N)-linked glycosylation is required for delivery of the major SV glycoproteins synaptophysin and SV2A to SVs. Despite this key role for N-glycosylation, the molecular compositions of SV N-glycans are largely unknown. In this study, we combined organelle isolation techniques and high-resolution mass spectrometry to characterize N-glycosylation at synapses and SVs from mouse brain. Detecting over 2,500 unique glycopeptides, we found that SVs harbor a distinct population of oligomannose and highly fucosylated N-glycans. Using complementary fluorescence methods, we identify at least one highly fucosylated N-glycan enriched in SVs compared with synaptosomes. High fucosylation was characteristic of SV proteins, plasma membrane proteins, and cell adhesion molecules with key roles in synaptic function and development. Our results define the N-glycoproteome of a specialized neuronal organelle and inform timely questions in the glycobiology of synaptic pruning and neuroinflammation.At mammalian neuronal synapses, synaptic vesicle (SV) glycoproteins are essential for robust neurotransmission. Asparagine (  N-glycosylation at the murine brain synapse. Their results define a glycobiological signature of brain synaptic vesicles and demonstrate that synaptic vesicle and plasma membrane proteins bear highly fucosylated N-glycans.Bradberry et al. combine organelle immunopurification and orthogonal chemical analysis methods to characterize protein  Post-translational modifications of SV proteins may play important roles in SV formation and function.N-glycome is rich in oligomannose and bisected N-acetylglucosamine (GlcNAc)-rich structures .23 The brain N-glycome is dynamic over the lifespan,24 and blocking neuronal N-glycan maturation results in a striking neurodegenerative phenotype.25 Moreover, evidence suggests that certain glycoepitopes may be important for brain development and function. For example, loss of the enzyme fucosyltransferase-9 (Fut9),26 which generates LewisX/SSEA-1/CD15 glycoepitopes28 in mouse brain by addition of antennary fucose in α(1–3) linkages to complex N-glycans,30 is associated with impaired neurite outgrowth in vitro31 and behavioral abnormalities in the mouse.29 Strikingly, the corresponding gene FUT9 has been linked to schizophrenia in human genome-wide association studies, along with several other glycosylation-related genes.34 Aberrant glycosylation has long been detected in the brains of patients with schizophrenia, though largely through the use of indirect methods such as immunoblotting and immunostaining.35 These findings suggest that glycobiological processes may contribute to the development of schizophrenia, but specific pathophysiologic mechanisms remain poorly understood in this context.Compared with plasma and other tissues, the brain 40 While these studies have improved our understanding of the heterogeneous distribution of N-glycosylation in the brain,39 their broad scope has limited opportunities for insight into roles for specific glycans in specific biological processes. Examining protein glycosylation in particular cell types or organelles, by contrast, offers an opportunity to link glycosylation with function. In particular, the abundance of glycoproteins in SVs43 makes these organelles attractive for the study of glycans in membrane protein trafficking. Moreover, nerve terminals represent a brain tissue component vulnerable to injury and degeneration, especially in the context of neuroinflammation,46 schizophrenia,47 and age-related cognitive decline.50 Changes in glycoprotein trafficking at the synapse may thus influence a broad range of clinically important processes. We also note a large body of evidence suggesting specialized extracellular matrix glycans at nerve terminals.51 A meaningful understanding of glycobiology at the synapse, however, has been limited by the lack of a deeply characterized presynaptic N-glycoproteome.Recent advances in mass spectrometry-based glycoproteomics methods have enabled the detection of hundreds to thousands of unique glycopeptides obtained from brain tissue.N-linked glycoproteome of SVs and their milieu. Using organelle immunoisolation techniques41 and complementary analytical methods, we demonstrate that SVs are enriched for specific N-glycans bearing antennary fucosylation. Highly fucosylated glycans were found primarily on SV proteins, cell adhesion molecules with known synaptic functions, and other proteins with key roles at the plasma membrane, including transporters and neurotransmitter receptors. Our results define the glycoproteome of a neuronal organelle, suggest a common glycosylation pathway shared by plasma membrane and SV proteins, and provide protein-level evidence to inform hypotheses linking protein glycosylation and synaptic biology.In this study, we establish a molecular foundation for synaptic glycobiology by defining the N-linked glycomic studies were conducted using synaptic material from whole mouse brain prepared by two approaches. Synaptosomes, which represent a “classical” crude preparation containing mostly preand post-synaptic elements, were prepared by differential centrifugation  with magnetic beads conjugated in-house to a monoclonal antibody (mAb) against SV2,13 according to recently described procedures  using an Orbitrap Eclipse mass spectrometer41 , N-methyl-D-aspartate (NMDA), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits were present in synaptosomes but were largely undetectable in SV samples  linkage to the N-linked core GlcNAc before the addition of antennary fucose in α or α linkages 56 56  or steppD)56  prior to nLC-MS.58 SV samples were characterized using sceHCD both with and without glycopeptide enrichment   . DigestiPLC-FLD)  enabled glycomes –5L. The glycomes . Among mglycomes , and 5I,glycomes  or in thglycomes .N-linked glycans. Strikingly, a late-eluting antennary fucosylated species bearing two antennary fucose residues, denoted φ, was significantly enriched in the SV samples  fucosidase among synaptosome or SV glycans  fucosidase or α mannosidase  were also found in synaptosome samples , consist-glycans . In agre-glycans .66 neuroplastin,69 and NCAM170 -galactose and the enzymes that catalyze its addition74 (74Gene Ontology (GO) analysis demonstrated that cell adhesion was among the most enriched biological process terms in highly fucosylated proteins . Other hdition74 . Despite41 The combination of SV-IP, glycan-focused MS, and fluorescence methods revealed that SVs bear a distinctive N-glycan signature marked by oligomannose and highly fucosylated glycans  spectroscopy,84 and their study may thus yield insights into the biology of schizophrenia and other developmental processes across the human lifespan.Our findings are particularly striking given the recent identification of subunits  and S78286 and schizophrenia,87 and lectins can directly activate the complement cascade.88 Among the best-known complement-activating lectins is mannose-binding lectin, which is activated by mannosylated glycans typically found on microorganisms but not on circulating glycoproteins.88 Mannose-binding lectin is a well-known mediator of inflammatory brain injury after ischemia or trauma.90 Strikingly, mannosylated glycans are common on synaptic glycoproteins22 ,94 may enable the characterization of glycans not detected in the present study.Sialic acid, a biologically important sugar that commonly decorates peptides  and S3, glycan φ . Finallymazdak.bradberry@nyspi.columbia.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the This study did not generate new unique reagents.https://doi.org/10.25345/C5TB0Z526. Relevant MSFragger output tables are included in Raw LC-MS data are publicly available online via the MassIVE repository at https://doi.org/10.5281/zenodo.7659070All original R scripts used to process the MSFragger output have been deposited at Zenodo and are available at Any additional information required to re-analyze the data reported in this paper is available from the 24 All work was conducted according to protocols approved by the University of Wisconsin Institutional Animal Care and Use Committee.C57B/6J mice of either sex between 15 and 20 days of age were used for all experiments. Juvenile mice were used with the objective of capturing both immature and mature glycosylation patterns.41 For each coupling reaction, 10 mg of Dynabeads stored in DMF were collected with a magnetic stand and resuspended in 200 μL borate buffer . To this suspension was added 200 μL mAb solution, followed by 200 μL 3 M ammonium sulfate in borate buffer, with mixing by pipetting up and down after each addition. This coupling mixture was incubated with rotation at 37°C overnight. The beads were then collected with a magnetic stand, the supernatant was discarded, and the beads were washed six times by trituration in 1 mL wash buffer followed by collection with the magnetic stand. The wash buffers were 500 mM NaCl, 50 mM ammonium acetate pH 4.5 and 500 mM NaCl, 50 mM Tris-HCl pH 8.0, used in an alternating manner . The beads were then resuspended in 1 mL KPBS , transferred to a fresh tube, and collected on a magnetic stand. The supernatant was removed and the beads were resuspended at 30 mg/mL using 300 μL KPBS. Beads stored in this manner at 4°C remained effective for at least 2 months.Anti-SV2 mAb  and anti-syt1 mAb  were purified from ascites stocks generated prior to 2010 using protein G chromatography  and dialyzed extensively against phosphate-buffered saline . Single-use aliquots  of mAb in PBS were kept frozen at −80°C. Dynabeads M-270 Epoxy  were coupled to mAb according to published procedures.All buffers, tubes, and centrifuge rotors were cooled to 0–2°C prior to use. One or two mice were anesthetized with isoflurane, euthanized, and the brains including cerebellum and brain stem were rapidly removed. Each brain was placed in a tight-fitting Teflon-glass Dounce homogenizer with 3.8 mL ice-cold potassium homogenization buffer  containing protease inhibitors  and homogenized with ten strokes using an overhead mixer rotating at 900 RPM. The homogenate was then centrifuged at 35,000 x g for 20 min at 2°C. During centrifugation, 5 mg α-SV2 Dynabeads per brain were washed in KPBS and resuspended in two 2-mL microcentrifuge tubes (2.5 mg beads in 100 μL in each tube). The supernatant from each brain homogenate was added to two tubes containing Dynabeads (1.9 mL/tube), and the tubes were placed inside 50-mL conical tubes packed with ice and incubated with rotation for 25 min in a cold room. The supernatants were then discarded and each 2.5 mg portion of beads, corresponding to SVs from ½ mouse brain, was washed 3 times by gentle trituration in 1 mL ice-cold KPBS followed by collection on a magnetic stand. The beads were then resuspended and transferred to a fresh tube using KPBS, with beads bearing SVs from the same brain combined into the same tube (5 mg/tube), and the supernatant was removed. For proteomics and glycoproteomics studies, the beads were eluted using 50 μL 2% SDS containing 25 mM Tris-HCl pH 8.0 with heating to 50°C for 5 min, and the eluates were frozen at −80°C prior to use. For HPLC studies, the beads were eluted using 45 μl 0.5% SDS with heating to 50°C for 5 min, followed by a second elution with 45 μL 2% n-β-dodecylmaltoside (DDM) (Gold Biotechnologies) for 5 min at room temperature. These eluates were combined and 2 μL 1 M triethylammonium bicarbonate (TEAB) was added prior to storage at −80°C.Synaptosomes were prepared by removing and homogenizing brains