Veterinary drugs are widely used in animals to prevent diseases and are a complex set of drugs with very different chemical properties. Multiclass and multi-residue methods for simultaneous detection of residues from veterinary drugs and contaminants in urine are very rare or non-existent. Therefore, the aim of this study was to develop and validate a sensitive and reliable quantitative LC-MS/MS method for simultaneous determination of a wide range of veterinary drug and pesticide residues and mycotoxins in bovine urine. This involved 42 veterinary drug residues , 28 pesticides and 2 mycotoxins. Stable isotopically labelled internal standards were used to facilitate effective quantification of the analytes. Analysis was performed in both positive and negative ionization modes with multiple reaction monitoring transitions over a period of 12 min.2) from 0.991 to 0.999. The LODs were from 0.01 to 2.71 µg/L, while the LOQs were from 0.05 to 7.52 µg/L. The CCα and CCβ were in range 0.05–12.11 µg/L and 0.08–15.16 µg/L. In addition, the average recoveries of the spiked urine samples were from 71.0 to 117.0% and coefficient of variation (CV) < 21.38% (intraday and interday).The parameters validated included linearity, limit of detection (LOD), limit of quantification (LOQ), detection capability (CCβ), decision limit (CCα), stability, accuracy and precision. The process followed guidelines of the regulation 2021/808/EC. The calibration curves were linear with coefficient of correlation (RA new isotopic LC-MS/MS method has been developed, validated and applied for identification and quantification of 72 residues of veterinary drugs and pesticides and other contaminants such as mycotoxins in bovine urine. The most appropriated sample preparation procedures involved sodium acetate buffer, enzymatic hydrolysis using β-glucuronidase and cleanup solid phase extraction with OASIS SPE cartridges. The parameters were satisfactorily validated fulfilling requirements under Regulation 2021/808/EC. Consequently, the method could be used in routine analysis of bovine urine samples for simultaneous detection of veterinary drug and pesticide residues as well as contaminants such as mycotoxins. Veterinary drugs are widely used in livestock production to prevent or treat diseases. While in some countries certain veterinary drugs such as thyreostats, anabolic hormones and β-agonists may be used as a growth-promoting agents . This isAnimals are also often simultaneously exposed to other hazards such as mycotoxins pesticides or heavy metals, and since these may also end up in food consumption, multi-residue and multi-toxin exposure studies are therefore very relevant to public health. Appropriate analytical methods are therefore required . The detThe establishment of sensitive analytical methods to detect residues and contaminants in food from animal origin as well as live animals is important for safety and public health. Currently, more sensitive methods for the determination of residues and contaminants in food of animal origin have been developed, but there are few multi-residue and multiclass analytical methods for simultaneous detection of residues of veterinary drugs and other hazards such us pesticides and mycotoxins. Zhan et al., 2013) , develop , develoMulticlass and multi-residue methods for simultaneous detection of residues from veterinary drugs and contaminants in urine are very rare or non-existent. A range of methods for residues of veterinary drugs and contaminants have been published from different authors although these involve single class or groups –22. StanThe objective of this study was to develop and validate a sensitive and reliable quantitative LC-MS/MS method for simultaneous determination of a wide range of veterinary drug and pesticide residues and mycotoxins in bovine urine. This involved 72 analyses including 42 veterinary drug residues , 28 pesticides and 2 mycotoxins. Analysis was performed in both positive and negative ionization modes with multiple reaction monitoring transitions over a period of 12 min.Six extraction protocols for effective and rugged multiresidue extraction of 72 compounds from urine were investigated. Four of the protocols followed solid phase extraction (two with and two without enzymatic hydrolysis), while the other two protocol involved usage of liquid-liquid extraction . The parameters validated included: limit of detection (LOD), limit of quantification (LOQ), decision limit (CCα), detection capability (CCβ), linearity, accuracy and precision. The criteria prescribed in Regulation 2021/808/EC were followed .Methanol, acetonitrile and water (LC-MS/MS grade), ethyl acetate, dichloromethane, ammonium hydroxide, acetic acid, ammonium acetate (HPLC grade) were purchased from Carlo Erba Reagent S.A.S ; formic acid (LC-MS/MS grade), sodium acetate (p.a.), sodium dihydrogen phosphate hydrate (p.a.), disodium hydrogen phosphate dihydrate (p.a.), sodium chloride (p.a.), β-glucuronidase aryl sulfatase from Helix pomatia and trichloroacetic acid (≥ 99.5%), Discovery® DSC-MCAX cartridges (300 mg/6 ml) were from Merck  and Oasis HLB cartridges (500 mg/6ml) were from Waters .Thyreostats: thiouracil (100%), propylthiouracil (99.6%), methylthiouracil (≥ 98.0%) and tapazol (99.7%) were purchased from Sigma-Aldrich ; Anabolic steroids: 19 nortestosterone (99.8%), clostebol (99.1%), boldenone (99.1%), methyltestosterone (99.5%) and testosterone (100.0%) were purchased from Sigma-Aldrich , while stanozolol (99.8%) were obtained from Dr. Ehrenstorfer GmbH ; Lactones of resorcylic acid: taleranol (99.5%) was purchased from Sigma-Aldrich , while zeranol (99.9%) was obtained from Dr. Ehrenstorfer GmbH ; β-agonists: clenbuterol HCl (99.1%), isoxsuprine HCl (100%), salbutamol (99.4%), zilpaterol HCl (96.0%), ractopamine HCl (95.5%) and terbutaline hemisulfate salt (100.0%) were purchased from Sigma-Aldrich , while brombuterol (98.0%), mabuterol HCl (98.0%), cimbuterol (98.0%) and clenpenterol HCl (98.0%) were obtained from Witega ; Antimicrobials: Amoxicillin (99.6%), ampicillin (99.8%), benzylpenicillin (99.3%), cloxacillin (98.7%), oxacillin (98.4%), lincomycin (100.3%), tylosin (87.9%), trimethoprim (99.5%), tetracycline (96.8%) and cephapirin (98.5%) were purchased from Sigma-Aldrich  and the rest of antimicrobials: ceftiofur (98.01%), cephalexin (96.6%), oxytetracycline (96.5%), enrofloxacin (99.74%), ciprofloxacin (98.0%), sulfadimidine (99.6%), sulfamethoxazole (99.7%), sulfadiazine (99.8%), sulfachloropyridazine (99.1%) and sulfadimethoxine (99.7%) were obtained from Dr. Ehrenstorfer GmbH ; Pesticides: carbofuran (99.9%), carbaryl (99.9%), parathion (99.7%), malathion (99.2%), diazinon (98.3%), dimethoate (99.8%), atrazine (99.5%), cypermethrin (98.4%), permethrin (98.1%), deltamethrin (99.9%), coumaphos (99.7%), dicholphos (99.8%), chlorpyrifos (99.8%), boscalid (99.5%), fentoate (98.8%), fenthion (98.5%), fenvalerate (99.4%), monocrotophos (99.8%), malaoxon (99.0%), methamidophos (98.1%), metacrifos (96.1%), amitraz (99.8%), omethoate (98.4%), vamidothion (≥ 98.0%), phosmet (99.8%), heptenophos (98.7%), bifenthrin (99.0%), methomyl (99.0%) were purchased from Sigma-Aldrich ; Mycotoxins: Ochratoxin A (≥ 98.0%) and zearalenone (99.0%) were obtained from Trilogy Analytical Laboratory, Inc. .The isotopic labelled internal standards used were: anabolic steroid: 19 − 17 β Nortestosteron-d3 was obtained from the RIVM, Netherlands; lactones of resocylic acid: β-zearalenol-d4 (≥ 98.0%) was obtained from Toronto Research Chemicals Inc. ; β agonists: Clenbuterol-d6 HCl (98.0%), brombuterol-d9 HCl (98.0%), mabuterol-d9 HCl (98.0%), clenpenterol-d5 HCl (98.0%) and cimbuterol-d9 (98.0%) obtained from Witega , isoxsuprine-d5 hemifumarate (≥ 98.0%) and ractopamine-d6 HCl (≥ 98.0%) from the European reference laboratory (EURL) at RIKILT, The Netherlands, salbutamol -d9 (≥ 98.0%) from Dr. Ehrenstorfer GmbH , zilpaterol–d7 (≥ 98.0%) was obtained from Toronto Research Chemicals Inc. , while terbutaline-d9 acetate hemihydrate (99.3%) was obtained from Sigma-Aldrich ; antimicrobials: flunixin–d3 100.0%), penicillin G-d7 N-ethylpiperidinium (98.1%) salt and pesticides: atrazine-d5 (99.7%), chlorpyrifos-d10 (100%) and carbofuran-d3 (99.3%) were obtained from Sigma-Aldrich  0.0%, pen.To prepare the stock standards solutions, 5 to 10 mg of each standard and internal standard was weighed and transferred to a 10 ml volumetric flask which contained methanol. The concentration of individual stock solutions was in range from 0.5 mg/ml to 1.0 mg/ml. The solutions were stored at -20 °C.A total of five mixed working solutions were prepared in methanol from the standards stock solutions. According to the values for minimum method performance requirements (MMPRs) the standards and internal standards were divided in groups . The staThe mixed working solutions from internal standards were prepared in methanol from the internal standards stock solutions. The internal standards were also divided in two groups, the first one  consisted of: 19 − 17β Nortestosteron-d3, isoxsuprine-d5, ractopamine-d6, salbutamol-d9, zilpaterol-d7, terbutaline-d9, Clenbuterol-d6, brombuterol-d9, mabuterol-d9, clenpenterol-d5, cimbuterol-d9 and β-zearalenol-d4. The concentration of the mixed internal standard working solution was 10 µg/ml, from which additionally, for spiking of the samples and preparation of matrix match calibration curve, was prepared mixed internal standard working solution with concentration from 1000 ng/ml. The second group  consisted of: flunixin–d3, penicillin G-d7, atrazine-d5, chlorpyrifos-d10 and carbofuran-d3. The concentration of this mixed internal standard working solution for spiking of the samples and preparation of matrix match calibration curve was 10 µg/ml.Matrix-matched calibration prepared in blank bovine urine and internal standard were utilized for quantification to compensate the matrix effects that influence analytical response. Due to aspects of availability, cost, and convenience the use of internal standards was feasible only for β-agonists, 7 antibiotics, 3 pesticides, 1 anabolic steroid and 1 mycotoxin (Table Six extraction protocols during the optimization of the extraction method were investigated for extraction of the 72 compounds from urine. In two extraction protocols liquid-liquid extraction (LLE) was applied, while solid phase extraction (SPE) was applied to the remaining four protocols. This characterization was as follows: LLE without enzymatic hydrolysis (protocol 1); LLE with enzymatic hydrolysis (protocol 2); SPE using OASIS HLB cartridges without enzymatic hydrolysis (protocol 3); SPE using OASIS HLB