eksigent).Flp-In T-REx 293 cells harbouring hFFAR2-DREADD-eYFP were cultured to confluence. Receptor expression was induced with 100 ng/ml of doxycycline for 24 hr, followed by 5 min stimulation at 37°C with sorbic acid or MOMBA (100 μM). The cells were washed with ice-cold PBS, and TE buffer containing protease and phosphatase inhibitor cocktails was added to the dishes followed by transfer of cells into pre-chilled tubes. The cells were homogenised and centrifuged at 1500 rpm for 5 min at 4°C. The supernatant was further centrifuged at 50,000 rpm for 45 min at 4°C. The resulting pellet was solubilised in RIPA buffer  and the protein content was determined using a BCA protein assay kit. hFFAR2-DREADD-eYFP was immunoprecipitated using GFP-Trap Agarose resin (Proteintech) followed by separation on a 4–12% polyacrylamide gel. The gels were stained with Coomassie blue and the bands corresponding to hFFAR2-DREADD-eYFP (~75 kDa) were excised. Gel pieces were destained in 50% EtOH and subjected to reduction with 5 mM DTT for 30 min at 56°C and alkylation with 10 mM iodoacetamide for 30 min before digestion with trypsin at 37°C overnight. The peptides were extracted with 5% formic acid and concentrated to 20 µl. The peptides were subjected to phosphopeptide enrichment using TiOPeptides were loaded onto the trap column for 5 min at a flow rate of 5 µl/min of loading buffer (98% water/2% ACN/0.05% trifluoroacetic acid). Trap column was then switched in-line with the analytical column and peptides were eluted with a gradient of increasing ACN, containing 0.1% formic acid at 300 nl/min. The eluate was sprayed into a TripleTOF 5600+electrospray tandem mass spectrometer  and analysed in Information Dependent Acquisition mode, performing 120 ms of MS followed by 80 ms MS/MS analyses on the 20 most intense peaks seen by MS.The MS/MS data file generated was analysed using the Mascot search algorithm , against SwissProt as well as against our in-house database to which we added the protein sequence of interest, using trypsin as the cleavage enzyme. Carbamidomethylation was entered as a fixed modification of cysteine and methionine oxidation, and phosphorylation of serine, threonine, and tyrosine as variable modifications. The peptide mass tolerance was set to 20 ppm and the MS/MS mass tolerance to ±0.1 Da.Scaffold  was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 20.0% probability. Peptide Probabilities from X! Tandem and Mascot were assigned by the Scaffold Local FDR algorithm. Peptide Probabilities from X! Tandem were assigned by the Peptide Prophet algorithm with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least two identified peptides.All data presented represent mean ± SEM of at least three independent experiments. Data analysis and curve fitting were carried out using the GraphPad Prism software package v.10 (GraphPad). Concentration-response data were fit to three-parameter sigmoidal concentration-response curves. In the case of inhibition experiments with antagonists, an equivalent analysis was followed to fit an inverse sigmoidal curve. Statistical analyses were carried out on data derived from at least three independent experiments by performing one-way ANOVA followed by Dunnet’s or Tukey’s post hoc test as indicated. important tools for monitoring distinct tissue-specific patterns of agonist-induced Free Fatty Acid receptor 2 phosphorylation. The work includes several validation experiments, which provide convincing evidence that will be beneficial for the scientific community.In this study, the authors present  Summary:Very systematic generation of phosphosite-specific antisera to monitor FFA2 phosphorylation in native cells and tissues. Provides evidence that FFA2 phosphorylation is tissue-specific.Strengths:Technical tour de force, rigorous experimental approaches taking advantage of wt and DREADD versions of FFA2 to make sure that ligand-and receptor-dependent phosphorylations are indeed specific to FFA2.Weaknesses:In this reviewer's opinion, the only shortcoming is that the implications of tissue-selective phosphorylation barcoding remain unexplored. However, I understand that tool development is required before tools are used to provide insight into the functional outcomes of receptor regulation by phosphorylation. The study is a technical tour de force to generate highly valuable tools. I have no major criticisms but suggest adding an additional aspect to the discussion as specified below.Arrestins are highly flexible and dynamic phosphate sensors. If two arrestins have to recognize 800 different phosphorylated GPCRs, is it possible that any barcode serves the same purpose: arrestin recognition followed by signal arrest and internalization? Because phosphorylation barcoding is linked to G protein-independent signaling, which is claimed by some but is experimentally unsupported, and because arrestins don't transduce receptor signals on their own , I would also include this option in the discussion, i.e. that the different barcodes are a way nature may have chosen to regulate the location of 800 GPCRs by only 2 arrestins. The strengths of this paper begin with the topic. Specifically, this approaches the question of how GPCR signals are directed to different outcomes under different conditions. There is rich complexity within this question; there are potentially billions of molecules that could interact with >800 human GPCRs and thousands of molecular effectors that may be activated. However, these outcomes are filtered through a small number of GPCR-interacting proteins that direct the signal.Experimentally, strengths include the initial experimental controls employed in characterizing their ever-important antisera, on which their conclusions hinge. In showing strong agonist-dependent and phosphosite-dependent recognition, as well as the addition of GRK inhibitors and eventually an antagonist and phosphatase treatment, the authors substantiate the role of the antiserum in recognizing their intended motifs. When employed, those antisera overall give clear indications of differences across variables in immunoblots, and while the immunocytochemical studies are qualitative and at times not visually significantly different across all variables, they are in large part congruent with the results of the immunoblots and provide secondary supporting evidence for the author's major claims. One confounding aspect of the immunocytochemical images is the presence of background pThr306/pThr310, like in Figures 4C and 6A and B. In 4A and C, while the immunoblot shows a complete absence of pThr306/pThr310, Figure 4C's immuno image does not. In 6A and B, a similar presence of pThr306/pThr310 is seen in the vehicle image, which is not strikingly over-shown by the MOMBA-treated image. In addition, only Ser/Thr residues of the C-terminus were investigated, while residues of ICL3 have long been known to direct signaling in many GPCRs. Because of the presentations of sequences, it was not clear whether there were residues of ICL3 that have the possibility of being involved.It may be possible and further testable to show whether the residues that maintain basal phosphorylation could also be tissue-specific, especially considering the presence of pThr306/pThr310 detection in both the Figure 6A immunoblot's vehicle lane (but not MOMBA lane). The aforementioned detection in the immunocytochemical vehicle image could support differential basal phosphorylation in the enteroendocrine cells. Should this be the case, it could have confounded the initial mass-spec screen wherein the Ser residues were basally active in that cell type, while in a distinct cell type that may not be the case. Lastly, should normalized quantification of these images be possible, it may help in clearing up these hard-to-compare visual images.It is noted that aspects of the writing and presentation may lead to confusion for some readers, but this does not affect the overall significance of the work.Nevertheless, in terms of the global goal of the authors, the indication of differences in phosphorylation states between tissues is still evident across the experiments. Accordingly, the paper is overall strongly well-researched, well-controlled, and the conclusions made by the authors are data-grounded and not overly extrapolated. Providing direct evidence for the tissue-based branch of the barcode hypothesis is both novel and significant for the field, and the paper leaves room for much more exciting research to be done in the area, opening the