The gut microbiome changes in response to a range of environmental conditions, life events and disease states. Pregnancy is a natural life event that involves major physiological adaptation yet studies of the microbiome in pregnancy are limited and their findings inconsistent. Pregnancy with type 1 diabetes (T1D) is associated with increased maternal and fetal risks but the gut microbiome in this context has not been characterized. By whole metagenome sequencing (WMS), we defined the taxonomic composition and function of the gut bacterial microbiome across 70 pregnancies, 36 in women with T1D.Women with and without T1D exhibited compositional and functional changes in the gut microbiome across pregnancy. Profiles in women with T1D were distinct, with an increase in bacteria that produce lipopolysaccharides and a decrease in those that produce short-chain fatty acids, especially in the third trimester. In addition, women with T1D had elevated concentrations of fecal calprotectin, a marker of intestinal inflammation, and serum intestinal fatty acid-binding protein (I-FABP), a marker of intestinal epithelial damage.Women with T1D exhibit a shift towards a more pro-inflammatory gut microbiome during pregnancy, associated with evidence of intestinal inflammation. These changes could contribute to the increased risk of pregnancy complications in women with T1D and are potentially modifiable by dietary means.Video abstractThe online version contains supplementary material available at 10.1186/s40168-021-01104-y. Actinobacteria and Proteobacteria phyla increased along with an overall decrease in bacterial richness . In studies in germ-free mice, they observed that inoculation with third compared to first trimester feces led to greater weight gain, insulin resistance and gut inflammation and suggested this was an adaptive proinflammatory response to defend the fetus from pathogens and provide it with nutrients. In contrast, after analysing fecal samples weekly across pregnancy, DiGiulio et al. ) and species of Lachnospiraceae bacterium 1157FAA (FDR 0.055) and an unclassified species of Veillonella genus (FDR 0.083) decreased during pregnancy  and their corresponding species (FDR 0.065 and 0.075), unclassified strains of Streptococcus thermophilus (FDR 0.06) and Bifidobacterium_animalis (FDR = 0.06) and their corresponding species (FDR 0.06 for both) increased throughout pregnancy  , a marker of short-term glycemic control  Table 1. Mode ofmester 3 . After c= 0.004) . FinallyBacteroides caccae and Bacteroides vulgatus, in the same cohort of T1D and non-T1D mothers in trimester 3. Relative abundances obtained by metagenomic sequencing and qPCR were strongly correlated . By fitting linear models in lmer with conception age, BMI, parity and HLA type introduced as fixed effects, and ‘woman ID’ and processing batches as random effects, qPCR confirmed the increase in relative abundance of B. caccae (P = 0.00005) and B. vulgatus (P = 0.04) in women with T1D  and MetaCyc reaction functional categories. A total of 451 complete pathways, 5628 KO and 3204 MetaCyc reaction categories were obtained. No significant interaction in richness was detected between factors T1D status and time. In the model in which time was considered as a continuous variable, richness was significantly higher in women with T1D for all three functional categories Figure . For betd-manno-octulosonate biosynthesis I), 17 KO gene categories and two MetaCyc reactions (DARAB5PISOM-RXN and UDPGLCNACEPIM-RXN) involved in the synthesis of bacterial lipopolysaccharides (LPSs) were enriched in women with T1D  involved in the degradation of mucin was also significantly reduced in women with T1D, but again only in trimester 2  involved in the deoxyxylulose 5-phosphate (DXP)-independent pathway for vitamin B6 synthesis, one pathway, five KO categories and six metaCyc reactions related to vitamin B12  synthesis and five pathways, 11 KO categories and 13 MetaCyc reactions involved in SCFA synthesis, including pyruvate and acetyl-CoA production and butyrate synthesis from acetate or lactate, were decreased in women with T1D 15 involv. The abu 2 Table .Differentially abundant functional features derived from HUMAnN2 were contributed not by a single species but rather a combination of species. Therefore, relative abundances of the principal contributing species in each of the six selected functions could be grouped into clusters Table .P value 0.04; Mann-Whitney test). Serum I-FABP was also increased in women with T1D compared to women without T1D   with any of the individual taxa that were differentially abundant between T1D and non-T1D women.Because the composition and function of the gut microbiome of women with T1D was suggestive of a pro-inflammatory state, we sought evidence for gut inflammation in women with T1D. Fecal calprotectin, released from neutrophils and monocytes, is a marker of intestinal inflammation that may result in increased epithelial permeability . Serum i) Figure . However.  * sqrt(fitc$s2.post). Note that after normalization, the data are on the log2 scale. For testing for differences in specific taxa due to HLA type, the limma model included, similarly to the previous model, ‘women IDs’ as blocks and adjustments for sample processing batches, conception age, BMI and parity. Four contrasts were fitted: (1) High (DR34) vs. low  risk HLA types, (2) DR34 vs. DR3X and DR4X grouped into a factor, (3) DRXX vs. DR3X and DR4X grouped into a factor and (4) DR34 vs. DRXX. P values were adjusted with the Benjamini and Hochberg method to control the FDR. FDR < 0.1 were considered significant. Taxa or functions significantly different with an abundance logFC greater than 0.5 or less than − 0.5 and present in at least 50% of the samples in either of the groups being compared were regarded as biologically significant. For identifying the ‘principal bacterial contributors’ to each differentially abundant function, first, the HUMAnN2-generated files with functions  stratified by contributing species were obtained. Next, the functions of interest with contributing species were disaggregated into individual files using grep. Finally, limma was applied as explained above to each subset of function with contributing species and only species with a larger log2-FC in the group of interest for the specific function were considered principal contributors belonging to the same bacterial cluster. The significance of the difference between measurements of serum markers was tested with a Wilcoxon rank sum test equivalent to the Mann-Whitney test using the wilcox.test function from the stats R package with parameter paired set to FALSE.Differential abundance of taxa from MetaPhlan2 and gene categories and metabolic pathways from HUMAnN2 was analysed with the R package limma . First, Bacteroides vulgatus (BV-1) 5′-GCATCATGAGTCCGCATGTTC-3′, BV-2 5′-TCCATACCCGACTTTATTCCTT-3′; Bacteroides caccae (BaCA-1) 5′-GGGCATCAGTTTGTTTGCTT-3′, BaCA-2 5′-GAACGCATCCCCATCTCATA-3′; universal 16S V4 primers Univ-1 5′-GTGYCAGCMGCCGCGGTAA-3′, Univ-2 5′-GGACTACNVGGGTWTCTAAT-3′. Standard curves were generated by 2-fold dilutions ranging from 10 to 0.02 ng of a pooled human fecal DNA.The qPCR reaction comprised 10 μL Sybr Green GoTaq qPCR Master Mix (2×) (Promega), 0.3 mM of each primer, 8.4 μL of water and 1 ng of DNA in 20 μL. Assays were performed in triplicate using the QuantStudio 12K Flex Real-Time PCR System (Thermofisher) with the following protocol: one cycle at 95 °C for 10 min, followed by 40 cycles of a two-stage temperature profile at 95 °C for 15 s and 60 °C for 1 min. Primers were E = (10−1/slope)-1. Efficiencies ranged from 97 to 102%. The abundances (N) for Bacteroides vulgatus and Bacteroides caccae were determined relative to the total bacterial load measured with the universal 16S primers, where N (B. vulgatus) = (Efficiency_B. vulgatus + 1)B.vulgatusCt_, N (B. caccae) = (Efficiency_B. caccae + 1)B.caccaeCt_, N universal = Ct_Universal, B. vulgatus relative abundance = N (B. vulgatus)/ N Universal, B. caccae_relative abundance = N (B. caccae)/ N Universal.Data from each triplicate fell within a 0.5 threshold cycle (Ct); outliers (> 1 standard deviation) were removed before calculating the average Ct of each sample. Amplification efficiency (E) was determined from the slope of the standard curves for each primer pair using the formula Bacteroides caccae and Bacteroides vulgatus and T1D status was determined using a linear mixed effects model (lmer) with conception age, BMI, parity and HLA type as fixed effects, and ‘woman ID’ and processing batches as random effects.Relative abundances were log10-transformed and used as input for the regression models. The association between the relative abundance of Additional file 1: Figure S1. Taxonomic composition of the 25 most abundant species as measured by WMS in fecal samples collected in trimesters 1, 2 and 3 of 70 pregnancies from 66 women (35 with T1D). X-axis depicts the non-informative study ID in the format womanID_pregnancy number_trimester. LCBD: local contribution to beta diversity (a measure of the uniqueness of communities). T1D: women with type 1 diabetes, non-T1D: women without T1D. Figure S2. Alpha diversity (Richness), by T1D status of women across trimesters. Figure S3. Beta diversity analysis by T1D status of women. PCoA ordination plots based on Bray-Curtis distances between samples at the Genus and Family taxonomic levels separated by trimesters in pregnancy. Figure S4. Beta diversity analysis by T1D status of women. PCoA ordination plots based on Bray-Curtis distances between samples at the Order and Phylum taxonomic levels separated by trimesters in pregnancy. Figure S5. Relative abundance and species composition of A) two orders differentially abundant between women with and without T1D, and B) one genus differentially abundant between trimesters in women with T1D. Figure S6. Log2 transformation of the relative abundance (1000 + 0.01) of bacterial species within the Enterobacteriales and Bifidobacteriales orders in women with and without T1D (mean ± SEM). P: P-value. Figure S7. Taxa differentially abundant between trimesters in women with and without T1D (mean ± SEM). Transformed data are on a log2 scale with the mean from fitted abundances shown as a point in each trimester for each group of women. The between trimesters denotes a significant difference between those trimesters while the after the trend line denotes significant difference between trimesters 1 and 3. The color denotes if differences between trimesters are within women with or without T1D. Figure S8. Boxplots representing the abundance distribution obtained by real-time quantitative PCR (qPCR) in women and with without T1D. P: P-value. Figure S9. Boxplots representing the functional alpha diversity distribution in women with and without T1D). P: P-value. Figure S10. Taxa contributing to pathways that are differentially more abundant in women with compared to women without T1D and non-T1D. A) PWY1269: CMP-3-deoxy-D-manno-octulosonate pathway I involved in LPS biosynthesis, and B) PWY−5838: Superpathway of menaquinol−8 synthesis involved in vitamin K2 synthesis. Y-axis: log2 of CPM (counts per million). Figure S11. Taxa contributing to functional features that are differentially more abundant in women with compared to women without T1D. A) Pyridoxal 5'-phosphate synthase (K06215) involved in vitamin B6 synthesis, B) COBALSYN−PWY: Adenosylcobalamin salvage from cobinamide involved in vitamin B12 synthesis, C) 3−hydroxybutyryl−CoA dehydrogenase (K00074) involved in short chain fatty acid (SCFA) production, and D) beta−N−acetylhexosaminidase (K01207), only differentially abundant in trimester 2, involved in mucin degradation. Y-axis: log2 of CPM (counts per million). Figure S12. Log2 transformed relative abundance (+ 0.01) of bacterial clusters based on broader functions in women with and without T1D across trimesters. P: P-value. Figure S13. Boxplots representing the distribution of measures for calprotectin and I-FABP in women with and without T1D. P: P-value.Additional file 2.