as above except that the homogenization buffer contained 125 mM NaCl, 25 mM HEPES-NaOH, and 5 mM EGTA, pH 7.4. The brain homogenate was centrifuged at 3,500 x g for 2 min, the pellet was discarded, and the supernatant transferred into two 2-mL microcentrifuge tubes and centrifuged for 12 min at 14,000 x g at 4°C. The supernatant was discarded and the pellet was resuspended in 1530 μL 50 mM TEAB per 2-mL tube. 90 μL of 10% SDS and 180 μL 10% DDM  were added to each tube, which was incubated for 1 h with rotation at 4°C prior to aliquoting and freezing at −80°C.SV2 and syt1 were immunoprecipitated from synaptosomes prepared as above but resuspended in 1.6 mL synaptosome homogenization buffer followed by the addition of 180 μL 10% DDM  per one-half brain. The samples were incubated with rotation for 1 h at 4°C followed by pelleting of insoluble material by centrifugation at 20,000 x g for 20 min at 4°C. The supernatants were transferred to new tubes, 3 mg α-syt1 or α-SV2 Dynabeads were added, and the tubes were incubated with rotation for 30 min. The beads were then washed with cold synaptosome resuspension buffer (4 × 1 mL) and eluted with 50 μL 2% SDS containing 25 mM Tris-HCl pH 8.0 with heating to 50°C for 5 min. Eluates were stored frozen at −80°C prior to use.vide infra). For synaptosomes, a similar procedure was followed but scaled up 10-fold, using 500 μL synaptosomal lysate as input, washes of 3 × 1 mL 80% ethanol, and elution in 250 μL trypsin solution.For immunopurified SV, syt1, and SV2 samples, ~60 μL bead eluate in SDS was combined with dithiothreitol  for a final concentration of 5 mM DTT and incubated at 50°C for 25 min. Iodoacetamide (200 mM freshly prepared aqueous stock solution) was added to a final concentration of 15 mM and the reaction incubated in the dark at room temperature for 30 min. More DTT was then added  to quench iodoacetamide. Dynabeads M-270 carboxylic acid  were then added  and the tubes were mixed well, followed by the addition of 1 volume of absolute ethanol and brief incubation on a thermomixer  to drive protein adsorption to the beads. The beads were washed three times with 200 μL 80% ethanol and transferred to a fresh tube with the final wash. The supernatant was removed and tryptic peptides were eluted from the beads by overnight digestion in 50 μL trypsin solution  with shaking in a thermomixer . Eluates from this step were used directly for LC-MS or subject to glycopeptide enrichment  were washed with acetonitrile (3 × 1 mL), 100 mM triethylammonium acetate in water (3 × 1 mL), 1% trifluoroacetic acid (TFA) in water (3 × 1 mL), and 1% TFA in 95:5 MeCN:H2O. Tryptic peptides  were brought up to 95:5 MeCN:H2O with the addition of 19 volumes of MeCN and applied to the column twice. The column was then washed with 95:5 MeCN:H2O containing 1% TFA (6 × 1 mL). Glycopeptides were eluted with 50:50 MeCN:H2O containing 1% TFA (850μL + 500 μL) followed by 95:5 H2O:MeCN containing 1% TFA (850μL + 500 μL). The eluates were dried in a speedvac and stored at −80°C. For each sample, 30 μL 0.2% formic acid in water was used to redissolve all dried eluates prior to LC-MS analysis.Glycopeptides were enriched by strong anion exchange-electrostatic repulsion chromatography (SAX-ERLIC) according to recently published procedures.95 and held at 50°C using a custom-built column heater. For standard proteomics experiments  with HCD energy 36%; resolution, 30,000; maximum injection time, 60 ms; AGC target, 50,000; normalized AGC target, 100%. A second fragmentation scan of the same precursor was triggered if one of the top 20 most abundant ions in the first MS2 spectrum was one of the following: m/z 204.0867, 138.0545, 366.1396, 274.0921, 292.1027, 126.055, 144.0655, 168.0654, 186.076. This second, triggered scan activated ion with either used stepped HCD (sceHCD) or activated ion-electron transfer dissociation (AI-ETD). SV samples were analyzed using sceHCD, while both sceHCD and AI-ETD were employed for synaptosome samples given their greater complexity and abundance of material for analysis. For AI-ETD, an Orbitrap Fusion Lumos mass spectrometer  was retrofitted with a 60 W CO2 laser to allow for photoactivation.56 For sceHCD, energies of 20%, 35%, and 50% were used . For these runs, mobile phase A was 0.2% formic acid in H2O, mobile phase B was 90:10 isopropanol:acetonitrile with 0.2% formic acid and 5 mM ammonium formate, and peptides were separated using a 2-h gradient as follows: 0–13 min, 0% B; 13–18 min, 0–3% B; 18–88 min, 3–22% B; 88–100 min, 22–70% B; 100–101 min, 70–85% B; 101–105 min, 85% B; 105–106 min, 85–0% B; 106–120 min, 0% B. In this method the column was kept at 60°C and the flow rate was 225 nL/min except during periods of high %B (88–106 min), when it was reduced to 200 nL/min.Data were collected using two systems, each comprising a hybrid Orbitrap mass spectrometer  interfaced to a nanoflow HPLC system  via a nanospray ionization source . Samples were separated using a column with integrated spray tip  packed in-house with C18 particles  at ultra-high pressureeriments  and 2, mics runs  and 2, Mics runs , 4 and 6ere used . For AI-https://github.com/mazbradberry/public/tree/glycoproteomics. For proteomics experiments with label-free quantification (LFQ), SV and synaptosome experiments were analyzed separately. Spectra were searched with MSFragger (v.3.4)59 using a mouse proteome database downloaded from Uniprot on 11 October 2021 and the following settings: precursor mass tolerance, ±20 ppm; fragment mass tolerance, ±20 ppm, mass calibration and parameter optimization enabled; isotope error, 0/1/2; enzymatic cleavage, strict trypsin with up to 2 missed cleavages; peptide length, 7–50; peptide mass range, 500–5000 Da. Methionine oxidation and N-terminal acetylation were allowed as variable modifications and cysteine carbamidomethylation was included as a fixed modification. Validation was performed with PeptideProphet using closed search defaults for peptides and ProteinProphet for proteins. LFQ was performed with IonQuant96 with match-between-runs, normalization, and MaxLFQ enabled. The following settings were used: feature detection m/z tolerance, ±10 ppm; feature detection RT tolerance, 0.4 min; match between runs (MBR) tolerance 5 min, MBR ion FDR, 0.01; MBR peptide and protein FDR, 1; top 3 ions used for quantification with a minimum frequency of 0.5 and detection in at least 1 experiment. For glycoproteomics experiments, runs were grouped by sample type  prior to analysis. The same mouse protein database was used and spectra were searched with MSFragger (v.3.4) using glyco mode60 with the following settings: precursor mass tolerance, ±20 ppm; fragment mass tolerance, ±20 ppm, mass calibration and parameter optimization enabled; isotope error, 0/1/2; enzymatic cleavage, strict trypsin with up to 2 missed cleavages; peptide length, 7–50; peptide mass range, 400–5000 Da. Methionine oxidation and N-terminal acetylation were allowed as variable modifications and cysteine carbamidomethylation included as a fixed modification. The default 183 mass offsets corresponding to possible N-glycan compositions were included and restricted to asparagine residues. Labile modification search mode was set to nglycan and diagnostic Y ion and fragment masses were left as defaults. PTM-Shepherd was enabled, diagnostic ion search was enabled with default settings, and glycans were assigned in N-glycan mode with an FDR of 0.025, mass tolerance of ±50 ppm, and isotope error range of −1 to 3. PeptideProphet and ProteinProphet were used for peptide and protein validation, respectively, with default settings. The same parameters were used for glycoproteomic studies of immunoprecipitated SV2 and syt1. Glycopeptide spectral matches (GlycoPSMs) were obtained from the psm output file, filtered for Q-values of ≤0.025, and annotated using the table shown in N-and O-glycosylation on the same peptide.22 Tetra-fucosylated glycans and compositions tentatively identified as singly fucosylated oligomannose glycans were also detected but were omitted from analysis given their uncertain identification and small contribution (<5%) to the total glycan pool. Relative frequencies of unique annotated and included N-glycans, along with glycoprotein LFQ intensity quintiles .98 Organelle markers  with the addition of widely used neuronal surface and SV markers.Raw files from LC-MS runs were analyzed using the FragPipe software suite (v.17.1) with further processing of FragPipe output performed in R. All R scripts used are available via Github at uintiles , were deuintiles , gene li markers  were det2O, and the sample (~3.2 mL) was applied. The cartridge was washed (2 × 1 mL 95:5 MeCN:H2O) and procainamide-labeled glycans eluted with 500 μL 50:50 MeCN:H2O. The eluates were dried using a speedvac, resuspended in Milli-Q water , and centrifuged to remove insoluble material.For synaptosome samples, 50 μL synaptosomal lysate in 1% DDM and 0.5% SDS was combined with 50 μL 2% DDM and 5 μL1 M DTT and heated to 50°C for 15 min. For SV samples, 100 μL of SDS-DDM eluate  was combined with 5 μL 1M DTT and heated to 50°C for 15 min. For each sample, 1 μL PNGase F solution  was then added, and the mixture was incubated at 37°C for 2 h. Each sample was allowed to cool to room temperature, combined with 50 μL of freshly prepared procainamide solution (40 mg/mL in 70:30 DMSO:acetic acid) and incubated on ice for 5 min. Samples were then centrifuged  and the supernatants (150 μL) transferred to fresh PCR tubes. 10 μL of sodium cyanoborohydride solution  was added to each reaction, which was then incubated at 65°C for 2 h in a fume hood using a miniature PCR block. All steps involving sodium cyanoborohydride, including sample cleanup, were carried out in a fume hood. The samples were then combined with 20 volumes of MeCN  in 2-mL microcentrifuge tubes and subjected to cleanup by solid-phase extraction using a vacuum manifold. For each sample, a solid-phase extraction cartridge  was equilibrated with 1 mL 95:5 MeCN:H2+ buffer , 1 μL 10x BSA , and 1 μL of each enzyme used as described in 4 μL procainamide-labeled glycans were combined with 1 μL 10x sodium acetate – CaAn Agilent HPLC system (Infinity 1260 Bio-inert) equipped with a fluorescence detector , amide HILIC column  and manual injector was used for analysis of procainamide-labeled glycans. 10 μL glycan digest was injected to overfill a 5 μL home-cut sample loop and the column was kept at 40°C. Mobile phase A was 100 mM ammonium formate, pH 4.4, mobile phase B was 100% acetonitrile. Samples were separated with a 90-min gradient as follows: 0–60 min, 75–62.5% B; 60–62 min, 62.5–15% B; 62–82 min, 15% B; 82–85 min, 15–75% B; 85–90 min, 75% B. The flow rate was 0.25 mL/min except during periods of lower % B (62–85 min), when it was reduced to 0.175 mL/min 30 min was allowed between runs for re-equilibration. Peak areas were determined by automatic integration in Agilent ChemStation software with the following settings: tangent skim mode, new exponential; tail peak skim height ratio, 5.00; front peak ski height ratio, 5.00; skim valley ratio, 20.00; baseline correction, advanced; peak to valley ratio, 500. Peaks eluting between 19 and 60 min were considered for analysis.Synaptosome and SV samples were combined with 4X SDS sample buffer containing DTT and heated to 50°C for 15 min except as shown in In 1345678910