cartridges with enzymatic hydrolysis (protocol 4); SPE using DSC-MCAX cartridges without enzymatic hydrolysis (protocol 5) and lastly SPE using DSC-MCAX cartridges with enzymatic hydrolysis (protocol 6). Enzymatic hydrolysis was performed with β-glucuronidase aryl sulfatase (Helix pomatia). Sample preparation steps for all protocols are given below. The initial preparation of the samples prior to extraction, as well as the spiking of urine samples with standards and internal standards are the same for all protocols.In the first step, 30 ml urine sample was centrifuged 5 min, at 2000 rpm and room temperature. Тhis step was used to remove proteins from the matrix. After centrifugation, 5 ml of urine sample was fortified with the standards and internal standards. Prior to extraction the samples were left to stand for 10 min. Next, the samples were prepared with different protocols, as follows.5 ml of 0.2 M sodium acetate buffer (pH = 5) and 5 ml 0.02 M Phosphate buffer (PBS) (pH = 7.2)  were added to the samples, and the samples were shaken for 1 min on a vortex. After this step samples were centrifuged 5 min, at 2000 rpm, at room temperature followed by LLE. 10 ml methanol:acetonitrile:acetic acid  was added and the samples were shaken for 1 min on a vortex and centrifuged again for 5 min, at 2000 rpm and room temperature and the supernatant was transferred to new test tubes. Тhe LLE was repeated with 10 ml of ethyl acetate: hexane . The samples were shaken for 1 min on a vortex and centrifuged 5 min, at 2000 rpm and room temperature. Тhe supernatant was collected, added to the first supernatant and the mixture was evaporated under nitrogen to near dryness at 35 °C. The residue was reconstituted with 1 ml of the mobile phase , filtered through a 0.45 μm membrane filter into 2 ml autosampler vials and analysed on the LC-MS/MS after separation on a reverse phase column.5 ml of 0.2 M sodium acetate buffer (pH = 5) and 5 ml 0.02 M phosphate buffer (PBS) (pH = 7.2)  were added to the samples, and the samples were shaken for 1 min on a vortex. Further, 20 µl of β-glucuronidase aryl sulfatase was added and the samples were incubated for 17 h at 37 °C. After cooling to room temperature, the samples were centrifuged for 5 min, at 2000 rpm and room temperature, followed by LLE. The LLE and the next steps for sample preparation were the same as in protocol 1.5 ml of 0.2 M sodium acetate buffer (pH = 5) and 5 ml 0.02 M phosphate buffer  were added and the samples were shaken for 1 min on a vortex before spinning of a centrifuge for 5 min at 2000 rpm and room temperature. This was followed SPE extraction. Oasis HLB cartridges were activated and conditioned with 5 ml of methanol and 5 ml of water. The extract was passed through the cartridges at one drop per second and the cartridge dried, washed with 5 ml of water and dried again. The residues were eluted first with 4 ml of methanol:acetonitrile:ammonium hydroxide  and then with 4 ml of methanol: dichloromethane . The eluent was evaporated to dryness at 35 °C under nitrogen, the residues were reconstituted with 1 ml of the mobile phase  pressed through 0.45 μm membrane filter into 2 ml autosampler vials prior to LC–MS/MS analysis after separation on a C-18 column.5 ml of 0.2 M sodium acetate buffer (pH = 5) and 5 ml 0.02 M Phosphate buffer  were added and the samples were shaken for 1 min on a vortex before spinning of a centrifuge for 5 min at 2000 rpm and room temperature. In the next step, βeta-glucuronidase aryl sulfatase (20 µl) was added and the samples incubated for 17 h at 37 °C followed after cooling, by centrifugation for 5 min at 2000 rpm and room temperature. Further, the same approach was applied as well as in protocol 3.In this protocol, DSC-MCAX cartridges instead of Oasis HLB cartridges were used and the rest of the conditions remaining the same as in the protocol 3.In this protocol, Oasis HLB cartridges were replaced by DSC-MCAX cartridges and the rest of the conditions remaining the same as in the protocol 6.The sample preparation steps for all protocols are given in the Table The LC-MS/MS  used for identification and quantification of the analytes was equipped with a binary pump, vacuum degasser, thermostated autosampler, thermostated column manager. A Kinetex C18 LC column  was used for chromatographic separation. MassLynx version 4.1 software  was used for instrument control, data acquisition and processing of results.The LC conditions were as follow: mobile phase flow rate: 0.2 ml/min; column temperature: 40 °C, elution program: 0–1 min, 95 − 80% A; 1–4 min, 80 − 60% A; 4–8 min, 60–95% A; 8–12 min, 95% A; mobile phase A contains: water with 5 mM ammonium acetate, 0.01% formic acid and 0.01% trichloroacetic acid; mobile phase B contains: methanol with 0.1% formic acid, oven temperature: 4 °C; injection volume: 5 µl. The MS/MS conditions were optimized as follows: capillary voltage of 3.0 kV; source temperature of 150 °C; desolvation temperature of 400 °C; cone gas at 100 L/h; desolvation gas at 300 L/h. Both positive and negative electrospray ionization were used along with multiple reaction monitoring (MRM) .Validation of the method was performed according to the European Commission Regulation 2021/808 . The par2) was calculated by least squares linear regression analysis.Matrix-matched calibration curves were prepared to determine the linearity. The blank urine samples were fortified at six concentration levels . The calibration curves were prepared every day with each series of analyzed samples, each concentration level performed injected in triplicate. The coefficient of correlation (RDecision limit (CCα). The CCα means the concentration of target analyte at and above which it can be concluded with an error probability of α (α = 1%) that a sample is non-compliant and the value 1 – α means statistical certainty in percentage that the permitted limit has been exceeded. CCα was calculated according to 2021/808/EC .Detection capability (CCβ). The CCβ means the smallest concentration of the target analyte that may be detected or quantified in a sample with an error probability of β (β = 5%). CCβ was calculated according to 2021/808/EC .The LOD and LOQ were determined as the lowest spiked concentration evaluated that gave a signal to noise (S/N) ratio of 3:1 for LOD and 10:1 for LOQ –30.r) and coefficient of variation for reproducibility (CVR)) of precision were estimated with spiking of the urine samples with standards and internal standards at the same concentration levels as well as recovery. The spiking levels are given in Tables The intraday  and interday  recovery values were calculated at three spiking levels , the samples were fortified according to the sensitivity of the method) , as well as selection of appropriate diagnostic ions are summarized in Table The recovery test results for the different sample preparation protocols are summarized in Table 2) from 0.991 for methylthiouracil and fenvalerate to 0.999 for sulfadimidine, phosmet and ochratoxin A. The range of calibration curve and R2 values for all compounds are presented in Table Very good linearity was attained with coefficient of correlation R from 0.9The results for LOD, LOQ, CCα and CCβ are shown in the Table Recovery, repeatability (intraday) and reproducibility (interday) were used for evaluation of the accuracy of the method. The intraday recovery range was from 71.0% for mabuterol spiked at 0.1 µg/l to 117% for cimbuterol at 0.1 µg/L, while the interday recovery range was from 76.2% to 19 nortestosterone spiked at 0.25 µg/L to 109.3% for coumaphos spiked at 7.5 µg/ L. The CV for repeatability ranged from 1.69% for tetracycline to 16.76% for sulfadiazine, while the CV for reproducibility was from 5.65% for tetracycline to 21.38% for methomyl. The results are summarized in Tables Stock standards of the anabolic hormones, lactones, beta agonists, thyreostats, mycotoxins and most antibiotics  were stable for 6 months at -20 °C, while pesticide standards were stable for 1 year, and amoxicillin, ampicillin, benzylpenicillin, cloxacillin as well as oxacillin were stable for 3 months. Working standard solutions were stable for 1 month when kept at -20 °C. The standards in the final extract were stable 3 days.The testing of forbidden substances in media such as urine is an important task of regulatory authorities and laboratories. In the European Union, the use of antimicrobials, thyreostats, anabolic hormones and β-agonist drugs for fattening slaughter animals has been banned since 1981 under Council Directive 81/602/EEC . For proUrine is widely used to monitor the illegal use of growth-promoting agents and veterinary drugs with a good number of these substances showing high clearance rates in urine –35. Whil2 cartridges for analysis of anabolic steroids [Six extraction protocols were investigated. In the first and second protocols involving LLE, thyreostats were not detected due to poor recovery. The results were comparable with previous studies by Kellman et al. (2009) and Eeckhaut et al. (2009) who concluded that while the LLE is simpler and easier than SPE, matrix (urine) interferences lead to low extraction efficiency hence the need for SPE , 40. Gómsteroids , 38, 41.Furthermore, Ho et al. (2006), used C8-SCX mixed-mode cartridge for analysis of anabolic steroids, corticosteroids and acidic drugs, while Leon et al. (2012), used Oasis HLB cartridges in a method for 87 analytes in different families of banned or unauthorized substances , 43. In A total of 83 bovine urine samples were collected from local farms and tested using the developed and validated method during 2021–2022. In five samples of urine, methyl- and propylthiouracil were found. No other veterinary drug residues and contaminants were found. Methylthiouracil (veterinary drug) residue amount was found between 1.08 and 1.31 µg/L in 5 samples and propylthiouracil (veterinary drug) amount was found to be 1.67–2.63 µg/L in 4 samples. In Fig. A new isotopic LC-MS/MS method has been developed, validated and applied for identification and quantification of 72 residues of veterinary drugs and pesticides and other contaminants such as mycotoxins in bovine urine. The most appropriated sample preparation procedures involved sodium acetate buffer, enzymatic hydrolysis using β-glucuronidase and cleanup solid phase extraction with OASIS SPE cartridges. The parameters were satisfactorily validated fulfilling requirements under Regulation 2021/808/EC. Consequently, the method could be used in routine analysis of bovine urine samples for simultaneous detection of veterinary drug and pesticide residues as well as contaminants such as mycotoxins.


