Female pattern hair loss (FPHL) is a non-scarring alopecia resulting from the progressive conversion of the terminal (t) scalp hair follicles (HFs) into intermediate/miniaturized (i/m) HFs. Although data supporting nutrient deficiency in FPHL HFs are lacking, therapeutic strategies are often associated with nutritional supplementation. Here, we show by metabolic analysis that selected nutrients important for hair growth such as essential amino acids and vitamins are indeed decreased in affected iHFs compared to tHFs in FPHL scalp skin, confirming nutrient insufficiency. iHFs also displayed a more quiescent metabolic phenotype, as indicated by altered metabolite abundance in freshly collected HFs and release/consumption during organ culture of products/substrates of TCA cycle, aerobic glycolysis, and glutaminolysis. Yet, as assessed by exogenous nutrient supplementation ex vivo, nutrient uptake mechanisms are not impaired in affected FPHL iHFs. Moreover, blood vessel density is not diminished in iHFs versus tHFs, despite differences in tHFs from different FPHL scalp locations or versus healthy scalp or changes in the expression of angiogenesis-associated growth factors. Thus, our data reveal that affected iHFs in FPHL display a relative nutrient insufficiency and dormant metabolism, but are still capable of absorbing nutrients, supporting the potential of nutritional supplementation as an adjunct therapy for FPHL. Female pattern hair loss (FPHL) is one of the most common causes of hair loss in women. The prevalence is known to increase with age and is estimated to be >25% in women above 49 years of age , which aBy definition, FPHL is a non-scarring form of alopecia, characterized by a diffuse distribution of hair loss on the scalp , which eOf note, an early diagnosis is important , as currHowever, it remains unknown whether and how nutritional deficiencies occur within human FPHL-associated HFs. Here we attempted to elucidate this aspect of FPHL pathogenesis by investigating whether affected iHFs  and tHFsTaken together, our data show that affected iHFs in FPHL display a relative nutrient insufficiency and relatively dormant HF metabolism, but are still capable to absorb nutrients.Occipital and parietal human scalp skin or follicular units were obtained from six healthy donors (aged 24–56 years) undergoing cosmetic surgery and thirteen FPHL patients (aged 35–70 years)  45,46] ,46 45,46n = 3 FPHL patients were microdissected and measured. For each donor, based on hair bulb diameter, hair shaft diameter, hair follicle length  using KEGG as metabolite set library. Enrichment ratio is computed by observed and expected hits as described in  fro fron = 3escribed ,50. Day n = 4 FPHL patients were microdissected and individually cultured for 24 h ex vivo at 37 °C with 5% CO2 in WCM medium. After a 3 h starvation period in PBS, the hair follicles were cultured for 1 h at 37 °C with 5% CO2 in WCM medium containing a combination of three fluorescent labeled metabolites as follows: 0.25 mM L-Cystine-Cy3 + 0.6 mM glutamic acid-Cy5 + 0.2 mM pantothenic acid-FAM. The culture was terminated by embedding the HFs in OCT and freezing them in liquid nitrogen. A total of 6 μm cryosections were obtained with a Leica cryostat. Images were taken using a Keyence fluorescence microscope BZ9100  maintaining a constant set exposure time throughout imaging.Full length terminal and i/m occipital and parietal anagen VI HFs  15] fro fro15] fFor in situ investigations, punch biopsies of intact skin were directly frozen after explant in OCT embedding medium (Thermo Fisher Scientific) and cryosectioned in 7 µm thick sections with a Leica cryostat ,46,51.Tissue cryosections were fixed in acetone, pre-incubated with 10% goat serum in TBS, and incubated with the primary antibodies (mouse anti-human CD31 (PECAM), DAKO. 1:30 and rabbit anti-human Collagen IV, Novus biologicals, 1:100) at 4 °C overnight. Secondary antibodies incubation was performed at RT for 45 min. Counterstaining with 4′,6-diamidino-2-phenylindole (1 µg/mL) was performed to visualize nuclei.Tissue cryosections were fixed in acetone, pre-incubated with 10% of goat serum in TBS, and incubated with the primary antibodies (mouse anti–human CD31 (PECAM), DAKO. 1:30 and rabbit anti-human VEGF  at 4 °C overnight. Secondary antibodies incubation was performed at RT for 45 min. Counterstaining with 4′,6-diamidino-2-phenylindole (1 µg/mL) was performed to visualize nuclei.Tissue cryosections were fixed in acetone, pre-incubated with 10% of goat serum in TBS, and then incubated with the primary antibody against human CD31 (DAKO. 1:30) at 4 °C overnight followed by secondary antibody incubation at RT for 45 min. Sections were then incubated with a primary antibody against human TSP-1 (Thermo Fisher. 1:50) at 4 °C overnight followed by secondary antibody incubation at RT for 45 min. DAPI (1 µg/mL) counterstaining was performed to visualize nuclei.Images were taken using a Keyence fluorescence microscope BZ9100  maintaining a constant set exposure time throughout imaging for further analysis. The number of CD31+ blood vessels lumen was counted in the perifollicular tissue and in the dermal papilla (DP) . VEGF stt-test with Benjamini and Hochberg 5%FDR correction, were used. If the data were not normally distributed, the non-parametric Kruskal–Wallis test, followed by Dunn’s multiple comparison test and multiple Mann–Whitney test with Benjamini and Hochberg 5%FDR correction, were applied. Data are expressed as mean ± SEM; p values < 0.05 were considered significant as indicated in the figure legends.Statistical analyses were performed with Graph Pad Prism 9 . The normality of the distribution(s) of the data was analyzed with the D’Agostino & Pearson omnibus test. Whenever datasets followed a normal distribution, the parametric one-way ANOVA and Tukey’s multiple comparison and multiple unpaired Student’s n = 4 experimental groups: tOCC, tPAR, iOCC, iPAR per patient, n = 3 patients). In light of clinical reports stating that supplementation with certain nutraceuticals may increase terminal hair count, anagen rate, and hair shaft thickness in FPHL patients ,26,53, wBetween 144 and 345 metabolites were identified in the HFs from each FPHL patient regardless of the HF type a, also rhttps://www.metaboanalyst.ca/MetaboAnalyst/home.xhtml, accessed on 30 March 2022) were associated with several anabolic and biosynthetic pathways broadly associated with cellular proliferation, such as aminoacyl-tRNA biosynthesis, glutamine and glutamate metabolism, biosynthesis of several amino acids, glycolysis, and citrate cycle (TCA cycle)  (A cycle) b. ParticA cycle) c. In addition, essential amino acids or molecules often found in nutricosmetics, such as pantothenic acid (vitamin B5), L-tryptophan, L-carnitine, and L-valine were reduced in iHFs, vs tHFs in both scalp sites c. In conTherefore, our data indicate that affected iHFs from FPHL patients reveal changes consistent with nutrient and metabolite insufficiency.Given our metabolomics findings revealing decreased levels of lactate, the main product of aerobic glycolysis, as well as of citric acid and malic acid, products of the TCA cycle, along with enrichment of L-glutamine, the substrate of glutaminolysis b,c, we hFirst, we compared the concentrations of the different metabolites in the culture medium of occipital healthy and FPHL tHFs. While the release of lactate  and consInterestingly, lactate secretion into the medium was significantly decreased by both occipital and parietal FPHL iHFs when compared to their terminal counterparts a. This dTaken together, our results suggest that iHFs from patients with FPHL are characterized by a lower degree of aerobic glycolysis and glutaminolysis.Because growing HFs produce and secrete vascular endothelial growth factor (VEGF) in the surrounding tissue to enhance vascularization providing nutrients ,54,55,56Surprisingly, only a trend toward a decrease in mesenchymal VEGF expression was detected in occipital tHFs from FPHL patients in comparison to healthy donors . OccipitVEGF expression and angiogenesis can be inhibited by thrombospondin 1 (TSP-1) ,60,61. TTaken together, our data show that VEGF expression is decreased in parietal iHFs from FPHL patients. Despite a lower TSP-1 expression in parietal FPHL iHFs, our data also reveal the interesting observation that intrinsic differences may exist in the level of TSP-1 in occipital tHFs from FPHL patients and healthy donors, and also comparing tHFs from the relatively unaffected occipital scalp and hair thinning predisposed parietal scalp follicles in FPHL patients. Thus, our findings further point toward differences in expression of modulators of perifollicular vascularization associated with the progression of FPHL.+ blood vessel cross sections (lumina) in the perifollicular CTS of intermediate and terminal HFs from different scalp regions of patients with FPHL and healthy donors (Given that parietal iHFs from patients with FPHL were characterized by different expressions of VEGF and TSP-1, we postulated that perifollicular vascularization may be impaired in intermediate HFs, thus explaining the decreased metabolic activity and intrafollicular nutrient/metabolite abundance. To probe this hypothesis, we quantified the area of CD31y donors , as an iy donors . Whilst y donors , this way donors a,b. Yet,y donors a,b.These data suggest that intrinsic differences are present between the vascularization of occipital versus parietal HFs, and that iHFs do not show significant impairment in perifollicular vascularization when compared to their terminal counterparts.Having established that iHFs from patients with FPHL did not exhibit vascularization abnormalities but lower relative abundance of specific nutrients, we considered that the nutrient uptake mechanisms  may be lThrough fluorochrome detection, we revealed that iHFs were able to take up those exogenously supplied nutrients, at a similar or faster rate than tHFs a–c. TherConventional treatment for FPHL and other forms of alopecia are often associated with nutraceutical supplementation, but scientific causal and mechanistic indications supporting this strategy are still poorly understood. In the present study, we present evidence that in patients with FPHL, the metabolic profile and metabolism of iHFs are different from that of tHFs. Specifically, affected iHFs are characterized by nutrient and metabolite insufficiency which highlight a relatively quiescent metabolic activity. In an attempt to clarify the mechanisms involved, we reveal that changes in perifollicular vascularization and thus access to blood-carried nutrients does not appear to be a contributory factor. However, we detected differences in the production and release of two important growth factors associated with angiogenesis and hair cycle, VEGF and TSP-1, between intermediate and terminal HFs from FPHL patients. Intriguingly, we also identified reduced perifollicular vascularization in parietal versus occipital tHFs from FPHL patients, which may explain the higher susceptibility of parietal scalp. Finally, we demonstrate that nutritional deficiencies in iHFs might be compensated by administration of exogenously supplied nutrients, placing nutraceuticals as ideal adjuvant in lessening hair loss and the progression of HF miniaturization in patients.During the growth phase, HF keratinocytes in the hair matrix undergo high rates of proliferation and cell division to produce the hair shaft . The higContrary to the expectations, we found increased glucose concentrations in the medium of parietal tHFs as compared to occipital tHFs. This might be a consequence of glycogenolysis, another important metabolic process occurring in the ORS of HFs . It is wThe amino acid L-glutamine is a critical fuel for HF regeneration  and its Interestingly, glutamate secretion into the extracellular space occurs concomitantly with the cellular import of L-cystine via an antiporter ,77. IntrThe overall slower metabolism of iHFs from FPHL patients is also reflected by the lower abundance of essential amino acids and vitamins, including pantothenic acid. Pantothenic acid is a precursor to coenzyme A and is known to promote keratinocytes, fibroblast, and DP cell proliferation ,80,81. ITaken together, our data suggest that the detected nutrient deficiency in iHFs from FPHL patients may result from a slower metabolism in comparison to tHFs. Given the importance of an active metabolism to maintain the high rate of proliferation required for hair growth, and to provide the metabolites necessary for hair shaft production, it is conceivable that these defects may be involved in HF miniaturization in FPHL patients.+ blood vessel density between parietal scalp skin from healthy controls and FPHL patients [The bulge region of the HF is permanently neighbored by a vascular structure termed upper venule annulus ,51,52, apatients . TherefoYet, very intriguingly, our data suggest intrinsic differences regarding angiogenesis in HF of FPHL patients, which may explain the faster progression of hair loss and miniaturization, especially in the parietal region in FPHL. While decreased VEGF and increased TSP-1 expression were identified in FPHL tHFs versus healthy tHFs, perifollicular blood vessel density remained unaltered. Interestingly, VEGF and TSP-1 are not only involved in angiogenesis, but respectively also in maintaining HF size  and in mFinally, we show that the intrafollicular concentrations of selected nutrients and nutricosmetics could be increased by exogenous supplementation ex vivo, indicating that iHFs retain the intrinsic capacity to absorb nutrients from the surrounding environment, and that uptake mechanisms are not impaired in iHFs . This suDespite the preliminary nature of this study, we show for the first time that nutrient insufficiency is a characteristic of iHFs in patients with FPHL, which is not caused by impaired nutrient uptake or poor perifollicular vascularization, these findings highlight nutraceutical supplementation as an inexpensive and safe adjunct therapy for FPHL management.