Ins1 and Ins2) in INS-1E cells and pancreatic islets. Moreover, GIP_HUMAN [22–51] promoted the translocation of NF-κB from cytoplasm to the nucleus. In the presence of a pharmacological inhibitor of NF-κB, GIP_HUMAN [22–51] was unable to suppress Ins2 mRNA expression. Moreover, GIP_HUMAN [22–51] downregulated insulin secretion at low (2.8 mmol/L) but not high (16.7 mmol/L) glucose concentration. By contrast, GIP_HUMAN [22–51] failed to affect cell proliferation and apoptosis. We conclude that GIP_HUMAN [22–51] suppresses insulin expression and secretion in pancreatic β cells without affecting β cell proliferation or apoptosis. Notably, the effects of GIP_HUMAN [22–51] on insulin secretion are glucose-dependent.GIP_HUMAN [22–51] is a recently discovered peptide that shares the same precursor molecule with glucose-dependent insulinotropic polypeptide (GIP). In vivo, chronic infusion of GIP_HUMAN [22–51] in ApoE−/− mice enhanced the development of aortic atherosclerotic lesions and upregulated inflammatory and proatherogenic proteins. In the present study, we evaluate the effects of GIP_HUMAN [22–51] on insulin mRNA expression and secretion in insulin-producing INS-1E cells and isolated rat pancreatic islets. Furthermore, we characterize the influence of GIP_HUMAN [22–51] on cell proliferation and death and on Nf-kB nuclear translocation. Rat insulin-producing INS-1E cells and pancreatic islets, isolated from male Wistar rats, were used in this study. Gene expression was evaluated using real-time PCR. Cell proliferation was studied using a BrdU incorporation assay. Cell death was quantified by evaluating histone-complexed DNA fragments. Insulin secretion was determined using an ELISA test. Nf-kB nuclear translocation was detected using immunofluorescence. GIP_HUMAN [22–51] suppressed insulin ( Blood glucose levels are regulated by numerous endocrine factors. Two major pancreatic hormones, insulin and glucagon, maintain glucose homeostasis through the complex regulation of glucose synthesis, intracellular glucose uptake and metabolism . It is wStudies on the biological role of GIP_HUMAN [22–51] showed that continuous infusion of GIP_HUMAN [22–51] for four weeks in mice potentiated the development of aortic atherosclerotic lesions and atheromatous plaque formation and increased proatherogenic proteins in an ApoE−/− mouse model. In contrast, infusion of the neutralizing GIP_HUMAN antibody blocked the effects on plaque formation. Therefore, the authors of this study suggested that GIP_HUMAN [22–51] can be considered as a new proatherogenic peptide .It is important to note that abnormalities in cardiovascular systems are common complications in patients with type 1 and 2 diabetes ,10. GIP GIP_HUMAN [22–51]  was synt2, humid atmosphere). Cells were cultured in a RPMI 1640 medium supplemented with 10% fetal calf serum FCS, 2 mmol/L glutamine, 100 kU/L penicillin, 100 mg/L streptomycin, 10 mmol/L HEPES buffer, 1 mmol/L sodium pyruvate, and 50 μmol/L β-mercaptoethanol.Rat insulin-producing INS-1E cells (a well-established β cell model to study insulin secretion and β cell biology ) were cuPancreatic islets were isolated from male Wistar rats weighing 300–350 g, as we previously described . Animals5 cells/well) were cultured in 6-well plates. After 24 h, the standard incubation medium was replaced by a serum-free medium, containing 0.1% free fatty acid and BSA, and the cells were incubated for an additional 3 h. Thereafter, GIP_HUMAN [22–51] peptide was added at a concentration of 0, 10 or 100 nmmol/L, and cells were then incubated for 3 and 24 h. The RNA was isolated using Extrazol . The quality and quantity of isolated RNA were determined using a NanoDrop 1000 spectrophotometer . RNA was stored at −86 °C for further analysis. Next, the RNA (0.5 µg) was reverse transcribed to cDNA using a FIREScript RT cDNA Synthesis KIT . The cDNA obtained in the previous step was diluted 10-fold. The PCR reaction mix was prepared using specific primers and HOT FIREPol EvaGreen qPCR Mix Plus . A PCR reaction was performed using QuantStudio 12 K flex (ThermoFosher Scientific). To calculate relative gene expression, the Hprt1 reference gene was used. PCR product specificity was confirmed using melting curve analysis. The following sequences of primers were used: Hprt1 (NM_012583.2): cagtcaacgggggacataa (left), ttggggctgtactgcttga (right), Ins1 (NM_019129.3): gaggctctgtacctggtgtg (left), ccaaggtctgaagatccccg (right), Ins2 (NM_019130.2): gtgaccagctacagtcggaa (left), accacaaaggtgctgtttgac (right), Pdx1 (NM_022852.4): cctttcccgaatggaaccga (left), and ttttccacgcgtgagctttg (right).After seeding, cells , cell membranes were permeabilized using 0.2% Triton X-100 in PBS  and washed . Non-specific bounds were neutralized  and incubated with a primary antibody (NF-kB p65/RelA Rabbit pAb (cat. no. A2547), Woburn, MA, USA). This antibody (diluted 1:160) was added for overnight incubation at 4 °C. PBS-Tween washing was performed twice, and membranes were incubated with a secondary antibody  for 1.5 h at RT. After being washed three times for 3 min each at RT, cells were mounted using a Fluoroshield with DAPI . Fluorescence was measured using a 488 nm excitation filter, and emissions were observed using a 520 nm emission filter under a LSM 510 inverted microscope . Images were processed using Axio vision v. 4.6 software.Nuclear translocation of Nf-kB was determined using immunofluorescence staining . INS-1E 4 cells/well) and cultured for 24 h. Then, cells were washed with a PBS buffer, and a 500 μL Krebs glucose-free solution was added . Incubation was conducted for 1 h. The role of GIP_HUMAN [22–51] peptide (10 mmol/L or 100 nmol/L) in insulin secretion was studied at low (2.8 mmol/L) or high (16.7 mmol/L) glucose concentrations. After incubation for 1 h, the KRB buffer was collected and centrifuged (200× g for 5 min) and samples were stored at −20 °C for the determination of released insulin. Insulin secretion was measured using rat insulin ELISA . Insulin secretion was normalized to protein content, which was measured using a Pierce™ BCA Protein Assay Kit . Insulin secretion from isolated pancreatic islets was determined, as previously described  peptide was added at a concentration of 0 or 100 nmol/L, and INS-1E cells were then incubated for 24 h. After the incubation, cells were washed with PBS, 1 mL of 75% ethanol containing 1.5% HCl was added, and cells were incubated overnight at −20 °C, as previously described  at 1, 10, or 100 nmol/L. Cells were then cultured for 24 and 48 h. The BrdU incorporation was tested using the Cell Proliferation ELISA BrdU (colorimetric) Kit . In brief, after the incubation with the GIP_HUMAN [22–51] peptide, a BrdU solution with a final concentration of 10 µmol/L was added to each well, and cells were cultured in a cell culture incubator for an additional 2 h. Next, the cell culture medium was removed, 200 µL of FixDenat solution was added, and cells were incubated for 30 min at RT. Afterwards, the FixDenat solution was removed and 100 µL of Anti-BrdU-POD working solution was added. After the incubation (70 min at RT), plates were washed three times using the washing buffer. Next, a substrate solution (100 µL) was added, and plates were incubated for approximately 10 min. The reaction was stopped by the addition of 25 µL HCl. The absorbances of samples were measured at 450 nm (reference wavelength was 690 nm) using a Synergy 2 Multi-Mode Microplate Reader .First, INS-1E cells were cultured in 96-well plates (2 × 104 cells per well (24 well-plates) were cultured in a serum-free medium for 3 h. Afterwards, a new medium supplemented with GIP_HUMAN [22–51] at 10 or 100 nmol/L concentration was added. Then, cells were incubated for 48 h in the presence or absence (control) of GIP_HUMAN [22–51]. Thereafter, cells were lysed in the Lysis Buffer, and apoptotic cells were measured using the Cell Death Detection ELISA PLUS kit according to the manufacturer’s protocol. Absorbances of samples at 405 nm (reference wavelength: 690 nm) were evaluated using the Synergy 2 Multi-Mode Microplate Reader (BioTek).Apoptosis was measured by the Cell Death Detection ELISA PLUS Kit (Roche Diagnostics), which is suitable for determining cytoplasmic histone-associated DNA fragments (mono- and oligo-nucleosomes) during cell death. Before the test, cells at a density of 8 × 10p < 0.05 (*) was considered as statistically significant difference.Results are presented as the mean ± standard error of the mean. Statistical analyses were performed using GraphPad Prism version 8.0 . One-way ANOVA followed by the Tukey post hoc test or if the normality test failed, non-parametric Kruskal–Wallis test were used. Ins1 and Ins2). Ins2 is an ortholog to the human insulin gene  may impact insulin mRNA expression in INS-1E cells. Due to a limited number of data, molecular mechanisms through which GIP_HUMAN [22–51] affects cellular functions has been poorly characterized so far. Nevertheless, a previous study reported that, in human aortic endothelial cells (HAoECs) or THP1-derived macrophages, GIP_HUMAN [22–51] induces translocation of NF-κB from cytoplasm to the nucleus  lowers insulin mRNA expression ( nucleus . Of note nucleus . Importa nucleus . Therefo–51 lowerIns1 and Ins2 mRNA expression is known from previous studies that evaluated the transcriptional regulation of Ins1 and Ins2 mRNA expression -treated β cells should be helpful for answering this question.This differential effect of pression ,24. Furtpression  found thpression . However1E cells . FurtherFurthermore, we observe that GIP_HUMAN [22–51] suppressed basal, but not glucose-induced, insulin secretion from INS-1E or from isolated rat pancreatic islets. In the present study, we did not evaluate the intracellular mechanism by which GIP_HUMAN [22–51] may suppress insulin secretion selectively at the low glucose concentration. It is well known that high levels of glucose-stimulated insulin secretion results from the closure of ATP-sensitive potassium channels, which leads to plasma membrane depolarization and the opening of voltage-sensitive calcium channels. Increases in intracellular calcium levels consequently cause insulin exocytosis . In contIn summary, these results indicate that GIP_HUMAN [22–51] is not involved in controlling postprandial insulin exocytosis. Nevertheless, it needs to be pointed out that our experiments are limited to in vitro incubation of insulin-producing cell lines and isolated islets only. Therefore, the potential role of GIP_HUMAN [22–51] in controlling insulin secretion remains to be investigated in vivo or in a setting that allows for the preservation of islet microcirculation.An additional finding in our study is that GIP_HUMAN [22–51] failed to affect insulin cell proliferation and viability. When discussing this point, it is worth noting that several research groups imply that NF-κB is involved in controlling pancreatic β cell death. For example, some studies indicate that in pancreatic β cells cytokines or pro-diabetic agents regulate cell viability and death via an NF-κB-dependent mechanism ,20. MoreIn the present study, we did not evaluate the mechanisms through which GIP_HUMAN [22–51] activates Nf-kB. Nevertheless, Masaki et al. reported that GIP_HUMAN [22–51] causes the degradation of IκB-α , which iIn summary, we show here that GIP_HUMAN [22–51] may contribute to glucose metabolism via the regulation of insulin mRNA expression. GIP_HUMAN [22–51] is a potent proatherogenic peptide . AtherosIn conclusion, we found that GIP_HUMAN [22–51] suppresses insulin expression and secretion in INS-1E cells and rat pancreatic islets, potentially through an NF-κB-dependent mechanism.


