Heligmosomoides polygyrus significantly reduced colorectal cancer cell proliferation in vitro. Using a range of approaches, we demonstrate that antigen-dependent reductions in cancer cell proliferation and viability are associated with increased expression of the critical cell cycle regulators p53 and p21. Interestingly, H. polygyrus-derived antigens significantly increased murine colorectal cancer cell migration, which was associated with an increased expression of the adherens junction protein β-catenin, whereas the opposite was true for human colorectal cancer cells. Together, these findings demonstrate that antigens derived from a gastrointestinal nematode can significantly alter colorectal cancer cell behavior. Further in-depth analysis may reveal novel candidates for targeting and treating late-stage cancer.As the global incidences of colorectal cancer rises, there is a growing importance in understanding the interaction between external factors, such as common infections, on the initiation and progression of this disease. While certain helminth infections have been shown to alter the severity and risk of developing colitis-associated colorectal cancer, whether these parasites can directly affect colorectal cancer progression is unknown. Here, we made use of murine and human colorectal cancer cell lines to demonstrate that exposure to antigens derived from the gastrointestinal nematode  Globally, colorectal cancer (CRC) is the third most commonly diagnosed malignancy and the second leading cause of cancer deaths . In 2018Notably, many soil-transmitted helminths (STH) establish life-long infections associated with chronic inflammation within the gastrointestinal tract. During persistent infection, parasitic helminths release excretory/secretory products containing components that can modify systemic and local inflammatory responses . Both heOpisthorchis viverrini and Clonorchis sinensis are classified as group one biological carcinogens and are conclusive causes of cholangiocarcinoma (CCA), also known as bile duct cancer [Certain helminth infections have been shown to confer an advantage to the host, by reducing autoimmune and allergic symptoms in infected individuals ,16,17,18t cancer ,23. Opist cancer .Chronic infection with Schistosoma has been associated with the development of bladder cancer, increased chromosomal aberrations, and increased DNA copy number ,26. IncrSchistosoma mansoni and Heligmosomoides polygyrus to determine how STH antigens impact CRC cell proliferation and migration. We reveal that antigen derived from H. polygyrus significantly decreased murine and human CRC cell proliferation, which was associated with increased expression of p53 and p21. Furthermore, exposure to these antigens reduced both murine and human CRC cell mitochondrial activity. H. polygyrus-derived antigens increased murine CRC cell migration, which was associated with increased expression of the adherens junction protein β-catenin. Interestingly, the opposite was true for human colorectal cancer cells. These data demonstrate the usefulness of in vitro assays in assessing how differing parasite antigens impact on CRC cell behavior and identify pathways that are altered in CRC cells following exposure to helminth antigens.Here, we made use of antigen derived from H. polygyrus adults and the excretory/secretory products (HES) and quantified the impact on cell viability using trypan blue exclusion. Our results reveal that, compared to untreated cells, there was a significantly decreased number of viable treated CT26.WT cells (H. polygyrus antigen treatment (Schistosoma mansoni ova (SEA) had no effect on CT26.WT cell proliferation  and at 18 h   and at 18 h   and at 30 h   and at 30 h  significantly reduced the in vitro proliferation of murine (CT26.WT) CRC cells, while HES alone significantly reduced the in vitro proliferation of human (HCT116) CRC cells. This result supports previous studies with a Trichinella spiralis-derived antigen, which inhibited the proliferation of human myeloid leukemia and hepatoma cells in vitro, as well as the proliferation of intestinal cells in vivo [O. viverrini, which increased the proliferation of fibroblasts in vitro [Accumulating reports suggest that helminth infections can exacerbate the initiation of CRC; however, whether infection or associated helminth antigens impact on CRC development is still unclear ,29,32,39 in vivo ,41,42. HH. polygyrus-derived antigens reduced CRC proliferation is unclear; however, BrdU incorporation and MTT assays demonstrated that HES significantly reduced DNA synthesis and cell viability, respectively, in CT26.WT cells. These data suggest that the excretory/secretory antigens are able to shift CRC cells from proliferation to a more quiescent phenotype. This is in accordance with the findings using Trichinella spiralis-derived antigen, which arrested cancer cell lines at G1 or S phase of the cell cycle [H. polygyrus antigen did not have a profound impact on CT26.WT DNA synthesis or cell viability of CRC cells, despite a significant impact on cancer cell proliferation. H. polygyrus-derived antigens contain different mixtures of proteins and glycoproteins, as well as small RNAs, all of which can modulate host cell behavior [H. polygyrus adult antigen and HES would need to be undertaken in order to determine whether the presence of varying components may account for the contrasting effects of these antigens on differing aspects of cell proliferation.The mechanism for how ll cycle . Interesbehavior ,44,45. FH. polygyrus-derived antigens, was associated with an upregulation of p53 and p21 expression; molecules that are associated with increased cell cycle arrest and apoptosis. Chronic infection with Taenia crassiceps and Trichinella spiralis differentially regulated local expression of p53 in vivo, whereas exposure of hepatic stellate cells to Schistosoma japonicum soluble egg antigen induced apoptosis following upregulation of p53 [Schistosoma japonicum soluble egg antigen induction of apoptosis was associated with inhibition of Akt signaling and subsequent upregulation of p53-dependent DR5 expression. HES has previously been described as anti-apoptotic because it significantly reduced annexin V and propidium iodine staining in proliferating CD4+ T cells [Western blot analyses showed that a significant reduction in proliferation of human and murine CRC cells, following exposure to n of p53 ,47,48. I T cells . Given oH. polygyrus antigen and HES caused an increase in CT26.WT cell migration, which was supported by changes in β-catenin expression. β-catenin (NCBI Gene ID: 1499) forms part of the adherens junctions between cells and is negatively regulated by adenomatous polyposis coli (APC) [H. polygyrus-derived antigens caused a significant decrease in human HCT116 cell migration, which corresponded with a decrease in β-catenin expression.Surprisingly, li (APC) . Signifili (APC) ,51. As ali (APC) ,50,52. Ili (APC) ,10,53,54H. polygyrus-derived antigens may alter cancer cell behavior in a number of ways, proteomics analysis of these antigens, combined with sequencing of the H. polygyrus genome, has led to the identification of several immunomodulatory molecules, including a structurally distinct equivalent of the mammalian transforming growth factor (TGF)-β cytokine, termed H. polygyrus TGF-β Mimic (Hp-TGM) [H. polygyrus-derived antigens, may drive the changes in cancer cell behavior that we report here through a common signaling pathway. Interestingly, although SEA was previously shown to inhibit CD4+ T cell proliferation and amplify TGF-β signaling [+ T cells [H. polygyrus-derived antigens, which contrasted with increased migration seen in antigen-exposed murine CT26.WT cells. Analysis of human HCT116 CRC cells demonstrated that they have a mutated type II TGF-β receptor gene and cannot respond to TGF-β in order to undergo EMT, invasion, or migration [H. polygyrus-derived antigens and the TGF-β mimic contained within HES.While it is possible that multiple components of (Hp-TGM) ,55. Stud(Hp-TGM) , which lignaling , we foun T cells ,55,57. Iigration . A subseigration . It is tH. polygyrus can alter human and murine CRC cell behavior. Further work would be required to test our hypothesis that Hp-TGM orchestrates this effect. This data provides an important basis for determining how parasite antigens influence cancer cell development in vivo and may have important implications for the etiology of colorectal cancer within parasite-endemic regions.Our data provides the first evidence that antigen derived from H. polygyrus somatic antigen (referred to as H. polygyrus antigen) and H. polygyrus excretory/secretory products (HES) were prepared using established methods described by Hewitson et al. and Johnston et al. [H. polygyrus larvae by gavage, and adult worms were recovered 14 days post-infection. Adult worms were washed extensively before incubation in RPMI 1640 medium, supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin, and 100 µg/mL gentamicin. Pooled culture supernatants were filtrated into phosphate buffered saline PBS over a 3000 MW Amicon membrane . The resulting HES was a well characterized preparation containing 374 proteins, novel O-linked glycoproteins, and small RNAs [H. polygyrus antigen was prepared by uniformly homogenizing adult worms in PBS on ice using a Polytron homogenizer . The resulting mixture of 446 proteins was centrifuged at 13,000 g for 30 min, from which the supernatant was collected [H. polygyrus antigen was quantified using a NanoDrop 1000 Spectrophotometer  before storing at −80 °C.n et al. ,60. In ball RNAs ,45,61. Hollected . The pro2 and 95% humidity.The colorectal cancer (CRC) cell line CT26.WT (a murine N-nitroso-N-methylurethane (NNMU)-induced, undifferentiated fibroblast colon carcinoma cell line) and HCT116  were purchased from the American Type Culture Collection (ATCC). CT26.WT and HCT116 were maintained in complete RPMI 1640 and McCoy’s 5A modified medium, respectively, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin. Cells were maintained at 37 °C in an atmosphere of 5% CO, and 5 × 104 cells were seeded per well, in triplicate, in a 24-well plate. Additionally, HES (10 µg), H. polygyrus antigen (10 µg), or 1× PBS was added to each of the required wells at the time of seeding. Live cells were grown in complete culture media and counted 24-, 48- and 72-h post-treatment using trypan blue exclusion.The short-term growth of cells was monitored over a 3-day period, as previously described , and 5 ×3 cells were seeded per well, in quadruplicate, in a 96-well plate. Additionally, HES (10 µg), H. polygyrus antigen (10 µg), or 1× PBS was added to each of the required wells for 24 h. A total of 0.5 mg/mL of MTT reagent was added to each well for 4 h, followed by the addition of 100 µL of solubilization solution. After an overnight incubation, the absorbance reading of each well was read at a wavelength of 595 nm. The absorbance of medium-only wells was subtracted from the absorbance of sample wells to obtain a final absorbance reading.CRC mitochondrial activity was determined using MTT assays , and 8 × 10H. polygyrus antigen on CT26.WT DNA synthesis and cell division, and 2 × 105 cells were seeded on a sterile glass coverslip in a 35 mm dish, treated with HES (10 µg), H. polygyrus antigen (10 µg), or 1X PBS, and allowed to adhere overnight. A total of 10 µm BrdU  was added to the medium for 8 h, followed by fixation with Carnoy’s fixative for 20 min at −20 °C. Cells were incubated in 2 M HCl for 1 h at 37 °C, neutralized in 0.1 M pH 8.5 borate buffer, and washed with PBS/0.5% Tween20. Cells were then incubated for 30 min at 37 °C in blocking buffer (5% sheep serum in PBS/0.5% Tween20). BrdU was detected with a mouse monoclonal anti-BrdU  for 30 min at 37 °C, followed by a secondary IgG coupled to Alexa488  for 30 min at 37 °C. Cells were washed with PBS/0.5% Tween20, incubated in 1 µg 4’6-diamidino-2-phenylindole (DAPI)  for 10 min at room temperature, in the dark, and washed with PBS/0.5% Tween20. The coverslips were then mounted onto microscope slides, stored in the dark in a humidifying chamber overnight at 4 °C, and visualized by fluorescence microscopy using an Axio Vert.A1 Fluorescent microscope .A BrdU incorporation assay was used in order to determine the effect of HES and g for 20 min at 4 °C and the supernatant was then ultracentrifuged at 100,000× g for 90 min and sterilized through a 0.2 μm filter, before protein concentration determined by bicinchoninic acid (BCA) assay. The final preparation was stored at −70 °C.SEA was kindly gifted by Associate Professor Benjamin Dewals (University of Liège). Briefly, the livers of Naval Medical Research Institute (NMRI) mice infected with 200 cercariae were cut into pieces and individually incubated overnight in 20 mL of PBS 100 μg/mL collagenase IV. In order to collect the egg pellet, the homogenates were filtered and the suspension on top of the final 45 μm strainer was collected and centrifuged at 1400 rpm for 5 min at 20 °C. This was then centrifuged through a 20% Percoll solution, washed in PBS 1 mM EGTA/1 mM EDTA, and centrifuged through a 25% Percoll solution, and a further wash in PBS 1 mM EGTA/1mM EDTA was performed. The purified eggs were resuspended in PBS at a concentration of 100,000 eggs/mL before homogenizing on ice. The crude mixture was centrifuged at 2000× 5 serum starved cells were seeded in triplicate in media containing 1% FBS, onto the apical surface of each hanging insert, and placed into wells containing 10% FBS. Following treatment with HES (10 µg), H. polygyrus antigen (10 µg), or 1× PBS, the plates were incubated for 24 h. Post-incubation, the lower surface of the insert was fixed with methanol and stained with crystal violet. Excess crystal violet was washed off the insert using distilled water before release of the crystal violet stain into 50% acetic acid. The absorbance of this solution was then quantified at 595 nm using a Rayto RT-2100C Microplate Reader .An established transwell migration assay, utilizing 24-well hanging inserts, fitted with an 8 µm pore size membrane , was used to investigate CRC cell migration . AdditioWestern blotting was performed as previously described . PrimaryDispensation to perform research at the Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, was granted on 19 October 2015 by the South African Department of Agriculture, Forestry, and Fisheries, in terms of Section 20 of the Animal diseases act, 1984 (Act no. 35 of 1984): reference 12/11/17 under the title of research: “Worm power: Can helminths modify the development of colorectal cancer? AEC 015/001”. Approval for all animal procedures was given on 18 March 2015 by the Faculty of Health Science Animal Ethics Committee (Project 015/001) and was performed by researchers accredited by the South African Veterinary Council (AR15/13922).p < 0.05, ** p < 0.01, and *** p < 0.001.Data were assessed for normality and equal variance using the GraphPad Prism software . For comparison between the three treatment groups, a parametric one-way analysis of variance with Tukey’s multiple comparison or a nonparametric Kruskal–Wallis with Dunn’s multiple comparison was used. *