G protein-coupled receptors (GPCRs) are important drug targets that mediate various signaling pathways by activating G proteins and engaging β-arrestin proteins. Despite its importance for the development of therapeutics with fewer side effects, the underlying mechanism that controls the balance between these signaling modes of GPCRs remains largely unclear. Here, we show that assembly into dimers and oligomers can largely influence the signaling mode of the platelet-activating factor receptor (PAFR). Single-particle analysis results show that PAFR can form oligomers at low densities through two possible dimer interfaces. Stabilization of PAFR oligomers through cross-linking increases G protein activity, and decreases β-arrestin recruitment and agonist-induced internalization significantly. Reciprocally, β-arrestin prevents PAFR oligomerization. Our results highlight a mechanism involved in the control of receptor signaling, and thereby provide important insights into the relationship between GPCR oligomerization and downstream signaling. The functional consequence of G protein-coupled receptor oligomerization remains debated. Here the authors show that platelet-activating factor receptor oligomerization enhances G protein coupling, and restrains β-arrestin recruitment and internalization. Many GPCRs can activate several intracellular signaling cascades through interactions with various types of G proteins2 and β-arrestins4. Distinct agonists acting on the same GPCR can engage different effector subsets, and modify cellular outcomes differently. This phenomenon is termed “biased signaling”6. The discovery of biased ligands that favor specific signaling pathways highlights the relevance of precise control of GPCR signaling for proper therapeutic action with fewer side effects10. Although significant effort has been spent in the past to understand how a ligand can orient GPCR signaling towards one or another pathway13, less is known about the physiological molecular mechanisms that orient GPCR signaling. For example, proteins that interact with GPCRs or lipid membrane composition are expected to control the balance between GPCR-mediated signaling pathways12.G protein-coupled receptors (GPCRs) form the largest family of cell surface receptors. GPCRs are targeted by approximately one-third of all approved drugs17 even in native tissues19, and such assemblies were suggested to play important roles in physiological and pathological processes21. Monomeric class A GPCRs are known to be able to activate multiple G proteins23 and β-arrestins29. However, a dynamic equilibrium between monomer and di/oligomer populations has also been observed for several GPCRs32. Di/oligomerization of class A GPCR receptors may affect their intracellular trafficking33 via either positive or negative cooperative ligand binding34, increase36 or decrease38 in G protein activation and modify G protein coupling selectivity40. However, the role of di/oligomerization of GPCRs in β-arrestin engagement31 remains unclear.Many GPCRs can assemble into dimers or oligomers41. Following its activation, PAFR couples to both Gq/11 and Gi/o and also possibly to G12/1342 and recruits β-arrestin1 (βarr1) to initiate receptor internalization44. Furthermore, PAFR plays an important role in many physiological and pathological events, including immune response, cardiovascular regulation, and tumor growth47. We recently reported the crystal structure of PAFR with antagonists, which included an unusual conformational arrangement of seven transmembranes (7TM) domains48. However, the structural basis of PAFR di/oligomer assembly, and its role in controlling signaling remains unclear.The platelet-activating factor (PAF) receptor (PAFR) is the natural receptor for an endogenous lipid, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF). PAFR is involved in platelet aggregation and inflammatory responsesHere, we report the formation of PAFR dimers and oligomers even at low densities in transfected cell lines. Using the cysteine (Cys) cross-linking strategy, we identify two types of symmetric PAFR dimer interfaces, one involving TM1 and the other TM4 and 5. Locking either one or both of these interfaces via Cys cross-linking stabilizes the PAFR dimers and oligomers, respectively. We also demonstrate increased G protein signaling as well as decreased β-arrestin recruitment and agonist-induced PAFR internalization due to the stabilization of PAFR di/oligomers. Such dimerization-induced biased signaling is also observed with a natural genetic variant of PAFR. Our findings thus reveal a mechanism by which GPCRs control signaling bias through oligomerization.2  of the dimers are labeled on both subunits.We first examined the formation of PAFR dimers or oligomers using single-molecule imaging of Halo-tagged receptors covalently labeled with non-cell-permeant fluorophores expressed at various densities Fig. . We dete51  to ~30% (+++: 0.76 ± 0.21 particles/μm2). Since the labeling efficiency of the fluorescent probe is about 80%, mono/di/oligomeric fraction calculated from the photobleaching step required further correction. After correction, as described in the “Methods” section, for the low expression level of the PAFR, a high percentage of particles corresponds to monomers and dimers (43.5% monomers and 46.7% dimers). But at high density, 56.0% correspond to dimers and 30.7% to oligomers (13.5% trimers and 17.2% tetramers), with a monomer fraction at 13.3%. We can also estimate that at low density, 25% of the PAFR receptor entities are in a monomeric state, while 55% of them are part of dimers. At high density, only 5% of the PAFR entities are in a monomeric state, while 46% of them are in a dimer, and 49% are part of oligomers (n ≥ 3).When performed with the PAFR, a mixture of monomers and di/oligomers were detected, and showed a density-dependent di/oligomerization behavior51 Fig. . When moBmax) is 7.0 and 26.5 pmol/mg of total proteins, respectively . In the low expression conditions (+), the mean density of receptors is about 2–14-fold higher than the physiological one, as measured by the endogenous expression of PAFR in a mesangial cell line (3.06 pmol/mg)53 or in hepatocyte membranes (0.5 pmol/mg)54. However, the exact receptor density within the specific membrane sub-compartment of these cells is expected to be higher.In these experimental conditions, the quantity of receptors  technology. The energy donor Renilla luciferase 8 (Rluc) and acceptor yellow fluorescent protein Venus were fused to PAFR C-terminus. A saturation curve with a high maximum BRET ratio was observed for PAFR-Rluc and PAFR-Venus, whereas a lower ratio was obtained for PAFR-Rluc and β55 as they form part of the protein–protein interaction interface. We thus used such peptides and performed a BRET assay to identify possible dimerization interfaces on the 7TMs of PAFR. The addition of the synthetic peptides TM1, TM4, TM5, and TM7 decreased the BRET signal between PAFR-Rluc and PAFR-Venus, whereas the remaining three peptides did not show significant effects  receptors58. Treatment with oxidative copper phenanthroline (CuP) was used to promote the cross-linking of two cysteines, which can be detected through the appearance of dimers by SDS–PAGE and protein transfer to membranes58.We then used a Cys cross-linking strategy to further characterize the dimeric interfaces. Cys cross-linking provides a better resolution of the proximity between two residues in protein–protein interactions Fig.  as it re58, we mainly considered receptor–receptor interaction within the extracellular part of the transmembrane helices, as this part is more prone to be oxidized, the intracellular compartment being reducing. We also quantified the change in the ratio of dimers to the total quantity of PAFR subunits detected on the blots to determine the efficiency of CuP-induced cross-linking between the two monomers  was used to enhance cell surface expression60 due to low expression levels of several cysteine mutants . As expected, the presence of CuP resulted in a significant increase in the oligomer ratio of all double cysteine mutants  assay4.59-I154C4.62 in TM4 could be cross-linked without CuP treatment  as evidenced by results of a diffusion-enhanced resonance energy transfer (DERET) internalization assay63 Fig. . This is63 Fig. . Indeed,63 Fig. . However64 and genetic mutations65 were previously shown to control GPCR dimer formation, and are therefore highly relevant for diseases. For example, mutations impairing rhodopsin dimerization have been found in retinitis pigmentosa65. We thus investigated whether natural genetic variation could also influence PAFR oligomerization66 , and the Gq inhibitor YM-25498069. YM-254980, but not PTX, totally suppressed the calcium response mediated by the WT, the mutant Y151C4.59-I154C4.62 and the variant E178K, demonstrating the Gq activation is needed for the calcium release effect ers Fig. . GRK2 aners Fig. . These d1 accumulation  within the βarr phosphorylation bar code in the C-terminal region of PAFR. This AA mutant is expressed similarly to the WT  HEK-293 cells, compared to the parental HEK-293 cells. We verified that these KO cells expressed neither βarr1 nor βarr2  in βarr1/2 KO cells exclusively, which can be reduced by overexpression of βarr1  dimerization and βarr recruitment differently, with full agonists favoring dimers and β-arrestin interactions, while biased ligands that do not recruit arrestin not favoring MOR dimers31. Such data further document the possible relationship between dimerization and the control of one signaling pathway over another, although dimerization and βarr recruitment are linked in an opposite way compared to what we observed with PAFR.Another interesting observation of our study is that βarrs limit oligomerization of the PAFR WT. This underlines the functional interplay between βarr and receptor quaternary structure. The influence of GPCR oligomerization on arrestin interactions has already been analyzed for GPCR heteromers, where either a decrease or increase in βarr recruitment was observed97. Here, we discovered a link between natural genetic variation and PAFR dimerization which leads to signaling bias. Further studies using conditional mouse models are needed to assess the physiological or pathological effects of this natural variation. Second, high expression of PAFR was observed in the invasion and metastasis of non-small cell lung cancer and colorectal cancer98. It might lead to a larger proportion of dimers and oligomers in these cells then potentially influencing PAFR signaling and internalization. Further studies to evaluate the effects of PAFR oligomerization on tumorigenesis remain to be performed.Finally, our data could be of physiological or pathophysiological relevance. First, natural genetic variations in the human genome are an underappreciated public health burden. To this end, GPCR variants can differentially impact individual