door for new questions and hypotheses. Summary:The authors generate and characterize two phosphospecific antisera for FFA2 receptor and claim a "bar code" difference between white fat and Peyers patches.Strengths:The question is interesting and the antibody characterization is convincing.Weaknesses:The mass spectrometry analysis is not convincing because the method is not quantitative . Figure 1 shows single tryptic peptides with one and two phosphorylation fragmentations as claimed, but there is no data testing the abundance of these so the differences claimed between cell treatment conditions are not established.The blot analysis cannot distinguish 296/7 but it does convincingly show an agonist increase. Can the authors clarify why the amount of constitutive phosphorylation is much higher in the example blot in Figure 2 than in Figure 3? It would be helpful to quantify this across more than one example, like in Figures 4 and 5 for tissue.Compound 101 is shown in Figure 2 to block barrestin recruitment. I agree this suggests phosphorylation mediated by GRK2/3 but this is not tested. The new antibodies should be good for this so I don't understand why the indirect approach.The conditions used to inhibit dephosphorylation are not specified, the method only says "phosphatase inhibitors". How do the authors know that low P at 306/7 in white fat is not a result of dephosphorylation during sample preparation? If these sites are GRK2/3 dependent (see above) then does adipose tissue lack this GRK? The following is the authors’ response to the original reviews.Thank you for the e-mail of 27th September that includes the eLife assessment and reviewers comments on manuscript eLife-RP-RA-2023-91861. We have considered these, added additional data and made various changes to the text as detailed below. We now submit a modified version that we would be happy to view as the ‘Version of Record’.We are very pleased to note the highly positive reports from the reviewers. The major change we have made is to alter the Introduction to include further consideration of the development of the ‘bar-code’ hypothesis. As highlighted by reviewer 2 the Lefkowitz/Duke University Group have been major proponents of this concept. However, as with many topics their views did not emerge in isolation. Indeed we  were developing similar ideas in the same period  Trends Pharmacol Sci 29, 413-420). Moreover, other groups, particularly that of Clark and collaborators at University of Texas, were developing similar ideas using the beta2-adrenoceptor as a model at least as early as this  Mol Pharmacol 65, 196-206). As such we have re-written parts of the Introduction to reflect these early studies whilst retaining information on more recent studies that have greatly expanded such early work. This has resulted in the addition of extra references and re-numbering of the Reference section. We have also provided statistical analysis of agonist-induced arrestin interactions with the receptor as requested by a reviewer and performed additional studies to assess the effect of the GRK2/3 inhibitor in agonist-regulation of phosphorylation of the hFFA2-DREADD receptor. This has led to an additional author  being added to the paper.To address first the ‘public reviews’Reviewer 11. We agree that we do not at this point explore the implications of the tissue specific barcoding we observe and report. However, as noted by the reviewer these will be studies for the future.2. The question of why these are only 2 widely expressed arrestins and very many GPCRs is not one we attempt to address here and groups using various arrestin ‘conformation’ sensors are probably much better placed to do so than we are.Reviewer 21. It is difficult to address the potential low level of ‘background’ staining in some of the immunocytochemical images versus the ‘cleaner’ background in some of the immunoblotting images. The methods and techniques used are very distinct. However, it should be apparent that the immunoblotting studies are performed (both using cell lines and tissues) post-immunoprecipitation and this is likely to reduce such background to a minimum. This is obviously not the case in the immunocytochemical studies. It is also likely, even though the antisera are immune-selected against the peptide target, there may be some level of immune-recognition this is not limited to the phosphorylated residues.2. Whilst this reviewer has commented in detail in the ‘recommendations’ section on the use of English, the other reviewers have not, and we do not find the manuscript challenging to follow or read.Reviewer 31. We agree that the mass-spectrometry presented is not quantitative. The intention was for the mass spec to be a guide for the development of the antisera used in the study. We have re-written the initial part of the Results section (page 7) to state that phosphorylation of Ser297 was evident in the basal and agonist-stimulated receptor whilst phosphorylation of Ser296 was only evident following agonist addition.2. Immunoblotting is intrinsically variable as parameters of antiserum titre in re-used samples is not assessed and although we are aware that FFA2 displays a degree of constitutive activity  J Biol Chem. 287(49):41195-209) we did not make any specific effort to supress this by, for example, including an inverse agonist ligand. Agonist-regulation of phosphorylation of the receptor, as detected in cell lines by the anti- pThr306/pThr310antiserum, is exceptionally clear cut in all the images displayed, and as we note for the pSer296/pSer297 antiserum this was always, in part, agonist-independent.The point about compound 101 not being tested directly in the immunoblotting studies performed on the cell line-expressed receptor is a good one. We have now performed such studies which are shown as Figure 2E. These illustrate that the GRK2/3 inhibitor compound 101 does not reduce substantially agonist-induced phosphorylation of the receptor at least as detected by the pThr306/pThr310antiserum or by the pSer296/pSer297 antiserum. Equally this compound had little effect on recognition of the receptor. As the PD2 mutations which correspond to the targets for the pThr306/pThr310antiserum have no significant effect on recruitment of arrestin 3 in response to MOMBA  this is perhaps not surprising. Moreover, the PD1 mutations that correspond to the pSer296/pSer297antiserum also, in isolation, only have a partial effect of MOMBA-induced interactions with arrestin 3.1. The use of phosphatase inhibitors is an integral part of these studies. As noted in Materials we used PhosSTOP . However, we failed to make it sufficiently clear that this reagent was present throughput sample preparation for both cell lines and tissue studies. This had been specified previously by two of us ) but we agree this was insufficient and we now correct this oversight by making this explicit in Results.RecommendationsReviewer 1Competing interest: We apologise for this typographic error. It is now corrected.Figures: We have upgraded the figure images to 300dpi and this markedly improves readabilityReviewer 2Revisiting writing: We thank the reviewer for their assessment of the text. However, we do not feel that ‘every sentence in the entire manuscript could be clarified’ is a reasonable statement. Neither of the other reviewers commented on this. Each of the authors read and approved the manuscript.Figures: see response to Reviewer 1. We have greatly enhanced image quality at this part of the process.Statistics on Figure 2: We apologise for this oversight. Although there were no significant differences in potency for MOMBA to promote interactions with arrestin-3 to each of the PD mutants versus wild type receptor, there were in terms of maximal effect. Statistical analysis was performed via one-way ANOVA followed by Dunnett’s multiple comparisons test. This is now detailed directly in Figure 2C and its associated legend. As noted by the reviewer there was indeed a highly significant effect of the GRK2/3 inhibitor compound 101 and this is now also noted in Figure 2D and its associated legend.Units on page 9: pEC50 is considered as Molar by default but we have now specified this.PD1-4: It would be cumbersome to write out (and to read) 8 mutations that make up PD1-4 and hence we think this is specified appropriately in the Figure.Reviewer 31. Mass spec: Please see comment point 1 to reviewer 3.2. Immunoblotting and compound 101: We have done so.3. Phosphatase inhibition: see public comments, reviewer 3.