Human CC tissue microarrays were used to analyze IGF2BP2 expression in CC. The knockdown of E6/E7 and IGF2BP2 attenuated the aerobic glycolytic capacity and growth of CC cells, while IGF2BP2 overexpression rescued this effect in vitro and in vivo. IGF2BP2 expression was higher in CC tissues than in adjacent tissues and was positively correlated with tumor stage. Mechanistically, E6/E7 proteins promoted aerobic glycolysis, proliferation, and metastasis in CC cells by regulating MYC mRNA m6A modifications through IGF2BP2. We found that E6/E7 promote CC by regulating MYC methylation sites via activating IGF2BP2 and established a link between E6/E7 and the promotion of aerobic glycolysis and CC progression. Blocking the HPV E6/E7-related metabolic pathway represents a potential strategy for the treatment of CC.Enhanced aerobic glycolysis constitutes an additional source of energy for tumor proliferation and metastasis. Human papillomavirus (HPV) infection is the main cause of cervical cancer (CC); however, the associated molecular mechanisms remain poorly defined, as does the relationship between CC and aerobic glycolysis. To investigate whether HPV 16/18 E6/E7 can enhance aerobic glycolysis in CC, E6/E7 expression was knocked down in SiHa and HeLa cells using small interfering RNA (siRNA). Then, glucose uptake, lactate production, ATP levels, reactive oxygen species (ROS) content, extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were evaluated. RNA-seq was used to probe the molecular mechanism involved in E6/E7-driven aerobic glycolysis, and identified IGF2BP2 as a target of E6/E7. The regulatory effect of IGF2BP2 was confirmed by qRT-PCR, western blot, and RIP assay. The biological roles and mechanisms underlying how HPV E6/E7 and IGF2BP2 promote CC progression were confirmed  Cervical cancer (CC) is the most common cancer among women in 28 countries and the fourth leading cause of cancer-related death in women globally Human papillomavirus  is a small, non-enveloped virus containing a circular double-stranded DNA genome. HPV mainly infects basal keratinocytes of poorly differentiated squamous epithelia and drives the tumorigenesis of CC, head and neck cancer, and oropharyngeal cancer, among others. Persistent infection with high-risk HPV is considered to be the main cause of CC Metabolic reprogramming due to oncogenic mutations is increasingly recognized as playing an important role in the tumorigenesis of several cancers, including CC RNA binding proteins (RBPs) are key effectors of gene expression, regulating RNA metabolism at multiple levels, and form a large regulatory network involved in maintaining cell homeostasis in vitro and in vivo. RNA-sequencing (RNA-seq) results showed that IGF2BP2 expression was significantly downregulated in E6/E7-knockout cells. Moreover, the expression of IGF2BP2 was significantly upregulated in tumor tissues compared with that in adjacent tissues and was positively correlated with CC stage. Mechanistically, we found that E6/E7 proteins regulate the activity of key enzymes in the aerobic glycolysis pathway by affecting the binding of IGF2BP2 to MYC m6A sites. We further established that the overexpression of IGF2BP2 in E6/E7-knockout CC cells rescued glycolytic flux. Combined, our results indicate that IGF2BP2 is a downstream target of E6/E7 and plays a role in the progression of CC.To date, the function of IGF2BP2 in CC has not been systematically investigated. Here, we found that the knockdown of E6/E7 in CC cells significantly attenuated their proliferative and metastatic abilities as well as their capacity for aerobic glycolysis 2. Both cell lines tested mycoplasma-negative and were authenticated using short tandem repeat (STR) profiling The human CC cell lines SiHa and HeLa were cultured in Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% FBS (Gibco) at 37 °C under 5% COSmall interfering RNAs (siRNAs) targeting HPV16 E6/E7, HPV18 E6/E7, IGF2BP2, and methyltransferase-like 14 (METTL14) were purchased from GenePharma . siRNA and negative control (NC) siRNA transfection was performed with Lipofectamine 3000 reagent  for 48 h. The transfection efficiency was verified by qRT-PCR and western blotting.The HPV16 E6/E7-shRNA lentivirus, HPV18 E6/E7-shRNA lentivirus, and NC-shRNA were constructed by GenePharma . LV17-NC and LV17-Homo IGF2BP2 were cloned into the pcDNA3.1 (+) vector. The CC cells were infected with lentiviruses and cultured in DMEM supplemented with 5 µg/ml polybrene. To rescue the effect of E6/E7 knockdown on CC cells, HPV16/18 E6/E7-knockdown cells were infected with the IGF2BP2-overexpression lentivirus. Stable cell lines were generated by selection with 10 µg/ml puromycin . The expression of HPV16 E6/E7, HPV18 E6/E7, and IGF2BP2 was validated by qRT-PCR and western blot. The siRNA, shRNA and plasmid sequences are listed in Total RNA was isolated from treated CC cells using an RNA extraction kit and reverse transcribed into cDNA by one-step RT-PCR. Real-time qPCR was performed using SYBR Green PCR Master Mix . The sequences of the primers used are shown in Differentially expressed genes in HPV16 E6/E7-knockout SiHa cells were identified by RNA-seq analysis which was performed by Lc-Bio Technologies (Hangzhou) Co., Ltd (China).Protein expression was measured by western blot as previously described 6A and IgG antibodies were added to the lysate with Protein A/G PLUS-Agarose, followed by overnight incubation. Finally, the beads were washed with IP buffer and analyzed by western blot.For co-IP, transfected SiHa cells were lysed using lysis buffer containing a protease and phosphatase inhibitor cocktail. After centrifugation of the lysed cells at 4 °C for 10 min, the supernatant was collected. Anti-mCell viability was monitored using a Cell Counting Kit-8 (CCK-8) assay . Cells transfected with siRNAs and their corresponding NCs were seeded into 96-well plates (5000 cells/well). After incubation with DMEM containing 10% CCK-8 reagent for 2 h, the absorbance was measured at 450 nm.For the colony formation assay, transfected cells were seeded in 60-mm dishes (400 cells/dish). After 2 weeks, the resulting cell colonies were fixed in 4% paraformaldehyde (PFA) and stained with 1% crystal violet (Solarbio) for 15 min. After washing with PBS, the number of colonies was counted using ImageJ software.EdU assays were performed using a BeyoClick EdU Cell Proliferation Kit with DAB . After incubation with 10 µM EdU for 2 h, the cells were fixed in 4% PFA for 15 min, permeabilized in PBS containing 0.3% Triton X-100, and stained using Click Additive Solution at 37 °C for 1 h. Nuclei were counterstained with DAPI (Beyotime). Images were captured using an EVOS M5000 Fluorescence Microscope .The real-time cell analysis (RTCA) system was used to continuously monitor cell migration for 30 h. Migration was measured using a 16-well CIM plate, which was divided into upper and lower chambers by 8-µm microporous membranes. The migration curves were recorded by RTCA software.The transwell invasion assay was performed in transwell chambers (Thermo Fisher Scientific) precoated with Matrigel. Transfected SiHa and HeLa cells were treated with mitomycin C, suspended in serum-free medium, and seeded into the upper chamber. DMEM supplemented with 10% FBS was placed into the lower chamber as a chemoattractant. After incubation for 24 h, the culture medium was discarded, and the cells were fixed, stained, and photographed.The TUNEL assay 7 SiHa cells were cross-linked in 1% formaldehyde; after 10 min, glycine was added to terminate the reaction. Adherent cells were scraped off in PBS containing a protease inhibitor cocktail and centrifuged at 2000 × g for 5 min to collect the precipitate. Micrococcal nuclease was added to digest the DNA to the optimal length and 0.5 M EDTA was used to stop the digest. The nuclear membrane was then disrupted by ultrasound and the supernatant containing the cross-linked chromatin sample was collected. Rabbit monoclonal anti-MYC  antibody, IgG antibody, and samples were incubated overnight at 4 °C for subsequent immunoprecipitation. ChIP-grade protein G magnetic beads were added to each IP reaction followed by shaking for 2 h. Then, the beads were cleaned with high/low salt solutions, the chromatin was eluted, and cross-linking was reversed. DNA was purified using spin columns and subjected to qRT-PCR. The primers used for ChIP-qPCR are listed in The ChIP assay RNA immunoprecipitation (RIP) 6A) antibody  was used to pull down m6A-modified MYC. Total RNA was extracted from treated SiHa cells and purified using a polyA Spin mRNA Isolation Kit . The IP buffer was prepared as previously described 6A antibody and IgG antibody in IP buffer. The purified RNA was then added to the above-mentioned microcentrifuge tubes containing protease and RNase inhibitors followed by incubation at 4 °C overnight. The RNA was extracted using an RNA extraction kit and analyzed by qRT-PCR, the primers used for qPCR are listed in 6A was detected by IP assay. Briefly, 100 µl of the mixture containing the antibody-immobilized beads-RNP complex was added to a new tube, resolved by SDS-PAGE, and subjected to western blotting.For Me-RIP, an N6-methyladenosine (mThe CC tissue microarrays (TMAs) FDU961 and FDU1921  were stained with antibodies targeting IGF2BP2 , MYC , and Ki-67  as previously described Flow cytometry was used to detect the effect of HPV16/18 E6/E7 and IGF2BP2 on cell apoptosis and the cell cycle in HeLa and SiHa cells transfected with siRNAs or plasmids. Cell apoptosis was assessed using an Annexin V-FITC/PI apoptosis double staining kit while the cell cycle was analyzed using PI/RNase staining buffer  according to the manufacturers' instructions. NovoExpress software  was used for data acquisition and analysis.Glucose uptake and lactate production in CC cell-culture medium were assayed using commercial kits  