drug responses by potentially changing ligand potencies and efficacies, receptor conformation, surface expression, and pathway preferences99, may behave as biased agonists. However, other factors that stabilize GPCR oligomers (i.e. lipids such as cholesterol and phospholipids101) might also play a role in biased signaling. Our model provides insights into the present concept of naturally biased signaling modulation and opens new possibilities for other physiological ways to control the balance between various GPCR signaling events.In conclusion, we reveal an interplay between receptor homo-oligomerization and downstream signals and provide a model for biased signaling modulation in GPCRs. According to this model, ligands that favor dimers, as reported previously3H-PAF (ART0727) was purchased from American Radiolabeled Chemicals . Lipofectamine 2000 (11668019) and Fluo4-AM (F14202) were obtained from Life Technologies . ABT-491 (A9227). Fluorescein sodium (46955), and dichloro  copper(II)  were purchased from Sigma-Aldrich . Coelenterazine-h (S2011), HaloTag® Alexa Fluor®660 (G8471), and HaloTag® Alexa Fluor® 488 (G1001) were purchased from Promega Corporation . Halo-Lumi4-Tb (SHALOTBC) labeling reagents, βarr1 cellular kit (64BAR1TPEB), βarr2 cellular kit (64BAR2TPEB), and IP-One Gq kit (62IPAPEJ) were purchased from Perkin Elmer Cisbio . Pertussis Toxin (PTX) inhibitor (HY-112779) was purchased from MedChemExpress  and YM-254890 (10-1590) from Focus Biomolecules .PAF  was purchased from Tocris Biosciences . Acetyl-2AR were tagged with a double tag, either Flag-SNAP or Flag-Halo, inserted immediately after the signal peptide57  were obtained from Addgene102. The EPAC1 cDNA was obtained from Dr. Lily Jiang . cDNA for the human GRK2 and GRK3 in pcDNA3.1 was fused to Renilla luciferase .The pRK5 plasmids encoding wild-type human PAFR and human β80 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 100 U/ml penicillin–streptomycin at 37 °C and 5% CO2 in a humidified incubator. Plasmids were transfected using Lipofectamine 2000 according to the manufacturer’s recommendations103. On-TARGETplus Smartpool siRNAs (L-011971-00-0020) from Horizon were used to target βarr1. A nontargeting pool (D-001810-10-05) was used as a control. 60–70% confluent cells were transfected with both siRNAs (100 nM) and plasmids  using Lipofectamine 2000. Data were collected in HEK-293, COS-7, or Δβ-arrestin1/2 cells, as stated in the figure legends.HEK-293 cells , COS-7 cells , and HEK-293 cells with targeted deletion of ARRB1 and ARRB2 55. Before use, the peptides were solubilized in pure dimethyl sulfoxide (DMSO) and diluted in the corresponding cell culture medium to a final concentration of 10 μM. Cells were incubated with the peptides mentioned above at 37 °C for 2 h before performing BRET analysis.Synthetic peptides representing each of the TM peptides for the human PAFR were obtained from the Jier  with ≥90% purity. The TM1 peptide consists of residues 15–41 (YTLFPIVYSIIFVLGVIANGYVLWVFA), TM2 peptide of residues 54–78 (IFMVNLTMADMLFLITLPLWIVYYQ), TM3 peptide of residues 88–114 (FLCNVAGCLFFINTYCSVAFLGVITYN), TM4 peptide of residues 130–156 (TRKRGISLSLVIWVAIVGAASYFLILD), TM5 peptide of residues 184–209 (VLIIHIFIVFSFFLVFLIILFCNLVI), TM6 peptide of residues 232–257 (WMVCTVLAVFIICFVPHHVVQLPWTL), and TM7 peptide of residues 270–296 (AINDAHQVTLCLLSTNCVLDPVIYCFL). Three basic sequences (KKK) were introduced at the N- and C-terminus to ensure their incorporation into the plasma membrane of cells, as demonstrated previously103. The bound antibody was detected by chemiluminescence using the SuperSignal substrate (Thermo Fisher Scientific) and Flexstation 3 .Flag-tagged subunits were transfected into the indicated cells and seeded in white transparent 96-well plates. 24 h after transfection, the HEK-293 cells were fixed in 4% paraformaldehyde. Cell surface expression was detected with a monoclonal mouse anti-Flag antibody M2 at 0.8 mg/ml  and a goat anti-mouse secondary antibody coupled to horseradish peroxidase at 0.25 mg/ml 2, 1 mM MgCl2 at pH 7.4). [3H]-PAF concentration in the range from 0.1 to 500 nM was used. Specific binding was determined as the total radioactivity bound minus the radioactivity bound in the presence of 10 mM unlabeled PAF as non-specific binding. The reaction mixture was incubated on ice for 2 h and then washed three times with binding buffer. Afterward, cells were lysed with 0.1 M NaOH and counted for radioactivity in a scintillation counter (Packard Instrument Co.). The total cell protein concentration was determined by the method of the bicinchoninic acid assay using bovine serum albumin as a standard. The maximal concentrations of binding sites (Bmax) were determined by Scatchard plots. Results were expressed as pmol [3H]-PAF/mg of total cell proteins53.24 h after transfection, cells were washed one time with binding buffer  for 30 min at 37 °C and fixed with 4% paraformaldehyde for 15 min. Then, the cells were washed thrice with PBS, and images were taken.A TIRF microscope, equipped with a high-NA TIRF objective  and an electron-multiplying charge-coupled device (AndoriXon DV-897 BV), was adopted to achieve single-molecule detection, and a solid-state 488-nm laser  with 1 mW was used. To avoid photobleaching before image acquisition, cells were searched and focused in a bright field, and a fine focus adjustment in TIRF mode was performed using only 2% laser power. This procedure results in negligible photobleaching. Afterward, the laser power was set to 83%, and image sequences (1000 frames) were acquired with an exposure time of 50 s, resulting in the acquisition of an image every 50 ms. Sequences of images (records) were stored directly on a computer hard drive for subsequent analysis.104. Briefly, an “à trous” wavelet filter was applied to each frame to extract all the single molecules. Each isolated molecule was fitted to a 2D Gaussian function to obtain the precise x–y location, as well as the intensity and background. To find the corresponding single molecule in successive frames, a well-established trajectory linking algorithm (http://site.physics. georgetown.edu/matlab/) was adopted. For each identified trace, the bleaching steps were determined manually by one investigator and rescored blindly by another.Imaging data were analyzed by a home-written MATLAB  codeN-order oligomer to be observed as an m-order is thusSince the labeling efficiency of the fluorescent probe is far from 100%, the mono/di/oligomeric fraction calculated from the photobleaching step required further correction. The binding between dye and protein units was treated as an independent random event for statistical analysis. The probability of a single subunit binding is mth-order oligomer Hence, the ratio of A system of linear equations o-phenanthroline), 1 mM CaCl2, 5 mM MgCl2, 16.7 mM Tris–HCl, pH 8.0, 100 mM NaCl) was added at 20 °C for 30 min. After incubation with 10 mM N-ethylmaleimide at 4 °C for 15 min to stop the cross-linking reaction, cells were lysed with lysis buffer  at 4 °C for 1.5 h. After centrifugation at 12,000 × g for 30 min, supernatants were warmed up with loading buffer  at 37 °C for 10 min. Electrophoresis was performed and the proteins were blotted onto nitrocellulose membranes. Membranes were imaged using an Odyssey infrared scanner  at 700 nm for Halo-Red57.24 h after transfection, adherent HEK-293 cells plated in 12-well plates were labeled with 300 nM Halo-660 in culture medium at 37 °C for 2 h. Then, cells were incubated with the drug (each at 10 μM) or PBS at 37 °C for 15 min. Afterwards cross-link buffer (1.5 mM Cu(II)- internalization assay was performed for measuring PAFR internalization in real timeLumi4-Tb was excited by a laser at 337 nm, and the emission fluorescence intensities were recorded for the donor  and acceptor  using a PHERAstar FS microplate reader . The ratio of 620/520 was obtained by dividing the donor signal (620 nm) by the acceptor signal (520 nm) and multiplying this value by 10,000. Data are expressed as the percentage of maximal internalization and were normalized as indicated.105.For cell surface quantification, SNAP-tagged PAFR was labeled with 100 nM SNAP-Lumi4-Tb at 4 °C for 1 h. After being labeled, cells were washed thrice with Tag-lite buffer. For measurement of PAFR oligomerization at the cell surface, SNAP-tagged PAFR were labeled, 24 or 48 h after transfection, by incubation at 4 °C for 1 h with a solution of 100 nM SNAP-Lumi4-Tb and 300 nM SNAP-green in Tag-lite buffer (Perkin-Elmer Cisbio). TR-FRET measurements were performed using a PHERAstar FS microplate reader. After excitation with a laser at 337 nm (40 flashes per well), the fluorescence was collected at 520 nm for a 50 μs reading after a 50 μs delay after excitation (window 1) or for a 400 μs reading after a 1200-μs delay (window 2). The acceptor ratio was determined by dividing the signal measured in window 1 by the signal measured in window 22+-sensitive Fluo-4 (Life Technologies). The fluorescence signals (excitation at 485 nm and emission at 525 nm) were measured for 60 s  and recorded using a Flexstation 3 microplate reader or FLIPR Tetra . The agonist was added after the first 20 s. The Ca2+ response is expressed as an agonist-stimulated increase in fluorescence103.Briefly, a plasmid of interest was transfected into the indicated cells seeded into black transparent 96-well microplates for at least 24 h. The cells were then preincubated for 1 h with Ca1 accumulation in HEK-293 cells was measured using the IP One HTRF kit (Perkin Elmer Cisbio), according to the manufacturer’s recommendations.IP106  were detected by Mithras LB940 . The BRET signals were plotted against the relative expression levels of each tagged receptor. netBRET ratio = [YFP emission at 530/Rluc emission 480] (where PAFR-Rluc or β2AR-Rluc are cotransfected with PAFR-Venus)−[YFP emission at 530/Rluc emission 480] , in the same experiment. The results were analyzed by nonlinear regression assuming a model with one-site binding  on a pooled dataset from three independent experiments.For the BRET measurements between protomers, increasing amounts of Venus-tagged receptors were co-expressed with constant amounts of Rluc-tagged receptors in HEK293 cellsh substrate was added at a final concentration of 5 μM, and BRET signals were detected by Mithras LB940 (Berthold Technologies) with stimulation by PBS or an agonist.For β-arrestin recruitment assay using ebBRET assay, βarr1-Rluc or βarr2-Rluc (BRET donor), Venus-CAAX (BRET acceptor), and PAFR were co-transfected into cells for at least 24 h. For BRET assay between βarrs and the receptor, Venus-tagged PAFR and βarr1-Rluc or βarr2-Rluc were co-transfected into cells for at least 24 h. Before detection, cells were starved with PBS at 37 °C for 30 min. Afterward, the coelenterazine 107, Venus-tagged PAFR and GRK2-Rluc or GRK3-Rluc were co-transfected into cells for at least 24 h. Before detection, cells