V2.2) are predominantly localized in presynaptic terminals, and are particularly important for pain transmission in the spinal cord. Furthermore, they have multiple isoforms, conferred by alternatively spliced or cassette exons, which are differentially expressed. Here, we have examined alternatively spliced exon47 variants that encode a long or short C-terminus in human CaV2.2. In the Ensembl database, all short exon47-containing transcripts were associated with the absence of exon18a, therefore, we also examined the effect of inclusion or absence of exon18a, combinatorially with the exon47 splice variants. We found that long exon47, only in the additional presence of exon18a, results in CaV2.2 currents that have a 3.6-fold greater maximum conductance than the other three combinations. In contrast, cell-surface expression of CaV2.2 in both tsA-201 cells and hippocampal neurons is increased ∼4-fold by long exon47, relative to short exon47, in either the presence or the absence of exon18a. This surprising discrepancy between trafficking and function indicates that cell-surface expression is enhanced by long exon47, independently of exon18a. However, in the presence of long exon47, exon18a mediates an additional permissive effect on CaV2.2 gating. We also investigated the single-nucleotide polymorphism in exon47 that has been linked to schizophrenia and Parkinson’s disease, which we found is only non-synonymous in the short exon47 C-terminal isoform, resulting in two minor alleles. This study highlights the importance of investigating the combinatorial effects of exon inclusion, rather than each in isolation, in order to increase our understanding of calcium channel function.N-type calcium channels (Ca  V) channels that provide Ca2+ for vesicular release from presynaptic terminals. The particular channels involved are predominantly CaV2.1 and CaV2.2.2 These channels are associated with auxiliary α2δ and β subunits, which optimize their trafficking and function.Synaptic transmission in the central and peripheral nervous system is dependent on voltage-gated calcium  analysis of Parkinson’s disease and schizophrenia, including a single-nucleotide polymorphism (SNP), Rs2278973, in CACNA1B.16 However, upon analysis, we identified that this SNP was only non-synonymous in an alternatively spliced exon47, which encodes a shorter C-terminal sequence than long exon47 (g exon47  and B. Tg exon47 .Ensembl17) of the short exon47 splice variant that all reported human CaV2.2 sequences containing this short exon47 variant were also associated with absence of cassette exon18a  were plated onto cell culture flasks or coverslips coated in poly-l-lysine, and cultured in Dulbecco’s Modified Eagle Medium supplemented with fetal bovine serum (5%), penicillin–streptomycin (1%), and GlutaMAX , in a 5% CO2 incubator at 37°C.Hippocampal neurons: Tissue was obtained from P0/P1 rat pups . All experiments were performed in accordance with the UK Home Office Animals (Scientific procedures) Act 1986, with UCL ethical approval. Briefly, hippocampi were dissected, chopped into small pieces, and treated for 40 min at 37°C with a papain solution containing 7 units/mL of papain, 0.2 mg/mL l-cysteine, 0.2 mg/mL bovine serum albumin (BSA), 1000 units/mL DNase-1, and 5 mg/mL glucose  in Hank’s basal salt solution medium (Thermo Fisher Scientific). Hippocampi were then washed with inactivation medium  and mechanically dissociated in serum medium . After centrifugation at 1000 x g for 10 min, cells were resuspended in serum medium and seeded onto coverslips precoated with poly-d-lysine hydrobromide . One hour later, cells were covered with serum-free neuronal plating medium comprising Neurobasal Medium, supplemented with B27 (2%) and GlutaMAX (1%) . Neurons were kept in a 5% CO2 incubator at 37°C and half the medium was replaced every 3-4 days. At 7 days in vitro and 2 h before transfection, half of the medium was removed, and fresh medium was added.V2.2 [huCaV2.2 (pSAD442-1) was a gift from Diane Lipscombe ], from which were derived GFP_CaV2.2-HA-C-long, GFP_CaV2.2-HA-C-short-Arg, GFP_CaV2.2-HA-C-long(Δexon18a), GFP_CaV2.2-HA-C-short(Δexon18a), GFP_CaV2.2-HA-C-short-Leu, and GFP_CaV2.2-HA-C-short-His. Other constructs used were β1b ,19 α2δ-1 , and the transfection markers, mCherry 20 and CD8 21 where stated. A double-HA tag was added to an extracellular loop in domain II of the human CaV2.2 in the same position as used previously in the rabbit CaV2.2  and GFP was added to the N-terminus. All the constructs were verified by Sanger sequencing (Source Bioscience) and have been submitted to Addgene. All cDNAs were used in either pcDNA3 vector for expression in tsA-201 cells or pCAGGS for hippocampal neurons.The following expression constructs were used: human CaAn anti-hemagglutinin  was used as primary antibody. For immunoblotting, secondary Goat Anti-Rat Horseradish Peroxidase  was used. For immunocytochemistry, secondary Anti-Rat Alexa Fluor 594 and Anti-Rat Alexa Fluor 647 antibodies  were used to visualize the HA-tagged channels.tsA-201 cells: The tsA-201 cells were transfected with PolyJet in a 3:1 ratio with DNA mix, according to the manufacturer’s protocol. Following incubation overnight at 37°C, culture medium was changed. The transfection mix consisted of plasmids containing cDNAs encoding each of the GFP_CaV2.2-HA variants, β1b, and α2δ-1 in a ratio of 3:2:2. For electrophysiological experiments, CD8 was used as a transfection marker (ratio 3:2:2:1).Hippocampal neurons: The transfection mix consisted of plasmids containing cDNAs encoding the GFP_CaV2.2-HA variants, β1b, α2δ-1, and mCherry in a ratio of 3:2:2:0.3. Neurons were transfected with Lipofectamine 2000 (Life Technology), according to the manufacturer’s protocol with 1 μL Lipofectamine and 2 μL DNA mix per sample.V2.2 currents in transfected tsA-201 cells were investigated by whole cell patch clamp recording. The patch pipette solution contained in m m: Cs-aspartate, 140; EGTA, 5; MgCl2, 2; CaCl2, 0.1; K2ATP, 2; HEPES, 10; pH 7.2, 310 mOsm with sucrose. The external solution for recording Ba2+ currents contained in m m: tetraethylammonium (TEA) Br, 160; KCl, 3; NaHCO3, 1.0; MgCl2, 1.0; HEPES, 10; glucose, 4; BaCl2, 1, pH 7.4, 320 mosM with sucrose. One millimolar extracellular Ba2+ was the charge carrier. Pipettes of resistance 2–4 MΩ were used. An Axopatch 1D or Axon 200B amplifier was used, and whole cell voltage-clamp recordings were sampled at 10 kHz frequency, filtered at 2 kHz, and digitized at 1 kHz; 70%-80% series resistance compensation was applied and all recorded currents were leak-subtracted using P/8 protocol. Analysis was performed using Pclamp 9 (Molecular Devices) and Origin 7 . To obtain the maximum conductance (Gmax), current–voltage (IV) relationships were fitted by a modified Boltzmann equation as follows: I = Gmax*(V − Vrev)//k)), where I is the current density (in pA/pF), Gmax is in nS/pF, Vrev is the apparent reversal potential, V50, act is the midpoint voltage for current activation, and k is the slope factor.Cag at 4°C for 10 min. Cell pellets were homogenized in PBS containing 1% Igepal, 0.1% SDS and protease inhibitors , pH 7.4, and then incubated on ice for 30 min to allow cell lysis. Whole cell lysates (WCLs) were centrifuged at 20 000 × g for 25 min at 4°C and supernatants were assayed for total protein . Aliquots of WCL  were diluted with 2× Laemmli buffer supplemented with 100 m m dithiothreitol and incubated at 60°C for 10 min.22 Samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 3%-8% Tris–Acetate gels (Thermo Fisher Scientific) and transferred to polyvinylidene fluoride membranes (BioRad). Membranes were blocked with 3% BSA, 0.5% Igepal in Tris-buffered saline  for 30 min at room temperature and then incubated overnight at 4°C with anti-HA  antibody in TBS containing 10% goat serum, 3% BSA, and 0.02% Igepal (pH 7.4). After washing in TBS containing 0.5% Igepal, membranes were incubated with the secondary antibody, Goat Anti-Rat HRP  for 1 h at room temperature. The signal was obtained using the enhanced chemiluminescence (ECL) 2 assay (Thermo Scientific) and membranes were scanned on a Typhoon 9410 phosphorimager .Immunoblotting was carried out on tsA-201 cells expressing the cDNAs as described. At 48 h after transfection, cells were rinsed with phosphate buffered saline , scraped into cold PBS, and centrifuged at 1000 × m 4′,6′-diamidino-2-phenylindole (DAPI), and mounted in VectaShield (Vector Laboratories). Imaging was performed on Zeiss LSM 780 confocal microscope.Cells were fixed with 4% paraformaldehyde in PBS for 5 min, incubated with blocking buffer  for 1 h at room temperature before being incubated with rat anti-HA (Roche) diluted either 1:250 (tsA-201cells) or 1:100  in 0.5× blocking buffer for 1 h at room temperature (tsA-201 cells) or at 4°C overnight . After washing, samples were incubated with secondary antibodies, anti-rat Alexa Fluor 594 (tsA cells) or anti-rat Alexa Fluor 647  at a dilution of 1:500 for 1 h at room temperature. Coverslips were washed, stained with 0.5 µThe tsA-201 cell images were obtained using a 63× objective at a resolution of 1024  ×  1024 pixels and an optical section of 0.5 μm. After choosing a region of interest containing transfected cells, the 3 × 3 tile function of the microscope was used to allow a larger area to be selected without bias. Images were then analyzed using Fiji. Every transfected cell was included. Surface labeling (HA staining) was measured using a freehand line tool of 5 pixels (0.65 μm) width and manually tracing the surface of the cells. Intracellular GFP staining was measured by drawing around the cell (omitting the nucleus and the plasma membrane). The value of the mean pixel intensity in different channels was measured separately and background was subtracted by measuring the intensity of an imaged area without transfected cells.Hippocampal neurons were imaged using a 20× objective with a 5-μm optical section. The fluorescence intensity along neuronal projections was assessed as follows: 2 concentric circles of 100 μm and 150 μm diameter were drawn around each neuronal cell body. A freehand line tool of 3 μm width tracing was used to draw, between the circles, the neuronal dendrites (3 to 5 averaged per neuron) and axons  in the mCherry images, which was then used as template for GFP and HA images .Hippocampal somata were imaged at 63× objective with a 1-μm optical section. For each neuron selected as positive for mCherry, immunofluorescence (HA and GFP) was measured by two ROIs, one ROI manually traced around the cell body, using a freehand line tool (0.4 μm width) to analyze the cell-surface staining and another ROI drawn between the cell surface and the nucleus to determine the intracellular staining. Total staining was calculated per each cell combining the 2 ROIs. The mean pixel intensity in the different channels were measured and the background was subtracted from each image.post hoc test for multiple comparisons or two-way ANOVA, followed by Bonferroni’s post hoc test for multiple comparisons, as stated.Quantitative data are presented by GraphPad Prism 8 software  or Origin-Pro 2021 as the mean ± standard error of the mean (SEM) and individual data points as described. Statistical significances were determined by either one-way analysis of variance (ANOVA) followed by Šidák’s V2.2 containing the following alternatively spliced exons: +exon 10a, +exon18a, Δexon24a, +exon31a, +exon37b, and + long exon47.10 We added an extracellular HA tag (2 HA sequences separated by a Gly residue) in an extracellular linker of domain II, in the corresponding position to that in the rabbit CaV2.2 we have used previously,6 and a GFP tag to the N terminus24 , compared to the absence of exon18a (V2.2-HA-C-short (containing short exon47), the inclusion of exon18a produced no increase in calcium currents  variant was increased by 4.5-fold compared with GFP_CaV2.2-HA-C-short (+exon18a) (V2.2 variants. Cell-surface expression of the GFP_CaV2.2-HA-C-long (Δexon18a) variant was still increased by 3.2-fold compared with GFP_CaV2.2-HA-C-short (Δexon18a) (In order to determine whether the marked increase in Caexon18a) . Howeverexon18a) , deletioexon18a) .V2.2-HA, as determined by GFP signal, associated with the presence of C-long exon47, compared to C-short exon47 (1.7-fold in the presence of exon18a and 1.2-fold in its absence)  . From th exon18a .V2.2-HA in tsA-201 cells but that trafficking to the plasma membrane is highly influenced by the presence of the long exon47 variant in the C-terminus. This is borne out by 2-way ANOVA analysis of the source of variation, showing there was no significant interaction between the presence of long versus short exon47 and the presence or absence of exon18a  variant was increased by 4.3-fold compared with GFP_CaV2.2-HA-C-short (+exon18a)  . For theP signal . In contP signal . However exon18a . The 2-wV2.2 in the dendrites relative to the short exon47 variant  in total CaP signal . The pre variant . Howeverendrites . Similarendrites .V2.2. In the short exon47 variant, however, this SNP results in three alternative amino acids, Arg, Leu, or His, at this position. Unlike the reference gene, Ensembl ENSG00000148408 (NCBI Reference Sequence: NM_000718.4), which would produce a Leu residue at this position, the Addgene plasmid #62574 that we used produces an Arg. Arg has a frequency of 91.7% in the population and, from comparison with other species, appears to be the ancestral gene (gnomAD genomes v3.1.2 database (Ensembl) https://www.ensembl.org/Homo_sapiens/Variation/Population?db=core;r=9:138121310-138122310;v=rs2278973;vdb=variation;vf=729552325).The Rs2278973 SNP gives no change in amino acid in the long exon47 variant of CaV2.2-HA-C-short . Intracellular GFP expression of GFP_CaV2.2-HA-C-long was increased by a smaller amount (1.7-fold) compared to all C-short variants , together with β1b and α2δ-1, were then expressed in tsA-201 cells, and CaV2.2 currents were recorded to examine whether there was any effect of the different SNP variants  are important in primary afferent transmission, including at nociceptor terminals in the dorsal horn. For this reason, they are a drug target in pain therapy.14,V channels and their auxiliary subunits, like most proteins, have multiple isoforms, conferred by one or more alternatively spliced or cassette exons, each of which may be differentially expressed in diverse tissues and developmental stages.29 It is therefore essential to understand which combinations of exons are actually included in transcripts in different conditions and tissues,31 and to examine their combined functional effects. For example, for CACNA1C encoding CaV1.2, a large number of novel full length transcript isoforms, many of which are predicted to be functional, were recently identified by long-range sequencing in human brain.32N-type calcium channels , compared to when exon18a is skipped, or compared to CaV2.2 containing short exon47. In contrast, for CaV2.2 containing short exon47, the inclusion of exon18a has no effect on calcium current amplitude. Surprisingly, this result is not mirrored by a similar pattern of changes in cell-surface expression. Here, the presence of long exon47 supports an ∼4-fold increase in CaV2.2 cell-surface trafficking in both tsA-201 cells and in hippocampal neurons, compared to CaV2.2 containing short exon47, irrespective of the presence or absence of exon18a. In parallel, there is an ∼2-fold increase in the full-length CaV2.2 protein level, conferred by long exon47, as determined by immunoblotting .23 All constructs in our study contained the most common isoform, exon37b. Of further relevance to the present study, it has also been found previously that protein–protein interaction motifs in the long C-terminal human CaV2.2 splice variant contain sequences, including a C-terminal postsynaptic density protein, Drosophila disc large tumor suppressor, and Zonula occludens-1 protein (PDZ) motif  binding domain  any specific effect to enhance synaptic targeting in neurons. It is therefore likely that other proteins are involved in the increase in CaV2.2 cell-surface expression. We have previously shown that increased cell-surface trafficking of exon37a-containing CaV2.2 requires α2δ,23 possibly because α2δ allows trafficking through the Golgi apparatus,46 where interaction with the adaptor protein (AP1) complex may occur.23 It will be important to examine whether the same is true for CaV2.2 containing long exon47.With respect to the mechanism of increased CaV2.2 function, expression, and trafficking of the SNP variants in short exon47, which encode three different amino acids. This indicates that the observed association of these variants with Parkinson’s disease and schizophrenia16 is not mediated by any grossly altered function. Instead, it is possible that the SNP variants might influence the splicing of exon47, and therefore the relative inclusion of the short and long exon47 variants in CaV2.2 transcripts. In this regard, it is of great interest that CaV2.2 channels play an important role in mediating dopamine release in striatum.47In this study, we observed no effect on CaFuture studies will be needed to determine the relative expression of the long and short exon47-containing isoforms in different human tissues and disease states, and whether the SNP alleles influence these properties.zqad060_Supplemental_FilesClick here for additional data file.