GNAS complex locus, encoding for the GTPase subunit Gsα. Here, we investigated how platelet phenotype and omics analysis can assist in the often difficult diagnosis. By coupling to the IP receptor, Gsα induces platelet inhibition via adenylyl cyclase and cAMP-dependent protein kinase A (PKA). In platelets from seven patients with suspected AHO, one of the largest cohorts examined, we studied the PKA-induced phenotypic changes. Five patients with a confirmed GNAS mutation, displayed impairments in Gsα-dependent VASP phosphorylation, aggregation, and microfluidic thrombus formation. Analysis of the platelet phosphoproteome revealed 2,516 phosphorylation sites, of which 453 were regulated by Gsα-PKA. Common changes in the patients were: (1) a joint panel of upregulated and downregulated phosphopeptides; (2) overall PKA dependency of the upregulated phosphopeptides; (3) links to key platelet function pathways. In one patient with GNAS mutation, diagnosed as non-AHO, the changes in platelet phosphoproteome were reversed. This combined approach thus revealed multiple phenotypic and molecular biomarkers to assist in the diagnosis of suspected PHP Ia.Patients diagnosed with pseudohypoparathyroidism type Ia (PHP Ia) suffer from hormonal resistance and abnormal postural features, in a condition classified as Albright hereditary osteodystrophy (AHO) syndrome. This syndrome is linked to a maternally inherited mutation in the  Patients diagnosed with pseudohypoparathyroidism type Ia  or with the related disorder PHP Ic also exhibit resistance to other hormones and show a variable set of clinical features, including as short stature, obesity, round face, subcutaneous ossification, brachydactyly, and other skeletal anomalies. Some patients also have mental retardation. Jointly these characteristics are classified as Albright hereditary osteodystrophy (AHO) syndrome. This infrequent and serious syndrome occurs with an estimated prevalence of 1:250,000, but it is likely underdiagnosed because of variation in the presentation and severity of the disease. In the past, PHP was also deduced from a defective activity of the erythrocyte Gsα protein, but the suitability of this test has been questioned2. This impairment is linked to dysfunctional mutations in the GNAS complex locus, encoding for the Gsα protein. The imprinted gene complex locus GNAS is located on chromosome 20q13.2-13.34. Importantly, genetic variation in this locus can also link to an altered regulation of blood pressure and an increased risk of cardiovascular disease5. In case of AHO, the syndrome characteristics are inherited from the mother, implicating that the affected tissues may preferentially express the maternal GNAS allele6. However, the disease can also be due to epigenetic variation6, in which case GNAS methylation studies are needed for a proper diagnosis of PHP patients7. On the other hand, paternal transmission of the mutated GNAS allele is not accompanied by hormone resistance, and is then classified as pseudopseudohypoparathyroidism (PPHP).Consistent among patients with established PHP (including AHO) is impairment in the Gsα-mediated signalling effects of tissues in response to multiple hormones8. Elevation in cAMP triggers intracellular signalling events via the broad-spectrum, cAMP-dependent protein kinase A (PKA)9. The platelet Gsα-AC-PKA pathway is by far most strongly triggered via the IP receptor (PTGIR)10, which causes global inhibition of platelet adhesion, shape change, cytoskeletal changes, secretion, aggregation and procoagulant activity12. The endothelium-derived prostaglandin I2  provides the main high-affinity trigger of IP receptors, along with the stable prostanoid prostaglandin E1 (PGE1) showing a somewhat lower affinity13. The other high-affinity receptor for prostacyclin, EP1, is not expressed on platelets14. Accordingly, both iloprost and PGE1 elevate platelet cAMP levels via Gsα, activate PKA and establish suppression of key signalling events including Ca2+ fluxes and integrin αIIbβ3 activation16. Earlier studies describe that in patients with PHP Ia (AHO) the Gsα functional defect is indeed linked to lower platelet responses to both iloprost and PGE117.In platelets like in other cells, the stimulation of G protein-coupled receptors (GPCR), interacting with the Gsα β/γ complex, causes activation of adenylyl cyclase (AC), which enzyme produces the second messenger cAMP18 and of the iloprost-induced platelet phosphoproteome19. In the platelets from healthy individuals, the (phospho)proteome appeared to be markedly stable. This offers the possibility to exploit the platelet proteome to find abnormalities on the protein level in patients with a congenital deficiency or other pathologies21.In our previous work, we have used protein mass spectrometry techniques for quantitative analysis of the global platelet proteomeHere, we studied seven rare patients of four families with confirmed or suspected PHP Ia, which is the largest cohort examined so far for this rare syndrome. In the platelets from these patients, we determined altered responses of the Gsα-AC-PKA pathway using various multiparameter function tests, and compared these with quantitative changes in the PKA-dependent (phospho)proteome. These proteome changes were: (1) linked to a defective Gsα and PKA activity, (2) related to changes in platelet function, and (3) evaluated for the potential of discriminating between AHO and non-AHO.In order to assess abnormalities of the Gsα-AC-PKA pathway, we analysed the platelets from seven rare patients (four families) with confirmed or suspected PHP Ia (Table GNAS. Family II (mother P2 and two children P3–4) had a heterozygous, dysfunctional mutation of GNAS in exon 3. Family III with unaffected mother P5 included a child (P6), showing all physical characteristics of PHP Ia, while no genetic mutation was found so far. Family IV consisted of a child (P7) with a heterozygous deleterious mutation in the GNAS locus and a differential diagnosis including PHP. On the days of measurement, blood samples were also obtained from healthy control subjects (C1–12). Throughout this paper, the numbering of individual patients and controls has been kept the same.Ia Table . Family 22. For this purpose, platelets were treated with a range of iloprost concentrations (0.5–10 nM) for 1 min, i.e. a time point reflecting the early phosphorylation activity of PKA. In the cells from control subjects, VASP phosphorylation increased dose-dependently with limited inter-individual variation  and 12 control subjects (C1–12) were used for the assessment of iloprost-induced VASP phosphorylation at Ser-239, which is a golden standard method to establish affected PKA-dependent phosphorylation events–7 and 1223. The LTA method can reveal defects in Gsα signalling activity, by establishing the inhibitory effects of IP receptor agonists iloprost or PGE1 on collagen-induced platelet aggregation17. From four patients, sufficient blood could be obtained to measure platelet aggregation responses. In the platelets from the day-control subjects, collagen-induced aggregation was fully inhibited by 100 nM PGE1  is commonly used to monitor platelet function abnormalities, requiring the use of day control samples24. To determine a role of Gsα in this assay, whole blood samples from patients and control subjects were preincubated with vehicle or PGE1, and then flowed at defined wall shear rate25. The thrombi formed on collagen were analysed for variables, indicative of the platelet activation state: platelet adhesion (V1), P-selectin expression (V2), platelet aggregation as integrated feature size (V3), a thrombus multilayer score (V4), and a thrombus morphological score (V5). Blood samples from control subjects indicated that 100 nM PGE1 was sufficient to cause an ~ 50% overall reduction in thrombus formation. Hence, this PGE1 concentration produced smaller-sized thrombi , which was accompanied by lower platelet adhesion (V1) and lower platelet secretion (V2)   resulted in a reducing effect of PGEmap Fig. A, constr27, we examined the protein composition of available platelets from all patients with suspected PHP Ia , again in comparison to the day control subjects (C1–4). Directly after isolation, the purified platelets were incubated with 0, 2 or 10 nM iloprost for 1 min at 37 °C, i.e. a condition known to identify the iloprost/PKA phosphoproteome19.By combined analysis of the proteome and phosphoproteomeBecause of the use of iTRAQ or TMT labels, proteome