were starved with PBS at 37 °C for 30 min. Afterward, the coelenterazine h substrate was added at a final concentration of 5 μM, and BRET signals were detected by Mithras LB940 with stimulation by PBS or an agonist.For GRK2 and GRK3 recruitment using BRET assay108, PAFR and EPAC1 were co-transfected into cells for at least 24 h. Before detection, cells were starved with PBS at 37 °C for 30 min. Afterward, the coelenterazine h substrate was added at a final concentration of 5 μM, and BRET signals were detected by Mithras LB940 with stimulation by forskolin (10 μM) to produce cAMP, and further stimulation by PAF to inhibit cAMP production.For the cAMP measurement by EPAC BRET sensorβarr1 and βarr2 expressions in cells were measured using a total βarr1 and βarr2 cellular kit (Perkin Elmer Cisbio), according to the manufacturer’s recommendations. TR-FRET measurements were performed using a PHERAstar FS microplate reader. After excitation with a laser at 337 nm (40 flashes per well), the fluorescence was collected at 620 and 665 nm for a 400 μs reading after a 60 μs delay after excitation. The ratio between 665 and 620 nm was determined.PDB 5ZKQ. Residues to be substituted with cysteine were retrieved from GPCRdb (www.gpcrdb.org), and substitutions were highlighted using PyMOL . Natural genetic variants of PAFR were also obtained from GPCRdb. The structures of the complexes between GPCR and arrestin were retrieved from the PDB 6U1N for muscarinic M2 receptor and βarr1 complex, PDB 6TKO for β1-adrenergic receptor and βarr1 complex, 5W0P for rhodopsin and arrestin-1 complex, 6UP7 for neurotensin NTS1 receptor and βarr1 complex), and analyzed using PyMOL.The PAFR 7TM structure was retrieved from the P-values were determined using unpaired two-tailed t-test, paired two-tailed t-test, or one-way ANOVA with Dunnett’s multiple comparisons test. Differences were considered to be not significantly different (ns) when P  > 0.05.All data in the figures and the supplementary figures are mean ± SEM of at least three independent experiments performed in triplicate unless stated differently in the figure legends. The curves were fitted using Prism software . Fluorescent images were analyzed using lmageJ . Single molecular imaging data were analyzed using MATLAB . Statistical differences were calculated using GraphPad Prism. Further information on research design is available in the Supplementary InformationReporting Summary


Evidence exists regarding the impact of flourishing in individuals living with chronic pain, but there are currently no reviews which collate the literature on flourishing in adolescents living with chronic pain and their parents. Therefore, the aim of this scoping review was to map and review the current literature, to document how flourishing is defined and understood in the literature, and to identify gaps in the field. Six databases were searched . In addition, a limited gray literature search was conducted. The resulting data were collated and reported in relation to the review questions, by examining the included papers to search for the presence of flourishing. Database searches resulted in 7326 papers after duplicate removal, with eight remaining papers being assessed for full‐text eligibility. Following full‐text screening, a final four papers were included in the review. Within the papers, flourishing was defined in relation to commonalities of benefit finding, enhanced maturity and growth, and social support. Gaps in the literature and directions for future research are considered. This review suggests that there is a dearth of knowledge and research regarding flourishing among adolescents living with chronic pain and their parents, despite aspects of flourishing identified in limited literature. This warrants further investigation. In addition, a limited gray literature search was conducted of conference abstracts as well as unpublished theses using EThOS. Although books were excluded, relevant book chapters were set aside for reference sections to be screened to augment the search. The search strategy involved searching for titles, abstracts, keywords, and MeSH terms as appropriate for each database, using a combination of four search strings. Search strings were developed following pilot searches in order to refine the search strategy and reduce irrelevant results, while retaining a broader focus. These search strings included 2.3www.zotero.org), where duplicates were removed. Remaining references were imported into Covidence (www.covidence.co.uk), a specialized software used for managing review data for screening. After further duplicates were removed, all remaining papers were screened against title and abstract by the first author (RP). A total of 60% of papers were double screened by trained research assistants , a process that has been followed in other research  using a data extraction template devised by Peters, Godfrey, et al.2.5To ensure rigor, data were subjected to quality assessment using a tool developed by Alderfer et al.3Following completion of all database searches, a total of 9086 references were imported in Zotero to screen for duplicates. Following this, 8814 remaining references were imported into Covidence for further screening. After another 1488 duplicates were removed, 7326 remaining papers were screened against title and abstract, resulting in 7318 papers being excluded and eight remaining papers being assessed for full‐text eligibility. Following full‐text screening, a further four papers were excluded, resulting in a final four papersA limited gray literature hand search of conference abstracts, unpublished theses and book chapter reference sections identified one potentially relevant book chapter reference, 17 potentially relevant conference abstracts and one potentially relevant result from an unpublished thesis. However, all of these were ultimately excluded as they did not meet the eligibility criteria. Reference sections of, and papers citing, the final four papers included in this review were also searched but produced no relevant results.M = 2.42 (SD = 0.12). The results of this scoping review are reported in accordance with Preferred Reporting Items for Systematic Reviews and Meta‐Analyses extension for Scoping Reviews (PRISMA‐ScR) guidelines.No papers were excluded from the review based on the scientific merit quality rating 1–3), as all papers were rated highly on average by both reviewers, ranging from 2.18 to 2.55, –3, as al3.1The results of the review indicate that the scope of the current literature on flourishing in pediatric chronic pain is highly limited, with only four papers matching the search criteria. The papers were included in the scoping review based on their characteristics, which are summarized in Table n = 3 papers originating in the United Kingdom and n = 1 in Canada. Three papers focused on adolescents living with chronic pain, while one papern = 6 and n = 10, and the two quantitative papers recruiting sample sizes of n = 110 and n = 145. The mean age of participants in the three adolescent focused papers was M = 14.43 years, while the mean age for fathers reported in Jordan et al.M = 37.5 years. Participants were predominantly female in all papers (70%), apart from the parent focused study (100% fathers). While three of the papers explored multiple types of chronic pain , one paperPapers were published in 2008, 2015, 2016, and 2018, with 3.2To address the research question regarding the definition and understanding of flourishing in pediatric pain literature, commonalities were identified among the four papers. These commonalities may prove useful in supplementing our definition of flourishing in adolescent chronic pain and included: benefit finding, enhanced maturity and growth, and social support.3.2.1A dominant commonality within the eligible papers was benefit finding in the context of living with adolescent chronic pain. Benefit finding may be considered an important aspect of flourishing and is defined by Soltani et al.Benefit and Burden Scale for ChildrenAll four studies observed benefit finding among participants. While Soltani et al.r = 0.32, p = <0.001), depression , and Post‐Traumatic Stress Disorder . Benefit finding was also positively associated with higher pain intensity  and pain interference , indicating that higher levels of benefit finding may be associated with negative outcomes. In addition, benefit finding was found to be negatively associated with quality of life . Although some of these results may appear surprising, Soltani et al.In the Soltani et al.A common finding relating to benefit finding in adolescent chronic pain was identified among two of the papers3.2.2A second common finding among two of the papers3.2.3Social support was identified as a commonality among three of the papers.3.3Numerous gaps in knowledge were identified when collating these results. Most importantly, only four papers were identified which matched the search criteria for flourishing in adolescent chronic pain. In addition, only two of these papers specifically focused on potential contributors to flourishing  societies for data collection, which are not representative of the global population.There were also some limitations in our review. Firstly, our search only included papers published in the English language. Due to a lack of capacity to translate papers in other languages, it is possible that relevant results to our searches may have been missed or overlooked. Secondly, we were unable to retrieve the full paper (if written) for a potentially relevant conference abstract which was identified as potentially eligible for inclusion in the review.Further studies examining flourishing could be used to explore and build on this overlooked but potentially valuable resource in supporting adolescents living with chronic pain. Results from such studies could be useful to inform treatment strategies based on Positive Psychology for adolescents with chronic pain and their parents, which draw on existing strengths and capabilities to support the self‐management of chronic pain among adolescents. Positive Psychology interventions have been shown to be beneficial for adults living with chronic painIn conclusion, this review, although based on a limited number of studies, suggests that there is a lack of research into flourishing among adolescents living with chronic pain and parents of such adolescents. Despite this, it is clear from the four papers identified in this review that adolescents living with chronic pain can indeed experience positive outcomes along with the predominant negative outcomes usually associated with living with chronic pain. This warrants further investigation to further explore flourishing, not only for the individual adolescent, but also for their wider social environment.This study was undertaken as part of a PhD Studentship funded by the Pain Relief Foundation.None of the authors have any conflicts of interest to declare.