M. tuberculosis challenge. In the circulation, Vδ2 T cell populations expanded after IV BCG vaccination, from a median of 1.5% (range: 0.8–2.3) of the CD3+ population at baseline, to 5.3% (range: 1.4–29.5) 4 weeks after M. tb, and were associated with TB protection. This protection was related to effector and central memory profiles; homing markers; and production of IFN-γ, TNF-α and granulysin. In comparison, Vδ2 cells did not expand after ID BCG, but underwent phenotypic and functional changes. When Vδ2 responses in bronchoalveolar lavage (BAL) samples were compared between routes, IV BCG vaccination resulted in highly functional mucosal Vδ2 cells, whereas ID BCG did not. We sought to explore whether an aerosol BCG boost following ID BCG vaccination could induce a γδ profile comparable to that induced with IV BCG. We found evidence that the aerosol BCG boost induced significant changes in the Vδ2 phenotype and function in cells isolated from the BAL. These results indicate that Vδ2 population frequency, activation and function are characteristic features of responses induced with IV BCG, and the translation of responses from the circulation to the site of infection could be a limiting factor in the response induced following ID BCG. An aerosol boost was able to localise activated Vδ2 populations at the mucosal surfaces of the lung. This vaccine strategy warrants further investigation to boost the waning human ID BCG response.Intravenously (IV) delivered BCG provides superior tuberculosis (TB) protection compared with the intradermal (ID) route in non-human primates (NHPs). We examined how γδ T cell responses changed in vivo after IV BCG vaccination of NHPs, and whether these correlated with protection against aerosol  M. tuberculosis (M. tb) infections [Mycobacterium bovis. The BCG vaccine provides up to 80% protection against the most severe forms of TB, including TB meningitis in children [Tuberculosis (TB) remains a leading cause of mortality due to an infectious disease, with COVID-19 halting progress to reduce the high global burden of TB [children . Howeverchildren . TherefoThere are several promising TB vaccines in various stages of the vaccine pipeline . Some ofM. tb infection by delivering BCG via the aerosol route may provide immunological advantages, as well as removing the requirement for using needles that require highly trained personal and safe disposal. There is evidence from work in the macaque model that primary vaccine delivery via the pulmonary route may be more protective than the intradermal (ID) route [M. tb challenge. However, other macaque studies indicate that many immune responses may be delayed after a single primary aerosol BCG vaccination [Aligning the vaccination route with the primary route of D) route ,14, and D) route . StudiesD) route  and greaD) route ,18 immunD) route  after M.cination , and boocination .Intravenous (IV) BCG vaccination has been shown to greatly enhance TB protection in non-human primate (NHP) models ,12,21, bIn addition to changes in conventional T cells, Darrah et al. also identified an increase in Vγ9Vδ2 T cells in the BAL fluid after IV BCG . γδ T ceWe aimed to utilise the macaque IV BCG vaccination and aerosol TB challenge model to confirm the role of Vδ2 cells in the IV BCG immune response, and to determine whether γδ T cell signatures correlated with TB disease protection. We used immunophenotyping and functional flow cytometry panels to characterise the Vδ2 cell response to vaccination, then determined how they may be involved in a protective response and whether this differed to responses defined after ID BCG. Furthermore, we investigated whether an aerosol BCG boost could induce changes to the Vδ2 cells locally that were similar to the IV BCG benchmark.Macaca mulatta) of an Indian genotype sourced from a UK breeding colony were used in this study. All animals were adult males, aged four—12 years. Animals were housed in compatible social groups in accordance with UK Home Office and NC3Rs guidelines, as described previously [M. tb-challenged animals  of aerosolised BCG achieved using an Omron U22 vibrating mesh nebuliser. Immune responses in samples of whole blood and PBMCs were measured after primary vaccination and boosting vaccination. BAL samples were collected for a local immune analysis prior to week 0, and at weeks 9, 12 and 14–16 (at necropsy) post ID BCG. All macaques in this study were males and six years of age.Lastly, in the third study described in M. tuberculosis Erdman strain K 01 (BEI Resources), prepared as previously described [Animals in the study outlined in escribed . A targeescribed ,26,27, tescribed .M. tb for the macaques in the study in The clinical outcomes following exposure to Peripheral blood mononuclear cells (PBMCs) were isolated from heparin anti-coagulated blood using standard methods, as previously described . MononucA surface staining antibody cocktail was developed using the following fluorochrome conjugated antibodies prepared in a brilliant stain buffer : CD3 AF700, CD195  PerCP-Cy5.5 ; TCR Vγ9 (7A5) FITC, TCR Vδ1 (TS8.2) PE-Cy7 ; NKG2D APC, CD27 BV711, CD69 BV510, CX3CR1 BV421, PD-1 BV605, CD103 BV785 ; CD45RA APC-Vio770 ; and TIM3 PE . Amine-reactive Live/Dead Fixable Red viability cell stain  was also included to enable separation of non-viable cells. Antibodies were titrated for use with mononuclear cells as well as whole blood.2 in RPMI + 10% FCS (R10) at a concentration of 1–2 × 106/mL.For evaluation, cryopreserved PBMC a samples6 cells were incubated at room temperature with the surface staining antibody cocktail above for 30 min in the dark. Then, samples were washed twice with the FACs buffer and fixed with a final concentration of 4% paraformaldehyde .For surface staining of incubated PBMCs or freshly isolated BAL mononuclear cells, samples were washed with a FACs buffer (PBS  +  1% FCS), counted and 1 × 10BD Compbeads  were labelled with the above fluorochromes for use as compensation controls. Cell data were acquired using a five-laser LSRII Fortessa instrument  and data were analysed using FlowJo software . Appropriate gating strategies were used to identify the lymphocyte population, exclude doublet events and gate on viable cells only. Vδ1 and Vδ2 subtypes were delineated using the expression of CD3, Vδ1 and Vγ9; their activation status using CD69 and NKG2D; their memory phenotype using CD45RA and CD27; their homing marker expression using CCR5, CX3CR1 and CD103; and their expression of the regulation/exhaustion markers using PD-1 and TIM3. Graphpad Prism  was used to generate graphical representations of flow cytometry data. The gating strategies for flow cytometry analyses are summarised in Whole blood immunophenotyping (WBIP) was carried out on blood samples collected from the macaques enrolled in the ID BCG, aerosol-boosted study, Intracellular cytokine staining (ICS) assays were utilised to determine the capacity of γδ T cells within the PBMCs or BAL mononuclear cells to produce inflammatory cytokine and degranulation/cytotoxicity markers following BCG vaccination.2 in the following conditions: without any stimulation; stimulation with BCG at a 1:1 ratio with mononuclear cells (chosen as the result of previous titration); or stimulation with PMA + Ionomycin (positive control). Two hours into the overnight incubation, the protein transport inhibitor Brefeldin-A  was added at a final concentration of 10 μg/mL, alongside the antibody for degranulation, CD107a (LAMP-1) Pacific Blue .Prior to staining, PBMCs or BAL mononuclear cells were incubated overnight with an R10 medium at 37 °C + 5% COAfter incubation, cells were washed with the FACS buffer and surface stained following the protocol for surface staining described above, using the antibodies CD3 AF700 , TCR Vγ9 FITC, TCR Vδ1 PeCy7  and Amine-reactive Live/Dead Fixable Red . Following surface staining, cells were washed and permeabilised by incubating with a cytofix/cytoperm reagent  for 15 min at room temperature in the dark. After permeabilization, cells were washed with a Permwash buffer  and incubated at room temperature for 30 min with an intracellular staining mix, consisting of the following fluorochrome conjugated antibodies prepared in the Permwash buffer: granzyme B APC, granulysin PE, IFN-γ BV510 ; TNF-α (MAb11) BUV395, IL-17 BV711 (both Becton Dickinson UK Limited). Cells were then washed twice with the Permwash buffer, and fixing and flow cytometry was carried out for the surface staining protocol.p-value of 0.031, and therefore the actual p-value is expected to be at or lower than 0.031, and therefore designated ‘p < 0.040’ throughout. γδ T cell profiles between groups were compared using Mann–Whitney U tests. The main conclusions from the paper are based on findings from pre-planned statistical analyses and therefore p-values were not adjusted for multiple comparisons.Raw flow cytometry FCS files were analysed using Flowjo v10.6.2  and data were transferred to Graphpad Prism v9.3.0  for graphical representation and a statistical analysis. Individual values were presented on scatter plots, box plots or histograms, depending on whether the comparison was between groups or timepoints, as well as the number of timepoints shown. Cytotoxicity data are presented using Log2-fold changes. γδ T cell proportion, phenotype and functional responses were compared before and after vaccination using a Wilcoxon-rank test. As one study had an N of 6 a, this tTo investigate any associations between the γδ T cells and the clinical and health outcomes, Spearman correlation tests were performed using Graphpad Prism v9.3.0 0 .To explore 3-dimentional relationships between phenotypic markers throughout the study, flow cytometry data from six different animals at five different timepoints were concatenated before the t-distributed stochastic neighbour embedding (tSNE) dimensionality reduction was performed using Flowjo v10.6.2 . A representative sample of live γδ T cells from each timepoint was taken using the Downsample gate function. tSNE parameters included a perplexity of 30 and 1000 iterations. Cell clusters were manually identified and gated. Heat map overlays represent the level of expression of each marker independently.p < 0.040) (p < 0.040). In contrast, the proportion of Vδ1 cells in the PBMCs showed a small, but significant, increase from baseline at 8 weeks after BCG (p < 0.040)  c. Furthe< 0.040) c.M. tb challenge 21 weeks after IV BCG vaccination, and clinical outcomes were monitored. TB disease outcomes were measured at necropsy 16 weeks later. Correlation analyses were undertaken to investigate relationships between the immune response post vaccination and clinical measures/disease outcomes. We found that possession of a higher proportion of Vδ2 cells in the PBMCs 12 weeks after IV BCG vaccination was associated with a lower lung consolidation score . This association was also present at baseline, prior to vaccination, and 8 weeks after vaccination, but did not reach significance , 8  and 12  weeks after IV BCG vaccination was associated with a worse TB pathology score, and a higher Vδ1:Vδ2 ratio was associated with more TB disease  monocytes , and mayM. tb challenge. We therefore sought to investigate this association in more detail, using phenotypic and functional assays.Overall, the proportion of Vδ2 T cells expanded in the blood after IV BCG vaccination, and a higher proportion of Vδ2 cells was associated with reduced TB disease severity after M. tb challenge, these cells were examined for phenotypic changes over the time course following IV BCG. Markers of memory, activation, homing and regulation were included in a flow cytometry panel  . There was also a trend for higher EM Vδ2 cells at week 12 after IV BCG, associating with a reduced lung consolidation score, although this did not reach statistical significance   a. There < 0.040) b. Cells  −0.829) c.p < 0.040 for both), although expression returned to baseline levels by 12 weeks after IV BCG . Vδ2 CX3CR1 homing marker expression 6 weeks after IV BCG was also associated with better clinical measures at the end of the study, specifically haemoglobin