analysis of the platelets was performed in four sets in 8-plex or 10-plex measurements, respectively , 4,845 , 5,540 , and 3,812   platelets was performed after TiO19.To establish the relevant iloprost-induced changes, we first listed the phosphopeptides that were present in multiple, ≥ 3 control samples (C1–4). For the found 2,516 phosphopeptides, the same cut-off was used as above, in order to define relevant changes induced by 2 nM iloprost (in brackets 10 nM iloprost), i.e. with ≥ 25% up- or downregulation. This provided an overall list of 453 regulated phosphopeptides, of which 146 (263) were upregulated and 192 (190) were downregulated (Suppl. Datafile By ordering the regulated phosphopeptides according to mean changes in (10 nM stimulated) control platelets, we importantly obtained highly similar patterns of iloprost-induced changes for the individual control subjects Fig. A. RaisinAssignment of the 453 phosphopeptides (regulated by 2–10 nM iloprost) to platelet function classes indicated a similar pattern for the up- and downregulated sites  for those phosphopeptides with PKA consensus site versus no such site  appeared to be accompanied by a consistent set of changes in the platelet protein phosphorylation pattern. Table This paper reveals a set of dysfunctional Gsα-mediated responses in platelets from seven rare patients with established or suspected PHP Ia , i.e. VASP phosphorylation, aggregation, and microfluidic thrombus formation. In all cases, the patient platelet were compared with day-control platelets from healthy control donors. These functional defects of platelets to Gsα stimuli . On the other hand, in the related disorder pseudopseudohypoparathyroidism (PPHP), mutations in the GNAS locus occur, which are usually paternally inherited and are designated as non-AHO. Molecular diagnosis of these syndromes is further complicated, as an AHO phenotype can also be accompanied by normal or hyper-activity of the Gsα protein29. Because of this complexity, platelet phenotyping for aberrant Gαs activity may assist in diagnosis.Patients with PHP-related disorders present with characteristic phenotypic features, such as a short stature and brachydactyly, but diagnosis only by physical examination is notoriously difficult. Genetic screening is usually performed to confirm a maternally inherited mutation in the 2), is a main mechanism by which the vessel wall prevents activation12. Accordingly, iloprost or PGE1 addition to platelets—similarly to PKA inhibition—potently suppresses agonist-induced responses including Ca2+ fluxes, integrin activation, adhesion, secretion, aggregation and thrombus formation30. Since the early recognition that the platelet inactivation is controlled by PKA-dependent phosphorylation31, extensive analyses have been performed to characterize the iloprost-induced phosphoproteome32. The present paper provides a first report to extend this work to patients with suspected Gαs defects, in order to explore how this technology can aid in new biomarker finding and diagnosis21.In platelets, the Gsα-AC-PKA pathway, induced by endothelial prostacyclin  with typical characteristics also obtained the ad-hoc diagnosis of AHO, although no mutation in GNAS was found . Patient P7 (family IV) was originally suspected for PHP Ia, as genetic screening revealed a heterozygous deletion mutation in GNAS. However, paternal inheritance is suspected, but not proven, which has led to a diagnosis of non-AHO. Markedly, our platelet phosphoproteome analysis is in accordance with a non-AHO phenotype.As indicated in overview Table 239, as a standard assay to check for Gsα-AC-PKA signalling34. Furthermore, for the same patients P1−4,6, we detected a dysfunctional response to PGE1 and iloprost, in terms of parameters of thrombus formation. Of note, due to the limited available blood samples, LTA could only be performed for two patients as a confirmation of this dysfunction. On the other hand, for patient P5  and patient P7 (concluded as non-AHO), the performed platelet function tests were within normal ranges.Table For platelets from six of the patients  we measured the iloprost-induced phosphoproteome as an alternative way to detect abnormalities in the Gsα-AC-PKA pathway. Therefore, we aimed to quantify per patient: (1) the overall changes in iloprost-induced protein phosphorylation patterns in patients versus controls; (2) the changes that can be linked to PKA dependent phosphorylation; and (3) shortlist of consistently changed phosphoproteins.GNAS mutation). This may be due to different thresholds in dose and time for Gsα-AC-PKA stimulation in the platelets from P3.In the five patients with established AHO , we noticed a dose-dependent increase in upregulated, but not in downregulated phosphopeptides in response to iloprost Table . Shortli32. Regarding the identified altered PKA-dependent phosphorylation events in confirmed AHO patients, several of the regulated proteins are of key importance for platelet signalling. These include the phospholipase C-β2 isoform (PLCB2); vasodilator-stimulated phosphoprotein (VASP) and the inositol-phosphate receptor regulator MRVI134. In addition, phosphorylated proteins were listed that control platelet shape change, including a myosin isoform (MYO9B) and the tight junction protein claudin-5 (CLDN5). Differentially regulated also were the regulatory subunit RIIβ (PRKAR2B) of the PKA-II holoenzyme, as well as BIN2, a protein with unknown function, but previously recognised as one of the most strongly regulated proteins in PKA signalling19.Reactome pathway analysis pointed to similar iloprost-induced changes in platelet functions, when evaluating the upregulated or the downregulated phosphoproteins. In either case, identified (sub)pathways were those of signal transduction, haemostasis, cell–cell interactions, and platelet activation. Jointly, this supports the presence of a coordinated mechanism of iloprost-induced platelet inhibition: directly mediated via PKA, and indirectly via a network of protein kinases as well as protein phosphatasesThe current data are in support of the use of platelet phosphoproteomics in the diagnosis of AHO and related diseases, for instance by checking a panel of biomarker phosphorylation sites as in Table Altogether, this work demonstrates an overall similarity in Gsα-AC-PKA mediated aberrations between functional responses and quantitative phosphoproteomics of platelets from patients with confirmed PHP Ia (AHO). The findings of aberrant upregulated as well as downregulated phosphopeptides in patients point to a change in the network of key protein kinases (starting from PKA) and phosphatases, which regulate multiple platelet functional properties.1 from Fluka-Sigma Aldrich . Horm type I collagen was purchased from Nycomed . Fluorescein isothiocyanate (FITC)-labelled antibody against phosphorylated vasodilator-stimulated phosphoprotein  was from nanoTools , Alexa Fluor (AF)647-labelled fibrinogen from Invitrogen Life Technologies , FITC-labelled anti-CD62P mAb against P-selectin from Beckman Coulter , and FITC-labelled PAC1 mAb against activated αIIbβ3 integrin from Becton Dickinson . The membrane probe DiOC6 came from Anaspec . Other materials were obtained from sources, as described before35.Iloprost was obtained from Bayer Schering Pharma , and PGEBlood was obtained from healthy controls and indicated patients, after full informed consent and in accordance with the Declaration of Helsinki. Experiments were approved by the Ethics Committee of Maastricht University and Maastricht University Medical Centre. Blood samples from patients (P1–7) were freshly taken, and always analysed in parallel with blood samples of unrelated day-control subjects (C1–12). The numbering of individual patients and control subjects in this paper is unchanged for all assays. Platelet samples for proteomics assays were stored at an internal biobank, until