However, their molecular mode-of-action is not fully understood. Here, we report the discovery of LEI-102, a CB2R agonist, used in conjunction with three other CBR ligands  to investigate the selective CB2R activation by binding kinetics, site-directed mutagenesis, and cryo-EM studies. We identify key residues for CB2R activation. Highly lipophilic HU308 and the endocannabinoids, but not the more polar LEI-102, APD371, and CP55,940, reach the binding pocket through a membrane channel in TM1-TM7. Favorable physico-chemical properties of LEI-102 enable oral efficacy in a chemotherapy-induced nephropathy model. This study delineates the molecular mechanism of CB2R activation by selective agonists and highlights the role of lipophilicity in CB2R engagement. This may have implications for GPCR drug design and sheds light on their activation by endogenous ligands.Cannabinoid CB 2 receptor (CB2R) agonists are investigated as therapeutic agents in the clinic. Here, authors report the discovery of LEI-102, a CB2R agonist, used in conjunction with three other CBR ligands  to investigate selective CB2R activation.Cannabinoid CB Cannabis sativa have been used for centuries in the treatment of various diseases, including cancer and neuropathic pain1. The synthetic version of its psychoactive constituent, Δ9-tetrahydrocannabinol , which have 68% sequence identity in their seven transmembrane (TM) domains11. Both receptors are activated by the endogenous signaling lipids anandamide (AEA) and 2-arachidonoylglycerol (2-AG)  is responsible for the psychotropic side effects of THC14. It plays a role in memory, learning, neurogenesis, neuronal migration, and synaptogenesis. Furthermore, its presence in many organ tissues belies more non-neurological functions15. The CB2R is mainly found on the cells of the immune system and is upregulated under pathophysiological conditions17. Its activation in general is associated with anti-inflammatory responses in tissue injury of the liver, heart, kidney, colon, and brain as determined in various preclinical models22. Based on preclinical studies, it is thought that selective CB2R agonists may retain and exceed certain therapeutic properties of THC without inducing psychotropic side effects23.THC exerts its therapeutic effects mostly via the G protein-coupled receptors (GPCRs) cannabinoid CB2R ligands24. HU308  of HU308, which has a calculated logarithm of octanol-water partition coefficient (cLogP) of 8.025, and its analogs prevented the successful clinical translation of this class of cannabinoid-based drugs.Various academic and industrial groups have developed selective CB308 Fig.  was the 2R ligands was developed with improved drug-like properties. For instance, Olorinab®  and the binding modes of diverse ligands and their activation mechanism were reported35. Remarkably, those structures revealed that CB1R and CB2R possess a highly similar, lipophilic orthosteric agonist binding pocket, which makes it challenging to explain the selective activation of CB2R. To date, no structural studies with selective CB2R agonists have been reported that could aid in understanding the molecular basis of CB2R selectivity.Recently, three-dimensional structures of the CB2R agonist with good physico-chemical and biological properties. LEI-102 is used in conjunction with CB2R selective agonists APD371 and HU308, and non-selective agonist CP55,940 to investigate the activation mechanism of CB2R. For this study, we combine ligand-target binding kinetics, site-directed mutagenesis, and cryo-EM methods. We find that CB2R has a distinct activation mechanism compared to CB1R. Additionally, we find that the physico-chemical properties of the ligands influence their entry pathway into the receptor. Highly lipophilic ligands, such as HU308 and the endocannabinoids, may reach the binding pocket through the membrane, whereas more polar ligands, such as LEI-102, APD371 and CP55,940, enter the receptor via an alternative route. Furthermore, we show that the favorable physico-chemical properties of LEI-102 and CB2R selectivity underscore its promising in vivo efficacy via oral administration in a chemotherapy-induced nephropathy model without inducing CNS-mediated side effects. Together, these studies enhance our insights into how certain physico-chemical properties of ligands translate to in vivo activity and changes their engagement to GPCRs.Here, we present the discovery of LEI-102, a potent and selective CB2R agonist with beneficial physico-chemical properties, LEI-102, a pyridinylbenzylimidazolidine-2,4-dione derivative, was designed and synthesized , potency (pEC50) and intrinsic activity (Emax) of LEI-102 were determined in [3H]RO6957022 displacement assays on stably expressing CB2R membranes and [35S]GTPγS G protein activation assays using HEK293T membranes transiently expressing recombinant hCB2R or hCB1R, respectively (Supplementary Table 2R (pKi = 8.0 ± 0.1) and was more potent than the selective CB2R agonists APD371 and HU308. LEI-102 did not bind CB1R, thereby showing at least 1000-fold selectivity (Supplementary Table max 76 ± 1%) with a pEC50 value of 6.9 ± 0.2  of all agonists and converted these into a residence time (RT). LEI-102 had a RT of 16 min, which was around half that of APD371 (45 min) and CP55,940 (32 min), whereas HU308 had the longest RT at the receptor of 71 min (Supplementary Table kon) varied greatly between the different agonists, ranking from fast to slow engagement CP55,940 > LEI-102 > 2-AG > APD371 > HU308 = AEA. The calculated engagement time (ET) to CB2R at 1 µM of each agonist further emphasized that CP55,940 arrived at CB2R within one second, whereas APD371, LEI-102, and 2-AG needed between 16 and 40 s to reach the CB2R binding site. Interestingly, HU308 and AEA took 143 and 152 s to bind CB2R, respectively. In view of the distinct target-binding kinetic profiles of the four synthetic CB2R agonists, we decided to elucidate their binding poses in CB2R using cryo-EM method.To quantify the ligand-target binding kinetic parameters of the agonists in more detail, we performed displacement and competition association assays with GTPγS functional assay, thereby allowing us to study the receptor activation mechanism  receptor . Next, we tested all four synthetic agonists and the two endocannabinoids on these four CB2R mutant receptors in [3H]CP55,940 and [35S]GTPγS assays. Only CB2R-L185ECL2H and CB2R-QuadrupleTM1,7 were evaluated in the [3H]CP55,940 displacement assays due to insufficient binding window for the other two mutant receptors (data not shown). The binding affinities of the agonists were not affected for mutant receptors CB2R-L185ECL2H and CB2R-QuadrupleTM1,7  induced marked elevations of serum creatinine and blood urea nitrogen levels  72 h following cisplatin injection in wild type mice compared with vehicle-treated control animals. LEI-102 showed a dose-dependent attenuation of the functional markers of cisplatin-induced kidney injury both when administered p.o.  or i.p.  did not produce any effects in the tetrad assay as compared with vehicle. There were no effects on nociceptive behavior assessed in tail withdrawal test nor on body temperature . Hence, the CB2R agonist LEI-102 maintained its selectivity over CB1R in vivo.To determine whether LEI-102 maintained its selectivity for CB38. In this study, we aimed to generate a better understanding of the binding and activation mechanism of CB2R-selective agonists. Therefore, we combined ligand-target binding kinetics, site-directed mutagenesis, and cryo-EM studies to investigate the activation mechanism of CB2R for the introduced CB2R selective agonist LEI-102 supplemented with agonists APD371, HU308, and CP55,940 on a molecular level. Furthermore, we investigated potential hotspots for CB2R/CB1R selectivity by creating swap mutants and discovered a ligand entry pathway for CBR agonists and endocannabinoids.So far, several crystal and cryo-EM CBR structures have been resolved in which non-selective agonists adopt a nearly identical binding position in the orthosteric pocket, regardless of the receptor2R-F1173.36 as replacement by alanine resulted in a complete loss of G protein activation by all tested agonists  and CB1R‑H270ECL2 (CB2R-L185)32. We observed improved binding of [3H]CP55,940 for LEI-102 and HU308 with the CB1R-H270ECL2L mutation, while the non-selective agonists showed no change  and cannabinoid receptors, might gain access to the binding pocket through lateral diffusion via a membrane channel between TM1 and TM751. We experimentally examined this membrane entry pathway by creating a CB2R quadruple mutant  for which we observed a significant loss of potency and a corresponding trend in reduced affinity, although not significant, for HU308 and the endocannabinoids  polar interaction network with H952.65 to activate CB2R, which is not observed for HU308. This flexibility of the CB2R binding pocket to be activated by a diverse set of chemotypes allows to select for a chemotype with more drug-like properties. This notion is supported by the oral efficacy of LEI-102 in the chemotherapy-induced nephropathy model and lack of CNS-adverse side effects  was custom synthesized at F. Hoffman-La Roche Ltd . [35S]GTPγS (specific activity 1250 Ci mmol−1 #NEG030H250UC), [3H]CP55,940 (specific activity 108.5 Ci mmol−1 #NET1051250UC) and GF/C filter plates (#6055690) were purchased from PerkinElmer . CP55,940 (#C1112), AM630 (#SML0327) and DL-dithiotreitol  were obtained from Sigma-Aldrich, HU308 (#H800010) was from LKT Laboratories , APD371 was provided by F. Hoffmann-La Roche Ltd, anandamide , 2-Arachidonylglycerol  and phenylmethylsulfonyl fluoride  were purchased from Tocris Bioscience  and GDP (#J61646) was from Thermo Fisher Scientific . All buffers and solutions were prepared using Millipore water (deionized using a MilliQ A10 Biocel with a 0.22 µm filter) and analytical grade reagents and solvents. Buffers are prepared at room temperature (RT) and stored at 4 °C, unless stated otherwise.Monoclonal M2 mouse anti-FLAG primary antibody (#F3165) was purchased from Sigma-Aldrich , while secondary goat anti-mouse HRP-conjugated antibody (#115-035-003) was bought from Jackson ImmunoResearch Laboratories . Bicinchoninic acid (BCA) ad BCA protein assay reagent was obtained from Pierce Chemical Company . +, 327.08 [M + H]+ (Br). 1H-NMR  δ 7.47 , 7.14 , 4.52 , 3.74 , 3.33 , 2.15–2.04 , 0.91 . 13C NMR  δ 169.57, 156.78, 134.41, 131.79, 129.48, 121.77, 60.01, 48.61, 45.98, 45.71, 28.57, 19.70.To solution of 1-(4-bromobenzyl)imidazolidine-2,4-dione  in anhydrous DMF (18 mL) were subsequently added K2  was added and the mixture was stirred at 75 °C for 20 h. The mixture was cooled to rt, diluted with EtOAc (100 mL) and water (10 mL) and the layers were separated. The water layer was extracted thrice with EtOAc (3 × 20 mL). The combined organic layers were extracted with sat. aqueous NaHCO3, water and brine, dried (MgSO4), filtered, and concentrated under reduced pressure. The raw product was co-evaporated with CHCl3 and used in the next step without further purification.A mixture of 1-(4-bromobenzyl)-3-isobutylimidazolidine-2,4-dione , KOAc  and bis(pinacolato)diboron  in DMF (10 mL) was sonicated for 15 min under argon flow. Subsequently, Pd(dppf)ClN-((6-bromo-3-fluoropyridin-2-yl)methyl)tetrahydro-2H-pyran-4-amine , 3-isobutyl-1-benzyl)imidazolidine-2,4-dione  and K2CO3  in toluene:ethanol  was added under argon atmosphere Pd(PPh3)4 . The resulting mixture was stirred for 18 h at 75 °C, subsequently cooled to rt, and filtered. The filtrate was diluted with EtOAc and washed with water and brine, dried (MgSO4), filtered, and concentrated under reduced pressure. The crude was purified with flash column chromatography (0‑20% MeOH in EtOAc) to yield 0.24 g of a white solid . Further purification with preparative HPLC resulted in a yield of 0.204 g . LCMS : tr = 5.32 min, m/z: 455.27 [M + H]+, 908.93 [2 M + H]+. HRMS (ESI+) m/z: calcd. for C25H32FN4O3 [M + H], 455.245; found, 455.245. 