Then, the inhibition of magnolol on TWIK1 was observed via a whole-cell patch clamp experiment, and it was found that magnolol had a significant inhibitory effect on TWIK1 (IC50 = 6.21 ± 0.13 μM). By molecular docking and alanine scanning mutagenesis, the IC50 of TWIK1 mutants G229A, T225A, I140A, L223A, and S224A was 20.77 ± 3.20, 21.81 ± 7.93, 10.22 ± 1.07, 9.55 ± 1.62, and 7.43 ± 3.20 μM, respectively. Thus, we conclude that the inhibition of the TWIK1 channel by magnolol is related to G229 and T225 on the P2- pore helix.TWIK1 (K2P1.1/KCNK1) belongs to the potassium channels of the two-pore domain. Its current is very small and difficult to measure. In this work, we used a 100 mM NH Magnolia officinalis is a Chinese herbal medicine. It is listed in “Shennong’s Herbal” and “Compendium of Materia Medica” and is used as a medicine with dry bark, root bark, and branch bark. In traditional medicine, M. officinalis plays many roles, including relieving asthma, eliminating phlegm, and relieving pain. In clinical application, its compatibility with various Chinese medicines is widely used to treat cough, asthma, abdominal distension, constipation, malaria, and other diseases )n}. In the equation, I% represents the percentage inhibition of current amplitudes, IC50 represents the concentration of half maximal inhibition, [D] represents the concentration of a compound, and n represents the Hill coefficient. Graphpad Prism 9.5 was also used for data statistics.All data were expressed as mean ± SEM (


They also initiate a diverse set of signaling events, including ERK1/2 activation, once thought to only occur by passive scaffolding and formation of multiprotein “signalosomes.” Herein, we demonstrate that β-arrestins and their GPCR-bound active states allosterically enhance ERK2 kinase activity. Our findings reveal a previously unappreciated mechanism by which β-arrestins function both as passive scaffolds and allosteric regulators to modulate downstream GPCR signaling pathways. β-arrestins are multivalent adaptor proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) to inhibit G protein signaling, mediate receptor internalization, and initiate alternative signaling events. β-arrestins link agonist-stimulated GPCRs to downstream signaling partners, such as the c-Raf–MEK1–ERK1/2 cascade leading to ERK1/2 activation. β-arrestins have been thought to transduce signals solely via passive scaffolding by facilitating the assembly of multiprotein signaling complexes. Recently, however, β-arrestin 1 and 2 were shown to activate two downstream signaling effectors, c-Src and c-Raf, allosterically. Over the last two decades, ERK1/2 have been the most intensely studied signaling proteins scaffolded by β-arrestins. Here, we demonstrate that β-arrestins play an active role in allosterically modulating ERK kinase activity in vitro and within intact cells. Specifically, we show that β-arrestins and their GPCR-mediated active states allosterically enhance ERK2 autophosphorylation and phosphorylation of a downstream ERK2 substrate, and we elucidate the mechanism by which β-arrestins do so. Furthermore, we find that allosteric stimulation of dually phosphorylated ERK2 by active-state β-arrestin 2 is more robust than by active-state β-arrestin 1, highlighting differential capacities of β-arrestin isoforms to regulate effector signaling pathways downstream of GPCRs. In summary, our study provides strong evidence for a new paradigm in which β-arrestins function as active “catalytic” scaffolds to allosterically unlock the enzymatic activity of signaling components downstream of GPCR activation. The m310183 and p-Tyr185 based on rat ERK2)  cascades are among the best-studied signaling pathways that regulate cellular responses to extracellular stimuli , 14. The151313at ERK2) . ActivatIn addition to their function in attenuating GPCR-mediated G protein signaling, βarrs act as molecular scaffolds in assembling signaling complexes, including RAF–MEK–ERK mitogenic signaling pathway components , 20–28. 20292V2R chimeric receptor that binds βarrs with higher affinity compared to wild-type β2AR   , leading to ERK1/2 activation A 15, 1616, 20–2320ype β2AR –38. As pFig. S1) –39. As iSI Appendix, Fig. S2). We found that ERK2 could phosphorylate itself linearly with regard to ERK concentration, suggesting a single phosphorylation event. Having demonstrated that ERK can phosphorylate itself, we next examined whether basal state βarr1/2 or their active state forms (bound to either V2Rpp or pβ2V2R) can allosterically regulate ERK2 autophosphorylation activity in the endpoint format kinase assay assessed by immunoblotting . Therefore, we performed in vitro kinase assays to determine autophosphorylation of ERK2 alone, as assessed by immunoblotting using a phospho-ERK1/2 antibody, with increasing ERK2 concentrations . The fluorescent-based real-time assay monitors Adenosine triphosphate (ATP) consumption by a kinase via measuring Adenosine diphosphate (ADP) formation using an ADP-specific fluorescence sensor. To quantify the allosteric effect of βarrs on ERK2 activity, we calculated initial rates from time-course ERK2 activity profiles . As shown in SI Appendix, Fig. S3C, the initial rates of ERK2 autophosphorylation activity are concentration-dependent, further demonstrating the utility of this assay for measuring real-time ERK2 activity in vitro. We next tested the effects of βarr1 or 2 alone or in their active state forms bound to either V2Rpp or pβ2V2R on ERK2 activity in this real-time in vitro kinase assay    A. Simila2 alone) B. Notabl183 and Tyr185 residues of rat ERK2; 183 or pTyr185 within the ERK2 activation loop showed that ERK2 autophosphorylation occurs at pTyr185 and that βarrs robustly enhanced this activity   in ERK2 subject to such modulation. As with most protein kinases, ERK2 activity is regulated by the phosphorylation state of residues within the activation loop “TEY” motif , 19, 40 activity B and C. C-MS/MS) D. Consisapproach B and C.i.e., in cis) or intermolecularly  (Left). Experiments were performed as above with or without βarr1 or 2 or their active states to determine their effects on Tyr-autophosphorylation. We expected that if Tyr-phosphorylation occurred intramolecularly, only wt-ERK2 would be phosphorylated; if phosphorylation occurred intermolecularly, both wt-ERK2 and KD-ERK2 would be phosphorylated. As shown in Right), Tyr185 phosphorylation occurred on wt-ERK2 but not on KD-ERK2, indicating that Tyr-autophosphorylation occurs in cis via an intramolecular mechanism.To gain further insight into the mechanistic determinants of the allosteric effect of βarrs on ERK2 activity, we next investigated whether Tyr-autophosphorylation occurs intramolecularly (n trans) E. Kinasen trans) E, Left. 185 residue (ERK2-pY). This was accomplished by examining ERK2-pY activity towards myelin basic protein (MBP), one of its common substrates, in a kinase reaction using [γ-32P]ATP as a radioactive phosphate source (185 and Thr183 (ERK2-pTpY). Monophosphorylated ERK2-pY was produced via an in vitro ERK2 autophosphorylation reaction in the presence of GST–βarr1 . Dually phosphorylated ERK2-pTpY was prepared in a kinase reaction by coincubating ERK2 with active human MEK1 . Subsequently, these ERK2 variants were purified using size-exclusion chromatography (SEC), and their phosphorylation status was assessed by immunoblotting using site-specific phospho-antibodies . Analysis of the kinase activities of these ERK2 variants shows that unphosphorylated ERK2 exhibited minimal activity toward MBP   . Interestingly, results from our fluorescence-based