and ATP production was measured using an ATP Assay Kit (Beyotime) following the manufacturers' protocols 4 cells per well. After allowing the cells to adhere overnight, the culture medium was discarded and fresh assay medium containing different detection reagents was added into each well. The cartridge was loaded with glucose (10 mM), oligomycin (1 μM), and 2-deoxyglucose  to detect ECAR at specified time points. Drug concentrations for OCR detection were as previously described Metabolic indicators  were used to evaluate glycolytic fluxes. Treated SiHa cells were evenly spread on a 24-well XF cell culture plate  at a density of 5×10n=7 per group) to establish a xenograft tumorigenesis model and observe survival time. SiHa cells stably expressing shNC, shIGF2BP2, shHPV16E6/E7+oe-NC, or shHPV16E6/E7+oe-IGF2BP2 were subcutaneously injected into nude mice (5×106 cells per mouse). The tumor volume was measured every two days after tumor formation. Tumor volume was calculated as V=1/2×length×width2. Images were acquired using a PerkinElmer IVIS preclinical in vivo imaging system. Survival data were also recorded for each mouse. The animal study was reviewed and approved by the Ethics Committee of the Fourth Military Medical University.Female BALB/c nude mice (4-6 weeks old) were randomly allocated to four groups  by qRT-PCR and western blot, and found that the decrease in the expression of E6/E7, IGF2BP2, and glycolysis-related genes was rescued in E6/E7-knockout cells transfected with oe-IGF2BP2 at both the mRNA Figure A and proy Figure D and coly Figure E of the The results showed that overexpressing IGF2BP2 could rescue the HPV16/18 E6/E7-knockdown-induced reduction in the invasive Figure F, migratin vivo, SiHa cells expressing shIGF2BP2, shHPV16 E6/E7+oe-NC, shHPV16 E6/E7+oe-IGF2BP2, or shNC were subcutaneously injected into 5-week-old nude mice. One week after the injection, the mice were observed daily, and tumors were imaged in vivo on day 20. We found that the injection of SiHa cells expressing shIGF2BP2 or shHPV16 E6/E7+oe-NC reduced the tumor size to a similar degree, whereas tumor size was slightly increased in animals injected with shHPV16 E6/E7+oe-IGF2BP2-expressing SiHa cells (Figure 6A modification by targeting IGF2BP2, thus promoting aerobic glycolysis and tumor progression in CC.To investigate the role of E6/E7 and IGF2BP2 th century, CC-associated incidence and mortality have declined significantly in the United States, mainly due to extensive screening practices and increased HPV vaccination rates in vitro and in vivo, while the effect of E6/E7 on aerobic glycolysis was explored for the first time. Our results identified possible therapeutic strategies for CC treatment involving the targeting of metabolic pathways.Since the mid-20Radiotherapy is an effective method for the treatment of CC, but radiation resistance inevitably develops 6A readers in vitro and in vivo. The results demonstrated that IGF2BP2 enhances the proliferative, metastatic, and aerobic glycolytic capacity of CC cells through binding to MYC m6A sites.IGF2BPs constitute a family of carcinoembryonic proteins that regulate cell function in cancer and exert carcinogenic effects as mIn cancer, active tumor cells use aerobic glycolysis as an energy source, even when mitochondrial function is intact 6A-MYC mRNA in vitro and in vivo. Given that cell metabolism is a complex process involving the activity of numerous genes, we cannot exclude that E6/E7 may also target aerobic glycolysis by influencing the methylation status of mRNAs other than MYC in an IGF2BP2-dependent manner. Our finding that the HPV E6/E7/IGF2BP2/m6A-MYC/glycolysis axis may play a key role in the pathogenesis of CC suggests that targeting HPV E6/E7 and downstream pathways may represent an attractive therapeutic option for the treatment of patients with this cancer.In summary, we demonstrated that E6/E7 can regulate the aerobic glycolysis of CC cells through IGF2BP2-mediated modulation of mSupplementary figures and tables.Click here for additional data file.


Hepatitis C virus (HCV) chronically infects 70 million people worldwide with an estimated annual disease-related mortality of 400,000. A vaccine could prevent spread of this pervasive human pathogen, but has proven difficult to develop, partly due to neutralizing antibody evasion mechanisms that are inherent features of the virus envelope glycoproteins, E1 and E2. A central actor is the E2 motif, hypervariable region 1 (HVR1), which protects several non-overlapping neutralization epitopes through an incompletely understood mechanism. Here, we show that introducing different HVR1-isolate sequences into cell-culture infectious JFH1-based H77 (genotype 1a) and J4 (genotype 1b) Core-NS2 recombinants can lead to severe viral attenuation. Culture adaptation of attenuated HVR1-swapped recombinants permitted us to identify E1/E2 substitutions at conserved positions both within and outside HVR1 that increased the infectivity of attenuated HVR1-swapped recombinants but were not adaptive for original recombinants. H77 recombinants with HVR1 from multiple other isolates consistently acquired substitutions at position 348 in E1 and position 385 in HVR1 of E2. Interestingly, HVR1-swapped J4 recombinants primarily acquired other substitutions: F291I (E1), F438V (E2), F447L/V/I (E2) and V710L (E2), indicating a different adaptation pathway. For H77 recombinants, the adaptive E1/E2 substitutions increased sensitivity to the neutralizing monoclonal antibodies AR3A and AR4A, whereas for J4 recombinants, they increased sensitivity to AR3A, while having no effect on sensitivity to AR4A. To evaluate effects of the substitutions on AR3A and AR4A binding, we performed ELISAs on extracted E1/E2 protein and performed immunoprecipitation of relevant viruses. However, extracted E1/E2 protein and immunoprecipitation of HCV particles only reproduced the neutralization phenotypes of the J4 recombinants. Finally, we found that the HVR1-swap E1/E2 substitutions decrease virus entry dependency on co-receptor SR-BI. Our study identifies E1/E2 positions that could be critical for intra-complex HVR1 interactions while emphasizing the need for developing novel tools for molecular studies of E1/E2 interactions. Hepatitis C virus (HCV) employs several mechanisms to avoid eradication by the host immune system. This includes the virus’ ability to evade the B-cell response by occluding conserved neutralizing antibody (NAb) epitopes on the HCV envelope protein 1 (E1) and 2 (E2). An important component of HCV NAb evasion is the E2 motif, hypervariable region 1 (HVR1), which broadly regulates HCV neutralization sensitivity. As HVR1 displays a high level of structural flexibility, it has not been possible to infer HVR1 localization from solved structures of E2. Consequently, little is known about its interaction with the remaining part of the envelope proteins. In this study, we describe substitutions at conserved E1 and E2 positions both within and outside of HVR1 that appear critical for HVR1-specific E1/E2 function. We showed that these substitutions regulated protection of conserved neutralization epitopes and influenced receptor dependency of viral entry. Finally, testing the effect of these substitutions on NAb binding to extracted protein and in HCV immunoprecipitation we demonstrated the challenges of studying these phenomena outside the context of neutralization assays of infectious virus particles. Overall, this study contributes to our understanding of HVR1-mediated NAb protection which must be overcome in HCV vaccine strategies. Globally, more than 70 million people are chronically infected with hepatitis C virus (HCV), and have an increased risk of developing liver cirrhosis and hepatocellular carcinoma. This is the cause of approximately 400,000 annual deaths, making HCV a global health concern . Since 2in vivo, suggesting that HVR1 function could directly impact infection outcome  HCV RNA) in 500 μl complete medium on a shaker for 1 hour at room temperature. Afterwards, the beads were washed five times in 200 μl washing buffer before the RNA was eluted in 22 μl elution buffer. The beads were removed by flicking the sample for 30 sec. Duplicates of 10 μl of the eluate were used for RNA extraction  together with a dilution series of controls with known HCV RNA concentrations of 0–5 x 106 IU/ml in 1-log increments. The extracted RNA was used for reverse-transcription quantitative PCR as described in [Dynabeads magnetic beads  were resuspended in 200 μl antibody binding and washing buffer and incubated with 5 μg of antibody  on a shaker for 30 min at room temperature. Subsequently, the beads were washed twice and incubated with the relevant virus .(TIF)Click here for additional data file.S2 TableHCV envelope gene sequences, including the presence of specific substitutions, were verified by direct sequencing of culture-derived HCV RNA. HCV infectivity titers are given in log10 FFU/ml per ml and represent a mean of three technical replicates. The substitutions are numbered according to H77 abs. ref. (GenBank #AF009606).(TIF)Click here for additional data file.S3 TableSequence analysis of amplified envelope protein sequences from cell culture adapted HVR1-swapped HCVcc recombinants. HCV-positive cells were passaged for the indicated number of days until the virus had spread to >80% of the cells. Only coding mutations are included. At positions with nucleotide mixtures; lower-case letters denote the minor sequence; upper-case letters denote the most prevalent sequence. Two letters written with upper-case letters denote two nucleotides present in comparably equal amounts. Dots indicate the original plasmid sequence. Amino acid positions are numbered according to H77 abs. ref. (GenBank #AF009606).