levels and weight gain following M. tb challenge    e. Conver= 0.886) e.p < 0.040 for both). Resuscitated PBMCs were stimulated overnight with BCG, intracellular cytokine staining was conducted, and flow cytometry  was applp < 0.040 for both) relative to those measured before vaccination. Correlation analyses indicated that the possession of more BCG-specific Vδ2 cells producing granulysin 4 weeks after IV BCG was associated with weight gain after M. tb challenge, indicating better health. In contrast, more BCG-specific Vδ2 cells expressing CD107a 8 weeks after IV BCG were associated with a higher total pathology score in animals that received aerosol M. tb challenge  assays applied to PBMCs at selected timepoints, as described in p = 0.004) 4 weeks post vaccination (p = 0.004 for both), although the extent of the change was markedly less than after IV BCG. Vδ2 TEMRA cells also increased at weeks 4 and 9 (p = 0.016 for both) after ID BCG. There was a trend towards a decrease in the Vδ2 CM population after ID BCG, although this did not reach significance  relative to pre-vaccination levels. In contrast to IV BCG vaccination, homing marker expression did not significantly change after ID BCG vaccination; however, a trend towards a decrease in CCR5 and CX3CR1 expression was seen  and 9 (p = 0.004) after ID vaccination, as well as TNF-α at week nine (p = 0.004) (p = 0.004) relative to baseline levels, and this further increased in response to ex vivo BCG stimulation (p = 0.008).When ICS assays were undertaken on PBMCs at selected timepoints, we found increased frequencies of BCG-specific IFN-γ-producing Vδ2 cells relative to baseline levels, at week 4 (= 0.004) e. The prM. tb infection.These data indicate that even though Vδ2 cells are not expanded in the circulation after ID BCG, as they are after IV BCG, they respond to vaccination, and show BCG-specific functional responses, specifically the production of IFN-γ and TNF-α and the expression of CD107a. Thus, we hypothesised that investigating the phenotype and function of Vδ2 cells in the lung would provide further insights into the differences caused by the altered vaccination routes, especially given IV BCG protection was demonstrated against aerosol p = 0.0004). IV BCG BAL Vδ2 cells also showed significantly higher expression of the CCR5 inflammatory homing marker (p = 0.026)  were collected from macaques 8 to 9 weeks after BCG vaccination to coincide with the peak in functional responses identified in the circulation after both IV and ID vaccination strategies. Comparison of Vδ2 cell phenotypes identified in BAL samples collected after IV or ID BCG vaccination revealed that the proportion of Vδ2+ cells did not significantly differ at this timepoint a, wherea= 0.026) b. CD69 i= 0.026) c.p = 0.002), and a trend towards higher expression of PD-1 (p = 0.066), than cells from ID BCG animals 8 to 9 weeks after vaccination  (p = 0.0004)  d. In comor both) e, only aor both) d. In con 0.0004) d.p = 0.005 and p = 0.012). This indicates that the BAL Vδ2 cells were degranulating in response to IV BCG vaccination, and this degranulation increased with a further ex vivo BCG exposure. The proportion of Vδ2 cells with intracellular granulysin and granzyme B did not differ between the two vaccination routes.Despite the induction of phenotypic and functional changes in the circulation with standard ID BCG vaccination, these did not translate to changes locally in the BAL, which may be important for TB protection. We therefore sought to understand whether an aerosol boost could induce changes in the Vδ2 cells locally in the BAL, as IV BCG vaccination had.p = 0.027), which corresponded to a decrease in the blood (p = 0.004) (p = 0.012), although levels were increasing prior and may still be a result of the ID BCG prime.The cohort of animals used to investigate the Vδ2 cell response to ID BCG vaccination  received= 0.004) c. CM Vδ2p = 0.004 and p = 0.031) (p = 0.004)  d. CD69 e= 0.004) d.p = 0.004), and a median of 1.84% (range: 0.58–7.31) to 17.2% (range: 7.91–19.5) (p = 0.004), respectively (p = 0.008). In contrast, CCR5 expression on BAL Vδ2 cells was low, with the suggestion of an upward trend between 3 and 5 weeks post boost. Lastly, 1 week after the aerosol boost, a large increase in the proportion of Vδ2 cells expressing PD-1 in the BAL (p = 0.004) was observed together with a smaller increase in the blood (p = 0.020).Homing marker expression by Vδ2 cells underwent considerable change after the aerosol BCG boost. The proportion of Vδ2 cells expressing CX3CR1 and CD103 in the BAL increased significantly 1 week post boost, from a median of 5.53% (range: 1.3–26.2) to 83.3% (range: 72.8–94.9) (ectively d. Vδ2 exp = 0.008 for both) and maintained 3 and 5 weeks (p = 0.004 and p = 0.008) after the boost. In contrast, there appeared to be an increase in the proportion of Vδ2 cells producing these cytokines in the BAL at the same timepoints, although this did not reach statistical significance , 1 week post boost (week 12 post ID BCG) and at necropsy 3 to 5 weeks post boost (14–16 weeks post ID BCG). A substantial decrease in the proportion of IFN-γ- and TNF-α-producing Vδ2 cells in the blood was observed 1 week (ificance a.p = 0.008) and 3 to 5 weeks post boost (p = 0.023) (p = 0.008) after the aerosol boost and there was a trend towards an increase between 3 and 5 weeks post boost (p = 0.055). A trend towards a further increase after ex vivo BCG stimulation was also observed.Examination of cytotoxicity markers after the aerosol BCG boost revealed that the proportion of Vδ2 cells displaying the CD107a degranulation marker significantly decreased in the blood 1 week (= 0.023) b. In conM. tb infection.Utilising the rhesus macaque model of IV BCG, we successfully investigated the role of γδ T cells in the IV BCG vaccine response, and determined that increased Vδ2 responses after vaccination are correlated with a decrease in TB disease severity, as outlined in M. tb infection and TB disease. The paradigm of a largely CD4+ response producing IFN-γ to enhance phagocytic cell activities is an important but limiting model. It is now recognized that there are many other important cell types in the TB response, including γδ T cells, MAIT cells and NK cells, as well as other secreted factors, such as IL-17 and other cytokines, cytotoxic molecules and antibodies, as reviewed by others [There is a lack of knowledge governing our understanding of what constitutes a protective immune response to y others ,33,34. Ty others .The BCG vaccine given using the standard route of intradermal injection offers limited immunity. However, when given using the IV route in preclinical models, it has been shown to induce far superior protection, even sterilising immunity ,12. HoweM. tb challenge  recognises small phosphoantigen build-up intracellularly via conformational changes occurring on the Butyrophilin receptors  [Vδ2 cells have many properties that may make them an important tool in the arsenal against lularly) . This bulularly) . Importalularly) . Once aclularly) . Dieli elularly) . As welllularly) ,41, as wlularly) .The switch in the Vδ2 memory phenotype towards EM and CM after IV BCG vaccination was associated with a better TB outcome e. There Our finding of a transient trend towards an increase in CD69 and CCR5 expression by Vδ2 cells in the blood after IV BCG d, followWe also found that IV BCG vaccination led to a high proportion of functional Vδ2 cells in the airways d,e, wherM. tb infection. Interestingly, expression of this marker in the blood later in the vaccination phase was associated with an increase in pathology  have taken place in Brazil, Malawi, Guinea Bissau and South Africa ,8,9,64,  studies ,65.We and others previously demonstrated that aerosol-delivered BCG is well tolerated in macaques ,20,66,67M. tb challenge, and thus the protective efficacy of the changes induced in the BAL cannot be determined. However, as similar changes were occurring to Vδ2 cells in the protective IV BCG response, these results are a promising indicator. Further investigation into this strategy with M. tb challenge to determine efficacy is warranted.A limitation of our study is that our macaques were ID BCG-vaccinated as adults and boosted 11 weeks later. In reality, it may be more feasible for an infant prime and adolescent aerosol boost when protection may be waning. We have now carried out this regime in a small pilot study of macaques, N = 3, and found that, as well as inducing an increase in PPD-specific IFN-γ cells and trained immunity signals, there was also a high proportion of γδ T cells in the BAL . AnotherM. tb, critical for accelerating the vaccine development pipeline.Despite protective efficacy, it is unlikely IV BCG will be rolled out in its current form due to safety concerns around the potential consequences of giving a live vaccine intravenously, especially to infants in rural settings. However, application through this route demonstrates the potential that BCG has in eliciting a potent immune response, allowing us to identify new immune correlates of protection and providing us with a benchmark against which we can measure new strategies. Here, we demonstrated the use of both these tools, discovering that Vδ2 T cells are associated with protection in this model, and that ID BCG plus an aerosol BCG boost induces similar changes to these cells in the BAL. These investigations represent pivotal steps in deciphering protective immunological mechanisms against


Repetitive transcranial magnetic stimulation (rTMS) is a promising intervention for late-life depression (LLD) but may have lower rates of response and remission owing to age-related brain changes. In particular, rTMS induced electric field strength may be attenuated by cortical atrophy in the prefrontal cortex. To identify clinical characteristics and treatment parameters associated with response, we undertook a pilot study of accelerated fMRI-guided intermittent theta burst stimulation (iTBS) to the right dorsolateral prefrontal cortex in 25 adults aged 50 or greater diagnosed with LLD and qualifying to receive clinical rTMS.Participants underwent baseline behavioral assessment, cognitive testing, and structural and functional MRI to generate individualized targets and perform electric field modeling. Forty-five sessions of iTBS were delivered over 9 days . Assessments and testing were repeated after 15 sessions (Visit 2) and 45 sessions (Visit 3). Primary outcome measure was the change in depressive symptoms on the Inventory of Depressive Symptomatology-30-Clinician (IDS-C-30) from Visit 1 to Visit 3.Overall there was a significant improvement in IDS score with the treatment  with 13/25 (52%) achieving response and 5/25 (20%) achieving remission (IDS-C-30 < 12). Electric field strength and antidepressant effect were positively correlated in a subregion of the ventrolateral prefrontal cortex (VLPFC) (Brodmann area 47) and negatively correlated in the posterior dorsolateral prefrontal cortex (DLPFC).Response and remission rates were lower than in recently published trials of accelerated fMRI-guided iTBS to the left DLPFC. These results suggest that sufficient electric field strength in VLPFC may be a contributor to effective rTMS, and that modeling to optimize electric field strength in this area may improve response and remission rates. Further studies are needed to clarify the relationship of induced electric field strength with antidepressant effects of rTMS for LLD. In partRepetitive transcranial magnetic stimulation (rTMS) is a FDA-approved therapeutic option for treatment-resistant depression that may be effective for LLD , 5. By g1.1.Unfortunately, increased age has been associated with diminished rates of response and remission in multiple studies since the initial demonstrations of rTMS for depression . A 2022 1.2.