assayed for this purpose on a later time point, such in accordance with the European data protection law.GNAS complex locus, resulting in a truncated Gsα protein36. This mutation is known to be linked to an impaired expression of functional Gsα, thus reducing the Gsα bioactivity in blood cells37. Family II consisted of a mother (P2) and affected two sons , all of whom carried a heterozygous single nucleotide substitution in exon 338 (c.1G → C), encoding for a dysfunctional Gsα protein, with an estimated reduction of 46%, 49% and 51% in Gsα activity in the erythrocyte membranes38. Family III consisted of two related subjects, a mother (P5) with an unremarkable phenotype; and an affected daughter (P6) with PHP diagnosis, of whom no mutation in the GNAS locus could be detected . In family IV, patient P7 obtained a diagnosis, not differentiating between PHP and POH (progressive osseous heteroplasia), while genetic analysis revealed a heterozygous deletion in the GNAS locus (c.565_568del), linked to nonsense-mediated decay of mRNA (unpublished). Both parents did not allow examinations. In all 7 patients, blood platelet counts were within the normal range  and fragmin (40 U/mL)39. For light transmission aggregometry, blood was collected into 0.1 volume of 129 mM trisodium citrate. Platelet-rich plasma (PRP) was prepared by centrifuging blood samples at 240 g for 15 min. Platelet counts were determined with a thrombocounter .For whole blood perfusion experiments, blood samples were collected into 0.1 volume of saline containing d-glucose)35. PRP was then obtained by centrifuging as above. After the addition of 0.066 volume of ACD, the platelets were pelleted by centrifugation at 870 g for 15 min. Pellets were resuspended in Hepes buffer pH 6.6 , while carefully excluding any bottom layer of red cells. The suspended platelets were then transferred to a clean Eppendorf tube. After the addition of 0.066 volume ACD and apyrase (1 U/mL), collected platelets were washed by centrifugation at 2,000g for 5 min. Final resuspension then was in Hepes buffer pH 7.45 , once more by excluding any residual erythrocytes, followed by transfer to a clean Eppendorf tube. Purity of the final platelet preparations (1–2 × 108/mL) for proteomics analyses was assessed by a thrombocounter and microscopic analysis. Contamination of platelets with red blood cells was < 1:15,000 and with leukocytes < 1:20,000.For measurements with washed platelets, including proteomics analyses, blood was collected into 0.1 volume of acid-citrate glucose solution  were incubated with vehicle or iloprost (0.5–10 nM) for 1 min, after which reactions were stopped with 2% formaldehyde . Fixed samples were centrifuged at 2,000g for 2 min, and pellets were washed twice with phosphate-buffered saline. The pelleted platelets were then resuspended in phosphate-buffered saline containing 0.1% saponin, to allow membrane permeabilization during 15 min. After addition of FITC-labelled anti-P-VASP mAb against phospho-Ser-239 , samples were incubated for 30 min, and analysed by flow cytometry , using a BD Accuri C6 flow cytometer .For analysis of VASP phosphorylation as a measure of Gsα-PKA activity, samples of purified platelet suspensions (2 × 108 platelets/mL) was measured with an automated Chronolog aggregometer . The platelets were preincubated with vehicle (ethanol), PGE1 or iloprost for 4 min, and then activated with collagen (5 µg/mL) at 37 °C. Platelet aggregation rate was determined from the slopes of curves (% transmission change per min). In earlier functional experiments PGE1 was used, which was later replaced by iloprost because of the meanwhile published iloprost phosphoproteome19. Cyclic AMP measurements in washed platelets were performed, as before40.Aggregation of platelets in plasma , PGE1 (100 nM) or iloprost (5 or 10 nM), were perfused over the collagen surface at a wall shear rate of 1,000 s-1 for 4.0 min. Thrombi on coverslips were post-stained with FITC-labelled anti-P-selectin mAb (25 μg/mL)25. Brightfield differential interference contrast and confocal fluorescence images were taken from the collagen surface, as described42. Microscopic images were analysed for five variables (V1-5) using Image J 25. Deposited platelets (V1) and P-selectin staining (V2) were quantified as percentage of surface-area-coverage (%SAC); integrated feature size of aggregated platelets (V3) was expressed as μm2; thrombus multilayer score (V4) was scaled 0–3, depending on the presence of multilayered thrombi; and thrombus morphological score (V5) was scaled 0–5, ranging from no or single platelet adhesion to full thrombus formation24.To measure whole blood thrombus formation, glass coverslips were coated with type I collagen and mounted into a Maastricht flow chamber, as described8/mL) in Hepes buffer pH 7.45 were incubated with vehicle or iloprost (1–10 nM) for 1.0 min at 37 °C. Reactions were stopped by addition of 50% lysis buffer , and incubated on ice. Directly after lysis, the samples were snap-frozen, and then stored at − 80 °C until use. The following sample sets were simultaneously analysed . Set II: four samples from patient P7 and four samples from control subject C4 . Set III: three samples for patient P2, three samples for patient P6, three samples for control subject C2 , and a calibration sample. Set IV: three samples for patient P3, three samples for patient P4, three samples for control subject C3,  and a calibration sample. In parallel, platelet samples were analysed for VASP-P determination. Platelets from asymptomatic patient P5 were not available for this analysis.Well-purified washed platelets 5 × 1008/mL in 44. The TMT labelling was performed according to the manufacturer's instructions. Quality control of all samples was as before19. Lysed samples of the purified platelets (5 × 108/mL) were diluted to the same protein concentration . Cysteines were reduced  and free sulfhydryl groups were alkylated  with 10 mM dithiothreitol and 30 mM iodoacetamide, respectively. Samples of sets I–IV were handled in equivalent manners, as described below.Sample preparation, proteolytic digestion and iTRAQ/TMT labelling were based on previously described methodsg, and supernatants were carefully removed. The precipitates were washed using 50 µL of ice-cold acetone, followed by a 15 min centrifugation. This step was repeated once. Precipitated proteins were re-solubilised into 6 M guanidine hydrochloride, and digested in-solution with trypsin  at a 1:20 enzyme:protein ratio, with a final concentration of 0.2 M guanidine hydrochloride, 2 mM CaCl2 and 50 mM triethylammonium bicarbonate . Digestion controls were performed, using a monolithic-RP HPLC system, as before45. Digests were individually labelled with iTRAQ 8-plex labels . After drying in vacuum, the samples were dissolved in iTRAQ 8-plex dissolution buffer , and labelled according to the manufacturer’s protocol. Samples per set were pooled at 1:1 ratios, were desalted by C18 solid phase extraction  and dried under vacuum.For sets I and II, aliquots containing 100 µg of protein were diluted tenfold with ice-cold ethanol and incubated for 1 h at − 40 °C. The mixtures were centrifuged for 30 min at 4 °C and 18,00047, with slight modifications. Proteins were digested  in 50 mM triethylammonium bicarbonate, 0.2 M guanidine hydrochloride, 2 mM CaCl2, pH 8.5 with trypsin 19. The peptides were collected by centrifugation at 14,000g for 20 min. To increase peptide yield, filters were additionally washed with 50 µL 50 mM triethylammonium bicarbonate, and subsequently with 50 µL water. Digestion performance and peptide yield was controlled, as previous48. Digests were individually labelled with TMT 10-plex labels . Equal peptide amounts of vacuum dried samples were reconstituted in 100 µL 100 mM triethylammonium bicarbonate, and labelled with 0.8 mg reagent according to the manufacturer’s protocol. Samples were then