1H NMR  δ 8.05 , 7.86 , 7.61 , 7.34 , 4.53 , 4.45 , 3.93 , 3.77 , 3.47 , 3.30 , 3.24 , 2.05 , 1.99 , 1.83 , 0.88 . 13C NMR  δ 171.54, 157.25 , 156.12, 153.03 , 140.51 , 138.83, 137.70, 129.13, 128.22, 125.56 , 122.81 , 118.38, 66.55, 55.55, 50.30, 46.81 , 42.95, 30.02, 28.32, 20.32.To a degassed mixture of 2R construction and co-expression of G protein for cryo-EM study were performed using the similar procedure as described before34. In brief, the WT human CB2R was modified to contain a fusion protein BRIL to improve the protein expression and thermostability, along with a 10×His-tag and a FLAG-tag at the N-terminal. The CB2R, Gαi1 and Gβ1γ2 subunits were cloned into the pFastBac vector separately using cloning kits.The N-BRIL fused wild type (WT) human CB34. The CB2R and Gi heterotrimer were co-expressed in Sf 9 insect cells using the Bac-to-Bac Baculovirus Expression System (Invitrogen). Cells were infected with three separate virus preparations for CB2R, Gαi1 and Gβ1γ2 at a ratio of 1:2:2 at a cell density of 2.5 × 106 cells/mL. After 48 h, the cell culture was collected by centrifugation and the cell pellets were stored at −80 °C until use. The cell pellets were thawed and lysed in the hypotonic buffer of 10 mM HEPES (pH 7.5), 10 mM MgCl2, 20 mM KCl with EDTA-free complete protease inhibitor cocktail tablets . The CB2R-Gi complex was formed in membranes by addition of 25 μM agonist  and 2 units of apyrase  in the presence 500 µg scFv16. The lysate was incubated for overnight at 4 °C and discard the supernatant by centrifugation at 186,000 × g for 30 min. Subsequently, the solubilization buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 0.75% (w/v) lauryl maltose neopentyl glycol , 0.15% (w/v) cholesterol hemisucinate  supplemented with 25 μM agonist and 2 units of apyrase (NEB) were added to solubilize complexes for 2 h at 4 °C. Insoluble material was removed by centrifugation at 186,000 × g for 30 min and the supernatant was immobilized by batch binding to TALON IMAC resin  including 20 mM imidazole over 6 h at 4 °C. Then, the resin was packed and washed with 15 column volumes (CVs) of washing buffer I containing 25 mM HEPES (pH 7.5), 100 mM NaCl, 10% (v/v) glycerol, 0.1% (w/v) LMNG, 0.02% (w/v) CHS, 30 mM imidazole and 20 μM agonist, and 15 CVs of washing buffer II containing 25 mM HEPES (pH 7.5), 100 mM NaCl, 10% (v/v) glycerol, 0.03% (w/v) LMNG, 0.006% (w/v) CHS, 50 mM imidazole and 20 μM agonist. After that, the protein was eluted using 3 CVs of elution buffer containing 25 mM HEPES (pH 7.5), 100 mM NaCl, 10% (v/v) glycerol, 0.01% (w/v) LMNG, 0.002% (w/v) CHS, 250 mM imidazole and 25 μM agonist. Finally, the complex was concentrated using the centrifugal filter with 100 kDa molecular weight cutoff and loaded onto a Superdex200 10/300 GL column  with buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 0.00075% (w/v) LMNG, 0.00025% GDN , 0.0001% (w/v) CHS, 100 μM TCEP. The fractions consisting of purified CB2R-Gi complex were collected and concentrated to 0.8–1.0 mg/mL for electron microscopy experiments.Methods of complex expression and purification in the current study have been described previously2R-Gi complexes, 3 μL of the concentrated protein was loaded to a glow-discharged holey carbon grid , and subsequently were plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). The chamber of Vitrobot was set to 100% humidity at 4 °C. The sample was blotted for 2.5 s with blot force 2. Cryo-EM images were collected on a Titan Krios microscope operated at 300 kV equipped with a Gatan Quantum energy filter, with a slit width of 20 eV, a Gatan K2 summit direct electron camera (Gatan). Images were taken at a dose rate of 8e−/Å2/s with a defocus range of −0.8 to −2.0 μm using SerialEM software64 in EFTEM nanoprobe mode, with 50 μm C2 aperture, at a calibrated magnification of 130,000 corresponding to a magnified pixel size of 1.04 Å. The total exposure time was 8.1 s and 45 frames were recorded per micrograph.For cryo-EM grids preparation of the CB65. For CB2R-Gαi-scFv16-APD371/LEI-102/HU308/CP55,940 dataset, a total of 7443, 5282, 7530, and 6473 movies were collected, respectively. For all datasets, patch motion correction was used for beam-induced motion correction. Contrast transfer function (CTF) parameters for each micrograph were determined by patch CTF estimation. Using Blob Picker in CryoSPARC to auto pick particles in the first 500 micrographs of CB2R-Gαi-scFv16-APD371 complex dataset and then 258,347 particles were extracted to conduct 2D classification. 9277 particles in good 2D patterns were selected as templates to pick better particles. 5,239,870, 3,398,611, 4,653,294, and 3,595,875 particles extracted, respectively, in a 256 Å box were divided into three hundred two-dimensional (2D) class averages with a maximum alignment resolution of 6 Å. Then, 1,152,146, 762,471, 355,832, and 440,292 particles were selected from good 2D classification after two round 2D classification, individually. Following 2D classification, these particles were subjected for ab initio reconstruction into four classes. After heterogeneous refinement, homogeneous refinement, non-uniform refinement and local refinement of the best-looking dataset in CryoSPARC, the final map has an indicated global resolution of 3.08, 2.98, 2.97, and 2.84 Å at a Fourier shell correlation (FSC) of 0.143, respectively. Local resolution was determined using the Bsoft package with half maps as input maps66.The cryo-EM data processing was performed with CryoSPARC2R-Gi-scFv16 complex, the CB2R-AM12033 cryo-EM structure and Gi protein in CB2R were used as the starting model. The model was docked into the EM density map using Chimera67, followed by iterative manual adjustment and rebuilding in COOT68 and phenix.real_space_refine in Phenix69. The model statistics were validated using MolProbity70. Structural figures were prepared in Chimera and PyMOL (http://www.pymol.org). The final refinement statistics were provided in Supplementary Table For CB1R and CB2R genes were subcloned into vector pcDNA3.1 with an N-terminal HA signal peptide and FLAG-tag. Mutations were introduced by QuikChange PCR (as described by supplier).The WT CB2R or hCB1R receptor, or mutant receptor using the calcium phosphate precipitation method71. In short, a DNA-calcium mix was made containing 270 mM CaCl2 and 10 µg plasmid DNA to which Hank’s Balanced Salt Solution  was added in a 1:1 (v/v) ratio and mixed by aeration to create consistent calcium phosphate precipitates. For transfection, 1 mL DNA-calcium mix was added per 10 cm ø plate, followed by a 48 h incubation under a humidified atmosphere at 37 °C with 5% CO2.24 h prior to transfection, HEK293T cells were seeded on 10 cm ø plates to reach approximately 50% confluence at the start of transfection. The cells were transfected with 10 µg plasmid DNA of WT hCB2. After an additional 24 h, cells were washed with PBS and fixed with 4% formaldehyde for 10 min at room temperature (rt). Cells were washed twice with Tris-buffered saline (TBS) and were blocked with TBS supplemented with 0.1% TWEEN 20 (TBST) and 2% BSA (w/v)) for 30 min at rt while shaking. Subsequently, the cells were incubated with monoclonal M2 mouse anti-FLAG primary antibody (1:4000) for 2 h at rt while shaking. After removal of the antibody, the cells were washed three times with TBST and incubated with the secondary goat anti-mouse HRP-conjugated antibody  for 1 h at rt while shaking. After a final wash with TBS, the cells were treated with 3,3’,5,5’-Tetramethylbenzidine  in the dark for maximally 10 min at rt to visualize immunoreactivity. The reaction was quenched with 1 M H3PO4, and absorbance was read at 450 nm with a Wallac EnVision 2104 Multilabel reader (PerkinElmer).Receptor expression after transfection was measured in an enzyme-linked immunosorbent assay (ELISA). After 24 h of transfection, HEK293T cells were detached with phosphate-buffered saline (PBS)/EDTA and seeded into a sterile 96-well poly-D-lysine coated plate at a density of 100,000 cells per well and kept under a humidified atmosphere at 37 °C with 5% COg for 5 min. Pellets were resuspended in ice-cold Tris buffer  and homogenized with an Ultra Turrax homogenizer . Cytosolic and membrane fractions were separated using a high-speed centrifugation step of 31,000 rpm in a Beckman Optima LE-80K ultracentrifuge with Ti70 Rotor for 20 min at 4 °C. After a second cycle of homogenization and centrifugation, the final pellets were resuspended in 50 mM Tris-HCl pH 7.4, 5 mM MgCl2 and stored in 100 µL aliquots at −80 °C until use. CHOK1_hCB2bgal cells were harvested when reaching 90% confluence in 15 cm ø plates after one week subculture at a 1:6 ratio. Membrane preparation followed a similar procedure as described above. Final membrane pellets were resuspended in 50 mM Tris-HCl pH 7.4 and stored in 100 µL aliquots at −80 °C until use. Membrane protein concentrations were determined using a BCA protein determination assay as described by the manufacturer72.For membrane preparation, HEK293T cells were harvested 48 h after transfection. Cells were detached by scraping into 3 mL of PBS and subsequently centrifuged at 2000 × 2R, [3H]RO6957022 competition association assays were executed. These assays were previously described with the main difference of incubation at 25 °C compared to 10 °C for identification of more distinct kinetic differences73. In short, prior to kinetic assessment of agonist binding, the affinity (IC50) of the agonists at the hCB2R was determined in [3H]RO6957022 displacement assays. CHOK1_hCB2bgal were thawed, homogenized, and subsequently diluted to 1 µg protein per well. When studying endocannabinoids, membranes were preincubated with 50 µM PMSF for 30 min. Membranes were incubated with ~1.5 nM [3H]RO6957022 and six increasing concentrations of competing agonists in a total volume of 100 µL assay buffer (50 mM Tris-HCl (pH 7.4), 0.1% (w/v) BSA). Incubations were done for 2 h at 10 °C to reach equilibrium. Subsequently, in competition association assays, agonists were incubated at their IC50 concentration in the presence of ~1.5 nM [3H]RO6957022 in a total volume of 100 µL assay buffer at 10 °C. Competition was initiated by addition of membrane homogenates at different time points for 2 h. Nonspecific binding (NSB) was determined with 10 µM AM630 and organic solvent (DMSO or acetonitrile) concentrations were <1% in all samples. Total radioligand binding (TB) did not exceed 10% of the amount added to prevent ligand depletion. Incubations were terminated by rapid vacuum filtration with ice-cold 50 mM Tris-HCl (pH 7.4), 0.1% (w/v) BSA buffer through Whatman GF/C filters using a Filtermate 96-well harvester (PerkinElmer). Filters were dried for at least 30 min at 55 °C, and subsequently 25 µL MicroScint scintillation cocktail (PerkinElmer #6013621) was added per well. Filter-bound radioactivity was measured by scintillation spectrometry using a Microbeta2 2450 counter (PerkinElmer).For assessment of kinetic agonist binding at hCB35S]GTPγS to the cannabinoid receptors as previously described25. In short, transient HEK293T membrane homogenates (10 µg/well) were diluted in assay buffer (50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 150 mM NaCl, 1 mM EDTA, 0.05% BSA (w/v) and 1 mM DTT, freshly prepared every day) and were pretreated with 10 µg saponin and 1 µM GDP. For endocannabinoid samples, the membranes were additionally pretreated for 30 min with 50 µM PMSF before agonist addition. To determine the G protein activation, the membranes were incubated with 10 µM or six increasing concentrations of agonist (ranging from 0.01 nM to 10 µM) for 30 min at rt. Basal receptor activity was determined in the presence of vehicle only (0.2% DMSO/acetonitrile). [35S]GTPγS (0.3 nM) was added and the mixture was co-incubated for an additional 90 min at 25 °C while shaking at 400 rpm. Filtration was performed, and filter-bound radioactivity was determined as described under [3H]RO6957022 Competition Association Assays except for using ice-cold 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2 buffer.G protein activation by agonists LEI-102, APD371, HU308, CP55,940 AEA, and 2-AG was measured by binding of radiolabeled [i) on WT and mutant receptors was determined in [3H]CP55,940 displacement assays. The amount of transient HEK293T membrane, ranging from 0.75 µg to 10 µg protein per well, was chosen to obtain a specific [3H]CP55,940 binding window of 1200-1500 disintegrations per minute (dpm) except for the CB2R-QuadrupleTM1,7 mutant, for which a window of ~500 dpm could be obtained using 20 µg protein per well. Membranes were thawed and subsequently homogenized using the Ultra Turrax homogenizer. For the endocannabinoid assays, the membranes were preincubated for 30 min with 50 µM PMSF. Homologous displacement assays were performed with 1.5 nM final concentration [3H]CP55,940 and when necessary supplemented with an additional concentration of 0.55 nM [3H]CP55,940 in the presence of competing CP55,940 (ranging from 0.01 nM to 1 µM) in assay buffer (50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 0.1% (w/v) BSA). Heterologous displacement assays were executed for LEI-102, APD371, HU308, AEA, and 2-AG using 1.5 nM final concentration [3H]CP55,940 with one concentration (10 µM) or six increasing concentrations (ranging from 0.1 nM to 10 µM) in assay buffer. For both assays, binding was initiated by addition of membrane homogenates to reach a final volume of 100 µL. NSB was determined using 10 µM CP55,940 and organic solvent (DMSO or acetonitrile) concentrations were <1% in all samples. TB did not exceed 10% of the amount added to prevent ligand depletion. Incubation was done for 2 h at 25 °C to reach equilibrium. Filtration was performed, and filter-bound radioactivity was determined as described under [3H]RO6957022 Competition Association Assays except for using ice-cold 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 0.1% (w/v) BSA buffer.Agonist affinity . CB2R knockout mice (CB2R−/−) and their wild-type littermates (CB2R+/+) were developed as described previously and had been backcrossed to a C57BL/6J background74. All animals were kept in a temperature-controlled environment (20–22 °C) with a 12 h light–dark cycle and were always allowed free access to food and water. All animal experiments reported in this manuscript complied with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” (NIH publication 86–23 revised 1985) and were approved by the Institutional Animal Care and Use Committee of the National Institute on Alcohol Abuse and Alcoholism .Ten to twelve-week-old male/female 63. Mice (CB2R−/− and CB2R+/+) were sacrificed 72 h after a single injection of cisplatin (cis-diamine platinum (II) dichloride (Sigma#P4394) 25 mg/kg i.p.; freshly dissolved in physiological saline) by cervical dislocation under deep anesthesia with 5% isoflurane, for collection of blood and tissue samples. LEI-102 was given i.p. or by oral gavage (p.o.) at 0.3, 3.0, and 10 mg/kg every day, starting 1.5 h before the cisplatin exposure. The drug was dissolved in a vehicle of DMSO:Tween 80:saline, 1:1:18. After administration of LEI‐102, mice were killed by cervical dislocation under deep anesthesia with 5% isoflurane, for collection of blood and tissue samples at the time described in the figure. The tetrad assay in mice has previously been described in detail29.The well-established model of cisplatin-induced nephropathy was used63. Tubular damage scores were determined based on the percentage of tubules showing epithelial necrosis where 0= normal; 1, <10%; 2, 10–25%; 3, 26–75%; 4, >75%. Tubular necrosis was defined as the loss of the proximal tubular brush border, blebbing of apical membranes, tubular epithelial cell detachment from the basement membrane, or intraluminal aggregation of cells and proteins. The morphometric examination was performed in a blinded manner. Ten fields were scored from each mouse kidneys at 200× magnification, and average scores were determined for each mouse. For final quantification graph, average tubular damage scores of six mice/group were plotted.Markers of kidney dysfunction (BUN and CREA), histopathology (PAS staining), immunostaining or ELISA for 3-nitrotyrosine  and 4-hydroxynonenal , and real-time PCR  for inflammatory cytokines were performed as previously describedAll experimental data were analyzed using GraphPad Prism 9.0 . All values obtained are means ± standard error of the mean (SEM) of at least three independent experiments performed in duplicate, unless stated otherwise.3H]RO6957022 competition association assays, the kon and koff were determined by non-linear regression analysis, using the “kinetics of competitive binding” model as described by Motulsky and Mahan :L] is the radioligand concentration per experiment (~1.5 nM), I is the IC50 concentration of agonist (nM), X is the time (s), and Y is the specific binding of the radioligand (dpm). Ka and Kb are the observed association rate constants (kobs) of the radioligand and the agonist of interest, respectively. k1 and k3 are the association rate constants (kon in M−1s−1) of [3H]RO6957022 (determined per experiment) and the agonist of interest, respectively. Similarly, k2 and k4 are the dissociation rate constants (koff in s−1) of [3H]RO6957022  and the agonist of interest, respectively. The engagement time (ET in seconds) of the agonists of interest was determined at 1 µM of agonist using the equation ET = 1/(kon · 1 × 10−6). The residence time (RT in min) was calculated using the equation RT = 1/(60 · koff)75. The association and dissociation rate constants were used to calculate the kinetic KD using: KD = koff/kon.From [35S]GTPγS agonist responses on hCB2R constructs were baseline-corrected for the individual mutant’s basal activity. The responses were normalized to the basal activity of the construct (0%) and top of the CP55,940 (for WT responses only) or WT curve . The potency (pEC50) and efficacy (Emax) values were obtained by non-linear regression to a sigmoidal concentration-effect curve with a Hill slope of 1 by using the “log(agonist) vs response (three parameters)” model. [35S]GTPγS data from hCB1R constructs were expressed as fold over the mutant’s basal activity to also quantify the effects of CB2R selective agonists.[D) of [3H]CP55,940 on different mutants were calculated from homologous displacements by non-linear regression analysis, using the “one-site homologous” model. The half-maximal inhibitory concentrations (pIC50) of the agonists in [3H]CP55,940 and [3H]RO6957022 assays were obtained by non-linear regression analysis of the homologous and heterologous displacement curves and further converted into inhibitory constant pKi using the Cheng-Prusoff equation76. In which the experimentally determined KD for each construct was used for [3H]CP55,940 assays or 0.78 nM for [3H]RO6957022 assays (data not shown).Displacement assays were baseline-corrected with NSB and normalized to this value (0%) and TB (100%). The equilibrium dissociation constants  followed by Tukey’s post hoc test for multiple comparisons or t-test if appropriate. The analysis was conducted using GraphPad Prism 9 software. p  <  0.05 was considered statistically significant.Differences in pECFurther information on research design is available in the Supplementary InformationReporting Summary


Mycoplasma pneumoniae  is an intracellular pathogen that causes pneumonia, tracheobronchitis, pharyngitis, and asthma in humans and can infect and survive in the host cells leading to excessive immune responses. Extracellular vesicles (EVs) from host cells carry components of pathogens to recipient cells and play a role in intercellular communication during infection. However, there is limited knowledge on whether EVs derived from M. pneumoniae-infected macrophages play as intercellular messengers and functional mechanisms. In this study, we establish a cell model of M. pneumoniae-infected macrophages that continuously secrete EVs to further asses their role as intercellular messengers and their functional mechanisms. Based on this model, we determined a method for isolating the pure EVs from M. pneumoniae-infected macrophages, which employs a sequence of operations, including differential centrifugation, filtering, and ultracentrifugation. We identified EVs and their purity using multiple methods, including electron microscopy, nanoparticle tracking analysis, Western blot, bacteria culture, and nucleic acid detection. EVs from M. pneumoniae-infected macrophages are pure, with a 30–200 nm diameter. These EVs can be taken up by uninfected macrophages and induce the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 through the nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPK) signals pathway. Moreover, the expression of inflammatory cytokines induced by EVs relies on TLR2-NF-κB/JNK signal pathways. These findings will help us better understand a persistent inflammatory response and cell-to-cell immune modulation in the context of M. pneumoniae infection. Mycoplasma pneumoniae  is a human pathogen that can cause upper and lower respiratory tract infections    was cultured at 37 °C with 5% CO2 in pleuropneumonia-like organism (PPLO) broth  containing 20% horse serum , 0.5% glucose, 800 U/mL penicillin, 0.2% sodium pyruvate , and 1% phenol red at pH 7.6 for at least seven days meeting. Color-changing units (CCU) (medium color changing from red to yellowish orange) were used to count M. pneumoniae [M. pneumoniae media at 1:10 with gradual dilution to ten tubes. The tenth tube was used as a negative control (tube without a test solution). The last tube of the medium was then used as the CCU of mycoplasma to determine the infectious dose for the subsequently infected cells. All tubes were incubated at 37 °C with 5% CO2 for seven days. M. pneumoniae organisms were harvested by centrifugation at 4 °C and 10,000× g for 30 min, then washed thrice with PBS . We designed a pair of detection primers according to the 16s rRNA sequence of M. pneumoniae   cells were cultured in an RPMI-1640 medium  supplemented with 10% fetal bovine serum (FBS)  and maintained at 37 °C in a humidified 5% COntiation  and the M. pneumoniae at a multiplicity of infection (MOI) for 0, 15, 30, 60, and 120 for 24 h. The cell viability was analyzed at each time point using cell counting kit-8 ) following the manufacturer’s protocol. The absorbance was read at 490 nm using a fluorescence microplate reader  , and cell differentiation was performed. MΦs were infected with MA, USA) A. Then, ng CCK-8 B. Uninfeng CCK-8 C. M. pneumoniae 1 × 107 CCU were resuspended with M. pneumoniae medium containing DIO dye , incubating at 37 °C for 12 h. The DIO-labeled M. pneumoniae was centrifugated at 10,000× g for 10 min and washed with PBS 5 times. THP-1 cells  were seeded in 15 mm laser confocal dishes following the above MΦ differentiated methods. The MΦs were infected with DIO-labeled M. pneumoniae, and MΦs were washed with PBS, fixed in 4% paraformaldehyde for 30 min, and washed again with PBS twice. The MΦs were then treated with 0.3% Triton X-100 in PBS for 20 min and washed thrice (5 min/time). Then, 100 μL rhodamine-phalloidin  was dripped to the MΦs for 30 min without light at RT. The dishes were washed three times and dripped with antifluorescence quenching mounting fluid , 100 ul, and were observed using a scanning confocal microscope (TCS SP5) .7 cell/dish and differentiated. The MΦs were infected with M. pneumoniae at the MOI of 15 for 24 h, and the total RNA was extracted with Trizol  following the manufacturer’s instructions. RNA purity was