|E|) of rTMS and correlation of its distribution and intensity with clinical and physiological outcomes. This capability provides a means for accurately and precisely assessing the effects of generalized and local atrophy on efficacy of rTMS in LLD. Electric field modeling has been used extensively in studies of the motor system, with strong correlations demonstrated between motor cortex |E|, coil-to-cortex distance, and motor threshold  to achieve the same |E| obtained in the motor cortex at 100% of threshold. A study by Deng et al. .2.1.via phone for inclusion and exclusion criteria.Recruitment took place through the UNM Treatment Resistant Depression Clinic, Geriatric Psychiatry Clinic, TMS Service, and ECT Service. All participants from the various clinics were referred for consideration of rTMS treatment for major depression, having failed various therapeutics such as oral antidepressants, esketamine, ECT, or traditional rTMS. Participants were screened 2.2.To be enrolled in the study, participants met the following inclusion criteria: 1) ages 50–79, 2) diagnosis of major depressive disorder of at least 6 months’ duration preceding study entry, confirmed by two independent board-certified psychiatrists according to DSM-5 criteria, 3) four or more adequate trials of antidepressants in the current episode, and 4) score of 10 or higher on the Quick Inventory of Depressive Symptomatology (16 item) (Self-Report) (QIDS-SR-16) at time of study entry. Exclusion criteria included: 1) history of seizure, 2) history of a major neurocognitive disorder or central nervous system disorder diagnosis, 3) implanted ferromagnetic material or contraindication to obtaining MRI, 4) pregnancy, 5) current incarceration, 6) inability to complete the protocol, 7) medical instability resulting in hospitalization or emergency department visit within the past month, and 8) psychotropic medication change or treatment with electroconvulsive therapy within the month preceding study entry.2.3.After screening and consent, participants underwent demographic survey ; assessment of depression history and treatment; mood and anxiety symptom assessment with the Inventory of Depressive Symptomatology for Clinicians ; Generalized Anxiety Disorder-7 (GAD-7); Snaith-Hamilton Assessment of Pleasure Seeking for Clinician Administration (SHAPS-C); Temporal Experience of Pleasure Scale (TEPS); and the Behavioral Inhibition System/Behavioral Approach System Scale (BIS/BAS). Select domains of cognition were assessed with the Delis-Kaplan Executive Function Scale (DKEFS), Wechsler Adult Intelligence Scale (WAIS), and Hopkins Verbal Learning Test-Revised (HVLT). These instruments were chosen in line with prior study protocols combining imaging and neuromodulation , 30.2.4.1- and T2-weighted structural images and two 6-min runs of resting-state functional MRI (rsfMRI) were obtained. For structural scans: repetition time (TR) = 2,530 milliseconds (ms), echo time (TE) = 1.64, 3.5, 5.36, 7.22, 9.08 ms, Inversion time (TI) = 1,200 ms, flip angle = 7.0°, slices = 192, field of view = 256, matrix 256 × 256, voxel size = 1.0 × 1.0 × 1.0 millimeter (mm). For resting-state scans: TR = 480 ms (multiband acceleration factor of 8), TE = 29 ms, flip angle (FA) = 75°, slices = 192, voxel size = 2.0 × 2.0 × 2.0 mm. The T1 was preprocessed by parcellating with Freesurfer 6.0.0 and then aligned to rsfMRI data  on a 3 T Siemens Prisma scanner. High-resolution TMRI data . The rsfMRI data . The fir2.5.1 images and the functional mask were then exported to the Localite neuronavigation system for registration during stimulation sessions.Resting-state fMRI analysis and determination of neuronavigation targets were based on the published method of Ning et al. . The use2.6.The 25 participants each received a total of 45 sessions  of iTBS to the cortical target with a MagPro X100 equipped with a Cool-B70 coil . The right DLPFC was chosen as the initial target region given its potential efficacy for depressive and anxious symptoms , 42, and2.7.After 15 sessions participants repeated all behavioral assessments as this corresponds to the timeframe for mid-course evaluation in a typical clinical rTMS course. They then received 30 more sessions. If there was minimal improvement (<10%) noted in IDS-C-30 score at Visit 2 or development of intolerable side effects, the participant was switched to stimulation of the left hemisphere for the remainder of treatment, in line with clinical practice. The day following completion of the 45th session, participants repeated behavioral assessments and cognitive testing (Visit 3). At 1 month and 2 months following the protocol, the subjects were contacted by phone and assessed with the IDS-C-30.2.8.ηp2) were calculated for Visit 1, 2, and 3 behavioral and cognitive outcomes using R v. 4.1.3 .Means and standard deviations for demographics and baseline behavioral and cognitive measures were calculated in SPSS Statistics 26 . Repeated-measures analyzes of variance and effect sizes expressed as partial eta squared (2.9.max|/2) or above half of the maximum induced standard deviation ], based on the entire cohort’s |E|. This restricted the analysis to 11 regions in the HCP-MMP atlas. Due to segmentation issues only 23 of 25 participants’ electric fields were included in the analysis.Using the T1 and T2 weighted images within Simulation of Non-Invasive Brain Stimulation (SimNIBS)  a segmen3.3.1.Baseline characteristics of the study population are displayed in 3.2.The most common reported side effects were scalp discomfort (68%), headache (48%), fatigue (40%), and sleep disruption (36%). All 25 participants completed all assessments for Visits 1, 2, and 3, and no participants discontinued involvement in the study. The average intensity of stimulation tolerated was 114.9% of RMT, with five subjects not able to tolerate the full dose of 120% of RMT.3.3.p values and effect sizes. Assumptions of normality and sphericity were met for the primary outcome measure of depressive symptoms, the IDS-C-30, and most behavioral and cognitive secondary outcome measures. For certain secondary outcome measures such as the GAD-7, Letter Fluency, and Color Word Score where assumptions of sphericity were not met, Greenhouse–Geisser correction was applied to the repeated measures ANOVA results. Mean depression scores for the entire cohort as measured by the IDS-C-30 improved significantly from Visit 1 to Visit 3  = 62.88, p = < 0.0001, ηp2 = 0.72) (t(24) = 5.90, p < 0.0001), Visit 1 and 3 (t(24) = 9.64, p < 0.00001), and Visit 2 and 3 (t(24) = 6.41, p < 0.00001). An exploratory analysis of long-term effects was undertaken with follow-up IDS-C-30 assessment via phone call to all participants at 1 month and 2 months after treatment. Two participants were not able to be reached for 1 month assessment; at three-month follow-up, five participants were not able to be reached; these were the only missing data points in the cohort. Mean IDS-C-30 scores and standard deviations at one-month follow-up were 22.4 +/− 15.0. At three-month follow-up, mean IDS-C-30 score and standard deviation were 25.5 +/− 12.3. = 0.72) . Clinica3.4.A total of six patients switched to left hemisphere treatment during the protocol due to lack of at least 10% improvement in the IDS-C-30 at Visit 2. Of the subjects that switched, by Visit 3 none met criteria for remission, two met criteria for response, two met criteria for partial response (25–50% improvement), and two patients did not respond .3.5.F = 12.74, p < 0.001, ηp2 = 0.35)  = 11.9; p < 0.0001; ηp2 = 0.33)  . Behavio = 0.33) .3.6.F = 7.85, p = 0.001, ηp2 = 0.25) and the SHAPS-C  = 10.47, p < 0.001, ηp2 = 0.3)  = 7.21; p = 0.002; ηp2 = 0.23) and Drive  = 4.51; p = 0.016; ηp2 = 0.16) (There were significant improvements observed in anhedonia symptoms from Visit 1 to Visit 3 as measured by the TEPS (2 = 0.3) . Behavio = 0.16) . After B3.7.There were no significant changes in any of the cognitive domains tested, including short term memory (HVLT-R), attention (WAIS), and executive function (DKEFS) from Visit 1 to Visit 3 see .3.8.via aggregative analysis of multiple imaging and brain stimulation data sets ; however, anticorrelation between the DLPFC targets and SgCC showed a moderate positive correlation with increasing age , i.e., anticorrelation magnitude decreased as age increased.The DLPFC target search region in ata sets , 49. Deg3.9.|E| found in the middle frontal gyrus and inferior frontal gyrus (|E| at the target (|target|E) for each patient was not significantly associated (p > 0.1) with change in IDS score, nor was |target|E correlated with the simulated electric field magnitude at the motor cortex . In whole-brain analysis, negative correlations were observed between |E| and change in IDS-C-30 between Visit 1 and 2 and Visit 1 and 3  in anterior and lateral regions, i.e., Brodmann areas 10, 47, and 45  was observed in posterior dorsolateral, dorsomedial, and motor regions. Of all areas meeting criteria for inclusion in electric field analysis, only posterior rostral Brodmann area 47 . In participants who received all 45 stimulation sessions to the right hemisphere, the degree of correlation was strong between |E| in p47r and change in IDS-C-30 from Visit 1 to 3   . After c4.In this open-label, single-arm pilot study, accelerated fMRI-guided iTBS significantly improved depressive and anxious symptoms in 25 patients with LLD. The protocol itself was well-tolerated, with no participants discontinuing treatments early. The most common side effects were scalp discomfort, mild headache, sleep disruption, and fatigue. There were no serious adverse events and no significant changes on any of the cognitive measures obtained, indicating that accelerated iTBS at clinical stimulation intensities (110–120%) is a safe form of neuromodulation even in a population at increased risk of cognitive side effects. A very large effect was observed in the primary outcome measure, the IDS-C-30, with a mean 17-point reduction in depressive symptoms observed by the end of 45 iTBS sessions over 9 days, equating to a 52% response rate and 20% remission rate. Large effects on generalized anxiety levels and anhedonia symptoms were also demonstrated in the cohort. Our results highlight the rapidity of clinical benefit seen with accelerated protocols , in whic4.1.This is the first rTMS study for LLD to target the right DLPFC with iTBS, an excitatory paradigm, and the first to use individualized fMRI guidance to the right DLPFC. The response rate of 52% is comparable to those described in uncontrolled non-accelerated studies of rTMS to the left DLPFC in general adult populations , 8, addi4.2.Our remission rate of 20% was lower than described by Cole et al.  in the SA fourth factor that may have contributed to lower response and remission rates is the accuracy of the functional targeting method used in our study. The maximum anticorrelated target in the DLPFC is theorized to be the subregion through which rTMS may most robustly modulate SgCC activity, which has been extensively linked to depressive symptomatology , 36. Sev4.3.|E| magnitude associates with clinical outcome for rTMS, the first such study in the right hemisphere. Greater |E| magnitude in anterior dorsolateral and ventrolateral regions was associated with greater antidepressant effect and was mirrored by the finding that greater |E| in posterior DLPFC was associated with less benefit. This anterior–posterior gradient agrees with theoretical work , a region categorized as ventrolateral prefrontal cortex (VLPFC), inferior frontal gyrus (IFG), and pars orbitalis. BA 47 has been implicated in language processing , emotion4.4.|E| given specific gyral and sulcal patterns  Pilot grant (GM122734).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Preliminary data from this study was previously presented at the American Association of Geriatric Psychiatry Annual Meeting, Orlando, FL, March 18–21, 2022.