kinase assay indicate that active state βarr2 exerts a more substantial allosteric effect on ERK2-pTpY activity compared with the equivalent active state form of βarr1 isoform. Further confirming these results, we found that βarrs robustly stimulate the kinase activity of dually phosphorylated ERK2-pTpY in terms of its ability to phosphorylate MBP as probed by a complementary approach in which 32P incorporation into MBP was visualized by autoradiography  A. On thecontrol) A and B. control) . To previography C and D.SI Appendix, Fig. S5). We initially confirmed that the MEK1/2 inhibitor effectively suppresses EGF-stimulated, MEK1/2-dependent ERK phosphorylation. Analysis of in-cell ERK2 kinase activity results , we performed our investigation using this isoform. Accordingly, we transfected CRISPR/Cas9 βarr1/2 knockout HEK293 cells with expression plasmids encoding ERK2 and increasing amounts of βarr2 . We util results  showed tβarrs are multivalent adaptor proteins that have been increasingly appreciated over the past two decades as playing broader roles in signal transduction by regulating a multitude of signaling pathways , 5, 27. 29Herein, we show that βarrs and their active states  allosterically enhance ERK2 autophosphorylation and phosphorylation of downstream substrate by dually phosphorylated ERK. This regulation of ERK2 activities is the result of βarrs acting as allosteric modulators exerting their effects by binding ERK2. We demonstrated these physical interactions between ERK2 and βarrs using pulldown and coimmunoprecipitation approaches. Quantitative measurement of kinase activity revealed that both βarr1 and 2 enhance the ERK autophosphorylation rate more than 10-fold. Moreover, receptor-mediated activation of βarrs further enhanced the ERK2 autophosphorylation rate. We conclude that βarr binding to ERK allosterically promotes robust enhancement of its autophosphorylation activity, both in their basal states and more robustly in their active states.185. Moreover, we demonstrated that this allosteric effect of βarrs on ERK2 Tyr-autophosphorylation occurs intramolecularly in a cis mechanism. Interestingly, we also found that the monophosphorylated ERK2-pY form of ERK2 exhibits moderately enhanced kinase activity towards MBP compared to its unphosphorylated form. This is consistent with the previously proposed idea that Tyr185 phosphorylation in ERK2 and similar monophosphorylation events in other kinases may serve as steps to establish a partially active conformation primed for subsequent full activation  of the regulatory proteins βarrs and ERK in various tissues may differentially influence the activation output. Taken together, these data strengthen our model that ERK2 can draw upon its allosteric interaction with βarr to increase its kinase activity, suggesting an alternative mechanism with distinct signaling outcomes compared to the ERK1/2 activation signal from upstream parallel MAP kinase cascades.We also identified the site at which ERK autophosphorylation activity is allosterically modulated by βarrs. Our data indicate that binding of βarrs to ERK2 induces its kinase activity by promoting phosphorylation of the ERK2 activation loop residue, Tyrtivation , 19, 32.tivation , 14, 19.tivation , 45. Thucis intramolecular mechanism, including Fus3, p38α, Raf, and GSK-3β Click here for additional data file.


In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure activity relationship and the selectivity mechanism.Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development.  Plasmodium falciparum, the most deadly of the malaria species, affected more than 200 million people and caused more than 600,000 deaths, mostly of African children1. Disruptions to funding and services due to the COVID-19 pandemic exacerbated the problems caused by widespread resistance of parasites to currently used therapies2, as well as resistance of the mosquito vectors to pyrethroid insecticides3. In particular, the recent emergence in Africa of artemisinin resistance-conferring K13 mutations5 is of great concern. There is an urgent need to develop new antimalarial compounds with novel mechanisms of action.Malaria is a devastating disease. In 2021, P. falciparum aminoacyl-tRNA synthetases (aaRSs) are susceptible to reaction hijacking by nucleoside sulfamates6. Tight-binding nucleoside sulfamate-amino acid adducts are generated in the active site, thereby blocking enzyme activity. A Plasmodium-specific reagent, ML901, was identified that hijacks P. falciparum tyrosine RS (PfTyrRS). By contrast, Homo sapiens Tyr-RS (HsTyrRS) does not catalyse formation of the adduct. X-ray crystallography revealed that differential flexibility of a loop over the catalytic site may underpin differential susceptibility to reaction-hijacking by ML901 6.A recent study showed that some P. falciparum cultures. OSM-S-106 was first identified as part of a screen of compounds from a GSK library . While OSM-S-106 is structurally divergent from the nucleoside sulfamates previously shown to target PfaaRSs via the reaction hijacking mechanism, mass spectrometry-based identification of covalent adducts and biochemical analyses revealed that Asn-tRNA-bound PfAsnRS is indeed susceptible to attack by OSM-S-106. By contrast, HsAsnRS is much less susceptible to hijacking by OSM-S-106.Here, we explored a new chemical class of reaction hijacking inhibitors. OSM-S-106  is an amHsAsnRS in complex with the natural intermediate, Asn-AMP, as well as with synthetically generated Asn-OSM-S-106, providing insights into ligand-induced changes in the enzyme structure. We generated an AlphaFold model of the PfAsnRS dimer. PfAsnRS harbours a large insert adjacent to motif I that is predicted to affect the dynamics of binding of substrates and release of products from the active site. This Plasmodium-specific structural feature may underpin differences in susceptibility to reaction hijacking. We generated a molecular model of PfAsnRS in complex with the native product, tRNA-Asn; and docked OSM-S-106 and derivatives into the AMP-binding site. This analysis provided insights into the structure-activity relationships that underpin the potency of OSM-S-106.AsnRSs are class II aaRSs, characterised by an α/β fold, with a highly conserved active site. We solved, for the first time, the crystal structure of P. falciparum cultures yielded the Tres Cantos Antimalarial Set (TCAMS) library with 13,500 active compounds7. One of these compounds, OSM-S-106  compounds against Synthesis of OSM-S-106 and its derivatives was achieved using the aminothieno pyrimidine synthesis protocol, as delineated in the P. falciparum (50% inhibitory concentration (IC50_72h) = 0.058 ± 0.017 μM; n = 8). Importantly, OSM-S-106 also prevented the development of P. berghei in liver cells , indicating a selectivity index (IC50HepG2/ IC50PbLuc) of over 140-fold ; but shows rapid intrinsic clearance in mouse microsomes   .50 = 4.4 ± 2.6 μM). Conversion of the sulfonamide to a sulfamate . No recrudescent parasites were observed over a 60-day selection period.5 Dd2-B2 parasites in each well of a 96-well plate with a Plasmodium-specific dihydroorotate dehydrogenase inhibitor  at 5 x IC50 (58 nM). This yielded 14 recrudescent wells (corresponding to a Minimum Inoculum for Resistance (MIR) of 1.4 × 106), with evidence of separate amplification events encompassing the dhodh locus consistent with increased IC50 values . This selection yielded 21 newly emerged coding variants in 10 unique core genes . In addition, parasites from both flasks contained mutations in the PF3D7_0211800 locus, which encodes