(TIF)Click here for additional data file.S4 TableS52-HVR1 HCVcc Click here for additional data file.S5 TableS52-HVR1 adapted in Huh7.5 cells. HCV-positive cell cultures were passaged for the indicated number of days until the virus had spread to >80% of the cells. Supernatant was collected at the peak of infection and used for sequence analysis of the full ORF. Only coding mutations are included. At positions with nucleotide mixtures; lower-case letters denote the minor sequence; upper-case letters denote the most prevalent sequence. Two letters written with upper-case letters denote two nucleotides present in comparably equal amounts. Dots indicate the original plasmid sequence. Amino acid positions are numbered according to H77 abs. ref. (GenBank #AF009606). Blue cells show cell culture adaptive mutations identified by Gottwein et al., 2009 [Full ORF sequence analysis of RT-PCR of J4 and J4l., 2009 .(TIF)Click here for additional data file.S1 Figin vitro transcribed HCV RNA of the indicated recombinants (A–C). Supernatants were collected 24, 48, 72 and 96 hours post transfection, and HCV infectivity titers were determined. At each timepoint, infectivity titers are represented by a mean of three technical replicates. Error bars show standard deviation. Lower level of quantification was 100 FFU/ml. The substitutions are numbered according to H77 abs. ref. (GenBank #AF009606). The data shown is a representative experiment out of at least 2.Huh7.5 cells were transfected with (TIF)Click here for additional data file.S2 Figin vitro transcribed HCV RNA of the indicated recombinants. Supernatants were collected 24, 48, 72 and 96 hours post transfection, and HCV infectivity titers were determined. At each timepoint, infectivity titers are represented by a mean of three technical replicates. Error bars show standard deviation. Lower level of quantification was 100 FFU/ml.Huh7.5 cells were transfected with (TIF)Click here for additional data file.S3 Fig50 value of the indicated recombinants. The broken line represents the IC50 value of unmodified J4. Error bars represent the 95% confidence intervals (IC95). The data was analyzed using three-parameter dose-response with the top value set to 100 and lower value set to 0, to calculate IC50 and IC95 using GraphPad Prism v8.0.0.Neutralization by the bNAb AR5A of the indicated recombinants. The viruses were incubated in four technical replicates with a 5-fold dilution series of AR5A, starting at 50 μg/ml, along with eight technical replicates of virus only. 48 hours post-infection the cells were immunostained for HCV antigen, and the number of FFUs/well was counted and normalized to the mean count of wells with virus only. Each dot represents the mean IC(TIF)Click here for additional data file.S4 Fig50 value of the indicated proteins. The broken line represents the OD50 value of unmodified H77, H77DH5-HVR1, J4 or J4S52-HVR1. The data was analyzed using three-parameter nonlinear regression using Graphpad Prism v8.0.0. The data shown is a representative experiment out of at least 2.Half-maximal binding concentration values for AR3A or AR4A binding to extracted E1/E2 protein, H77 (A and B) or J4 (C and D). Cell lysates containing E1/E2 protein were added to lectin-coated ELISA plates and incubated overnight. mAbs were added in two technical replicates in a 5-fold dilution series for a 2-hour incubation. The binding was detected with an HRP-conjugated anti-human IgG secondary antibody. TMB Stabilized Chromogen substrate (Thermo Fisher Scientific) was added and the reaction was stopped using HCl prior to absorbance measurement at 450 nm. Each dot represents the mean OD(TIF)Click here for additional data file.S5 FigHuh7.5 cells were incubated with a 5-fold dilution series of anti-SR-BI mAb, C16-71, which specifically blocks the interaction between HCV and SR-BI in four technical replicates with eight technical replicates of virus only and four technical replicates of control antibody D. 48 hours post-infection the cells were immunostained for HCV antigen, and the number of FFUs/well was counted and normalized to the mean count of wells with virus only. Error bars represent standard deviation. The data were analysed using three-parameter sigmoid dose-response curves using Graphpad Prism v8.0.0 with bottom constraints set to 0.(TIF)Click here for additional data file.


Histones and their posttranslational modifications facilitate diverse chromatin functions in eukaryotes. Core histones  package genomes after DNA replication. In contrast, variant histones promote specialized chromatin functions, including DNA repair, genome stability, and epigenetic inheritance. Previous studies have identified only a few H2B variants in animals; their roles and evolutionary origins remain largely unknown. Here, using phylogenomic analyses, we reveal the presence of five H2B variants broadly present in mammalian genomes. Three of these variants have been previously described: H2B.1, H2B.L , and H2B.W. In addition, we identify and describe two new variants: H2B.K and H2B.N. Four of these variants originated in mammals, whereas H2B.K arose prior to the last common ancestor of bony vertebrates. We find that though H2B variants are subject to high gene turnover, most are broadly retained in mammals, including humans. Despite an overall signature of purifying selection, H2B variants evolve more rapidly than core H2B with considerable divergence in sequence and length. All five H2B variants are expressed in the germline. H2B.K and H2B.N are predominantly expressed in oocytes, an atypical expression site for mammalian histone variants. Our findings suggest that H2B variants likely encode potentially redundant but vital functions via unusual chromatin packaging or nonchromatin functions in mammalian germline cells. Our discovery of novel histone variants highlights the advantages of comprehensive phylogenomic analyses and provides unique opportunities to study how innovations in chromatin function evolve. The genome and epigenome together determine form and function in all organisms. A significant component of the epigenome in eukaryotes comprises DNA-packaging units called nucleosomes. Eukaryotic nucleosomes typically contain ∼147 bp of DNA spooled around an octamer of four core histones—H2A, H2B, H3, and H4 . These hIn addition to RC histones, eukaryotes encode histone variants to promote functional diversity and specificity in cellular processes. Histone variants are commonly expressed throughout the cell cycle. As a result, they are also referred to as replication-independent (or RI) histones. RI or variant histones replace RC histones in nucleosomes to promote specialized functions like DNA repair, chromosome segregation, and gene regulation . TypicalSome histone variants arose in early eukaryotic evolution, whereas others have evolved more recently in specific lineages . ExampleAll four core RC histone proteins—H2A, H2B, H3, and H4—are present in stoichiometric ratios within nucleosomes. However, there is nonuniform diversification of RC histones into histone variants. For example, there are many H2A variants in mammals but comparatively fewer H3 variants and even fewer H2B and H4 variants , 2021. TThree H2B variants—H2B.1, H2B.W, and subH2B —have been previously described in mammals, where they appear to be specialized for roles in the germline. H2B.1  was one of the earliest H2B variants to be discovered in mammalian testes . This vaHere, we perform detailed phylogenomic analyses of mammalian histone H2B variants and describe five evolutionarily distinct H2B variants in mammals, including two novel H2B variants, which we named H2B.K and H2B.N following previously proposed nomenclature guidelines . Except To identify variants of histone H2B in mammals, we interrogated genome assemblies from 18 representative mammals. We performed comprehensive and iterative homology-based searches using both previously identified histone variants and new histone variants identified during our analyses  see Mat. AlthougH2B.E, which was originally identified through functional analyses of olfactory neurons in mice H2B.E, whg genome  online. g genome , we reco cluster . AligninH2B.O  albeit at low levels .Of the remaining broadly distributed H2B variants, three  have been previously described, whereas two  are newly identified by our analysis. Each of the three variants displayed unique evolutionary features. Most of the seven clades have high bootstrap support for the grouping of their orthologs (>50%) to the exclusion of other H2Bs. The only exception was H2B.1 orthologs that grouped together with low confidence (14%), likely due to their very high sequence similarity to RC H2B within the HFD used to generate the phylogeny  online. H2B.M, which is found in genomic proximity to human H2B.W, groups within the H2B.W clade  or whether they arose via independent duplications of RC H2B (polyphyletic). This poor resolution contrasts with the strong evidence for monophyly (single evolutionary origin) of the short histone H2A variants in mammals  share less than 50% amino acid identity with RC H2B in their HFD. For example, many residues in the HFD of H2B.W are conserved among orthologs, but diverged from RC H2B JSD∼1, A; these Even though H2B.L localizes to the subacrosome in sperm , it is nFinally, H2B.N shows the most dramatic differences from RC H2B in the HFD A. AlthouH2B.1 and H2B.W clades arose in the last common ancestor of eutherian mammals (∼105 Ma), whereas the H2B.L and H2B.N clades also contain marsupial and platypus (but not chicken) sequences, and therefore arose in the last common ancestor of all mammals (∼177 Ma)  A (diverg∼177 Ma) .H2B.K is the only H2B variant for which we could identify an ortholog in the shared syntenic location in chicken, a nonmammalian outgroup  but not in sea urchin cleavage-stage H2Bs challenges their orthology. Overall, our analyses suggests that four H2B variants arose in mammals, whereas H2B.K likely originated