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ulcerative colitis (UC) is a persistent and non-specific inflammatory condition that mainly affects the bowels and has challenging treatment. UC has a growing incidence and significantly affects the well-being of patients. Many medications used to treat UC can disrupt the metabolism and immune system homeostasis, frequently leading to significant adverse effects. Hence, exploring alternative therapies, such as traditional Chinese medicine and probiotics, has recently emerged as a primary research hotspot owing to their safety. Although the therapeutic mechanism of Shaoyao decoction has not been clarified, it has demonstrated a beneficial clinical effect on UC.This study aimed to assess the effect of Shaoyao decoction on a rat model of UC and investigate its underlying mechanisms.The rat model of UC was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS). The extent of damage to the intestines was assessed using the disease activity index (DAI), colonic mucosa damage index (CMDI), and histological scores. Immunohistochemistry was employed to detect the tissue levels of interleukin (IL)-17, transforming growth factor (TGF)-β1, and IL-10. Additionally, the proportion of Th17 and Treg cells was detected using flow cytometry. In colon tissue, the levels of forkhead box (Fox)p3, RAR-related orphan receptor (ROR)γt, IL-6, p-STAT3, and STAT3 proteins were quantified by Western blotting.Treatment with Shaoyao decoction enhanced the overall health of rats and reduced colonic damage. Additionally, Shaoyao decoction significantly alleviated the severity of DAI, CMDI, and HS. The proportion of Th17 cells was reduced, and the proportion of Treg cells was increased by Shaoyao decoction. The expression of IL-17 and RORγt was suppressed by Shaoyao decoction, while the expression of IL-10, TGF-β1, and Foxp3 was increased. The expression of IL-6, p-STAT3, and STAT3 was decreased by Shaoyao decoction.The Shaoyao decoction alleviates the symptoms of TNBS-induced UC by decreasing inflammation and mitigating intestinal damage while preserving the balance between Th17 and Treg. Shaoyao decoction modulates the IL-6/STAT3 axis, thereby regulating the balance between Th17 and Treg cells.The online version contains supplementary material available at 10.1186/s12906-023-04237-9. Ulcerative colitis (UC) is the long-lasting and non-specific inflammation of the digestive tract that greatly impacts the overall health of individuals by causing both physical and mental distress . TreatinCurrently, UC is mainly treated by conventional medications such as aminosalicylates, corticosteroids, immunosuppressants, and biological agents , 10. TheWith fewer negative side effects, traditional Chinese medicine (TCM) is a dependable option and supplementary treatment . The ShaWe purchased 60 male Sprague–Dawley rats from Hunan SJA Laboratory Animal Co, Ltd. . They aged 5 to 6 weeks and weighed between 150 and 170 g. The animal quality certificate number is 430727211101295026. The rats were kept in conventional cages in a controlled environment free from specific pathogens. The Animal Ethics Committee of the First Affiliated Hospital of Hunan University of Chinese Medicine approved all experimental procedures, with the ethics number ZYFY20210615.Sigma-Aldrich  provided 2,4,6-trinitrobenzenesulfonic acid (TNBS). The H&E staining kit was provided by Shanghai Biyuntian Biotechnology Company. Rabbit anti-mouse IL-17, TGF-β1, IL-6, and β-actin antibodies were purchased from Beijing Boaosen Biotechnology. We used sheep anti-rabbit IgG labeled with horseradish peroxidase as the secondary antibody, which was obtained from Beijing Boaosen Biotechnology. The Shaoyao decoction, comprising Radix Paeoniae Alba, Radix Angelicae Sinensis, Rhizoma Coptidis, Semen Arecae, Radix Aucklandiae, Radix Et Rhizoma Glycyrrhizae, Radix Et Rhizoma Rhei, Radix Scutellariae, and Cortex Cinnamomi , SYDL group (receiving 11.1 g/kg of low-dose Shaoyao decoction), SYDM group (receiving 22.2 g/kg of medium-dose Shaoyao decoction), and SYDH group (receiving 44.4 g/kg of high-dose Shaoyao decoction). To induce UC, 5 mg TNBS solution was prepared by dissolving it in 0.2 mL of 50% ethanol. All rats except for the control group were given a single intrarectal dose of TNBS after anesthesia with 3% pentobarbital sodium (40 mg/kg). A polyethylene tube with a diameter of 0.4 mm was coated with paraffin oil and introduced into the rectum, reaching a depth of 8 cm. Shaoyao decoction and mesalazine were administered orally for 14 days starting from day 7, while the control group received normal saline in the same period.We collected the blood samples of the control rats to obtain blank serum. We also collected blood samples from low-dose Shaoyao decoction  group. At 4 ֯C, the pure Shaoyao decoction samples, blank serum, and Shaoyao decoction-treated serum were centrifuged at 3000 rpm for 10 min. Next, 200 μL of the supernatant was collected and combined with 800 μL of methanol. The collected supernatant was obtained after centrifuging at a speed of 13,000 revolutions per minute for 10 min. Then, the supernatant was filtered using a microporous membrane with a pore size of 0.22 μm. The filtered samples were utilized for subsequent analyses. The Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC LC–MS/MS) analysis was conducted using a Vanquish UHPLC system from Thermo Fisher Scientific, equipped with a Waters UPLC BEH C18 column with 1.7 μm in diameter and 2.1*100 mm in length. The volume of the injected sample was adjusted to 5 µl. A flow rate of 0.5 ml per minute was established. The mobile phase was composed of 0.1% formic acid in aqueous solution (A) and 0.1% formic acid in acetonitrile (B). The program for the multistep linear elution gradient was as follows: from 0 to 11 min, the percentage of A ranged from 85 to 25%; from 11 to 12 min, it ranged from 25 to 2%; from 12 to 14 min, it remained at 2%; from 14 to 14.1 min, it increased from 2 to 85%; from 14.1 to 15 min, it remained at 85%; and from 15 to 16 min, it also remained at 85%. To acquire the MS and MS/MS data using the IDA acquisition mode, the combination of a Q Exactive Focus mass spectrometer and Xcalibur software was utilized. In each acquisition cycle, the mass range varied between 100 and 1500. The top three results of each cycle were selected, and the corresponding MS/MS data were obtained. The flow rate of the sheath gas was 45 Arb, while the auxiliary gas had a flow rate of 15 Arb. The capillary temperature was set at 400 ֯C. The full ms resolution was 70000, and the MS/MS resolution was 17500. In NCE mode, the collision energy was set at 15/30/45. The spray voltage could be either 4.0 kV (positive) or -4.0 kV (negative). During every data acquisition cycle, the molecular ions exhibiting the highest intensity and surpassing 100 were chosen to gather the corresponding secondary MS data. The original mass spectrum was imported using Progenesis QI software. Retention time correction, peak identification, extraction, and integration were performed using MAPS 1.0 BIOTREE software. Biotree's self-constructed secondary mass spectrometry database was used to identify the MS/MS data [From day 7, we monitored the animals' daily progress in developing experimental colitis using the DAI scoring system . The recOn the 23rd day, rats in each group were intraperitoneally given pentobarbital to induce anesthesia. Then, they were euthanized by cervical dislocation. Afterward, samples were collected from the colon (from the cecum to the anus) and spleen. The colon was sliced along its longitudinal axis and washed with normal saline. As per Luketal's criteria, the colonic mucosa damage index (CMDI) was evaluated .The colon tissue was fixed in 4% paraformaldehyde solution. Then, it was subjected to paraffin-embedded sections, xylene dewaxing, gradient ethanol solution dehydration, hematoxylin solution staining, 1% hydrochloric acid ethanol fractionation, and eosin solution staining. An expert pathologist, unaware of the study, evaluated the histological score . Part ofThe colon sections were sliced into 4 μm thick sections, then the sections were treated with xylene and ethanol to remove waxes and restore moisture. The sections were treated with a solution of citrate antigen repair, followed by permeabilization by a 0.5% Triton X-100 solution for 20 min. Subsequently, samples were exposed to a 3% H2O2 solution, and then blocked using a 5% bovine serum albumin for 20 min. Thereafter, the sections were incubated with IL-17 (1:500), TGF-β1 (1:500), and IL-10 (1:500) at 37 °C for 1 h. Finally, the sections were incubated with sheep anti-rabbit IgG (1:1 000) at 37 °C for 20 min. The DAB reaction was utilized to visualize the intestinal slides, which were then observed using an optical microscope.+CD25+ Foxp3+ cells, while Th17 cells were identified as CD4+ IL-17+ cells.To obtain individual cell suspensions, we gathered lymphocytes from the spleen and subjected them to centrifugation for 10 min. Initially, the cells were stained using anti-CD4 FITC and anti-CD25-PE-Cy7. Subsequently, they were fixed and permeabilized. Following this, anti-Foxp3 PE or anti-IL-17A PE antibodies were added, and the mixture was incubated at 4 °C for 30 min. Flowjo CE software was used to acquire staining data on a Cytek Dxp Athena flow cytometer. Treg cells were identified as CD4 Protease inhibitors and pre-chilled lysate buffer were used to lyse the colon tissues. We subjected the same quantity of protein (50 μg) to 12% SDS-PAGE. Then, the membrane was blocked with Tris-buffered saline Tween 20 buffer that included 5% skim milk. We added primary antibodies (1:1 000 dilution) at 4 °C overnight. The secondary antibodies (1:2000 dilution) were added to the membranes and incubated at room temperature for 1 h. Then, the ECL AB solution was mixed in equal volumes and incubated with the PVDF membrane for 1 min. β-Actin was employed as a control for the sample. Bio-Rad chemiluminescence instrument was used for exposure. We scanned the film with two-color infrared fluorescence scanning instrument, Odyssey. The optical density of the band of interest and the internal reference (β-actin) were determined using Image J software .The SPSS software (ver. 2.0) was utilized to conduct statistical analyses. Data were generated using SPSS , and graphs were designed using GraphPad Prism 8.0 software . Continuous variables are expressed as mean ± SD or median /CD4 + lymphocytes Fig. A, B and Compared to the control group, the TNBS group exhibited an increased expression of IL-6, p-STAT3, and STAT3 in the colon tissue Fig. . After aImbalanced immune response in UC is caused by impaired intestinal immune response and endangers the integrity of the intestinal mucosal barrier. Additionally, Th17/Treg imbalance is closely associated with impaired immune response in UC –25. The UC causes intestinal inflammation and disrupts the balance between pro-inflammatory and anti-inflammatory responses, overactivating Th17 cells and inhibiting Treg cell response in UC . MaintaiClinical studies demonstrated a notable increase in IL-6 expression and STAT3 phosphorylation in lamina propria lymphocytes and colonic epithelial cells . Recent The protective mechanisms of Shaoyao decoction may be related to its numerous active components. Shaoyao Decoction analysis by UHPLC LC–MS/MS revealed 49 blood constituents. Certain monomers have been demonstrated to possess protective properties in UC. Rhizoma Coptidis-derived berberine has been utilized for treating UC by modulating gut microbiota and regulating Treg/Th17 ratio . TreatmeCollectively, our findings demonstrate that the Shaoyao decoction not only ameliorates the overall health in UC but also suppresses IL-6/STAT3 signaling pathway and improves Treg/Th17 imbalance. Hence, we hypothesized that Shaoyao decoction can protect against intestinal ulcers by modulation Th17/Treg imbalance through the IL-6/STAT3 signaling pathway.Shaoyao decoction relieved the symptoms of UC, reduced intestinal inflammation and pathological damage, and maintained Treg/Th17 balance in TNBS-induced UC. The effect of Shaoyao decoction on Th17/Treg balance in UC was associated with the modulation of IL-6/STAT3 signaling pathway. Hence, we hypothesized that Shaoyao decoction improved Th17/Treg balance in rats with UC by modulating the IL-6/STAT3 signaling pathway.Additional file 1: Figure 4E. Loading order: control group, TNBS group, mesalazine group, SYDH group, SYDM group, SYDL group. Figure 5A. Loading order in the red circle: control group, TNBS group, mesalazine group, SYDH group, SYDM group, SYDL group.