Among novel strategies proposed in pest management, synergistic agents are used to improve insecticide efficacy through an elevation of intracellular calcium concentration that activates the calcium-dependent intracellular pathway. This leads to a changed target site conformation and to increased sensitivity to insecticides while reducing their concentrations. Because virus-like particles (VLPs) increase the intracellular calcium concentration, they can be used as a synergistic agent to synergize the effect of insecticides. VLPs are self-assembled viral protein complexes, and by contrast to entomopathogen viruses, they are devoid of genetic material, which makes them non-infectious and safer than viruses. Although VLPs are well-known to be used in human health, we propose in this study the development of a promising strategy based on the use of VLPs as synergistic agents in pest management. This will lead to increased insecticides efficacy while reducing their concentrations. Today, world agriculture faces a major challenge: increasing food production to feed about 9 billion people by 2050 . To imprIn this context, sustainable strategies have recently been developed. This includes the use of microbial pest-control agents  ; chemicaFor several years, we have proposed a new patented concept based on the use of chemical synergistic agents combined with a given insecticide, which are able to optimize, in both in vitro and in vivo, the efficacy of the treatment while reducing the concentrations ,23,24,25As already indicated, calcium ions play a key role on the membrane target sensitivity to insecticides. They generate versatile activation of specific calcium-dependent intracellular signaling pathways that determine a large variety of functions  known, fAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) induces intracellular calcium rise through the activation of CdCl2-sensitive ion channels or membrane transports in the insect cell membrane. The results have clearly indicated that AcMNPV interaction with insect cell line plasma membrane increases acetylcholinesterase sensitivity for the organophosphate insecticide chlorpyrifos-ethyl through elevation of intracellular calcium concentration [In the same context, insect viruses can also be used as biological synergistic agents ,36. In tFrom our results, we then propose another alternative, which is based on the use of virus-like particles (VLPs) as a novel synergistic agent. By contrast to viruses, VLPs present some advantages since they are composed of viral structural proteins expressed in heterologous systems that assemble spontaneously but are lacking the viral genome ,38. VLPsVLPs can be produced in a variety of systems, including bacteria, yeast, mammalian cells, insect cells, and plants  39,40,4,42,43. VEscherichia coli bacteria was the first established recombinant expression system. This expression system is characterized by high protein yields  L1 protein expression [Pseudomonas fluorescens for the cowpea chlorotic mottle virus (CCMV) coat proteins expression [E. coli expression platform [The expression system using n yields , rapid cpression  and Pseupression . Severalplatform .Saccharomyces cerevisiae, in Pichia pastoris, and more recently in Hansenula polymorpha [Pichia pastoris has been reported [This is the most popular expression system to produce VLPs is the yeast system due to its easy and high protein expression , its abilymorpha . The yealymorpha . Howeverlymorpha ,56. Morelymorpha . Severalreported .Although mammalian cells present lower yield for VLPs production compared to other systems , they haVLPs are produced in cells that are previously either transiently or stably transfected or transduced with viral expression vectors . AlthougSf21 and Sf9 derived from Spodoptera frugiperda and the BTI-TN-5B1-4 cell line (High-Five™) derived from Trichoplusia ni [The baculovirus/insect cells system is the most commonly used expression system for large-scale production of both non-enveloped and enveloped VLPs, simple or complex, comprising up to five proteins . This islusia ni ,60. It hlusia ni ,64. Althlusia ni ,65. Thislusia ni ,58,59. Olusia ni . To limilusia ni . Alternalusia ni .Solanum tuberosum (potato), Lycopersicon esculentum (tomato), Glycine max (soybean), Lupinus luteus L. , Arabidopsis thaliana, and Nicotiana benthamiana or Nicotiana tabacum L. (tobacco) [Plant expression systems for VLPs production allow the production of properly folded complex proteins and represent a cost-effective eukaryotic system  that is tobacco) . Plants tobacco)  but lacktobacco) . Stable tobacco) . On the tobacco)   or in mammalian cells (CHO) to express three HBV antigens , leading to higher immunogenicity (Sci-B-Vac®). The hepatitis E VLP-based vaccine, Hecolin®, only licensed in China, is produced using E. coli expression system, and another vaccine expressed in insect cells is under clinical trial  orst HBsAg .The propst HBsAg . HBV- anuberosum , L. escuculentum , and N. thamiana ,75,110. thamiana . Plant Vthaliana ,45,108. thaliana  Table 2® or in mFor a few decades, nanoparticle (NP)-based delivery agents, such as liposomes, polymers, dendrimers, magnetic nanoparticles, and protein-based NPs, have been used as carriers for drug delivery. These systems comprise the administration and controlled-release delivery of pharmaceutical compounds to a specific area in tissues, improving efficacy and safety . EmerginCompared to classical NPs, VLPs present several advantages, including specific cell targeting. VLPs show a natural tropism to certain tissue, and a more specific targeting function can be obtained through attaching receptor-recognizing domains to the drug carriers  Table 2Table 2. inset Acyrthosiphon pisum revealed that the mortality rate  produced by the insecticide indoxacarb is increased by about 25% when co-applied with VLPs (5 × 109 particles/µL) . VLPs are considered safer than viruses due to the absence of the virus genome and capability of triggering calcium concentration elevation. For these reasons, they could be proposed as alternative synergistic agents co-applied with a given insecticide for optimizing insecticide efficacy through the elevation of the intracellular calcium concentration and be exploited in pest management. Nevertheless, ecotoxicological and environmental stability studies should be required before further applications.Up to date, VLPs are not used in the context of pest management. However, VLPs have recently received our attention because (i) they have been used worldwide in human health for over 30 years for HBV vaccination with a safety and efficacy profile more than satisfactory, taking into account the available safety data ,120,121,


Histone deacetylase inhibitors (HDIs) are promising drugs for the treatment of inflammatory diseases. However, their therapeutical exploitation is slowed down by severe adverse manifestations that can hardly be foreseen, mainly due to incomplete knowledge of how HDIs impact the delicate balance of inflammatory mediators. In this work, we characterized the effects of the HDI trichostatin A (TSA) on the expression of TNFAIP3, which is a crucial inhibitor of the classical NF-kB pathway and an LPS-induced negative feedback regulator. The accumulation of TNFAIP3 mRNA after LPS stimulation showed biphasic behavior, with one wave within the first hour of stimulation and a second wave several hours later, which were both reduced by TSA. By using inhibition and knockdown approaches, we identified two temporally and mechanistically distinct modes of action. The first wave of TNAIP3 accumulation was directly blunted by the histone deacetylase (HDAC) blockade. By contrast, the second wave was decreased mainly because of the lack of endogenous TNF-α induction, which, in turn, depended on the intact HDAC activity. In both cases, class I HDACs appeared to play a nonredundant role, with HDAC3 required, but not sufficient, for TNF-α and TNFAIP3 induction. In addition to TNFAIP3, TNF-α is known to induce many response genes that orchestrate the inflammatory cascade. Thus, suppression of TNF-α may represent a general mechanism through which HDIs regulate a selected set of target genes. Histone deacetylases (HDACs) are key enzymes that regulate histone lysine acetylation, thus influencing the way in which DNA is wrapped around histones and packaged into chromatin . By acti2+-dependent deacetylase activity. Finally, class III HDACs, also called sirtuins (SIRT1–SIRT7), act via different mechanisms and require a co-factor NAD+ for activation [The 18 members of the mammalian HDAC family are classified into four groups based on their homology with yeast orthologs . Class I2+-dependent HDACs can be blocked by a large group of epigenetic drugs with rather different chemical natures and specificities known as HDAC inhibitors (HDIs) [Zns (HDIs) . The fous (HDIs) , plus a s (HDIs) ,10.Since HDACs decrease histone acetylation, which is associated with the repression of gene transcription, HDIs are expected to induce a generalized increase in gene expression. However, HDIs are now known to suppress as many genes as they induce ,12,13. TFrom a mechanistic point of view, many studies highlighted an intricate pattern of interactions and reciprocal regulation between HDACs and several immunomodulatory transcription factors. Regarding only what concerns the NF-κB p65 protein, acetylation of at least seven lysines  was shown to regulate its DNA-binding ability, transcriptional potency and duration of action . For exaTNFAIP3 locus [TNF-α-induced protein 3  is a zinc finger ubiquitin-editing inhibitor of the canonical NF-kB pathway that is recognized as a crucial negative regulator of inflammation and immunity ,45. TNFAP3 locus . Thus, uIn RAW264.7 cells, which are used as a well-established model of murine macrophages , the preFurther experiments were performed to better characterize the features of the TSA-induced gene inhibition. First, TSA was administered together or after the LPS challenge, in addition to the previously shown pre-treatment. In these conditions, all genes were inhibited, but with different efficacies. Indeed, IL-6 and TNFAIP3 were both profoundly reduced, irrespective of the timing of the TSA administration. By contrast, TNF-α inhibition was only marginally reduced, although still statistically significant, when TSA was given after the inflammatory challenge. Pulse–chase experiments B were peOverall, these data indicated that inhibition of the TNF-α/TNFAIP3 axis (and of IL-6) by TSA was rapid and transient, which was in line with TSA being a reversible inhibitor . HoweverThe regulation of these genes was further investigated by using different HDIs, namely, sodium butyrate , entinostat (a benzydamide selective for class I HADCs) and sirtinol (a class III HADC inhibitor). TSA treatment was reported as a comparison. The concentrations used in this set of experiments were devoid of toxic effects, as shown by the untouched cell viability A and by Because class II HDACs are tissue-specific enzymes , we inveOverall, these results indicated that inhibition of class I HDAC activity accounted for the effects of TSA on the selected set of mediators, in accordance with the restricted HDAC expression in these cells. As expected, class III HDACs were not involved