pooled at 1:1 ratios, and further treated similar to sets I + II.For sets III and IV, 150 µg protein per sample was loaded onto a 30 kDa molecular weight cut off spin filter to perform filter-aided sample preparation, as described elsewhere18 column; BioBasic-18, 0.5 mm ID × 15 cm, 5 µm particle size, 300 Å pore size, Thermo Scientific) using a linear gradient. For iTRAQ pools, this gradient was ranging from 3–50% solvent B . For TMT pools, a linear gradient ranged from 3–50% solvent B  to obtain 20 concatenated fractions for LC–MS analysis.The multiplexed iTRAQ or TMT pools were used to quantify the global proteomes and the phosphoproteomes of the individual samples. For global proteome analysis, 10% (iTRAQ) or 3.5% (TMT) of a pooled mixture was pre-fractioned on a U3000 HPLC (Thermo Scientific) by high pH reversed-phase chromatography , and incubated twice with TiO2 beads for 10 min. Incubations first had a peptide to bead ratio of 1:6, and then a ratio of 1:3. For set III or IV, an additional incubation at 1:1.5 ratio was performed. Subsequently, the beads of all incubation steps were combined in one Eppendorf tube, and washed and eluted, as previously49. In short, 80% acetonitrile, 1% trifluoroacetic acid was used for washing step 1, and 10% acetonitrile, 0.1% trifluoroacetic acid for washing step 2. The phosphopeptides were eluted by incubation with 1% NH4OH for 10 min. The eluates were acidified using formic acid (pH < 2). To obtain better phosphopeptide recovery, the enrichment procedure was repeated once with a slight variation, i.e. a loading buffer of 70% acetonitrile, 2% trifluoroacetic acid, and a washing buffer of 50% acetonitrile, 0.1% trifluoroacetic acid. Phosphopeptides were eluted as described above and then acidified.For phosphopeptide analysis, the remaining of the pooled iTRAQ or TMT samples were subjected to a TiO50, and fractionated on a U3000 RSLC system in hydrophilic interaction liquid chromatography (HILIC) mode , using a binary gradient ranging from 10–35% solvent B  in 40 min (flow rate: 4 µL/min). A total of 9 fractions per set were collected for subsequent LC–MS analysis.The acidified phosphopeptides were desalted using Oligo R3 micro-columnsin italic sets III and IV, if different): RP and HILIC fractions were individually analysed by nano-LC/MS–MS, using a Ultimate 3,000 RSLC-nano system online-coupled to a Q-Exactive Plus (iTRAQ) or a Q-Exactive HF (TMT) mass spectrometer (both Thermo Scientific). Individual fractions were loaded onto a trap column  with 0.1% trifluoroacetic acid; flow rate: 20 µL/min. This was followed by separation of peptides on the main column , using a binary gradient ranging from 3–42% (3–35%) solvent B  in 145 min (60 min). In the Q-Exactive, survey scans were acquired at resolution of 70,000  using an automatic gain control (AGC) target value of 3 × 106 (1 × 106). MS/MS spectra of the top 15 most intense ions were acquired with a resolution of 17,500 , an isolation width of 2.0 (0.8) m/z, a normalised collision energy of 35% (33%), an AGC target value of 1 (2) × 105 ions, a maximum injection time of 250 (200) ms and a dynamic exclusion of 12 (30) s with and underfill ratio of 10%. The first fixed mass was set to 105 (100) m/z. In order to compensate for the iTRAQ-induced increase of peptide charge states, reaction tubes with 10% ammonium water were placed in front of the ion source as described elsewhere51. For TMT samples, to compensate for a higher complexity of the global proteome fractions, the isolation width was reduced to 0.4 m/z to reduce the potential precursor co-isolation.Sets I and II (TMT 10-plex) at N-termini and lysines of + 304.2053 (+ 229.163) Da and carbamidomethylation of Cys + 57.0214 Da as fixed modifications, (3) oxidation of Met + 15.9949 Da as variable modification, (4) mass tolerances of 10 ppm and to 0.02 Da for MS and MS/MS, respectively. For HILIC fractions, phosphorylation of Ser/Thr/Tyr (+ 79.9663 Da) was selected as additional variable modification. False discovery rate (FDR) estimation on the level of peptide spectrum matches (PSM) was performed using the peptide validator node, filtering for high confidence 1% FDR. The reporter ion quantifier node was used for iTRAQ (TMT) reporter quantification.Raw data were processed with Proteome Discoverer 1.4 (Thermo-Fisher Scientific). Data were searched against the Uniprot human database  using Mascot and Sequest with the following settings: (1) trypsin as enzyme allowing two missed cleavages, (2) iTRAQ 8-plex  only unique proteins quantified with at least 2 unique peptides were considered. For phosphoproteome quantification (HILIC fractions), phosphorylation site localisation was determined using phospho-RS(TMT) channel. These factors compensated for systematic errors  and allowed to obtain normalised ratios per protein. The normalised ratios were divided by the median (log2 transformed) over all eight (ten) values to obtain scaled normalised abundance values (NAVs) for all proteins and channels. The NAVs (log2 transformed) allowed determination of inter-sample ratios for platelets from the patients and control subjects for each condition.Sets I + II (sets III + IV): as proteome discoverer only provided 7 (9) ratios for the 8 (10) samples, an artificial 113/113 (126/126) ratio was created and set to 1.0 per protein and all ratios were log2 transformed. Per channel, the median ratio over all proteins was calculated, from which the median of all eight (ten) values was determined to define normalisation factors per iTRAQ Inter-individual variation for the global proteome was estimated by separate analysis of the samples from all subjects (combined sets) in comparison to averaged control values (C1–4). Differences from average control values were thresholded based on altered ratios log2 transformed (≥ 25% up or downregulation: range of − 0.322 to + 0.322).https://ms.imp.ac.at/?goto=phosphors) was used. Data were normalised as described above. NAVs were calculated per phospho-site, as described for the global proteome. Average NAVs were calculated per iTRAQ or TMT channel, after grouping per phosphopeptide sequence, phosphorylation site and protein. Phosphopeptides present in ≥ 3 control samples were used to assess average control values, after normalisation per data set, and were log2 transformed. Treatment effects were estimated based on an arbitrary cut-off of ≥ 25% up- or downregulation (log2 range of − 0.322 to + 0.322). Thresholds for relevant changes in patient samples were more strictly set at 2 × (− 0.322 to + 0.322); comparisons were made to mean control NAVs, for up- or downregulation per phosphopeptide.For determination of confident phosphorylation sites at the peptide level, a ready-to-use Excel macro provided by Mechtler lab . Consensus sites for PKA-induced phosphorylation were as before19, using the GPS2.1 algorithm for kinase consensus sequence prediction53. Classification of proteins was as before27.Phosphorylation data were compared with a reference dataset of iloprost-induced changes in protein phosphorylation of healthy control platelets , as publishedLists of protein identifiers  were introduced in the Reactome pathway database (reactome.org), and analysed on biological pathways with n > 4 entities and false discovery rates of > 0.82. Lists contained phosphorylated proteins regulated by 2 and/or 10 nM iloprost: 196 upregulated and 159 downregulated. Weight factors were included, presenting the numbers of regulated phosphorylation sites per protein.Inclusion of the quite rare patients with suspected PHP Ia  occurred over the years 2014–2017. As required for diagnostic platelet function analyses, blood samples from the 8 patients were directly compared with samples from a healthy day-control subject .The common rationale in diagnostic laboratories is that the (isolated) platelets from all healthy subjects make up a normal pool  that, however, needs to be