An electrochemical biosensor was fabricated using nanoparticles of acetylcholinesterase (AChE) and choline oxidase (ChO)/Pt nanoparticles (PtNPs)/porous graphene oxide nanosheet (GONS) composite. A pencil graphite electrode (PGE) was used for the electrodeposition of nanocomposite and the determination of acetylcholine (ACh), a neurotransmitter. Various techniques such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectra and cyclic voltammetry (CV) were used for characterization. This biosensor (AChENPs-ChONPs/GONS/PtNPs/PGE) indicated a very short response time (3 s), a lower limit of detection (0.001 µM), good linearity (0.001–200 µM), longer storage stability (6 months) and better reproducibility. The percent analytical recoveries of added acetylcholine in serum (5.0 and 10 µM) were found to be 97.6 ± 0.7 and 96.5 ± 0.3 for the present biosensor. The coefficients of variation were obtained to be 8% and 3.25%, correspondingly. The biosensor was applied to measure the ACh amount in the serum of healthy individuals and patients with Alzheimer’s disease. The number of interferents had no effect on the biosensor at their physiological concentrations. ACh, scientifically referred to as 2-acetoxy-N, N, N-trimethylethanaminium, is an important neurotransmitter in which both the central and peripheral nervous systems (PNS) are involved [Neurotransmitters are substances that the body utilizes to communicate between cells. In the case of the human body, acetylcholine (ACh) is one of the most common neurotransmitters. It is secreted by cholinergic neurons. Choline (Ch) and its coenzyme A ester (acetyl CoA) are converted to ACh  have similar characteristics to platinum nanoparticles (PtNPs) in terms of more surface area, biocompatibility, better conductivity, good electrocatalytic features for oxidation/reduction toward HO2, etc. . In combGraphene is a 2D carbon material having different arrangements such as graphene oxide (GO) and reduced graphene oxide/graphene nanosheets. Characteristics such as large surface area, thermal stability, mechanical properties, conducting properties, electrochemical behavior, etc., were shown . TherefoFeatures such as better conductivity, selectivity and sensitivity have been shown by the fabrication of nanomaterials onto PG electrodes . During Acetylcholine esterase (EC 3.1.1.7) was obtained from electric eel. Choline oxidase (EC 1.1.3.17) was obtained from Alcaligenes sp. Acetylcholine chloride, hexachloroplatinic acid, graphite powder, acetone, sodium nitrate, concentrated sulfuric acid, potassium permanganate, ascorbic acid, ethanol and methanol were obtained from Sigma Aldrich . A 6B pencil (Make: Apsara) with graphite lead of 2 mm thickness was obtained from a local shop and used as the working electrode. In all experiments, deionized water (DW) and analytical reagent (AR)-grade chemicals were used. Serum samples were collected from the hospital of PGIMS, Rohtak.A potentiostat instrument designed by AutoLab  was used. It comprised three electrodes, i.e., Pt wire as the auxiliary electrode, a Ag/AgCl electrode (3.5 M KCl) as the reference electrode and AChENPs/ChONPs/PtNPs/GONS/PGE as the working electrode. Fourier-transform infrared spectroscopy (FTIR) (manufactured by Bruker), a JEM 2100-F Transmission electron microscope (TEM)  and a D/Max 2550 X-ray diffractometer (XRD)  were used.2PtCl6.6H20) under continuous stirring. After complete addition of the leaf extract, the mixture was kept for incubation at 30 °C for 24 h. The color of the solution changed from light yellow to dark brown, which revealed the synthesis of PtNPs. The colored solution was centrifuged for 30 min, followed by redispersion in DW to remove further unwanted biological impurities.The preparation of PtNPs was performed as described by Thirumurugan et al. , with mi2SO4 98%) were mixed in a reaction flask of 500 mL under continuous stirring for 15 min in a water bath with ice at a temperature between 0 and 5 °C. Then, 4.0 g of potassium permanganate was added slowly to the above solution within 15 min at 20 °C. Following that, DW (20 mL) was added slowly under stirring at 40 °C for 90 min in the water bath. A brown-colored suspension was formed, which was treated with 30% H2O2 solution (6 mL). A 5% amount of hydrochloric acid (HCl) was used to wash the GO suspension and was monitored by DW for the removal of excess manganese (Mn) until it reached neutrality. An oven was used to dry the purified GO at 60 °C for 24 h. To prepare the graphene oxide nanosheet (GONS), GO (0.1 g) and broccoli juice (240 µL) were added to a beaker (250 mL), producing a homogeneous dispersion. This dispersion was stirred for 24 h. After stirring, it was sonicated for 60 min and, lastly, dried at 60 °C for 24 h.Firstly, the improved Hummer method  was usedg . After that, a 0.12 g amount of cysteamine di-hydrochloride was added to the ENPs under continuous stirring (5–6 h), which provides an amino group to the ENPs. These functionalized ENPs were separated from the enzyme solution by centrifugation at 1200 rpm for 10 min and stored at 4 °C until use. The size and shape of the functionalized AChENPs/ChONPs were studied by TEM from AIRF commercially.For the preparation of AChENPs/ChONPs, the desolvation method was used by using ethanol . Firstly2SO4:1H2O2) for 10 min and then washed with DW. A PtNPs/GONS hybrid was prepared by taking both in an equal ratio (2 mg/2 mL) under continuous stirring for 12 h, followed by centrifugation at 1200 rpm to separate the nanocomposite (PtNPs/GONS). Cyclic voltammetry in a potentiostat/galvanostat was used for the electrodeposition of PtNPs/GONS on the surface of the PGE by immersing it in 23 mL of 2.5 mM K3Fe(CN)6/K4Fe(CN)6 (1:1) and 2 mL of PtNPs/GONS solution with 40 continuous polymerization circles (−1.1 to 0.1 V) at a scan rate of 20 mV/s. The subsequent PtNPs/GONS-modified PGE was washed thoroughly with DW to get rid of boundless matter  was manually polished with alumina slurry by using a polishing cloth. It was monitored by systematic washing with DW. The cleaned electrode was dipped in 4 mL of piranha solution 6/K4Fe(CN)6 (1:1) with 200 µL of AChCl (0.05 M), and a voltage range between −0.1 and +0.1 V at a scan rate of 20 mV/s was applied. Hence, the working of this biosensor is based on the following reaction:An amperometric acetylcholine biosensor was constructed using AChENPs/ChONPs/GONS/PtNPs/PGE (working electrode), Pt wire (counter electrode) and Ag/AgCl (reference electrode) saturated 3.5 M KCl. All measurements were performed on an electrochemical analyzer (AutoLab PGSTAT 30) at room temperature. The cyclic voltammetry experiments were performed in 25 mL of 2.5 mMKThe current measurement or flow of electrons in the galvanostat remains directly related to the acetylcholine concentration.The effects of various analytical parameters such as pH, incubation temperature, substrate (acetylcholine) concentration and reaction period were considered for the optimization of the AChENPs/ChONPs/GONS/PtNP-modified PGE-based nanosensor. For the determination of optimum pH, phosphate buffers (0.1 M) with pH between 5.0 and 10 at a break of 0.5 were prepared. Similarly, the incubation temperature range (20–50 °C) at an interval of 5 °C in a temperature-controlled water bath and at a time duration of 2–90 s was measured for the detection of the most favorable temperature and incubation time, respectively. The acetylcholine effect on the biosensor response was studied by changing the concentration of acetylcholine in the range of 0.001–200 µM.The acetylcholine biosensor was evaluated by measuring its lower detection limit (LOD), % analytical recovery of added acetylcholine in serum, within and between batch coefficients of variation (precision) and correlation with the standard method.The TEM study revealed that the mean size of the PtNPs is 20 nm, and they are predominantly spherical in shape, while some of the nanoparticles have a dodecahedron shape, as shown in The XRD study revealed a sharp diffraction peak at 2θ = 25°, which indicated that the porous graphene nanosheets existed individually with a highly disordered nature .−1, which was owed to the N-H enlarging vibration of the -NH2 group. The value at the 2332 cm−1 wavelength corresponds to C-N stretching, and the strong intense peak value at 1634 cm−1 may be due to the presence of –C=C stretching. The peaks at 1173 cm−1 were due to C-O bonding, as shown in −1, 1636 cm−1, 2344 cm−1 and 3309 cm−1, resulting from C-H bonding, C=C conjugation, C-N band and N-H extending vibrations. The lack of bands corresponding to the oxygen-functional groups in the porous graphene oxide nanosheet spectrum further confirms its formation (−1 and 1632 (C=C bonding), 2080 cm−1 and 2462 cm−1 (C≡C extending), and 3170 cm−1 (C-H bonding), as shown in −1, 1290 cm−1, 1632 cm−1 and 3222 cm−1, which confirms O-H bonding, C-N stretching and N-H, as well as C-H bonding. The AChENPs/ChONPs/PtNPs/GONS/PG electrode showed various bonds at different peaks of 1236–1955 cm−1, 2056–2979 cm−1 and 3138–3874 cm−1, which confirms the presence of enzyme nanoparticles.The FTIR analysis of PtNPs showed a broad band at 3341 cmormation B. The baThe bare electrode studied by SEM shows no deposition A onto PG2O3NPs/rGO/PEDOT/fluorine-doped tin oxide (FTO) electrodes [2/rGO/gold electrodes (AuEs) [The present acetylcholine biosensor showed an optimum current at pH 7.5 A and an ectrodes , AChE-Chs (AuEs)  and AChEs (AuEs) ; but lows (AuEs)  and biofs (AuEs)  and AChEs (AuEs) . The opts (AuEs) . The per–1), the current response also increased accordingly, which also confirms the stability in the performance of the present biosensor (The biosensor (AChENPs/ChONPs/GONS/PtNPs/PGE) showed that with varying scan rates  [The present AChENPs/ChONPs/GONS/PtNPs/PG electrode-based nanosensor was evaluated with different analytical parameters, such as linear range, limit of detection, coefficients of variation and analytical recovery. The present biosensor attained linearity between the current and acetylcholine (substrate) concentration range of 0.001–200 µM. By comparison with earlier biosensors, this nanosensor showed better linearity than those based on AChE-ChO/Fe–800 µM) , AChE-Ch–800 µM) , 0.1–1.0–800 µM) , and ACh–800 µM) . The lim–800 µM) , 0.1 µM,–800 µM) ).The percent analytical recoveries of added acetylcholine in serum (5.0 and 10 µM) were found to be 97.6 ± 0.7 and 96.5 ± 0.3 for the present biosensor, showing the accuracy of this biosensor.The coefficients of variation (within and between batches) obtained were 2.8% and 3.25%, correspondingly, indicating the reliability of the present biosensor. These results also revealed the improved analytic performance of the present biosensor in terms of better repeatability and reproducibility, which can be attributed to the excellent immobilization of AChENPs/ChONPs onto the GONS/PtNPs/PG electrode.n = 20), ranging from 9.0 to 12.0 nM within the normal range (8.2–11.3 nM) [n = 20) ranged from 1.0 to 6.5 nM, which is significantly lower (p < 0.01) than that in healthy individuals (The present biosensor measured the level of the acetylcholine (ACh) concentration in the serum of healthy individuals (11.3 nM) . The levividuals . Earlier2 = 0.989) showed a good correlation between the existing sensor with earlier methods, as shown in The levels of ACh in serum as determined by the present biosensor were compared with those by the standard colorimetric method. The calculated correlation coefficient . In all assays, the concentration of ACh (0.1 mM) was kept constant. The value of the current did not particularly change in the presence of these interferents. Therefore, it was concluded that the interferants impart minimal impact on the analysis of ACh by the present biosensor .2/rGO/Au (3 months) [The present nanosensor investigated the response of the acetylcholine concentration in 0.1 M PBS for a time period of 6 months. During this time, the original performance of the nanosensor decreased by 35% upon continuous use 250 times . Therefo months)  and AChE months) . VariousAn enhanced biosensor was constructed for the determination of acetylcholine in the serum samples of healthy patients and Alzheimer’s patients. The present ENP-based biosensor demonstrated better analytical performance, such as a lower detection limit (0.001 µM), shorter reaction time (3 s), good linearity (0.001–200 µM), longer storage stability (6 months) and better reproducibility, compared to earlier reports. Before this, an ENP-based biosensor for acetylcholine detection had not been prepared. Hence, this nanocomposite (GONS/PtNPs/PGE) also improved the performance of the present biosensor compared to the direct immobilization of native enzymes on the electrode surface. Therefore, it can also be used for the improvement of other biosensors.