assessed by electrophoresis on a 1% agarose gel. RNA quality assessment was determined using a NanoDrop 2000  and Agilent Bioanalyzer 2100 . The quality RNA samples (RNA-integrity number ≥ 7) were used for RNA-seq. The RNA libraries were sequenced on the Illumina sequencing platform, and the data were analyzed by Genedenovo Biotechnology Co., Ltd. . Finally, differential genes were screened based on conventional standards (|log2 (FC)| > 1, FDR < 0.05).First, RNA-seq was employed to analyze and understand the mechanism profiling based on the infected-cell model. Then, THP-1 cells were plated in 10 cm dishes with 1 × 10g and 4 °C overnight (16 h) using an angle rotor (Himac CP100WX ultracentrifuge) . The FBS supernatant was filtered using a 0.22 μm Millipore filter and stored at −20 °C. The THP-1 MΦs were infected with M. pneumoniae at MOI of 15 with 10% EVs-depleted FBS for 48–72 h. The supernatants from M. pneumoniae-infected, and uninfected MΦs were enriched, respectively. Following the classic isolation methods of EVs [g for 10 min, 2000× g for 20 min, and 3000× g for 30 min, respectively, and were filtered through a 0.22 μm Millipore filter. Then, the supernatants were ultracentrifuged at 120,000× g and 4 °C for 70 min, and the pellet was suspended with 100 μL PBS and stored at −80 °C. A BCA method for the total protein amount of EVs was taken to quantify EVs [We prepared a self-made FBS with EVs depleted, and FBS was ultracentrifuged at 120,000× s of EVs , the celtify EVs  followinEVs, 10 μL, were carefully dripped onto a carbon-coated copper grid and incubated for 2 min at room temperature (RT). The EVs liquids were absorbed from the copper grid by filter papers and 10 μL of 3% phosphotungstic acid , and pH was adjusted to 7.0 before use. Samples were then dripped on the copper grid for 5 min at RT for negative staining. The copper grid was dried for 10 min and then observed at 80–100 kV voltage with transmission electron microscopy (HT7700) .7–1 × 109 particles/mL. Meanwhile, the particle trajectory of the EVs was recorded.EVs’ concentration and size distribution were measured by ZETAVIEW PMX 110  and corresponding software ZetaView (version 8.0402 SP2). The size and quality of the EVs were detected under a 405 nm emission light; EVs were diluted with PBS to 1 × 10g for 70 min at least three times, and the pellet was suspended with 100 μL of PBS. THP-1 cells  were seeded in 15 mm laser confocal dishes, and PKH67-labeled EVs at 25 μg/mL concentration were added to the laser confocal dish at 37 °C for 8 h. The fluorescence staining of the recipient MΦs was done as described previously.According to the manufacturer’s protocol, two EV types (uninfected or infected MΦs-derived EVs) were labeled to examine EV uptake by PKH67 . The suspensions containing 100 μg of EVs were resuspended in 0.5 mL Diluent C. Synchronously, 4 μL of PKH67 were then added into another 0.5 mL Diluent C. Samples were mixed with each for 4 min lucifugally at RT, and 10 mL of medium with EVs-depleted were added. The labeled EVs were rewashed with PBS and re-ultracentrifuged at 120,000× 6 THP-1 cells were seeded in a 6-well culture plate to differentiate and rest. The MΦs were pretreated with GW4869 (10 μM) for 2 h before being infected with M. pneumoniae. The cell supernatants were then collected and proceeded with a series of differential centrifugation after infection 48–72 h, and then filtered using 0.22 μm. The prepared cell supernatants of EVs blocking and new 1640 media (1:1 volume ratio) were added to new prepared MΦs seeded in a 6-well culture plate. The MΦs were pretreated with Cytochalasin D (4 μM) for 2 h before being treated with EVs. All the cell proteins were extracted after 24 h.A total of 1.2 × 106 were seeded in a 6-well culture plate to differentiate and rest. The uninfected-MΦs were treated with 50 μg/mL EVs from uninfected and infected MΦs for 24 h, respectively. According to the manufacturer’s instructions, the total RNA was extracted by Trizol reagent . RNA concentration and integrity were determined using a NanoDrop 8000 spectrophotometer . Then, 1 μg of total RNA was reverse transcripted to the cDNA using HiScript® II 1st Strand cDNA Synthesis Kit . The reaction conditions were 50 °C for 30 min and 85 °C for 5 s. qPCR was conducted using the ChamQ™ Universal SYBR qPCR Master Mix Kit  with the following steps: 95 °C for 30 s; 95 °C for 10 s, 60 °C for 30 s, and recycle for 40; 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. mRNA levels were normalized to GAPDH  following the manufacturer’s instructions. The absorbances of samples were measured at 450 nm using a fluorescence microplate reader .The cell supernatants were centrifugated at 12,000× Mycoplasma pneumoniae antibody ab53600 (1:1000) ; TLR2 (1:1000), TLR4 (1:1000), Myd88 (1:1000), NF-κB p65 (1:1000), P-NF-κB p65 (Ser536) (1:1000), total ERK1/2 (4A4) (1:1000), P-ERK1 (Thr204)/ERK2 (Thr187) (1:1000), total p38 (5A1) (1:1000), P-p38 (Thr180/Tyr182) (1:1000), total JNK1/2/3 (1:1000), P-JNK1/2/3 (Thr183/Thr221) (1:1000), and GAPDH (1:8000) . The membranes were then washed five times with TBST buffer and incubated in the secondary antibody, HRP-conjugated goat antimouse or goat antirabbit antibodies , with the dilution at 1:6000. The membranes were washed five times with TBST buffer again and visualized with a chemiluminescence (ECL) kit  using Amersham Imager 6000 .The EVs pellets suspension was added with 6× protein loading buffer  and was heated and denatured at 100 °C for 10 min. The 6-well plates were added with ice-cold protein lysis  containing a protease inhibitor and phosphatase inhibitor, and PMSF. Following experimental steps according to the manufacturer’s protocol, a BCA protein assay measured the concentration of cell lysates supernatant. The protein samples were then separated on a 12% SDS-PAGE gel  with a constant voltage of 120 V and transferred to PVDF membranes  activated by methanol soaking at 0.3 A for 2 h and blocked in 5% skim-milk powder , dissolved in TBST  for 1 h at RT. These membranes were incubated overnight at 4 °C with the following primary antibodies: CD63 (1:1000), CD81 (1:1000), and CD9 (1:1000) ; TSG101 (1:1000) and Calnexin (1:1000) ; anti-p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ns, not significant.All data were expressed as the mean ± SD and analyzed by ANOVA. The graphs were performed using GraphPad Prism8.0. Significance is shown as * M. pneumoniae-infected MΦs induce the inflammation cytokines expression of uninfected-MΦs through triggering TLR2-NF-κB/JNK signal pathways. In addition, other recognized-PAMPs receptors may exist that deliver the signals and phosphorylate the ERK and p38, inducing the production of proinflammatory cytokines. However, the role of EVs in modulating the immune response in vivo is still unclear. We found that EVs released from M. pneumoniae infected-MΦs carry PAMPs and can act as the bridge between infected and uninfected cells in host immunity. Understanding the long-term inflammatory response and intercellular immune modulation in the host during M. pneumoniae infection will be helpful.This study demonstrated that EVs shed from


Scutellaria (Lamiaceae) have a wide variety of bioactive secondary metabolites with diverse biological properties, e.g., anti-inflammatory, antiallergenic, antioxidant, antiviral, and antitumor activities. The chemical composition of the hydroethanolic extracts, obtained from dried plants of S. incarnata, S. coccinea, and S. ventenatii × S. incarnata, was determined by UHPLC/ESI-Q-Orbitrap-MS. The flavones were found in a higher proportion. Baicalin and dihydrobaicalein-glucuronide were the major extract components in S. incarnata (287.127 ± 0.005 mg/g and 140.18 ± 0.07 mg/g), in S. coccinea (158.3 ± 0.34 mg/g and 51.20 ± 0.02 mg/g), and in S. ventenatii × S. incarnata (186.87 ± 0.01 mg/g and 44.89 ± 0.06 mg/g). The S. coccinea extract showed the highest antioxidant activity in the four complementary techniques employed to evaluate all extracts: ORAC (3828 ± 3.0 µmol Trolox®/g extract), ABTS+• (747 ± 1.8 µmol Trolox®/g extract), online HPLC-ABTS+• (910 ± 1.3 µmol Trolox®/g extract), and β-carotene (74.3 ± 0.8 µmol Trolox®/g extract).Plants of the genus  Aedes aegypti + and [M−H]− ions was studied using different energies  in a higher-energy collision dissociation cell (HCD). The collision energy values were chosen while keeping in mind the highest number of product ions registered without losing the [M+H]+ or [M−H]− signals of phenolic substances. A total of 17 compounds were identified by comparing their mass spectra with those found in the literature and in spectral databases +. This ion decays, generating fragments [(M+H)−C6H8O6−H2O]+ at m/z 255.06497 and [(M+H) −C6H8O6−C8H8]+ at m/z 169.01292 due to the loss of water and the retro Diels–Alder reaction suffered by the aglycone, respectively. The latter process has been reported as characteristic of flavanones +.Dihydrobaicalein-glucuronide and its aglycone, dihydrobaicalein, have previously been detected at low concentrations in other  species ,25,27. Iavanones . The proScutellaria species. In its (ESI/MS) mass spectrum, from the deprotonated molecule [M−H]− at m/z 563.13953, losses of 60, 90, and 120 units are observed, which generate fragments at m/z 503.12198, 473.10934, and 443.09860, respectively. According to Becchi and Fraisse [m/z 413.08817 is generated by the cleavage of the hexose [(M−H)−C5H10O5]−, which does not involve the rupture of the 1′C–6C bond between the aglycone and the glycoside (m/z 383.07773 corresponds to the successive loss of 60 (−C2H4O2) and 120 (−C4H8O4) units in the sugars; the product ion at m/z 353.06668 is similarly formed by the successive losses of 60 and 150 units (−C5H10O5).Apigenin-C-glucoside-C-arabinoside (schaftoside) was reported by Liu et al.  and Han  Fraisse  and Wu elycoside . The proScutellaria spp. extracts. The ion [M+H]+ appears in the mass spectrum at m/z 471.14970 and corresponds to the elemental formula of this substance, C21H27O12. The [M+H]+ ion dissociates, generating the product ion [(M+H)−2H2O]+ at m/z 435.12823 by loss of two water molecules. The elimination of a molecule of rhamnose [(M+H)−C6H10O4]+ generates the product ion at m/z 325.09137, which decays, eliminating a glucose to form the fragment [(M+H)−C6H10O4−C6H10O5]+ at m/z 163.03882. In turn, this ion loses water, forming the product ion [(M+H)−C6H10O4−C6H10O5−H2O]+ at m/z 145.07590. The protonated [M+H]+ molecule of umbelliferone-hexoside-pentoside undergoes the loss of a glucose molecule [(M+H)−C6H10O5]+ to generate the product ion at m/z 309.09671 , corresponding to a deprotonated [M−H]− molecule at m/z 329.17474 (C20H26O4) was not identified at a confirmatory level. The deprotonated molecule [M−H]− undergoes the loss of a methyl radical with formation of the product [(M−H) −CH3]−• at m/z 314.15262 (100%). The product ions at m/z 285.11313 (0.2%) and m/z 271.09641 (0.3%) correspond to the losses of the propane (C3H8) and butane (C4H10) molecules, respectively  mode. In the case of ions extracted from the TIC for which a reference substance was not available, quantification was performed using the calibration curve obtained for a structurally similar substance.Compound quantification was performed with the external standardization technique using [M+H]S. baicalensis [S. lateriflora [2O2 in HK-2 cells [In the three extracts, baicalin was the major compound . This cocalensis ,45 and Seriflora . Baicalieriflora  and has eriflora , reducin-2 cells , inducin-2 cells , and inh-2 cells . The flaScutellaria species were very similar. However, the amount and ratio of the compounds present in the extracts differed. This permitted distinguishing each one of the species studied. In all of the extracts, the glycosylated compounds were found together with their respective aglycones. Their extraction is explained by the type of solvent used (ethanol–water), which made it possible to isolate both very polar (glycosides) and moderately polar (aglycones) molecules and compounds with several −OH groups in their structures [The chemical compositions of the extracts of the three ructures ,52.