P. falciparum cytoplasmic asparaginyl tRNA synthetase (PfAsnRS). All four Dd2-OSM-2 clones harboured an R487S change in PfAsnRS, while both Dd2-OSM-3 clones and one of the Dd2-OSM-2 clones had a Copy Number Variant (CNV) across a genomic segment on chromosome 2 that contains PfAsnRS. This CNV has never been reported before nor have SNVs in PfAsnRS. Of interest, the precise boundaries of the CNV region varied between clones, suggesting independent events. The likelihood of missense mutations and CNVs in the same gene by chance is extremely low. Based on our experience with hundreds of selections we hypothesized that PfNT4 is more likely a drug resistance gene and that PfAsnRS is the target.We employed a gradual ramp-up exposure method, which has been reported to increase the success of evolving resistant parasitesPfAsnRS as a potential target, we examined the ability of OSM-S-106 to inhibit protein translation. We employed an in-cell assay of protein translation in P. falciparum trophozoites, monitored by incorporation of a clickable derivative of the puromycin homologue, O-propargyl-puromycin (OPP)15. Following a 6-h exposure, protein translation is inhibited with an IC50 value of 0.51 μM  exposure to WR99210, an inhibitor of PfDHFR, prevented replication into the next cycle, but had no short-term effect on protein translation , that have been shown to be pro-inhibitors of aaRSs6  and the anticipated masses were interrogated. The extract yielded a strong signal for Asn-OSM-S-106, with a precursor ion at m/z 421.0746, retention time at 4.9 min and fragmentation spectrum consistent with that of the synthetic Asn-OSM-S-106 conjugate  and PfGlyRS (PF3D7_1420400), given that we observed production of a low level of adducts of OSM-S-106 with these amino acids. OSM-S-106 contains a sulfonamide group that is predicted to bind tightly to carbonic anhydrase18, suggesting P. falciparum carbonic anhydrase  as another possible target, so, we also knocked down PfCA. Luminescence-based viability assays revealed that knockdown of the cytoplasmic aaRSs perturbs the growth of parasites . Following precipitation of the tRNA and protein, the supernatants were subjected to LC-MS analysis. For PfAsnRS, we detected a peak at m/z 421.0735 with retention time at 7.1 min, consistent with that of the Asn-OSM-S-106 standard , and kindly provided by Dr Steven Langston, Takeda Pharmaceuticals, as a positive control for reaction-hijacking inhibition of aaRSs. When added in the presence of all substrates, AMS inhibits ATP consumption by both PfAsnRS and HsAsnRS; although PfAsnRS appears to be more susceptible  . Here, we generated recombinant CDHsAsnRS and verified that it forms a dimer in solution  involved in ATP binding and dimerization, and a linker domain between motifs II and III 21 . Memberssolution ,H. We sosolution .HsAsnRS was incubated in the presence of Asn and the ATP analogue, AMPPNP. Diffraction quality crystals were obtained; and we solved the structure (refined at 2.2 Å resolution), revealing the presence of Asn-AMP in the active site  that lies between the two beta strands of Motif II 28, was chosen as a suitable template.Our previous studies provided evidence that reaction hijacking involves nucleophilic attack of an aromatic sulfate/sulfonamide on the amino acid charged tRNA product in the enzyme active site. For this reaction to occur, the pro-inhibitor needs to bind into the AMP vacated site. We therefore generated a model of the Asn-tRNA-bound  complex  to enablPfAsnRS shows that the residues around the active site pocket are closely aligned, except that in the PfAsnRS model the terminal CAA of the tRNA acceptor stem clashes with residues in the flipping loop, a region of the protein known to reposition to allow acceptor stem access on tRNA binding 28. Close alignment of residues lining the active site of HsAsnRS and our model provides further confidence in the active site structure of our PfAsnRS model . More recently, some aaRSs were found to be susceptible to reaction hijacking by nucleoside sulfamates, in this case, attacking the enzyme bound charged tRNA and forming an inhibitory adduct with the amino acid. The identification of the Plasmodium-specific pyrazolopyrimidine sulfamate pro-inhibitor, ML901, opened the possibility of developing bespoke pro-inhibitors that target different PfaaRSs.Nucleoside sulfamates are an exciting class of enzyme pro-inhibitors. They have been shown to induce certain ubiquitin activating (E1) enzymes to synthesise potent inhibitory adducts of the nucleoside sulfamate with enzyme-bound ubiquitin-like proteins. This reaction mechanism is powerful and has resulted in multiple new clinical candidates that target E1 enzymes  6, which may limit its oral bioavailability. Here, we explored an aminothienopyrimidine-based sulfonamide, OSM-S-106, which was first identified in a screen of GSK compounds and has since been explored as part of an Open Source Malaria initiative. OSM-S-106 exhibits drug-like properties with a synthetically accessible scaffold and favourable lipophilicity characteristics (AlogP 1.65). OSM-S-106 exhibits good activity against cultures of P. falciparum. Importantly, OSM-S-106 also prevents development of liver stage parasites, suggesting that this class of compound could be used for prophylaxis as well as treatment. One important characteristic of new antimalarial compounds is that they should exhibit a low propensity for resistance. We found that no resistant parasites emerged from an inoculum of 2.4 × 108 exposed parasites, which compares well with other compounds selected for development33. While OSM-S-106 is stable during incubation with human microsomes and rat hepatocytes, it shows rapid intrinsic clearance in mouse microsomes. This complicates studies of pharmacokinetic properties in mice. Here we focused on in vitro analyses.ML901 exhibits excellent potency and selectivity, and effects single-dose cure in a mouse model of PfAsnRS. We showed that transfectants harbouring the PfAsnRSR487S mutation have decreased sensitivity to OSM-S-106, while down-regulation of PfAsnRS enhanced sensitivity, validating PfAsnRS as a target.We used a gradual ramp-up method to evolve resistant parasites with a view to obtaining insights into the target of OSM-S-106. Following two months of selection we retrieved parasites exhibiting four-fold resistance that harboured an R487S mutation or a Copy Number Variant (CNV) in cytoplasmic P. falciparum nucleoside transporter 4 (PfNT4). Moreover, down-regulation of PfNT4 enhanced sensitivity to OSM-S-106. Indeed, enhanced sensitivity was observed even in the presence of anhydrotetracycline (aTC), potentially due to low level down-regulation. A similar base level sensitisation has been observed for a PfHsp70 inhibitor in an aptamer-regulated PfHsp70 line34. PfNT4 is a putative purine transporter that has been shown to be dispensable for blood stage growth but required for sporozoite colonization of salivary glands36. It is possible that PfNT4 transports OSM-S-106 away from its primary site of action and that mutations in PfNT4 enhance the accumulation of OSM-S-106. Further work is needed to test this possibility.Interestingly, some of the parasite clones also exhibited mutations in the 17, providing further evidence that OSM-S-106 exerts its activity by inhibiting tRNA charging. If OSM-S-106 indeed inhibits PfAsnRS via a reaction hijacking mechanism, Asn-OSM-S-106 adducts would be generated in the active site. Using targeted mass spectrometry, we detected a strong signal for Asn-OSM-S-106. Of interest, minor MS peaks were also detected for