in the common ancestor of bony vertebrates (∼435 Ma), although this may even be an underestimate of its age.outgroup A. We exta urchin . Cleavaga urchin , our phya urchin . HoweverH2B.L, have experienced additional lineage-specific gene duplications  found in humans is also present in chimpanzee, bonobo, and gorilla, suggesting that a true pseudogenization event occurred ∼9 Ma in Homininae  and plotted it as a function of species divergence time  to synonymous (dS) changes. Neutrally evolving sequences have dN/dS ratios close to 1, whereas strong purifying selection results in ratios near 0, with most nonsynonymous changes disallowed. Using the same set of species selected above  with that of model 0 with dN/dS estimated from the alignment. Using this test, we found strong evidence for purifying selection for all H2B variants across mammals  selection at a select number of sites, or relaxed constraint. For example, complete lack of constraint would imply no functional selection for protein-coding capacity . To test for neutral evolution, we evaluated H2B variants by examining the ratio of rates of nonsynonymous . Indeed, H2B variants in plants (H2B.L (13 species) due to its recurrent pseudogenization in many primates  that is ubiquitously expressed  and germline (testes and ovaries) tissues from a wide range of species—opossum, dog, pig, mouse, human, and chicken . We speculated that this inconsistency might be due to unusual RNA structures or tissue heterogeneity. Instead of poly-A tails, RC histone transcripts have unusual stem-loop RNA structures at their 3′ ends that bind stem-loop binding protein, which regulates their stability and translation  selection.We were concerned about the inconsistency between our and previous analyses about the expression of some H2B variants  in most eukaryotes. Because of their critical genome-wide functions, many RC histones evolve under extreme evolutionary constraint, permitting only limited changes in protein sequence even over nearly a billion years of divergence between protists and humans. In contrast, histone variants can acquire changes that enable them to elaborate new functions, contributing to the eukaryotic nucleosome’s remarkable structural and functional plasticity. Understanding the evolutionary history of histones can reveal how biological challenges faced by different organisms have been resolved by chromatin innovation.H2B.O, H2B.K, H2B.N). Two H2B variants are only found in a small subset of mammals—H2B.E in Muridae and H2B.O in platypus—whereas five variants  are found more extensively across eutherian mammals. We find that one of the newly discovered variants, H2B.K, arose prior to the origin of bony vertebrates. Given its age, slow divergence, and widespread retention, it is somewhat surprising that H2B.K has escaped detection until now. We attribute this to the difficulty of correctly classifying histone variants when interrogating single genomes . The atypical absence of H2B.K (and H2B.N) from the mouse genome, where the most extensive characterization of variant histones has been carried out, likely exacerbated this difficulty  are also detected in human embryos, suggesting the possibility of embryonic functions, which could be elucidated by future in vivo analyses.In contrast to spermatogenesis, oogenesis does not appear to involve dramatic chromatin changes analogous to the protamine transition. Yet maternal inheritance of certain histone variants is essential for embryonic viability and development . During The newly identified H2B variants also present some novel features that have not been previously observed in histones. Although H2B.K resembles RC H2B in its HFD, its highly diverged N-terminal tail includes a polyglutamine repeat and overall lower charge, suggesting it may confer different functionality and looser chromatin packing when incorporated into nucleosomes. Its near-ubiquitous presence across vertebrate genomes and strong sequence conservation motivates future functional studies. In contrast to H2B.K, H2B.N is dramatically different from RC H2B. For example, most H2B.N proteins have a significant C-terminal truncation that removes the αC domain, eliminating the important nucleosome acidic patch that mediates many other chromatin interactions . This feOur analyses not only reveal chromatin innovation in mammalian germlines but may also provide important clues for chromatin aberrations that can arise in cancer cells. For example, misexpression of other germline-specific histone variants and mutations in RC H2B can be detected in cancer cells . Recent Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), guinea pig (Cavia porcellus), rabbit (Oryctolagus cuniculus), pig (Sus scrofa), sheep (Ovis aries), cow (Bos taurus), horse , white rhinoceros (Ceratotherium simum), cat (Felis catus), dog (Canis lupus familiaris), panda (Ailuropoda melanoleuca), elephant (Loxodonta africana), armadillo (Dasypus novemcinctus), opossum (Monodelphis domestica), Tasmanian devil (Sarcophilus harrisii), and platypus (Ornithorhynchus anatinus), as well as a nonmammalian outgroup species, chicken     online.  gallus) , 1997 onllus)   online allus)   online.H2B.E, we performed a TBlastN search for H2B.E in rodent and lagomorph genomes—western wild mouse (Mus spretus), ryukyu mouse (Mus caroli), shrew mouse (Mus pahari), wood mouse (Apodemus sylvaticus), deer mouse (Peromyscus manuculatus), short-tailed field vole (Microtus agrestis), prarie vole (Microtus ochrogaster), golden hamster (Mesocricetus auratus), chinese hamster (Cricetulus griseus), jerboa (Jaculus jaculus), kangaroo rat (Dipodomys ordii), brazilian guinea pig (Cavia aperea), squirrel (Ictidomys tridecemlineatus), alpine marmot (Marmota marmota), and pika (Ochotona princeps) (see To determine the age of eps) see  online.H2B.K, we performed a TBlastN search for H2B.K in nonmammalian species—zebra finch (Taeniopygia guttata), western clawed frog , coelacanth , zebrafish (Danio rerio), elephant shark , lamprey (Petromyzon marinus), sea urchin (Strongylocentrotus purpuratus), and fruit fly (Drosophila melanogaster) (see To determine the age of ter) see  online.H2B variant orthologs in 29 primates were identified using TBlastN analyses of NCBI’s nonredundant nucleotide collection (nr/nt) and whole-genome shotgun contigs (wgs) databases see  online.H2B.K could not be identified in Tasmanian devil, likely because the syntenic region is split between two scaffolds: the intervening sequence may be missing from the assembly. Histones or flanking genes located on scaffolds labeled with Chr_UN were not included in our analyses.Once histone variants were identified, we used shared synteny (conserved genetic neighborhood) to identify putative orthologs in all representative mammalian genomes. We retrieved nucleotide sequences for all hits and their genomic neighborhoods, and recorded coordinates for syntenic analyses using the UCSC Genome Browser . For synH2B.W, H2B.K, and H2B.N were annotated based either on protein alignments with closely related species or on Ensembl, RefSeq, or GenScan predictions. Since the N- and C-terminal residues of H2B.W orthologs show high divergence in mammals, our current annotations in nonprimate species may need to be revised with further experimental evidence. Pseudogenes were annotated based on disrupted ORFs or the presence of gene remnants as determined by a TBlastN search of the histone variant sequence against its syntenic regions (as in the case of H2B.W and H2B.1). In three cases, H2B variant copies were also found on the same chromosome immediately outside the syntenic location—one cow and sheep H2B.W variant and a horse H2B.1 pseudogene. These are not annotated as other loci in Since RC H2Bs are present in numerous identical copies in mammalian genomes, we only used one copy of any identical RC H2Bs in our analyses. We used a copy of RC H2B that is present in six copies in the human genome (H2Bc4/H2Bc6/H2Bc7/H2Bc8/H2Bc10) and three copies in the mouse genome. Exons and introns in variants H2B.E, H2B.L, H2B.1, H2B.W, H2B.K, and H2B.N)  to classify ORFs and pseudogenes into H2B variant families (B.N) see  online.https://www.geneious.com) and all phylogenies were generated using maximum-likelihood methods in PhyML   .We analyzed selective pressures on H2B variants in diverse mammals or in simian primates using the codeml algorithm from the PAML suite  supplem online. N/dS value of 1  and model 0 with dN/dS estimated from the alignment. We determined statistical significance by comparing twice the difference in log-likelihoods between the two models with a χ2 distribution with 1 degree of freedom  and either model 7  or model 8a (where the eleventh class has dN/dS fixed at 1). We determined statistically significance by comparing twice the difference in log-likelihoods between the models (M7 vs. M8 or M8 vs. M8a) to a χ2 distribution with the degrees of freedom reflecting the difference in number of parameters between the models being compared , mouse lemur (Microcebus murinus), and rhesus macaque (Macaca mulatta). These species were selected because they possess at least one intact copy of every H2B variant. We calculated a two-way JSD metric .We used Phyre2  to consthttps://www.ncbi.nlm.nih.gov/books/NBK158900), and mapped reads to same-species genome assemblies using the STAR mapper  to divide those counts by the total number of mapped reads in each sample in millions, followed by the size of each transcript in kb to obtain RPKM values. A previously published housekeeping gene, human C1orf43  signals, we first extracted 600 bp of genomic sequence on each side of the stop codon of each H2B variant. We downloaded a model for the histone 3′-UTR stem loop (accession no. RF00032) from the RFAM database  searchedMolecular Biology and Evolution online.msac019_Supplementary_DataClick here for additional data file.