validated day-by-day to check for the quality of blood drawing and sample preparation. At a first visit, blood from patients and day controls was analysed for platelet functions with Gsα-stimulating agents . In addition, platelet samples were made for proteomics analysis . If possible and required, at a second visit, blood from patients  and controls was used for additional functional assays. Note that flow cytometry of VASP phosphorylation was performed with all samples. The total number of included controls was 12.19 and to retain an affordable work-flow, for the proteomic assays, for omics analyses we compared patient samples with the means of respective day-control samples. Sets I-II of platelet and control samples, obtained in 2014–2015, were analysed in 8-plex using iTRAQ labelling. Set III-IV samples, obtained in 2017, were analysed in 10-plex using TMT labelling.Given the earlier established high stability of the platelet proteome and phosphoproteome among healthy subjectsd, i.e. (M1 − M2)/SDM1+M2, in which M1 refers for means of patients and M2 to means of controls). For medium effect size, we used a d ≥ 0.5. Cohen's d calculates a standardised difference between both groups that are not affected by sample size. Heatmaps were created using the R package version 3.5.1. Unsupervised hierarchical clustering of datasets from patients and controls was also performed in R.Functional and proteomics data of control subjects are represented as medians ± interquartile ranges; data from patients are indicated individually. Differences of individual patients were considered to be statistically significant, when outside the normal ranges . Effect sizes were calculated from the Cohen’s Datafile S1Datafile S2Supplementary information


Understanding the cellular mechanisms of kilohertz (kHz) electrical stimulation is of broad interest in neuromodulation including forms of transcranial electrical stimulation, interferential stimulation, and high-rate spinal cord stimulation (SCS). Yet, the well-established low-pass filtering by neuronal membranes suggests minimal neuronal polarization in respond to charge-balanced kHz stimulation. The hippocampal brain slice model is among the most studied systems in neuroscience and exhaustively characterized in screening the effects of electrical stimulation. High-frequency electric fields of varied amplitudes (1–150 V/m), waveforms  and frequencies (1 and10 kHz) were tested. Changes in single or paired-pulse field EPSPs (fEPSP) in CA1 were measured in response to radial-directed and tangential-directed electric fields, with brief (30 s) or long (30 min) application times. The effects of kHz stimulation on ongoing endogenous network activity were tested in carbachol-induced γ oscillation of CA3a and CA3c. Across 23 conditions evaluated, no significant changes in fEPSP were resolved, while responses were detected for within-slice control direct current (DC) fields; 1-kHz sinusoidal and pulse stimulation (≥60 V/m), but not 10 kHz, induced changes in oscillating neuronal network. We thus report no responses to low-amplitude 1-kHz or any 10-kHz fields, suggesting that any brain sensitivity to these fields is via yet to be-determined mechanism(s) of action which were not identified in our experimental preparation. There a large mismatch between enthusiasm for clinical treatments using kilohertz (kHz) frequency electrical stimulation and the understanding of kHz mechanisms of action. Indeed, the well-established low-pass properties of cell membranes should attenuate any response to kHz stimulation. This study presents the largest and broadest characterization of the cellular effects of kHz stimulation using the most established animal model to detect CNS sensitivity to electric fields: our work systematically evaluated sensitivity of hippocampal synaptic function and oscillatory network activity in response to kHz. Only at low kHz (1 but not 10 kHz) with high intensity and during oscillations, responses were detected. This systematic and largely negative experimental series suggest kHz neuromodulation operates via yet to be determined mechanisms.Electric fields at low frequencies (<100 Hz) are highly effective in changing firing rate and timing of neuronal population , includide novo generation of action potentials/pacing).Across this proliferation of techniques and application of kHz neuromodulation, the cellular mechanisms of kHz electrical stimulation remain unclear . While, Our goal was to systematically evaluate the sensitivity of hippocampal synaptic function and oscillatory network activity to kilohertz (kHz) frequency extracellular electrical stimulation. For assessing the sub and supra-threshold effects of electric stimulation on brain excitability, the application of uniform electric fields across the rodent slice preparation is among the longest-standing and most exhaustively studied animal models . fEPSPs,Here, we use fEPSP and oscillations to test the effect of 1- and 10-kHz electrical stimulation using sinusoidal symmetric and asymmetric pulse waveforms. We used direct current (DC) electrical stimulation as a within-slice control to confirm the sensitivity to low-frequency stimulation. Our data suggest the presence of diminished neuronal sensitivity in response to kHz stimulation consistent with the dramatic low-pass filtering property of the neuronal membrane. Oscillatory networks  are more sensitive to electrical stimulation but only to 1-kHz stimulation at ≥60-V/m intensity. Thus, consistent with results using sub-kHz electric fields, the structure of ongoing network oscillations would determine maximal sensitivity and effects of stimulation . If the All animal experiments were conducted in accordance with guidelines and protocols approved by the Institutional Animal Care and Use Committee at The City Collage of New York (CUNY).−1) and xylazine (0.7 mg kg−1) applied intraperitoneally and killed by cervical dislocation. The brain was quickly removed and immersed in chilled (2–6°C) dissecting solution containing the following: 110 mm choline chloride, 3.2 mm KCl, 1.25 mm NaH2PO4, 26 mm NaHCO3, 0.5 mm CaCl2, 7 mm MgCl2, 2 mm sodium ascorbate, 3 mm sodium pyruvate, and 10 mm D-glucose. Transverse hippocampal slices (400 μm thick) were cut using a vibrating microtome (Campden Instruments) and transferred to a recovery chamber for 30 min at 34°C with a modified artificial CSF (ACSF) containing the following: 124 mm NaCl, 3.2 mm KCl, 1.25 mm NaH2PO4, 26 mm NaHCO3, 2.5 mm CaCl2, 1.3 mm MgCl2, 2 mm sodium ascorbate, 3 mm sodium pyruvate, and 25 mm D-glucose. Slices were then transferred to a holding chamber for at least 30 min (or until needed) at 30°C with ACSF containing the following: 124 mm NaCl, 3.2 mm KCl, 1.25 mm NaH2PO4, 26 mm NaHCO3, 2.5 mm CaCl2, 1.3 mm MgCl2, and 25 mm D-glucose. For fEPSP experimental recordings, slices were then transferred to a fluid–gas interface recording chamber  perfused with warmed ACSF (30.0 ± 0.1°C) at 1.0 ml min−1. For γ oscillation experiments, slices were transferred to a fluid–gas interface recording at 34°C. All solutions were saturated with a gas mixture of 95% O2–5% CO2. γ Oscillations were induced by perfusing the slices with ACSF containing 20 μm carbachol (carbamoylcholine chloride). All reagents were purchased from Sigma-Aldrich.Hippocampal brain slices were prepared from male Wistar rats aged three to five weeks old, which were deeply anaesthetized with ketamine  pulled by a Sutter Instruments P-97 and filled with ACSF (resistance 0.5–2 MΩ) were placed in stratum radiatum of CA1, ∼400 μm from the stimulating electrode and within 100 μm from stratum pyramidale . fEPSPs n per slice per waveform.For acute experiments, fEPSPs were evoked every 30 s, alternating between control and kHz (or Direct current stimulation (DCS)) conditions. Waveforms were applied for 1 s and fEPSPs were evoked midway  through the stimulation . Where