the adducts of glycine and alanine, when cultures were treated with a high concentration of OSM-S-106 (10 μM). This suggests that PfGlyRS and PfAlaRS, both of which are also class II aaRSs, are susceptible to hijacking by OSM-S-106. Our findings that down-regulation of PfGlyRS and PfAlaRS did not enhance susceptibility to OSM-S-106, and that glycine and alanine adducts were not detected in the extracts with 1 M OSM-S-106 treatment suggest that PfAsnRS is the main target. However, even partial inhibition of PfGlyRS and PfAlaRS may enhance the action of OSM-S-106 and underpin the difficulty of evolving resistance.We showed that treatment of cultures with OSM-S-106 inhibits protein translation and triggers eIF2α phosphorylation, which is diagnostic of the presence of uncharged tRNAPfAsnRS, PfAsnRSR487S and HsAsnRS. ATP consumption by the three enzymes is greatly enhanced by addition of tRNA, consistent with productive aminoacylation. We found that commercially available E. coli tRNA was effective as a substrate for all three enzyme preparations, which facilitated the comparison. These biochemical data suggest that the PfAsnRSR487S mutation does not affect enzyme activity, consistent with the lack of any obvious growth phenotype.We generated recombinant PfAsnRS, but only in the presence of tRNA. This is consistent with the reaction hijacking mechanism. The concentration of OSM-S-106 (~1 μM) needed to induce 50% inhibition of ATP consumption is much higher than the amount needed to kill parasite cultures (~60 nM). This may be due to the fact that, in our biochemical assay, the enzyme first needs to generate the charged tRNA product, which is then attacked by the pro-inhibitor to generate the Asn-OSM-S-106 adduct. Tight binding of the adduct prevents the enzyme from undergoing further catalytic cycles. By contrast, in cells, tRNAs are generally fully loaded37; and may be able to rebind onto the aaRS, which may promote adduct formation. In addition, sulfonamide- and sulfamate-containing compounds are known to bind to red blood cell carbonic anhydrase38 which may facilitate accumulation of OSM-S-106 into parasitised red blood cells.OSM-S-106 inhibited ATP consumption by PfAsnRSR487S is less susceptible to inhibition by OSM-S-106 than wildtype PfAsnRS. It is interesting to consider how this mutation might decrease the sensitivity of the enzyme to hijacking by OSM-S-106. Residue R487 lies in a helix that partially caps the active site; and is close to the tRNA binding site. Of interest, analysis of a hybrid structural mode of tRNA-bound AsnRS from the filarial nematode Brugia malayi24 revealed an important role for the equivalent residue, R425; this residue is involved in a salt-bridge interaction that needs to be broken to allow access of the 3’ end of tRNA to the active site. Thus, the R487S mutation may affect the stability of the complex of PfAsnRS with the Asn-tRNA product, which may in turn affect residence time of the bound Asn-tRNA and therefore susceptibility to reaction hijacking.Recombinant HsAsnRS, and targeted mass spectrometry revealed that HsAsnRS produces very little Asn-OSM-S-106 adduct. A major structural difference between PfAsnRS and HsAsnRS is the presence of a large Plasmodium-specific insert, adjacent to the flipping loop. This flipping loop is known to lock the activated Asn-AMP intermediate in place but to ‘flip’ out of the way to allow the tRNA acceptor stem to insert adjacent to the active site39. The presence of the large insert in PfAsnRS may increase the time the Asn-tRNA product remains bound to the enzyme. This may enable AMP to vacate the active site and OSM-S-106 to bind to the site and mount a nucleophilic attack on the susceptible carbonyl group in Asn-tRNA. By contrast, the Asn-tRNA product may be released more rapidly from HsAsnRS, thus limiting the opportunity for reaction hijacking.OSM-S-106 does not inhibit ATP consumption by HsAsnRS in the presence of ATP and Asn, with a view to capturing the complex with the activated Asn-AMP intermediate were not successful. Therefore, we employed the more slowly hydrolysable ATP analogue, AMPPNP. Diffraction quality crystals were obtained under these conditions. A comparison of the apo and Asn-AMP bound structures reveals stabilisation of two flexible regions. A four amino acid stretch between the beta hairpin and Motif I is stabilised by the formation of a contact between E279 and the Asn part of the ligand. Motif II, which lies further towards the C-terminus, is intersected by a second flexible loop. In the presence of bound Asn-AMP, R322 and E324 in Motif II are stabilised by an interaction with the adenylate part of the ligand, further contributing to binding the activated Asn-AMP intermediate.Initial attempts to crystallize CDHsAsnRS is unable to generate the Asn-OSM-S-106 complex, it is able to bind the synthetic adduct, as evidenced by inhibition of the consumption of ATP in the presence of Asn-OSM-S-106. We generated a high-resolution structure of HsAsnRS in complex with synthetic Asn-OSM-S-106. The OSM-S-106 is positioned in the adenylate binding pocket with the sulfonamide-carbonyl bond overlaying the position of the phosphate-carbonyl bond of Asn-AMP and the sulfamate-carbonyl bond of Asn-AMS. Interestingly, however, the benzine ring of OSM-S-106 lies in the same plane as the thienopyrimidine ring. By contrast, the ribose of the adenylate containing structures is twisted with respect to the nucleoside.While PfAsnRS at high resolution were not successful, possibly due to the presence of the large insert and an extended N-terminal domain. Previous work has shown that these domains are needed for correct folding of PfAsnRS26. We therefore generated a molecular model of PfAsnRS bound to Asn-tRNA, building on structural information for HsAsnRS and EcAspRS/tRNA. The model represents the product-bound form of the enzyme primed for binding of OSM-S-106 or other potential AMP mimics. OSM-S-106 docks into PfAsnRS with the benzene ring twisted toward planarity with the thienopyrimidine ring, in a pose similar to that observed in the crystal structure of HsAsnRS/Asn-OSM-S-106 complex. The position of the sulfonamide nitrogen overlaps with that of the AMP phosphate and the AMS sulfamate nitrogen. Similarly, the amino groups on the pyrimidine rings of OSM-S-106, AMP and AMS all align closely. These docking studies illustrate that potent pro-inhibitors must bind in a manner that precisely positions the reactive sulfonamide to attack the carbonyl carbon of Asn-tRNA.Our attempts to solve the crystal structure of Interrogation of the lower energy docked conformations of the lower potency OSM-S-106 derivatives reveals a failure to position correctly either the sulfonamide nitrogen or the pyrimidine nitrogen. The work provides insights into the very subtle positioning requirements for reaction hijacking to occur; and provides a basis for the design of new compounds with improved activity.PfAsnRS as a P. falciparum aaRS that can be specifically targeted by reaction hijacking; and identifies OSM-S-106 as an exemplar of a new chemical class of species-specific reaction hijacking inhibitor. The ability to selectively hijack particular aaRSs provides a new way to inhibit a class of enzymes that are considered good drug targets in Plasmodium and other infectious organisms41. Our biochemical, structural, and modelling studies reveal the molecular correlates of potent antimalarial activity. This work will help in the development of new, much needed, antimalarial therapies.In summary, this work identifies Supplement 1