Osmr knockdown and Lifr overexpression attenuated the OSM-mediated effect on proliferation in both cell lines indicating that mOSM affected the proliferation signaling mainly through the OSMR. Furthermore, mOSM induced activation of the JAK-STAT, PI3K-AKT, and MAPK-ERK pathways in OP9 and NIH/3T3 cells with differences in total protein levels between the two cell lines. Our findings offer new insights into the regulation of proliferation by mOSM.The IL-6 family cytokine Oncostatin M (OSM) is involved in cell development, growth, hematopoiesis, inflammation, and cancer. Intriguingly, OSM has proliferative and antiproliferative effects depending on the target cell. The molecular mechanisms underlying these opposing effects are not fully understood. Previously, we found OSM upregulation in different myeloproliferative syndromes. However, OSM receptor (OSMR) expression was detected on stromal cells but not the malignant cells themselves. In the present study, we, therefore, investigated the effect of murine OSM (mOSM) on proliferation in stromal and fibroblast cell lines. We found that mOSM impairs the proliferation of bone marrow (BM) stromal cells, whereas fibroblasts responded to mOSM with increased proliferation. When we set out to reveal the mechanisms underlying these opposing effects, we detected increased expression of the OSM receptors OSMR and LIFR in stromal cells. Interestingly,  The IL-6 family cytokine Oncostatin M (OSM) was first described as a factor released by U937 lymphoma cells with the ability to inhibit the growth of the human melanoma cells A375 in vitro . NumerouPhysiologically, OSM is released by activated leukocytes including macrophages ,14, monoGenerally, the cellular responses and downstream pathway activation after OSM binding to its receptors are highly dependent on the target cell type. Activation of type I and II receptors by OSM induces downstream pathways such as JAK-STAT, MAPK-ERK, and PI3K-AKT ,34,35. MAlthough OSM was initially described as an antiproliferative cytokine inhibiting the growth of melanoma, breast cancer, chondrosarcoma, lung adenocarcinoma, and glioblastoma cells ,41,42,43In previous studies, OSM was found to be upregulated in different myeloproliferative syndromes ,51. Its In the present study, we, therefore, investigated the molecular mechanisms leading to the opposing effects of OSM on proliferation in murine bone marrow stromal cells and fibroblasts.Based on its upregulation in various cancer entities ,11,12,13It has previously been demonstrated that mOSM promotes the proliferation of NIH/3T3 fibroblasts . We confLifr mRNA expression in OP9 cells compared to NIH/3T3 cells in microarray analyses, whereas Osmr mRNA expression was not altered between both cell lines  surface expression was not possible despite using different flow cytometry antibodies supposed to bind mOSMR .We next investigated whether mOSM treatment affects the expression of the OSMR and LIFR in OP9 and NIH/3T3 cells. We detected an initial reduction of total cellular OSMR in both cell lines one hour after mOSM treatment, followed by a return to basal levels after 8 h of mOSM exposure. For the LIFR, a similar regulation pattern expression in OP9 cells was observed with initial downregulation followed by upregulation. In contrast, NIH/3T3 cells retained low-level expression of the LIFR during mOSM treatment D. These Osmr or Lifr in both OP9 and NIH/3T3 cell lines. Knockdown efficacy of four Osmr-specific shRNAs and five shRNAs targeting the Lifr were evaluated  and p53 in our cell lines. Interestingly, RB was phosphorylated in NIH/3T3 cells and dephosphorylated in OP9 cells upon mOSM treatment, consistent with the mOSM effect on proliferation. However, total RB levels were not altered between OP9 and NIH/3T3 cells before or after OSM treatment B indicatTo identify gene and protein classes up- or downregulated upon mOSM treatment we performed functional enrichment analysis of the global transcriptomes of OP9 and NIH/3T3 cells in the presence or absence of mOSM. OP9 cells showed gene set enrichment according to the antiproliferative effect of mOSM: “apoptotic signaling pathway” was positively enriched in response to mOSM, whereas “response to growth factors” and different developmental processes were negatively enriched . In contWe next analyzed mOSM-induced cytokine secretion in OP9 and NIH/3T3 cells, which might affect proliferation in an autocrine manner. For this purpose, supernatants of OP9 cells (+/− mOSM treatment) were added to NIH/3T3 cells and vice versa for 24 h, and cell cycle analysis was performed. OP9 cells treated with the supernatant of mOSM-stimulated NIH/3T3 cells showed impaired proliferative capacity indicated by lower S phase percentage. Conversely, NIH/3T3 cells treated with the supernatant of mOSM stimulated OP9 cells showed increased proliferation A, similap < 0.0001). NIH/3T3 cells also showed increased production of IL-6 upon mOSM treatment (13.9 pg/mL vs. not detectable), however significantly less than OP9 cells , albeit to a lesser extent than mOSM (2.7-fold). The proliferation of OP9 cells was not affected by mIL-6 C. IntereIn the present study, we investigated the OSMR dependent regulation of proliferation in OP9 and NIH/3T3 cells by mOSM. In line with previous reports ,61, we uThe contribution of OSM in tumorigenesis has been controversial: Depending on the tumor entity, OSM was shown to promote tumor progression and epithelial-mesenchymal transition ,46,47,62Lifr overexpression was associated with attenuated effects on proliferation by mOSM in both cell lines, indicating that Lifr overexpression might act to decrease levels of free mOSM for binding to the OSMR leading to reduced effects on proliferation. In support of this hypothesis, general receptor-ligand studies showed that increased receptor surface density was associated with increased ligand occupancy [Lifr knockdown attenuated the OSM-mediated response on proliferation in NIH/3T3 but not in OP9 cells. We assume that Lifr knockdown might have been more efficient in NIH/3T3 cells due to per se lower LIFR expression. Consequently, Lifr knockdown impaired the mOSM effect on proliferation in NIH/3T3 cells. In contrast, OP9 cells showed higher LIFR expression compared to NIH/3T3, therefore Lifr knockdown possibly did not sufficiently impair LIFR expression to detect altered mOSM effects on proliferation. Whereas our study revealed only marginal effects of mOSM-LIFR interaction on proliferation, recent studies identified the LIFR to be important in mOSM-regulated bone metabolism [Our study described an inferior role for the LIFR in the regulation of cellular proliferation by mOSM. ccupancy . Furthertabolism ,31. Thertabolism  and mighAs previous research showed that huOSM affects the proliferation of chondrosarcoma and lung adenocarcinoma cells in a JAK-dependent manner ,56, we aIn NIH/3T3 cells, ruxolitinib reduced OSM-induced proliferation only partly, indicating the involvement of additional mechanisms. These might include an altered abundance of signaling proteins, as we detected increased levels of total TYK2 and ERK1/2 in NIH/3T3 cells, whereas OP9 cells contained higher levels of total JAK2 und STAT5 proteins.Stat1 mRNA levels in OP9 and increased expression of Stat3 and Stat5 in NIH/3T3 cells. Active STAT3 and STAT5 signaling are known to increase proliferation in different cellular contexts [Downstream of JAK activation, we found phosphorylation of STAT1, 3, and 5 in both cell lines in response to mOSM treatment. Interestingly, transcriptome analysis revealed upregulation of contexts ,69,70,71contexts ,73,74. Icontexts , whereascontexts . Anothercontexts . Based ocontexts ,76.In our study, ERK expression was found to be lower in OP9 compared to NIH/3T3 cells. Interestingly, mOSM induced downregulation of ERK expression in NIH/3T3 whereas OP9 cells retained constant ERK levels. This supports previous research showing OSM-mediated downregulation of ERK expression that correlated with increased proliferation .Transcriptome analysis of OP9 and NIH/3T3 cells highlighted enrichment of specific gene sets corresponding to the antiproliferative and proliferative effects of mOSM. This result was corroborated by differential RB activation in OP9 versus NIH/3T3 cells. Furthermore, another study showed downregulation of the cyclin D1/CDK4 complex and reduced pRB levels upon OSM stimulation inducing growth arrest in mouse skeletal muscle cells .Future studies should further investigate the differences in signaling proteins using specific inhibitors and knockdown experiments. Moreover, OSM was shown to activate c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase  and protWe detected mOSM-induced IL-6 secretion in OP9 and NIH/3T3 cells, which is consistent with previous reports showing IL-6 release in response to OSM in different contexts ,60. InteOf note, findings from studies on mOSM might not be transferred completely to the structural different huOSM. In contrast to mOSM, huOSM was shown to bind to the type I and type II OSM-receptors with high affinity ,27. WhilTaken together, our study has demonstrated that both proliferative and antiproliferative effects of mOSM were mediated mainly through the OSMR. Based on our findings, mOSM seems to bind the LIFR with marginal but synergistic (compared to the OSMR) effects on proliferation. Furthermore, mOSM induces activation of the JAK-STAT, PI3K-AKT, and MAPK-ERK pathways in OP9 and NIH/3T3 cells with differences in total protein levels between the two cells lines. RB activation and gene set enrichment clusters correspond with the proliferative or antiproliferative response to mOSM.Due to its multifaceted activities in various cancer entities, OSM is currently considered a novel therapeutic target, and a number of targeting antibodies and other compounds have been developed in preclinical research . Our finBa/F3 cells and NIH/3T3 cells were obtained from the German Resource Centre for Biological Material (DSMZ). Phoenix E helper-virus free ecotropic packaging cells were a kind gift from G. Nolan, Stanford, USA. OP9 and M2-10B4 cells were a kind gift from Christine Dierks, University Hospital Freiburg, Germany. EL08-1D2 cells were kindly provided by R. A. Oostendorp, Munich, Germany. Primary murine stromal cells were isolated from C57BL/6 mice. Primary human stromal cells from healthy donors have been obtained from the stem cell transplant department of the University of Freiburg, Germany.2, 5% CO2.Ba/F3 cells were maintained in RPMI 1640 medium  containing 10% fetal calf serum , 1% Penicillin-Streptomycin , and 2 ng/mL murine interleukin-3 . Phoenix E and NIH/3T3 cells were maintained in DMEM (Thermo Fisher) supplemented with 10% FCS. NIH/3T3 cells were cultivated in presence of 1% PS. OP9 cells and M2-10B4 cells were maintained in alpha-MEM (Thermo Fisher) supplemented with 10% FCS and 1% PS. EL08-1D2 cells were maintained in alpha-MEM supplemented with 7.5% FCS, 2.5% horse serum (Thermo Fisher), 1% PS und 200 µL ß-Mercaptoethanol . All cells were cultured at 37 °C with 95% OMurine recombinant OSM was purchased from Cell Signaling . Human recombinant OSM and murine recombinant IL-6 were purchased from Peprotech. Cytokines were used at a final concentration of 10 ng/mL. Ruxolitinib was purchased from ChemieTek, Indianapolis, IN, USA, the neutralizing anti-IL-6 antibody (MM600C) from Thermo Fisher.Phoenix E cells were transiently transfected using Turbofect (Thermo Fisher) and retroviral stocks were collected twice at 12-h intervals beginning 24 h after transfection. OP9 and NIH/3T3 cells were stably transduced with retroviral supernatant supplemented with 4 µg/mL polybrene  for 3 times every 12 h.® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit (Thermo Fisher) according to the manufacturer’s instruction. DNA content was quantified using FxCycle™ Violet Stain (Thermo Fisher) according to the manufacturer’s instruction. EdU was used at a concentration of 20 µM.Proliferation was assessed using Click-iTCells were treated for 24 h with mOSM or huOSM, respectively. EdU was added during the last hour of treatment or the last 12 h of treatment for primary stromal cells. For experiments comparing OP9 and NIH/3T3 cells, cells were serum-starved with 1% FCS for six hours before and during treatment. Ruxolitinib was added at the same time as mOSM for proliferation assays.Lifr cDNA (MR221059) was obtained from OriGene . The retroviral vector was constructed by cloning the Lifr cDNA into the MigRI retroviral vector coexpressing the enhanced green fluorescent protein  [Lifr overexpression. RIEP (rtTA-IRES-EcoReceptor-PGK-PuroR), TREBAV (TRE-dsRed-miRE-PGK-BSDr-2A-Venus), and pSuper plasmids were kindly provided by C. Miething, Freiburg, Germany. For doxycycline-inducible knockdown of Lifr or Osmr respectively, OP9 and NIH/3T3 cells were stably transduced first with RIEP and subsequently with TREBAV vectors containing the appropriate shRNA. All cell lines including control cell lines were cultured in presence of 1 µg/mL doxycycline. Doxycycline was added at least 48 h before starting experiments. The following shRNA sequences were used for the knockdown experiments: Lifr 5‘ TGCTGTTGACAGTGAGCGCTACCATGTTGCTGTAGACAAATAGTGAAGCCACAGATGTATTTGTCTACAGCAACATGGTAATGCCTACTGCCTCGGA3‘ (here named: Lifr 1), Osmr 5′ TGCTGTTGACAGTGAGCGCAAGCATCTTCTTGTAAACTTATAGTGAAGCCACAGATGTATAAGTTTACAAGAAGATGCTTTTGCCTACTGCCTCGGA3‘ (here named: Osmr 5) and Renilla (control) 5′ CGCGACTCCTATAATTTCTAATTAGTGAAGCCACAGATGTAATTAGAAATTATAGGAGTCGCT3′. Further shRNA sequences are described in Murine wildtype PA, USA) . Empty MTM  cytometer and FlowJo 10.4 was used to analyze the data.Flow cytometry was performed using an LSRFortessaTM (Becton-Dickinson) as described before [Antibodies used were: LIFRalpha  and pSTAT3 . Cells were sorted on an Aria IIId before ,83. AntiTM X-100, 20 mM sodium phosphate (pH 7.5), 10 mM sodium pyrophosphate (pH 7.0), 50 mM NaF, 1 mM sodium orthovanadate, 1 mM glycerolphosphate, and protease inhibitors . Blotting was performed on polyvinylidene fluoride (PVDF) membranes . Antibodies against pAKT (cs9271), AKT (cs9272), pERK1/2 (cs9101), ERK1/2 (cs4695), pJAK2 (cs4406), JAK2 (cs3230), pJAK3 (cs5031), JAK3 (cs8863), pRB (cs8516), pSTAT1 (cs7649), pSTAT3 (cs9131), STAT3 (cs9132), pSTAT5 (cs9314), STAT5 (cs94205) and pTYK2 (cs9321) were purchased from Cell Signaling. TYK2 (sc5271), LIFR (sc515337), OSMR (sc376511), RB (sc50) and Vinculin (sc73614) were from Santa Cruz, Dallas, TX, USA. pJAK1 (44-422G) was from Thermo Fisher. STAT1 (21120) was from Becton Dickinson. ß-actin (A5316) was from Sigma. Vinculin and ß-actin were used as loading controls. Secondary antibodies used were mouse IgG HRP-linked (cs7076) and rabbit IgG HRP-linked (cs7074) from Cell Signaling. Blots were developed using SuperSignalTM chemoluminescent substrates (Thermo Fisher). Quantification of Western Blots was done using LabImage 1D Sofware .Immunoblot analysis was performed as previously described . Cells wTM  cytometer.Cytokine secretion was measured using LEGENDplex™ Mouse Inflammation Panel (Biolegend) according to the manufacturer’s instruction. The cytokine array was performed using an LSRFortessap-values were calculated by Transcriptome Analysis Console (Affymetrix) software. Data were analyzed using Metascape analysis  [p < 0.05 in control cells compared to mOSM treated cells. Microarray data have been deposited to the Gene Expression Omnibus (GEO) and will be accessible through GEO Series accession number GSE185646.RNA was isolated with the RNeasy Mini Kit (Qiagen) and transcribed into cDNA with the First Strand cDNA Synthesis Kit (Thermo Fisher). RNA quality was assayed using an Agilent 4200 TapeStation . Mouse Clariom S Assay (Thermo Fisher) was used to analyze mRNA expression according to the manufacturer’s instructions. The er 2021)  with genp-Values were determined by Student’s t-test and one-way ANOVA with Bonferroni post-test for comparison of more than two groups. Data are represented as means ± SEM.Statistical analysis was performed using GraphPad Prism software.


N-docosahexaenoylethanolamine (synaptamide) mediating the anti-neuroinflammatory function of synaptamide. In this study, we evaluated the effect of an endogenous and a synthetic ligand of GPR110, synaptamide and -N-(2-hydroxy-2-methylpropyl) docosa-4,7,10,13,16,19-hexaenamide , on the mTBI-induced long-term optic tract histopathology and visual dysfunction using Closed-Head Impact Model of Engineered Rotational Acceleration (CHIMERA), a clinically relevant model of mTBI.Repetitive mild traumatic brain injury (mTBI) can result in chronic visual dysfunction. G-protein receptor 110  is the target receptor of in vitro was evaluated by the cyclic adenosine monophosphate (cAMP) production in primary microglia isolated from adult WT or KO mouse brains.The brain injury in wild-type (WT) and GPR110 knockout (KO) mice was induced by CHIMERA applied daily for 3 days, and GPR110 ligands were intraperitoneally injected immediately following each impact. The expression of GPR110 and proinflammatory mediator tumor necrosis factor (TNF) in the brain was measured by using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) in an acute phase. Chronic inflammatory responses in the optic tract and visual dysfunction were assessed by immunostaining for Iba-1 and GFAP and visual evoked potential (VEP), respectively. The effect of GPR110 ligands CHIMERA injury acutely upregulated the GPR110 and TNF gene level in mouse brain. Repetitive CHIMERA (rCHIMERA) increased the GFAP and Iba-1 immunostaining of glia cells and silver staining of degenerating axons in the optic tract with significant reduction of N1 amplitude of visual evoked potential at up to 3.5 months after injury. Both GPR110 ligands dose- and GPR110-dependently increased cAMP in cultured primary microglia with A8, a ligand with improved stability, being more effective than synaptamide. Intraperitoneal injection of A8 at 1 mg/kg or synaptamide at 5 mg/kg significantly reduced the acute expression of TNF mRNA in the brain and ameliorated chronic optic tract microgliosis, astrogliosis, and axonal degeneration as well as visual deficit caused by injury in WT but not in GPR110 KO mice.Our data demonstrate that ligand-induced activation of the GPR110/cAMP system upregulated after injury ameliorates the long-term optic tract histopathology and visual impairment caused by rCHIMERA. Based on the anti-inflammatory nature of GPR110 activation, we suggest that GPR110 ligands may have therapeutic potential for chronic visual dysfunction associated with mTBI.The online version contains supplementary material available at 10.1186/s12974-021-02195-y. The white matter of brain, mainly composed of myelinated axons that relay a coordinating communication of grey matter of brain, is known to be susceptible to the impact of the acceleration/deceleration forces –3. The oN-docosahexaenoylethanolamin) is a metabolite of docosahexaenoic acid, an omega-3 fatty acid enriched in the brain [Synaptamide  with free access to standard food and water under a 12-h light–dark cycle. GPR110 heterozygous mice on C57BL/6J genetic background were generated by the Knockout Mouse Project (KOMP) Repository (MMRRC_046507-UCD). Heterozygous GPR110 male and female mice were bred to produce GPR110 knockout offspring. The WT littermates were used as controls. Adult male and female mice at an age of 2–4 months were used for all experiments. GPR110 KO mice developed normally and showed no visible abnormal phototype at this age. All animal experiments were conducted according to the NIH guidelines for the health and use of laboratory animals (LMS-HK-13).N,N-dimethylacetamide : Solutol H15 : 1 × PBS (1:1:2). The investigator who performed the experiment was blinded with respect to the compound identity for treatments until all data analyses were completed.The mild closed traumatic brain injury was induced by rCHIMERA (rCHI) as previously described . Briefly-ΔΔCt value.Mice were anesthetized with isoflurane and transcardially perfused with 0.1 M phosphate buffer (pH 7.4). The cerebral cortex was rapidly dissected, and total RNA was extracted by Trizol reagent , reverse transcribed to cDNA with High Capacity cDNA Reverse Transcription kit . Quantitative RT-PCR was performed using TaqMan™ probes (Thermo Fisher Scientific) run on the QuantStudio 3 Real-Time PCR system (Applied Biosystems by Thermo Fisher Scientific). cDNA samples were reacted with the TaqMan™ probe for GPR110  or TNF  using TaqMan™ Fast Advanced Master Mix  in the presence of the TaqMan™ probe for HPRT (hypoxanthine guanine phosphoribosyl transferase)  which was used as an internal control. Data were analyzed using comparative Ct method. The relative mRNA level of target gene normalization to HPRT was calculated as 2The animals were deeply anesthetized with isoflurane and transcardially perfused with 0.1 M phosphate buffer (pH 7.4) and fixed with 4% paraformaldehyde. The brains were carefully removed and post-fixed in 4% paraformaldehyde solution for overnight at 4 °C and subsequently transferred into 30% sucrose solution at 4 °C. The brains were embedded with O.C.T. compound medium  and frozen on dry ice and stored at – 80 °C. Coronal sections (25 μm thickness) were sliced by Leica Cryostat  and stored in cryoprotective solution at – 20 °C. Three sections per mouse (approximately bregma 1.68 to − 2.2 mm) were selected for staining.2) using Metamorph software .The immunofluorescence double staining for Iba-1 and GFAP was conducted according to the procedure previously described . The secThe silver staining was performed using FD NeuroSilver™ Kit  according to the manufacturer's protocol. The 