in LPS-dependent gene induction; however, their blockade appeared to selectively potentiate the expression of TNF-α and CCL5.Based on the observation that HDAC2 and HDAC3 were the most expressed HDACs in our model cell line, their role in the transcriptional activation of inflammatory mediators was investigated using siRNA-mediated gene targeting. We reasoned that the blockade of HDACs other than HDAC3 may have contributed to the dramatic reduction in TNF-α and TNFAIP3 mRNAs observed with TSA. Thus, RAW246.7 cells transfected with the control (scr) or HDAC3 siRNAs were treated with TSA prior to a time-course LPS challenge. Overall, these results indicated that, in murine macrophages, HDAC3 was required, but not sufficient, for the transcriptional activation of selected inflammatory genes and that HDACs other than HDAC3 were required to induce TNF-α and TNFAIP3, but presumably not IL-6.Previous kinetic experiments revealed a bi-phasic regulation of TNFAIP3 in HDAC3-deprived cells. Indeed, while they confirmed that TNFAIP3 was not inhibited by HDAC3 knockdown at an early time point (60 min), they also showed that the amount of TNFAIP3 decreased at a later time point (240 min). Because TNFAIP3 is a direct TNF-α-responsive gene , we hypoThese results suggested that inflammatory TNFAIP3 mRNA induction was indirectly blocked by TSA via the suppression of endogenous TNF-α at later time points, while direct HDAC inhibition may have been responsible for the earlier time point blockade.The present paper reports the blocking of the anti-inflammatory TNF-response protein TNFAIP3 using HDI TSA and investigates the involvement of endogenous TNF-α regulation in this phenomenon. TNFAIP3 induction using LPS showed biphasic behavior, with an early peak at 60 min of stimulation, followed by a sharp decrease and a second wave of induction. By blocking soluble TNF-α, we confirmed that the second peak, but not the first one, depended on the induction of endogenous TNF-α ,47. Not One obvious question concerns which HDAC isoform is required for the induction of the TNF-α/TNFAIP3 axis. In accordance with the current view that class I HDACs are mainly involved in the regulation of inflammation and class II HDACs are mainly involved in the mechanisms of adaptive immunity , the comAnother question is the mechanism through which HDAC influences the expression of the TNF-α/TNFAIP3 axis. HDACs regulate gene expression both via histone lysine deacetylation and via the deacetylation of lysines contained in hundreds or even thousands of other substrates ,13,14,15Of course, our findings do not completely unravel the mechanisms underlying target gene regulation by TSA. In particular, it remains to be determined how HDACs promote the expression of TNF-α and the first wave of TNFAIP3. We hypothesize that this may depend on promoter-specific mechanisms rather than bulk modifications of histones or inflammatory transcription factors based on previous studies that demonstrated that many inflammatory genes are not affected by HDAC inhibition and on the untouched upregulation of CCL5 that we have shown here ,33. For In summary, our results unveiled two levels of TNFAIP3 regulation by HDIs, which were characterized by different mechanisms and kinetics. One acted at early time points after inflammatory stimulation via direct inhibition of HDACs (different from HDAC3); the other acted on the second wave of TNFAIP3 induction and depended on the lack of endogenous TNF-α, whose production, in turn, required intact HDAC3 deacetylase activity. These findings identified a novel mechanism underlying the multifaceted regulation of inflammation by HDIs, which may help to better understand the complex results of HDI therapy in inflammatory conditions and pave the way for the future development of novel drug combinations to treat human diseases.E. coli 055:B5, Sigma, St. Louis, MO, USA) was used at 1 μg/mL. Murine recombinant TNF-α  was used at 10 ng/mL. TSA  was used at 100 ng/mL unless differently specified. Sirtinol  was used at 60 μM. Sodium butyrate and entinostat  were used at 5 mM and 1 μM, respectively. Neutralizing anti-TNF-α antibody  was used at 10 μg/mL. Cell viability was assessed using ViobilityTM 488/520 Fixable Dye  according to the manufacturer’s instructions. Samples were read on a MACSQuant 16 Instrument  and analyzed using FlowJoTM v10.8 Software .RAW264.7 and THP1 cells were purchased from American Type Culture Collection (ATCC) and cultured in DMEM complemented with 10% FBS. For all the experiments described here, RAW264.7 cells were used between the 4th and the 8th passage after thawing. Cells were pre-treated with TSA for 30 min before LPS stimulation unless differently indicated in figure legends. For pulse–chase experiments, cells were pre-treated with TSA for 30 min, followed by extensive washings before the addition of LPS. LPS  . Preliminary expression of inflammatory mediators and HDAC1-11 was evaluated using a set of custom PCR-array-based screening plates . The SsoAdvanced Universal SYBR Green Supermix  was used according to the manufacturer’s instructions. The expression of TNF-α, TNFAIP3, IL-6, CCL5 and 18S in further experiments was evaluated using commercial TaqMan™ Gene Expression Assays and TaqMan Fast Advanced Master Mix according to the manufacturer’s instructions . Reactions were run on a StepOne Plus Real-Time PCR System  and analyzed using StepOne Plus Software . The results are expressed either using the 2RAW264.7 cells were transfected with two different HDAC3 and one HDAC2 Silencer Select Validated siRNAs or with a control (scramble) siRNA  using Opti-MEM I reduced serum medium and Lipofectamine RNAiMAX transfection reagent  as previously described ,61. Tran3OV4, 2 mM DTT, 1 mM NaF, 1 mM PMSF and protease inhibitor cocktail; all from Millipore Sigma, St. Louis, MO, USA). Equal amounts of extracts were analyzed through SDS-PAGE, followed by blotting with antibodies against HDAC1, HDAC2, HDAC3 , HDAC8 , TNFAIP3  and β-actin . Protein bands were detected with Pierce SuperSignal West Pico Chemiluminescent Substrate  and quantified via computerized image analysis using Image Lab 6.1 software . Data were normalized based on the β-actin content.Human and mouse TNF-α and IL-6 were measured using ELISA  in cell-free supernatants according to the manufacturer’s instructions. For the Western blot analysis, cells were lysed in NP-40 lysis buffer  with inhibitors  vs. response-equation. A p-value < 0.05 was considered statistically significant. The GraphPad Prism program was used for the calculations.Comparisons between treatments were performed using a one-sample


In TNF-α-treated HUVECs, the expression of ALKBH5 was significantly decreased. ALKBH5 overexpression promoted the proliferation and inhibited the apoptosis in TNF-α-treated HUVECs, suggesting that ALKBH5 had a protective effect on cell damage induced by TNF-α. Importantly, ALKBH5 promoted the expression of Bcl-2 in HUVECs. Bcl2 overexpression reduced the expression of Gadd45, Bax, and p21, which are transcriptionally activated by p53. But the expression of p53 has not been significantly affected, indicating that Bcl2 might regulate the apoptosis by inhibiting p53 downstream targets. In addition, ALKBH5 overexpression significantly increased the level of pri-miR-7 and decreased the level of miR-7. In conclusion, ALKBH5 attenuated the TNF-α-induced cell injury via promoting Bcl2 expression. Our research expands the understanding of the progression mechanism of atherosclerosis and provides a potential strategy for the protection of vascular endothelial injury.The dysfunction and apoptosis of vascular endothelial cells are the initiating links in the formation of atherosclerosis. N6-methyladenosine (m6A) is an extremely extensive RNA methylation modification and its abnormality leads to the occurrence of various human diseases. In this study, we explored the effects of demethylase  The inflammatory damage, dysfunction, and apoptosis of vascular endothelial cells are the initiating links in the formation of AS . Therefoα-Ketoglutarate-dependent dioxygenase ALKB homolog 5 (ALKBH5) is a primary m6A demethylase, a member of the AlkB family, and also a homolog of obesity-related genes (FTO) [N6-methyladenosine (m6A) is an extremely extensive RNA methylation modification that participates in the transport, shearing, translation, and processing of mRNA ,3,4. Itses (FTO) . ALKBH5 es (FTO) ,8. In ades (FTO) ,10,11. Ses (FTO) ,13. Curres (FTO) ,15,16. Hin vitro. Here we explored the effects of ALKBH5 on TNF-α-induced apoptosis of HUVECs and its specific pathways in an in vitro cell model, trying to provide a potential strategy for the protection of vascular endothelial injury.In many studies, TNF-α-treated human umbilical vein endothelial cells (HUVECs) were used to simulate arteriosclerosis 22.12 at 37℃. The model of endothelial cell injury was constructed with 10 ng/mL TNF-α for 24 h. Overexpression plasmids, pcDNA3.1-ALKBH5 and pcDNA3.1-Bcl2 were purchased from YouBio . The siRNA-Bcl2 and siRNA-control were synthetized from Sangon Biotech . Lipofectamine 2000  was used for the transfection. miR-7 mimic (5′-TGGAAGACTAGTGATTTTGTTG-3′) and negative control (NC) mimic (5′-GGUUCGUACGUACACUGUUCA-3′) were synthetized from GeneChem Co., Ltd .HUVECs were purchased from KeyGen BioTECH Corp., Ltd  and grown in DMEM  with 5% CO2.2Total protein was extracted from HUVECs after the treatment or transfection for 24 h using a radio immunoprecipitation assay  buffer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to separate protein samples. Then, the protein was transferred into a PVDF membrane. After being blocked with non-fat milk for 1 h, the membrane was incubated with the primary antibody for 1 h, and then with the secondary antibody for 1 h at room temperature. The protein was immunoluminescent with ECL kit . Antibodies against ALKBH5 , Bcl2 , p21 , Bax , and Gadd45  were purchased from Abcam .2.3−ΔΔCT method. Primers for Bcl-2 , miR-7 (5′-TGGAAGACTAGTGATTTTGTT-3′) and pre-miR-7 (5′-CGCTTCGTGTTGTATGGAAGAC-3′) were used in this research.Total mRNA was extracted from HUVECs using miRNA Purification Kit and Ultrapure RNA Kit . Reverse transcription was performed using cDNA Synthesis Kits . Then, the expression of miRNA was detected using qPCR Assay Kits . Relative expression levels were calculated using 22.4Cell viability was detected using CCK8 assay. After the transfection or treatment, cells were seeded into a 96-well plate at a density of 5000 cells/well. Cell viability was measured every 24 h. 10 µL of CCK8 reagent  was added to the cells and maintained for 1.5 h at 37°C. The cell absorbance was measured at 450 nm using a spectrophotometer.2.5The apoptosis was detected using Flow cytometry analysis. After the transfection or treatment for 24 h, cells were incubated with Annexin V/FITC  at room temperature for 5 min and incubated with PI at room temperature for 15 min. FlowJo software was used to analyze the flow results.2.6t test, and differences between three and four groups were analyzed using the one-way ANOVA. All data were generated in three replicates. P < 0.05 was considered statistically significant.Data in this research was presented as mean value ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 7 software. Differences