+•, online HPLC-ABTS+•, and β-carotene discoloration assays of hydroalcoholic extracts of Scutellaria spp. Results from different antioxidant assays were analyzed by ANOVA followed by Tukey’s post hoc test in order to establish significant differences (p < 0.05) between samples. The S. coccinea extract had the highest antioxidant activity in all the trials; this may be due to the amounts of baicalin and dihydrobaicalein-glucuronide . The even higher antioxidant activity of the S. coccinea extract may be due to the presence of isoliquiritin, which was found only in this extract. Isoliquiritin acts as an “activator” of superoxide dismutase and catalase enzymes [curonide , which a profile . This ag enzymes ,55 to coScutellaria spp. extracts can act antagonistically; that is, they do not increase the antioxidant activity of the mixture but decrease it.No extract surpassed the antioxidant activity value of quercetin in any trial. However, when studying the standard compounds individually, it was found that scutellarein and apigenin presented a response very similar to that of quercetin in all techniques employed. This indicates that some compounds in the +• method, a relationship was established between the amount of compound present in each extract and its antioxidant activity. The higher the amount of dihydrobaicalein-glucuronide present in the extracts, the higher the measured antioxidant activity  had the highest sum, followed by S. coccinea  and S. ventenatii × S. incarnata . This indicates that, apart from the antagonistic interactions mentioned above, the antioxidant response was dose dependent. This coincides with what was reported by Seo et al. [S. baicalensis, analyzed by online HPLC-ABTS+•.Using the online HPLC-ABTSactivity . When tho et al.  for the The antioxidant activity of phenolic compounds depends on their structure. The degree of glycosylation, as well as the position and number of hydroxyl groups, contribute to the antioxidant response related to the donation of hydrogen atoms or electrons to form more stable intermediates . Some stScutellaria spp. extracts . Cotelle [In the present study, it was observed that the flavanone dihydrobaicalein-glucuronide, a molecule without a double bond in positions 2 and 3 of the C ring, was the main contributor to the antioxidant response of  Cotelle  attribut Cotelle  discoverO-glycosidic bond. Since the studied extracts were rich in glycosylated compounds, they could be used eventually in a phytotherapeutic product with high antioxidant value.Among the standard substances evaluated individually , the aglScutellaria spp. contains approximately 50,000 plants, which can be harvested four times a year, ca. a total of 250 kg of plant material could be obtained per hectare per year. Therefore, the annual quantity of dry extract, considering a percentage of humidity of the material of ca. 80%, would be 13.5 kg, for a yield of 27%, with high amounts of baicalin (ca. 3.7 kg/ha/year) and dihydrobaicalein-glucuronide (ca. 1.8 kg/ha/year), substances of commercial interest due to their special biological activity.Bearing in mind that a 1 ha plantation of ® (≥97%) were purchased from Sigma-Aldrich . Salvigenin (≥98%), scutellarin (≥98%), scutellarein (≥98%), baicalin (≥98%), apigenin-7-glucuronide (≥98%), wogonin (≥98%), wogonoside (≥98%), eriodictyol-7-glucoside (≥98%), and norwogonine (≥98%) were obtained from Chemfaces . Galangin (≥95%), kaempferol-3-O-rutinoside (≥98%), and vitexin (≥98%) were purchased from Phytolab GmbH . HPLC-grade acetonitrile, HPLC-grade formic acid (FA), isopropanol (98%), ammonium formate (AF) (≥99%), LC/MS-grade methanol, and potassium persulfate (≥98%) were obtained from Merck . Type I water was obtained from a Millipore Direct-QTM purification system.2,2′-Azino-bis-83-ethylbenzothiazolin-6-sulfonic acid (ABTS) (≥98%), gallic (≥97%) and linoleic (≥99%) acids, β-carotene (≥ 93%), chloroform (≥99%), baicalein (≥95%), apigenin (≥95%), and TroloxScutellaria plant material was provided by the Colombian state through the No. 270 contract for access to genetic resources and derived products between the Ministry of Environment and Sustainable Development and the Industrial University of Santander. Leaves and stems of S. incarnata (voucher number UIS219783), S. coccinea (voucher number UIS219784), and S. ventenatii × S. incarnata  were collected during flowering in experimental plots of the Pilot Agroindustrial Complex of the National Research Center for the Agro-industrialization of Tropical Aromatic and Medicinal Plant Species, CENIVAM , in April 2021.The Scutellaria species, a 23 experimental design with a central point was developed in order to determine the best extraction conditions. An ultrasound-assisted solvent extraction (SE) technique was used; each point was performed in triplicate, while the central point was performed in quintuplicate. The modification factors were temperature (A), ethanol–water (solvent) ratio (B), and time (C). The combined response was determined by normalizing the yield and antioxidant activity data and summing their values. The data were processed with Statgraphics Centurion XVI software, version 16.1. For the extraction, dried and ground plants (1 g) were used, which were placed in a 100 mL amber bottle, to which an ethanol–water solution (20 mL) was added. The mixture was subjected to ultrasonication  and filtered (Wattman No. 1 paper), and the residue was extracted once more with ethanol–water (10 mL). The extracts were vacuum rotoevaporated at 50 °C in Heidolph equipment , dried in a VirTis AdVantage Plus tray lyophilizer  and stored at 4 °C in the absence of light.For the three Samples were weighed (0.5 mg) and dissolved in mobile phase (1 mL) 50:50 methanol–water (0.1% formic acid and 5 mM ammonium formate) to a final concentration of 500 mg/L.2) were set to 40 and 10 arbitrary units, respectively. Nitrogen (>99% purity) was obtained from a generator . The ions injected into the HCD cell, coming from the C-trap, were fragmented, using normalized collision energies in steps from 10 to 70 eV. Data were acquired and analyzed using Thermo Scientific™ Dionex™ Chromatography Data System (CDS) software, version 7.2 and Thermo Xcalibur 3.1 software . Identification of compounds was carried out by comparing their retention times (tR), exact ion masses, isotopic ratios, and fragmentation patterns with those of standard substances and databases [Phenolic compounds were analyzed on a VanquishTM ultra-high performance liquid chromatograph (UHPLC) , equipped with a thermostatically controlled column compartment (40 °C). Chromatographic separation was performed on a 50 mm L × 2.1 mm I.D. Zorbax Eclipse XDB C18 column, 1.8 µm particle size . The flow rate of the mobile phase containing (A) water (0.1% FA + 5 mM AF) and (B) MeOH (0.1% FA + 5 mM AF) was 300 μL/min. The initial gradient condition was A-100%, which linearly changed to B-100% in 8 min, held for 4 min, returned to A-100% in 1 min, and remained so for 3 min. The injection volume was 2 μL. The UHPLC was connected to a Q-Exactive Plus Orbitrap mass spectrometer  with a heated electrospray ionization (HESI-II) source. The capillary voltage was 3.5 kV. The nebulizer temperature was set at 350 °C and the capillary temperature at 320 °C. The sheath gas and auxiliary gas . ABTS (7 mM in acetate buffer) and PDS (2.45 mM) were mixed with sonication (30 min) and stored at 4 °C (24 h) in the absence of light. ABTS+• (900 μL) was taken and diluted in acetate buffer until an absorbance of 0.71 ± 0.02 at λ = 750 nm was obtained. Each extract (2 mg) was dissolved in methanol (2 mL) and diluted in acetate buffer . The diluted extract (10 μL) and ABTS+• solution (190 μL) were deposited in each well of the plate, and the absorbance was measured for 60 min.The ABTSe et al. , with so+•. Chromatographic analysis was performed using the method established by Sierra et al. [18 chromatographic column of 100 mm length × 4.6 mm I.D. × 5 μm particle size , AT G1315D diode array detector, AT G1311C quaternary pump, and AT G1329B autosampler. Ten microliters of each extract (10 g/L) were injected. The column temperature was maintained at 25 °C. An aqueous solution of FA at 0.5% (v/v) (A) and HPLC grade acetonitrile (B) were used as the mobile phase, with a flow of 1 mL/min and a programmed elution gradient of 98% A (0 min), 88% A (15 min), 88% A (15–23 min), 60% A (46 min), 10% A (71 min), 10% A (71–75 min), 98% A (80 min), and 98% A (80–85). The detector was set to the following wavelengths: λ = 245, 270, 290, and 515 nm.The extract was analyzed in a high-efficiency liquid chromatograph, HPLC 1200 Infinity , coupled to a Pinnacle PCX post-column derivatizer  in order to determine its ability to decolorize the cation–radical ABTSa et al.  in a liq+• was generated by the oxidation of ABTS with PDS in a 2.9:1 molar ratio. The ABTS+• stock solution was prepared by direct contact of ABTS  and PDS (0.7 mM). The cation–radical concentration in the reactor was 79 μM, which allowed for achieving an absorbance (0.71 ± 0.02) equal to that established in the cation–radical decolorization assay ABTS+• carried out in the microplate reader  and linoleic acid (10 mg); the mixture was shaken at 3000 rpm for 30 s. The chloroform was evaporated under a nitrogen flow (3 min), Type I water (1 mL) was added, and it was stirred at 3000 rpm for 1 min. Subsequently, a 1% PDS solution (3 mL) was added as a co-oxidizing agent, and it was brought up to 25 mL with Type I water. The absorbance reading was performed at a wavelength of λ = 450 nm at 45 °C in a 96-well ModulusTM II Microplate Multimode Reader The antioxidant activity was evaluated following the methodology described by Panovska et al. , with soThe oxygen radical absorbance capacity (ORAC) was determined according to Huang et al. , with soScutellaria species native to Colombia allowed us to identify that for S. incarnata, a temperature of 50 °C, an ethanol–water percentage of 70%, and an extraction time of 5 min were the parameters that provided its highest extraction yield and antioxidant activity, while for S. coccinea and S. ventenatii × S. incarnata, these values were 50 °C, 70% and 40%, and 15 min, respectively. For the first time, the nonvolatile secondary metabolite composition of three Scutellaria species native to Colombia was determined, as well as the presence of umbelliferone-hexoside-pentoside in their extracts, a compound not found in other species of the genus. Dihydrobaicalein-glucuronide and baicalin were the major components of the S. incarnata (287.127 ± 0.005 mg/g and 140.18 ± 0.07 mg/g), S. coccinea (158.3 ± 0.34 mg/g and 51.20 ± 0.02 mg/g), and S. ventenatii × S. incarnata (186.87 ± 0.01 mg/g and 44.89 ± 0.06 mg/g) extracts. The highest antioxidant activity, measured by different techniques, was found for the S. coccinea extract (3828 ± 3.0 µmol Trolox®/g extract). The online HPLC-ABTS+• method allowed the identification of dihydrobaicalein-glucuronide and baicalin as the compounds that mainly contribute to the total antioxidant activity of Scutellaria spp. extracts, with 70%, 60%, and 50% of the activity for S. incarnata, S. coccinea, and S. ventenatii × S. incarnata, respectively. The Scutellaria spp. studied could be used to obtain antioxidant extracts with potential use in the pharmaceutical sector and would also be good candidates for the isolation, by advanced fractionation techniques (prep/HPLC), of individual bioactive compounds as natural ingredients for different phytotherapeutic products.The experimental design carried out to extract phenolic compounds from three