Basophils normally make up <2% of the white blood cells (WBC). There is no clear consensus for basophil identification by flow cytometry. The increased demand for basophil activation test (BAT) to identifying and monitoring allergic patients highlights the need for a standardized approach to identify basophils.Using flow cytometry we analyzed whole blood stained with antibodies against: IgE, CD123, CD193, CD203c, CD3, HLADR, FcɛRI, CRTH2 and CD45. We examined unstimulated blood as well as blood stimulated with Anti-IgE and fMLP. Finally, we compared the results to a complete blood count (CBC) from an FDA approved hematological analyzer.Basophil identification relying on just one surface marker performed worse than approaches utilizing two identification markers. The percentage of basophils from WBC determined by flow cytometry results had a good correlation with the CBC results even though the CBC results were generally higher. Stimulating whole blood with the basophil activators did not interfere with the basophil identification markers.negative or FcɛRI performed the best.In flow cytometry assays, two surface markers should be used for identifying basophils and if a very pure basophil fraction is desired a third marker can be considered. In our hands the approaches that included CD123 in combination with either CD193, HLADR Historically basophils have not been given much attention. The rapid development in flow cytometry combined with the discovery that CD63 is translocated to the basophil surface after activation lead to the development of the flow cytometry-based basophil activation test (BAT) and has revitalized the interest in basophils –5. The B/CD203c+ –16. In tAll clinical investigations were conducted according to Declaration of Helsinki principles. All human studies were approved by the Western Institutional Review Board (IRB 1285028). A total of 79 donors, age range 5 to 87, were utilized for the study. The donors were recruited from patients and employes at Amerimmune LLC, CBC was done by Quest Diagnostics using the FDA approved Sysmex XN11 automated hematology analyzer .Whole blood was collected in a heparin and an EDTA tube. The EDTA tube was used for external CBC analysis. The heparin tube was kept at 18–25°C. Basophil identification was done using unstimulated blood (PBS) as well as blood stimulated with either Anti-IgE-FITC  or fMLP . The samples were incubated for 20 min at 37°C followed by 10 min at 4°C , 21–24. The samples were acquired on a 3 laser/10 color BD FACSCanto. CS&T beads  were acquired daily to ensure consistent performance of the cytometer. The instrument has been CAP and CLIA validated for clinical diagnostic studies. All samples were acquired for 5 min at the highest acquisition speed setting.Data analysis was performed using FCS Express software . The gating strategy is to gate on singlets using FSC-A/FSC-H plot. A CD45/SSC plot is used to identify WBC. This is followed by an FSC/SSC gate to gate out eosinophils and majority of neutrophils. The basophils are subsequently identified in 13 different plots: CD123/CD193, IgE/FcɛRI, HLADR/CD123, CD203c/FcɛRI, FcɛRI/CD193, CD3/CD294, CD3/CD193, FcɛRI/CD193, CD123/FcɛRI, IgE/SSC, FcɛRI/SSC, CD193/SSC and CD203c/SSC . The % bt-test to determine significance. A p-value less than 0.05 is considered significant, *p < 0.05, **p < 0.01. Correlation and Bland-Altman analysis and plots were performed using GraphPad Prism .The results show a slight reduction in the % basophils to 89%–92% at Day 1 compared to Day 0. The absolute number of basophils collected dropped to 83%–87% of the value at Day 0. The results are very similar for all the tested gating combination with no method detecting a significant different percentages or absolute number of basophils . Based o− gave a clear separate population in all analysis. CD203c/FcɛRI, CD193/FcɛRI and CD123/FcɛRI worked for all but one donor. In the other end of the spectrum are IgE/SSC, FcɛRI/SSC and CD3−/CDTH2 which frequently could not detect any basophils or often did not give a clear separate population . To easier appreciate the performance of each gating strategy we calculated the average for the R square values for each gating strategy, The average R square ranking are (high to low): IgE/SSC, CD123/CD193, CD123/FcɛRI, CD123/HLADR− and CD203c/CD193 > CD203c/FcɛRI > IgE/FcɛRI > CD203c/SSC, CD193/FcɛRI, CD3−/CRTH2 > CD193/SSC > CD3−/CD193 > FcɛRI/SSC. The graphs are all shown in R square and the slope are summarized in Next, we used linear correlation to see if the % basophils of WBC results are similar using the different gating approaches. The R square value ranges from 0.69 (FcɛRI/SSC vs. FcɛRI/IgE) to 0.99 : CD123/HLADR− > CD193/FcɛRI > CD123/CD193 > CD123/FcɛRI > CD3−/CD193 > CD193/SSC > CD203c/CD193 > IgE/FcɛRI > FcɛRI/SSC > CD3−/CRTH2 > CD203c/FcɛRI > CD203c/SSC.An essential question is if the basophils identified by one gating strategy will also be identified as basophils by the other gating strategies. We addressed this by displaying the basophils obtained by one gating strategy in each of the 12 other strategies and recording the percentages of the cells that were then identified as basophils. The individual box plots are shown in A CBC with differential test is a well-established method that among other results provides the percent basophils of whole blood. For each donor we collected a tube for CBC analysis at an external reference laboratory. The CBC were all run within 24 h of sample collection on an FDA approved hematology analyzer. The linear correlation analysis between the CBC and each of the flow methods show some degree of correlation with an R square value between 0.59 to 0.8. The highest being IgE/SSC, CD193/SSC, CD123/CD193 followed by CD123/FcɛRI, CD123/HLADR- > IgE/FcɛRI, CD203c/FcɛRI > CD203c/CD193 > CD193/FcɛRI > CD203c/SSC > CD3-/CRTH2 > FcɛRI/SSC. The sloop is between 0.74–0.84 indicating that the flow values generally are lower than the CBC values. To better understand the difference between the methods we visualized the data in two different Bland-Altman plots, Difference vs. Average and Ratio vs. Average , 33. TheThe BAT involves stimulating whole blood with positive controls that activates the basophils in both a IgE/FcɛRI dependent and independent manner. The activation of basophils might affect the expression of the identification markers.We stimulated whole blood with Anti-IgE-FITC or fMLP before staining for basophil identification and compared it to unstimulated blood (PBS control) to see if stimulation affects our ability to identify basophils.We observed that fMLP activation decreases CRTH2 expression slightly. Anti-IgE activation reduce the FcɛRI signal. The antibody used for stimulation was the same used for detection which result in an increase in IgE signal after Anti-IgE stimulation. Both anti-IgE and fMLP activation increases CD203c expression. The rest of the markers were unaffected by stimulation .