between two groups were analyzed using the student’s Ethics approval and consent to participate: This research study was approved by the Institutional Review Board of First School of Clinical Medicine College.3m6A methylation is involved in the transport, shearing, translation, and processing of mRNA. Its abnormality leads to changes in the corresponding downstream target proteins and the occurrence of human diseases. Here we detected the expression of methyltransferases and demethylases in TNF-α-treated HUVECs. Downregulated ALKBH5 was observed in TNF-α-induced inflammatory HUVECs. In the present research, we proved that ALKBH5 inhibited TNF-α-induced apoptosis through Bcl-2 pathway in HUVECs.3.1HUVECs were treated with 10 ng/mL TNF-α for 24 h to generate the vascular endothelial cell injury model . As showTo determine the effect of cell injury on ALKBH5 expression, the protein was extracted from HUVECs after the treatment of 10 ng/mL TNF-α for 24 h. As shown in 3.2To investigate the specific effect of ALKBH5 on vascular endothelial cell injury, the overexpression plasmid of ALKBH5 was transfected into TNF-α-treated HUVECs with the empty plasmid as a negative control and TNF-α-treated HUVECs as the blank control. Western blot was performed to detect the level of ALKBH5. As shown in 3.3Previous studies have shown that ALKBH5 inhibits Bcl2 degradation by reducing m6A modification, thereby regulating cell apoptosis in ovarian cancer, hepatocellular carcinoma, and cerebral ischemia-reperfusion injury ,18,19. TTo investigate the pathway by which Bcl2 inhibits the apoptosis, Bcl2 overexpression plasmid was transfected into HUVECs. As shown in 3.4To verify the hypothesis proposed above, the overexpression plasmid of ALKBH5 and Bcl2-siRNA were co-transfected into TNF-α-treated HUVECs. As shown in 3.5Previous study has shown that ALKBH5 promoted the expression of Bcl2 through regulating miR-7 in ovarian cancer . Therefo4ii) and α-ketoglutarate-dependent manner. The modification and regulation of mRNA by M6A involves a variety of cellular processes, including mRNA splicing, export, localization, translation, and stability [In the present research, we investigated the specific role of demethylase ALKBH5 on TNF-α-induced vascular endothelial injury. ALKBH5 was identified as an RNA demethylase in 2013 . It belotability . When metability . Studiestability . In normtability . M6A mettability ,25,26. Itability ,17,27.According to our results, the expression level of demethylase ALKBH5 was significantly decreased in TNF-α-treated HUVECs. Importantly, ALKBH5 overexpression promoted proliferation and inhibited the apoptosis in TNF-α-treated HUVECs, suggesting that ALKBH5 could protect cells from TNF-α-induced cell injury. At present, there are few reports on the role and mechanism of m6A methylation related genes in AS. The expression of METTL14 and m6A level is significantly upregulated in atherosclerotic vascular endothelial cells (ASVEC). METTL14 promotes cell viability through promoting the processing of mature miR-19a . In anotWe further found that ALKBH5 could promote the expression of Bcl-2 in HUVECs. At the same time, high expression of Bcl2 reduced the expression of GADD45, Bax, and p21, which are transcriptionally activated by p53, but the expression of p53 has not been significantly affected. Based on the above results, we hypothesized that Bcl2 can regulate apoptosis by inhibiting the transcriptional activation of p53. However, there is no more evidence that the transcriptional activity of p53 is directly inhibited by ALKBH5, which still needs further experimental research. Several studies have shown that ALKBH5 inhibits Bcl2 degradation by reducing m6A modification, thereby regulating cell apoptosis in ovarian cancer, hepatocellular carcinoma and cerebral ischemia-reperfusion injury ,18,19. HIn the present research, we also explored the mechanism by which ALKBH5 promotes the expression of Bcl2. Previous studies have shown that ALKBH5 can regulate the expression of Bcl2 through miR-7, but the specific mechanism is still unclear . In this5ALKBH5 was downregulated in TNF-α-treated HUVECs and attenuated the TNF-α-induced cell injury through promoting the expression of Bcl2. This research expands our understanding of the progression mechanism of AS and provides potential treatment strategies for AS.


Background:  Blockade of tumour necrosis factor (anti-TNF) is effective in patients with Crohn’s Disease but has been associated with infection risk and neurological complications such as demyelination. Niemann-Pick disease Type C1 (NPC1) is a lysosomal storage disorder presenting in childhood with neurological deterioration, liver damage and respiratory infections. Some NPC1 patients develop severe Crohn’s disease. Our objective was to investigate the safety and effectiveness of anti-TNF in NPC1 patients with Crohn’s disease.Methods: Retrospective data on phenotype and therapy response were collected in 2019-2020 for the time period 2014 to 2020 from patients in the UK, France, Germany and Canada with genetically confirmed NPC1 defects and intestinal inflammation. We investigated TNF secretion in peripheral blood mononuclear cells treated with NPC1 inhibitor in response to bacterial stimuli.Results: NPC1 inhibitor treatedperipheral blood mononuclear cells (PBMCs) show significantly increased TNF production after lipopolysaccharide or bacterial challenge providing a rationale for anti-TNF therapy. We identified 4 NPC1 patients with Crohn’s disease (CD)-like intestinal inflammation treated using anti-TNF therapy . Anti-TNF therapy was associated with reduced gastrointestinal symptoms with no apparent adverse neurological events. Therapy improved intestinal inflammation in 4 patients.Conclusions: Anti-TNF therapy appears safe in patients with NPC1 and is an effective treatment strategy for the management of intestinal inflammation in these patients. Variants in theNPC1 gene account for 95% of the genetic defects in patients with NPC. Patients experience progressive neurological impairment with reduced muscle tone, seizures, speech impairment and early onset dementia as well as hepatosplenomegaly, interstitial lung disease with recurrent pneumonia. Niemann-Pick disease Type C (NPC) is an autosomal recessive lysosomal storage disorder presenting most typically throughout childhood or adolescence. The NPC1 defect disrupts auto-phagosome maturation which impairs antibacterial responses towardsSalmonella typhimurium andAdherent-invasive E coli (AIEC). Mice with NPC1 deficiency can develop mild intestinal inflammation.Around 7% of patients with NPC1 develop inflammatory bowel disease (IBD), in particular Crohn’s disease with weight loss, diarrhoea, perianal fissures and fistula formation. However, anti-TNF treatment can cause neurological side effects such as demyelination and non-demyelinating central nervous system (CNS) inflammation including vasculitis. Since NPC1 patients could be particularly vulnerable to adverse neurological events due to their underlying progressive neurological disease we investigated if anti-TNF is a safe and effective treatment strategy for intestinal inflammation in NPC patients.In paediatric and adult patients with Crohn’s disease anti-TNF agents  are standard of care to induce and maintain remissionIn this retrospective cohort study, we identified patients with NPC1 and intestinal inflammation by contacting specialist centres involved in their care. We recruited patients with NPC1 and intestinal inflammation who had been on anti-TNF agents as part of their management. Data collection took place in 2019 –2020 with data collection from 2014 until 2020. Selection bias was reduced by the use of pre-determined inclusion criteria, and as a consequence, patients with incomplete datasets were excluded. Data was collected from historical medical records to calculate wPCDAI scores to reduce memory bias.Patients with genetically confirmed NPC1 and confirmed intestinal inflammation  treated with anti-TNF agents were identified by contacting specialist facilities responsible for their care in Europe and Canada. We identified 5 patients and retrospective data was collected. Data was obtained from local hospital notes by the patients’ lead clinician. Out of five patients, one was excluded due to incomplete data being available. De-identified patient information was provided by the clinicians responsible for their care. was calculated at baseline, 4 weeks, 10 weeks, 6 months and a year after starting anti-TNF agents. A change in wPCDAI of >17.5 was used to indicate a response to treatment with a wPCDAI <12.5 suggesting remission, wPCDAI > 40 suggesting moderate disease and wPCDAI > 57.5 suggesting severe disease. Potential side effects including unexpected changes in neurological function and infectious complications (e.g. increased frequency of respiratory infections) since starting anti-TNF therapy were recorded.Information on previous treatment and responses  were obtained in addition to response to anti-TNF therapy. The mathematically weighted paediatric Crohn’s disease activity index (wPCDAI)Peripheral blood mononuclear cells (PBMCs) from healthy volunteers supplied from the NHS blood bank as leukocyte cones were obtained via the Oxford GI biobank.Salmonella typhimurium (Multiplicity of infection (MOI): 10) or Adherent invasic E. Coli (AIEC) (MOI: 10). Supernatant was collected and frozen at 0, 2, 4 and 6 hours after stimulation. TNF quantification was performed using an eBioscience ELISA kit  as per manufacturer’s protocol.PBMCs were separated from whole blood by using a Ficoll gradient. PBMCs  were either treated with 2ug/ml U18666A (Sigma Aldrich) drug for 24 hours or were left untreated. After U18666A treatment, PBMCs  were either stimulated with lipopolysaccharide (LPS) (200 ng/ml) orinfected withGraphPad Prism 9 software .R is an open access alternative. Statistical significance was calculated using a Mann-Whitney U test or ANOVA for multiple test comparison. P < 0.05 were considered significant .Data analysis was performed usingWritten informed consent was obtained locally by the patients’ lead clinician from the patients or their guardians for deidentified data to be used for research purposes. Patient data were collected as part of the Oxford IBD cohort study and a sub-project to investigate rare diseases . Anonymous patient data were analyzed. Healthy donor samples were obtained via Oxford GI illnesses biobank. caused a TNF associated inflammatory cytokine response. Healthy volunteer PBMCs were incubated with U18666A before being stimulated with LPS,AIEC or Salmonella. At baseline (0 hour) we did not observe any significant differences in TNF levels between U18666A treated and control cells   . This su. The mean age of Crohn’s disease onset was 8.1 years with a mean treatment period of 27.75 months. Therapy used for control of intestinal inflammation was exclusive enteral nutrition (EEN) (n=1), local steroids (n=1), systemic steroids (n=4), 5-ASA (n=2), adalimumab (n=2), infliximab (n=4), methotrexate (n=1), anti-IL12/23p40 targeting ustekinumab (n=1) and the integrin antagonist vedolizumab (n=1). Two patients received both infliximab and adalimumab (patients 1 and 4) and patient 3 stopped infliximab and was switched to ustekinumab due to loss