i−1 min−1) was required to be <0.33% of the mean baseline fEPSP slopes . For the control condition, the same stability criteria were used, but no stimulation was applied. To quantify long-term effects, fEPSP slopes were normalized to the mean of the 20 min immediately preceding high-frequency stimulation. Sampling frequency was reduced to 10 kHz during long-term experiments in both 1- and 10-kHz stimulation because of technical limitations. The responses were compared between sham and control condition in three different times .For long-term experiments, fEPSPs were evoked every 30 s and fEPSP slope was monitored online. After at least 30 min of stable baseline fEPSP recordings, 1 and 10 kHz waveforms were applied parallel to the somato-dendritic axis  at 80 V/m for 30 min. fEPSPs were continuously evoked every 30 s throughout the kHz and for 60 min after kHz ended. To determine stability before stimulation, a least squares linear fit was applied to the baseline fEPSP slopes. The slope of the linear fit  and one-way repeated measure ANOVA for different intensities used in kHz waveforms. Bonferroni correction was used for multiple comparison correction. All the analysis was performed in R (RStudio).All data are reported as the mean ± SEM. Reported n values represent the number of slices used in each condition. Statistical analysis was performed using unpaired, one sample t test as implemented in JASP v0.13.1.0 using default effect size prior /p(H0|80 v/m = baseline). Effect size estimates are reported as median posterior Cohen’s δ with 95% credibility interval (CI) using a two-tailed H1 in order not to bias estimates in the expected direction. Bayesian ANOVAs were conducted using JASP with default priors, and effects are reported as Bayes factor for the inclusion of a particular effect, calculated as the ratio between the likelihood of the data given the model with versus the next simpler model without that effect.Difference across highest electric field intensity and baseline were analyzed using the Bayesian paired samples kHz and DCS extracellular electric fields were applied to slices via two parallel Ag–AgCl wires  placed in the recording chamber on opposite sides of the brain slice with the recording site approximately equidistant from each wire. Slices were oriented so that the resulting electric field was either parallel  or perpendicular  to the somato-dendritic axis of CA1 pyramidal neurons . In CA3 kHz was applied at 1 and 10 kHz using the following kHz waveforms : sinusoid, pulse (40–10–40 μs for 1 and 10 kHz), and an asymmetric pulse waveform with the shorter duration pulse at 2× the amplitude of the longer duration pulse  field with ACSF. Data acquisition and electrical stimulation were controlled by Power1401–625 kHz hardware and Signal software version 6.0 (CED). Voltage signals were amplified (10×), analog low pass filtered  and digitized . To reduce noise and stimulation artifacts, the voltage recordings were always performed relative to an iso-potential electrode placed in bath A.1. Fieln = number of slices. For statistical analysis paired t test was used to compare poststimulation and prestimulation in each electric field intensity and significance level (p) was corrected using Bonferroni for multiple . All the analysis was performed in R (RStudio).Signals were recorded in frames of 5 s (1.5 s before and 1.5 s after stimulation) and stimulation was applied for 2 s. Stimulation artifacts were minimized by subtracting the voltage in an iso-potential refence electrode from the recording electrode in the slice . SpectroField EPSPs (fEPSPs) measured at dendrites reflect the aggregate postsynaptic current entering to a population of neurons, which is a measure of synaptic input. fEPSPs are sensitive to low-frequency electric fields . Using rF = 0.5835, ns) in any of intensities tested . However, DC stimulation significantly modulated fEPSP slope [–DC  +DC ]. Neither DC nor 1-kHz sinusoidal stimulation affected PPF. Increasing stimulation frequency from 1 to 10 kHz  = 0.86, ns; PPF, 10 kHz: F = 2.8, ns), or changing recording time from during stimulation to immediately after the field was turned off  = 1.21, ns; PPF F = 0.88, ns; fEPSP, 10 kHz F = 2.2, ns, PPF F = 1.316, ns) did not modulate fEPSP over the range of electric field intensities tested , sinusoidal stimulation with 1 kHz did not produce significant effects  = 0.788, ns; 10 kHz, F = 1.03, ns) or PPF  = 1.30, ns; 10 kHz, F = 0.68, ns; F = 0.63, ns; 10 kHz, F = 1.022, ns; PPF: 1 kHz, F = 0.72, ns; 10 kHz, F = 0.86, ns; Symmetric and asymmetric charge-balanced waveforms are ubiquitous in implanted stimulators including DBS and SCS. Stimulation with radially-directed symmetric pulse waveforms at 1- and 10-kHz electric fields did not modulate fEPSP  = 0.231, ns, PPF, F = 0.58, ns; 10 kHz: fEPSP F = 1.52, ns; F = 0.08, ns, PPF, F = 0.52, ns), and asymmetric waveforms  = 1.71, ns, PPF, F = 1.30, ns), at 1 kHz or 10 kHz, did not modulate fEPSPs.When electric field was applied in tangential direction , sinusoidal waveform  = 0.113, p = 0.739; 10 kHz: F = 0.09, p = 0.767), time  = 0.024, p = 0.97; 10 kHz: F = 1.01, p = 0.375), and no interactions  = 1.01, p = 0.37; 10 kHz: F = 1.92, p = 0.158).Whereas all the prior results used brief application of electric fields, we further tested whether stimulation for a longer period  can induce lasting effects on fEPSP under the hypothesis that small effects could be amplified with longer stimulation duration. Stable baseline fEPSP was recorded every 30 s for over 20 min before stimulation and 60 min after stimulation. Electrical stimulation was done using sinusoidal 1- and 10-kHz stimulation with 80-V/m electric field intensity  and effe+0 = 0.34 with median posterior +0 = 0.63 with median posterior Since these negative results may support either evidence of absence (provide support for null hypothesis) or absence of evidence because of lack of statistical power, we performed Bayes factor hypothesis testing for fEPSP evoked during 80 V/m stimulation applied in radial direction  for 1- and 10-kHz sinusoidal, symmetric, and asymmetric waveforms. Moderate evidence was found for the absence of effect using 80 V/m, 10-kHz sinusoidal waveform, meaning that the observed data were ∼ 3× more likely to be under the null hypothesis than the alternative  in support of the null hypothesis regarding effect of time  and moderate evidence  in support of the null hypothesis regarding effect of stimulation condition . Regarding interactions, Bayesian analysis revealed moderate and anecdotal evidence in support of the null hypothesis for 1 and 10 kHz, respectively .N = 14; CA3c, N = 12, ns; Uniform unmodulated 1- and 10-kHz electric fields were applied across hippocampal slices exhibiting γ oscillations under carbachol perfusion A.1. OsciB.1). This muted sustained effect is presumably reflecting homeostatic network regulation to bring the network back toward equilibrium . Moreover, stimulation produced a poststimulation suppression of oscillation , intensities, 1- and 10-kHz frequencies, electric field direction , stimulation duration , and during and PF effects. While impractical to test all combinations, our overall experimental strategy was intended to identify responses. We focused (number of slices) on 80 V/m but tested a range of intensities in case responses are not monotonic. Given established sensitivity to DC fields of slice prep neurons , we condOur results are limited by several factors. It is never possible to exclude β errors, though our use of a high SNR experimental system, with multiple slices and numerous repetitions per condition per slice, as well as within slice positive DC controls, together suggest such undetected effects would be variable or small in any case. Alternative mechanisms of electric fields such as ion concentration changes , fiber bFollowing the quasi-uniform assumption , we appl