−. The lowest correlation with an R square below 0.9 was seen for FcɛRI/SSC, CD193/SSC, CD193/CD3-, CD294/CD3- and CD203c/CD193  Reliability/easiness of gating, (2) Specificity vs. inclusiveness, (3) Correlation between the different approaches, (4) Correlation with CBC results, (5) Stability of the markers, (6) Effect of stimulation.negative in which case the correlations were low. The single surface markers approaches did not perform well except for IgE/SSC that was among the best. It is worth noting that for 10 of the samples IgE/SSC could not identify any basophils at all. All these patients have elevated levels of circulating IgE that will compete with the surface bound IgE for anti-IgE antibody binding. This approach does not provide us information on the extent the gates captures all the basophils or how basophil specific they are. To understand which strategies are best at capturing the highest percentages of basophils with the least contamination we investigated to what extend a population identified by a given gate would also be identified as basophils using the other gating strategies. The results of this approach are very similar to what we saw in our initial correlation analysis, and it confirms that best gating strategies utilizing two surface markers unless one of the markers is CD3negative. It is important to remember that this form of comparison has a bias towards higher specificity and inclusiveness when the same marker is present in both strategies such as comparing CD123/HLADRnegative vs. CD123/CD193 or IgE/SSC vs. IgE/FcɛRI.All the strategies used in this paper to identify basophils utilizing published markers and marker combinations. We started by examining if we get similar results between the different methods. The linear correlation analysis showed the highest correlation between gating strategies utilizing two surface markers unless one of the markers is CD3R square value at 0.68  post collection , 22. OurA recurring dilemma in designing a BAT flow panel is on its simplicity and cost-effectiveness. We included CD45 because it is essential for a good WBC gate. We would recommend including it rather than relying solely on FSC and SSC to narrow in on the basophil population.−/CRTH2 and CD3−/CD193 performed the worst with respect to inclusiveness and specificity. The IgE/FcɛRI gate has too many instances difficult/impossible to gate. This is typically observed in individuals where the IgE staining does not work. The CD193/FcɛRI approach also have some instances of difficult to gate issues, especially after anti-IgE stimulation, even thou the specificity and inclusiveness is among the best. Not having a well-defined population can cause variation between technologists analyzing the samples and problems if using automatic gating. CD203c expression is low in resting cells and increase after basophil stimulation. This can cause the gate used for identification to shift between samples. Our identification was mainly done on resting cells and for both CD203c/CD193 and CD203c/FcɛRI we saw a high specificity but the inclusiveness we low reflecting that is a problem capturing all the basophils. The remaining three approaches, CD123/CD193; CD123/HLADRnegative; and CD123/FcɛRI all performs as the best in all our tests, and we recommend choosing one of these. If the flow panel allows for one more parameter, it is possible to combine two methods such as CD123/CD193 and CD123/HLADRnegative. All three methods include CD123 as one of the parameters and CD123 expression has been reported to be reduced after stimulation (We do not recommend relying on just one marker for identifying basophils as those strategies did not perform very well in our study. Three of them were not very precise or accurate. IgE/SSC works very well when it does not completely fail which it did more than 13% of the time. Among the gating strategies utilizing two surface markers CD3mulation . This stmulation –39. We d


Gigantidas vrijenhoeki. The G. vrijenhoeki protein was hydrolyzed by various hydrolytic enzymes. The peptic hydrolysate exhibited the highest ACE-inhibitory activity and was fractionated into four molecular weight ranges by ultrafiltration. The <1 kDa fraction exhibited the highest ACE inhibitory activity and was found to have 11 peptide sequences. Among the analyzed peptides, KLLWNGKM exhibited stronger ACE inhibitory activity and an IC50 value of 0.007 μM. To investigate the ACE-inhibitory activity of the analyzed peptides, a molecular docking study was performed. KLLWNGKM exhibited the highest binding energy , which was mainly attributed to the formation of hydrogen bonds with the ACE active pockets, zinc-binding motif, and zinc ion. These results indicate that G. vrijenhoeki-derived peptides can serve as nutritional and pharmacological candidates for controlling blood pressure.The objective of this study was to prepare an angiotensin I-converting enzyme (ACE)-inhibitory peptide from the hydrothermal vent mussel,  Hypertension is a major healthcare concern that increases the risk of death, stroke, myocardial infarction, arteriosclerosis, cerebral hemorrhage, and other vascular diseases and is affected by various factors, such as salt intake, smoking, stress, and obesity ,2. As paSeveral synthetic inhibitors, such as captopril, enalapril, lisinopril, and alacepril, have been developed and used extensively for the management of hypertension and cardiovascular disorders ,9. HowevTakifugu bimaculatus The peptide was chemically synthesized at the peptide synthesizer facility of PepTron Inc. . The peptides were synthesized using the Fmoc solid-phase method with a peptide synthesizer . The synthetic peptides were purified using HPCL  on a Capcell Pak C18 column . The column was developed at a flow rate of 1.0 mL/min by a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid. The identity of synthetic peptides was confirmed by liquid chromatography-mass spectroscopy (LC-MS) , and the purity of the synthetic peptide was confirmed to be over 95%.controlA is the absorbance of the positive control, blankA is the absorbance of the blank containing distilled water, and sampleA is the absorbance of the sample. The IC50 value was determined as the concentration of inhibitor required to inhibit 50% of the ACE activity.ACE inhibitory activity was measured using the Dojindo ACE Kit-WET kit , according to the manufacturer’s instructions. The ACE-inhibitory activity was calculated as follows:https://www.rcsb.org/, accessed on 11 October 2022). The structures of the 11 peptides derived from GVH were drawn using the CDOCKER tool. Docking of bioactive peptides to ACE was performed using the Lib Dock tool in Discovery Studio 2022 .The molecular docking analysis was performed according to the method described by Kang et al.  with slip < 0.05, ** p < 0.01.All quantitative data are presented as means ± standard deviation and represent at least three individual experiments conducted using fresh reagents. Statistical comparisons of the mean values were performed using analysis of variance (ANOVA) followed by Duncan’s multiple range test using SPSS software v29. Differences in mean values were considered statistically significant at * G. vrijenhoeki protein was hydrolyzed using alcalase, neutrase, flavourzyme, protamex, pepsin, trypsin, α-chymotrypsin, bromelain, and papain, and their evaluated ACE inhibitory activity. Among enzymatic hydrolysates, peptic hydrolysate showed the highest ACE inhibitory activity compared to other hydrolysates. Subsequently, the peptic hydrolysate was fractionated by ultrafiltration and their fractions significantly improved the ACE inhibitory activity compared to hydrolysate. The low molecular weight fraction (>1 kDa) showed the highest ACE inhibitory activity and identified eleven ACE inhibitory peptides. Among the identified peptides, GVP-10 (KLLWNGKM) exhibited the strongest ACE inhibitory activity with an IC50 value of 0.007 μM. Molecular docking studies indicated that GVP-10 was able to bind to residues in the ACE-active pockets (S1 and S’2), interact with zinc-binding motifs, and coordinate with zinc ions. Based on these results, we propose that the ACE-inhibitory peptide isolated from the G. vrijenhoeki protein has a beneficial effect in regulating blood pressure.In this study,