of effect before restarting both agents on family request. Complete remission was temporarily achieved in one patient (patient 1) using infliximab induction therapy. All other patients achieved a partial response (patients 2–4)  . Two patts 2–4) . In all 4 patients who received anti-TNF therapy, over the 1 year of follow up, we did not observe any adverse neurological events or worsening of neurological function suggesting anti-TNF therapy does not accelerate neurological decline in this patient group. Furthermore, there was no increase in respiratory complications or other serious infection in patients reported in this cohort at 1-year of follow up. Neurological decline was seen in some patients after the first year follow up period however, clinicians felt this was consistent with NPC1 progression. Patient 2 died whilst on anti-TNF therapy due to neurological and respiratory complications including inability to clear secretions. The clinicians felt this was due to NPC progression rather than anti-TNF treatment. However, this patient experienced an initial honeymoon period of improving motor skills, better respiratory function, nutrition, pain control  during the first year of treatment with a significant improvement in quality of life. Patient 1 had an improvement in both a large perianal wound pocket and a vaginal fistula and became able to swallow small amounts again. Unfortunately, the patient did not have an endoscopy prior to starting treatment so it is unclear if this was due to improvement in neurological function or upper gastrointestinal manifestations of Crohn’s disease.Four patients had a partial response to infliximab therapy. One patient had poor disease control on infliximab. Despite this, infliximab appeared to achieve better reported disease control on clinician global assessment than prior immunosuppression, steroids and exclusive enteral nutrition.We assessed patient response to treatment using the wPCDAI scoring system to indicate severity of intestinal symptoms at baseline, 4 weeks, 10 weeks, 6 months and a year. Patient 2 initially had severe Crohn’s disease with a wPCDAI of 82.5 and showed a significant improvement in score to 17.5 over the first 90 days of treatment with anti-TNF therapy . This imin-vitro cell model increased levels of TNF upon LPS and bacterial stimulation suggesting that TNF is a differentially expressed cytokine in NPC1 and providing a molecular rationale of the treatment.NPC1 patients can present with severe Crohn’s-like disease in particular debilitating perianal disease. Anti-TNF therapy in NPC1-IBD patients results in symptom improvement without neurological deterioration at 1 year of follow up. We show in an.Patients with NPC1 suffer from a severe life-limiting disease, the potential risks of TNF therapy (demyelination and increased infection susceptibility) must be balanced against the improvement in quality of life due to effective IBD therapy and symptom control. Our data has shown improved management of Crohn’s-like disease in patients with NPC following anti-TNF therapy without worsening neurological function or infectious complications. Our data suggest that anti-TNF is safe to use in NPC from a neurological perspective. The improvement in IBD activity in NPC1 patients  was asso). Elevated levels of TNF within demyelinating plaques has been associated with the development of multiple sclerosis (MS). Polymorphism in both TNFR1 and TNFR2 have been linked to an increased risk of MS. Similarly, anti-TNF therapies have been linked to CNS demyelination and increased disease burden in patients with multiple sclerosis with a dose-dependent increase in relapse rate found in a trial of the TNF antagonist lenecept. Despite this, anti-TNF associated demyelination occurs independently to the classical risk factors for MS. Cases of anti-TNF associated demyelination have been described in patients with Crohn’s disease, psoriasis, rheumatoid arthritis and ankylosing spondylitis potentially hinting to a rare but relevant safety signal. For example, in 75 patients starting anti-TNF therapy for rheumatoid arthritis or spondyloarthropathies, three patients who developed neurological symptoms demonstrated new cortical lesions consistent with demyelination on MRI. Another case series of four patients with neurological symptoms on anti-TNF therapy demonstrated MRI changes and CSF oligoclonal bands with three patients meeting the diagnostic criteria for multiple sclerosis. However, pathophysiological mechanisms behind these effects are not completely understood. Several mechanisms have been proposed, including poor cerebral penetration of anti-TNF agents due to large molecular size which could prevent immunosuppressive actions in the brain. Another theory is based on the multiple forms and receptors for TNF. Soluble TNF signals through TNFR1 which is a ubiquitous receptor whose signalling can result in both pro and anti-apoptotic effects but more commonly results in inflammation and increased apoptosis through caspase 8 activation. In contrast, signalling from transmembrane TNF is largely through TNFR2. TNFR2 is found on immune cells, endothelial cells and some cells of the CNS and is thought to be neuroprotective and stimulate cell survival through phosphatidylinositol 3-kinase (PI3K) activity. These opposing responses combined with variable receptor expression results in complex consequences of TNF manipulation and could explain why TNF may have both beneficial and deleterious role in multiple sclerosis.TNF can promote neuro-inflammation and neuronal damage as well as having a protective role in some diseases .Data are available under the terms of the et al. aim to investigate the safety and effectiveness of anti-TNF therapy in NPC patients with Crohn's disease symptoms. To do so, they evaluate retrospective data from four patients with confirmed NPC1 mutations and intestinal inflammation and provide in vitro evidence suggesting that TNF expression may be inherently higher in NPC.In this report, Williams This is a well-written article, and I agree with the authors' conclusion that anti-TNF therapy appears to be safe in patients with NPC1 mutations and may be an effective strategy for the management of intestinal inflammation in these patients. I have a couple of suggestions:in vitro model of NPC, does not increase TNF-alpha by itself . They then quantify changes in TNF-a expression in response to bacterial exposure , and show that the response of NPC-like cells is stronger than that of control cells. The authors suggest that this increase is consistent with an increased TNF production in response to LPS and gut bacteria contributing to mucosal inflammation in NPC patients. This, of course, would provide a mechanism for the reported improvement in quality of life in response to anti-TNF in the study patients. While I agree with the authors' overall interpretation of their data, I would suggest expanding the discussion to include the article by Iftakhar-E-Khudaet al. who reported that another U18666A model does display TNF-a increase in the absence of bacterial challenges, albeit within a different time frame. How do those findings fit into the authors' conclusions? The authors show that the treatment of PBMCs with U18666A, which is a widely used approach to generate a chemically-inducedet al., Vincent et al.), especially in view of the link between liver dysfunction and Crohn's disease. The Discussion incorporates published work on the use of anti-TNFa therapies in other diseases. I think it would also be appropriate to include citations reporting the effects of anti-TNFa therapy on NPC liver disease .in vitro experiments on PBMCs from healthy donors, including small molecule inhibition of NPC1 and infection models.The case series is backed up byResults are described clearly.Tables and figures are helpful to the reader and complementary to the information provided in the text.The discussion is well articulated and leads to a relevant conclusive message.It would be relevant and interesting to examine infliximab level and anti-infliximab antibody level in the four patients described. Therapeutic drug monitoring would add further insight into how patients with NPC1 respond to infliximab. Three out of the four patients described had a partial response to IFX, and it’d be relevant to know whether loss of response was due to the development of anti-IFX antibody as opposed to low IFX trough levels. Is this data available and can it be analysed? If not, could the authors discuss whether anti-TNF therapeutic drug monitoring could/should be evaluated in future studies of patients with NPC1 and IBD?Minor commentsMethods, Page 4 – Statistical analysis: “R is an open access alternative” – Can the authors clarify whether R was utilised or not for data analysis in this specific work?[5] Ruemmele FM, Veres G, Kohlo KL, et al. Consensus guidelines of ECCO/ESPGHAN on the medical management of pediatric Crohn’s disease. J Crohns Colitis. 2014; 8(10): 1179-207. These guidelines were updated in 2020 and you may want to add the following updated version:.[5] van Rheenen PF, Aloi M, Assa A, et al. The Medical Management of Paediatric Crohn's Disease: an ECCO-ESPGHAN Guideline Update. Journal of Crohns and Colitis 2020: 1-24References: Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Paediatric gastroenterology with sub-specialist interest in paediatric IBDI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Introduction: Mental health literacy (MHL) is defined as “one's knowledge and beliefs about mental disorders” . Additionally, it includes the capability to recognise specific disorders, distinguish risk factors and causes, recognise self-treatments and available professional help, and it is an attitude that encourages recognition and appropriate help-seeking . Background: In the UK mental health problems are one of the main public health issues as it affects one in four individuals. It specifically has a high prevalence among university students which is between the ages of 16–24 years The study design used for this study was a systematic literature review in which data were collected from PubMed, EBSCO and ScienceDirect using specific keywords in the advanced search. The amount of papers found were 953 after abstract screening for keywords only 34 papers were left and then final abstract and full-text article screening for the inclusion and exclusion criteria was done leaving it with only 8 papers. A quality assessment was done for the eight papers using CASP tool for RCT papers and EPHH tool for cross-sectional papers.The findings showed that there are different levels of MHL between university students depending on their gender, education year, faculty and ethnicity. It was also found that the professional help seeking behaviour is not significantly different between genders but there was a difference regarding those identifying themselves as bisexuals despite them having high MHL scores.In conclusion the MHL of university students in the UK was found to be lower than those in Australia thus stating that more attention should be drawn to the issue and the need for easily accessible counselling services should be provided and promoted by universities. So that students can know about their existence and make good use of it whenever needed.