20×-magnified images of the optic tract were acquired in bright field with an Olympus 1 × 81 microscopic system. The staining intensity of images was quantified using Image J .2 repeatedly with each set including 100 sweeps. Three sets of readings were recorded and averaged to obtain the amplitude and latency (implicit time) of the N1 component (the first negative peak: P1–N1) as described earlier [The visual evoked potential (VEP) and electroretinogram (ERG) were recorded with the Epsion Visual Electrophysiology System . All procedures were performed under dim red light after the mice were acclimated in the room at least for 1 h. Mice were anesthetized with an intraperitoneal injection of ketamine and xylazine cocktail solution (100 mg/kg ketamine + 10 mg/kg xylazine). Each pupil of the mouse was dilated with a drop of 2.5% phenylephrine hydrochloride solution before being placed on a heated platform of color dome. A reference electrode was placed in the lower lip of the mouse and a ground electrode placed on the tail. For VEP measurements, the active electrode was subcutaneously inserted in the middle of the two ears. Both eyes were stimulated by the flash stimuli of white light (6500 K) at an intensity of 3.0 cd s/m earlier . For ERG earlier . After t4-anandamide and d6-A8 was added to 300 μL of homogenate, which was then brought to BHT-methanol/water (7:3) and centrifuged for 20 min at 4 °C. Supernatants were loaded onto a Strata-X polymeric C18 reverse-phase SPE cartridge  that was wetted with BHT-methanol and equilibrated with water. After washing with water, samples were eluted with 2.5 mL BHT-methanol into glass tubes, dried under N2, and resuspended in a small volume of BHT-methanol. Separation was made on an Eclipse C18 HPLC column  using a tertiary gradient consisting of water (A), methanol (B), and acetonitrile  (C), with all solvents containing 0.01% acetic acid (Thermo Scientific). After pre-equilibration of column with A/B (60%/40%), 5 μL extract was injected, and the solvent composition was linearly changed to A/B/C (36.3%/15%/48.7%) in 5 min, followed by a linear gradient to A/B/C (13.5%/68.4%/18.1%) over 22 min. The mass transitions of 376.3 to 66.085, 400.3 to 72.081, 352.3 to 66.085, and 406.4 to 78.118 were used to detect d4-synaptamide, analog 8, d4-anandamide, and d6-A8, respectively. Quantitation of d4-synaptamide and A8 was made using d4-anandamide and d6-A8 as the respective internal standard. Results were normalized to protein amount and presented as fmol per microgram protein.Synaptamide and A8 were analyzed by reversed phase liquid chromatography coupled to high-resolution tandem mass spectrometry using a Thermo Scientific Q-Exactive mass spectrometer. Mice were intraperitoneally injected with a mixture of d4-synaptamide and A8. At 1, 2, and 24 h after injection, mice were perfused with 1× PBS, and the cerebral cortex was collected and homogenized in 500 μL of water/methanol (1:1) mixture containing 2 μM URB597 (a fatty acid amide hydrolase inhibitor) and 50 μg/mL butyl hydroxytoluene (BHT) . Protein concentration of the homogenate was determined by the bicinchoninic acid protein assay kit . A mixture of deuterated internal standards of dThe brain tissue from GPR110-WT or KO mice at 4 months of age were dissociated into single cells using the Adult Brain Dissociation Kit  according to the manufacturer’s instruction. To isolate the microglia, the single-cell suspension was incubated with CD11b Microbeads for 30 min at 4 °C. The cells were resuspended in MACS buffer and passed through the LS column (Miltenyi Biotech). The column was washed 4 times with MACS buffer and then magnetically labeled CD11b-positive cells were flushed out of the columns twice using MACS buffer. Enriched CD11b-positive microglia cells were pelleted by centrifuging at 300 × g at 4 °C for 10 min and resuspended in DMEM media supplemented with 10% fetal bovine serum (FBS). Isolated CD11b-positive microglia cells were cultured in poly-D-lysin coated 384-well white/clear bottom plate for 7 days by changing 50% of DMEM media supplemented with 10% FBS every other day. On the day of the cAMP assay, media was changed to the stimulation media consisting of phenol red-free DMEM supplemented with 0.5 mM IBMX , a phosphodiesterase inhibitor. Microglia cells were treated with different concentrations of synaptamide or A8 complexed with 0.05% fatty acid-free BSA (Sigma-Aldrich) in the presence of 40 μM vitamin E for 10 min. Separately, microglia cells were treated with 10 μM Forskolin  for 10 min. The cAMP production was measured by a homogenous time-resolved fluorescence assay with a cAMP Gs Dynamic Kit  using the FlexStaion 3 device (Molecular devices)p < 0.05 was considered statistically significant.The data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism  for one-way or two-way analysis of variance (ANOVA) followed by the Tukey’s post hoc test for multiple comparisons, and p < 0.0001, n = 3-4/group, Fig. gpr110 expression responds quickly to the brain injury.To investigate the possible role of GPR110 activation in improving TBI outcome, we first examined the GPR110 mRNA expression in adult mouse brain using quantitative RT-PCR at 1 and 24 h after single or multiple CHIMERA given daily for three consecutive days. Both single and multiple CHIMERA significantly upregulated GPR110 gene level at 1 and 24 h after injury compared to the sham group -N-(2-hydroxy-2-methylpropyl)docosa-4,7,10,13,16,19-hexaenamide  . Intraperitoneal administration of A8 or synaptamide immediately following each CHIMERA dose-dependently suppressed the TNF expression. Compared with the vehicle-treated injured group (rCHI + V), synaptamide at 5 mg/kg or A8 at 1 mg/kg significantly suppressed TNF expression . Synaptamide at 2 mg/kg or A8 at 0.1 and 0.5 mg/kg also showed the trend to reduce the TNF mRNA expression although the reduction was not statistically significant. The time–course of synaptamide and A8 in the brain was examined by injecting d4-synaptamide at 5 mg/kg and A8 at 1 mg/kg  than synaptamide , even though the injected dose of A8 was 5-fold less compared with synaptamide. Both compounds were no longer detectable in the brain cortex after 24 h, indicating rapid metabolism and clearance of these compounds. Considering these results, we selected 5 mg/kg of synaptamide and 1 mg/kg of A8 for treatment for in vivo experiments.To determine the proper dose of GPR110 ligands for p < 0.0001 vs. rCHI + V) and GFAP expression in OT  but N-oleoylethanolamine (OEA), an inactive lipid control, showed no effect, indicating that the suppression of glia cell activation was a specific effect of synaptamide . Treating WT injured mice with synaptamide or A8 significantly reduced both Iba-1 and GFAP expression ; however, this effect was not observed in GPR110 KO injured mice  and GFAP expression  in the OT of injured WT mice; however, injured GPR110 KO mice did not respond to the treatments . The silver staining intensity of OT from the injured mice treated with synaptamide or A8 was significantly reduced compared with the injured group treated with vehicle only . However, no such effect was observed in GPR110 KO mice after treatment with synaptamide or A8 . The treatment with the GPR110 ligand A8 ameliorated this reduction in WT (p < 0.05 vs. rCHI + V) but not in GPR110 KO mice  at 3 months post injury  or A8  compared to the vehicle treatment after injury (21.88 ± 3.37 μV) in WT but not in GPR110 KO mice  along with the electroretinogram (ERG). At 2 weeks after injury, rCHIMERA significantly decreased N1 amplitude of VEP compared with the Sham group regardless of the genotype . In some cancer conditions such as prostate, liver, and breast cancer, upregulated expression of GPR110 has been reported –30. Signin vivo and in cultured microglia where GPR110 is expressed [G protein-coupled receptors (GPCRs) have been the targets for potential therapeutic agents in diverse fields , 32. Manxpressed , 17. Recxpressed , 36. In xpressed . AlthougNeuroinflammation is an important mechanism underlying the pathogenesis of traumatic brain injury , 38, whiin vivo stability of A8. Enhanced in vivo stability and biological effectiveness of A8 suggest that A8 may have better translational potential than synaptamide.A8, a chemical analogue of synaptamide with improved stability, has been recently described as a better ligand to GPR110 compared with the endogenous ligand synaptamide . Indeed,Persistent gliosis caused by repetitive mild TBI is often associated with a functional deficit in animals and humans , 40–42. Our results demonstrate that synaptamide and A8 attenuate optic tract histopathology and visual impairment caused by rCHIMERA by activating the GPR110/cAMP system that is upregulated after injury. This study provides new insight for the translational potential of targeting GPR110 using its ligands for improving the chronic outcome after repeated mild TBI.Additional file 1: Figure S1. Synaptamide-specific suppression of glia cell activation in optic tract (OT) at one day post-rCHIMERA. A. Representative micrographic images of Iba-1 and GFAP immunofluorescence. WT mice were injected with synaptamide or oleoylethanolamine (OEA) at 5 mg/kg (i.p.) after each CHIMERA, and brains were collected for immunostaining at 1 day after the last injury. B, C. Quantitative analysis of Iba-1 (B) and GFAP expression (B), showing that synaptamide suppressed Iba-1 and GFAP expression induced by rCHIMEA while oleoylethanolamine (OEA) had no effect. The synaptamide treatment without injury did not affect the GFAP and Iba-1 expression in the brain. The data are expressed as mean ± SEM (n=3). *p<0.05, **p<0.01, ****p<0.001 vs. Sham. Figure S2. GPR110-dependent inhibition of glia cell activation in corpus callosum by synaptamide and A8 at 7 days after injury. A, C. Representative micrographic images of Iba-1 (A) and GFAP (C) immunofluorescence in the corpus callosum (CC) from WT and GPR110 KO mice at 1 week after injury (rCHI). WT and GPR110 KO mice were injected with synaptamide  or A8  after each CHIMERA, and brains were collected for immunostaining at 7 days after the last injury. B, D. Quantitative analysis showing significant suppression of Iba-1 (B) and GFAP expression (D) by the treatment with synaptamide or A8 compared to the vehicle-treated group (rCHI + V) in WT but not in GPR110 KO mice after rCHIMERA. The corpus callosum region (CC) is outlined with dashed lines. The data are expressed as mean ± SEM (n=3). *p<0.05, **p<0.01, ***p<0.001 vs. Sham-WT. Figure S3. Increases in N1 amplitude of VEP by A8 at 2 weeks after rCHIMERA. A: Average traces of VEP evaluated at 2 weeks post injury. Full-field flash VEP was elicited at a constant intensity of 3.0 cd·s/m2 with the active electrode subcutaneously inserted in the middle of the two ears. B, Quantitative analysis of the N1 amplitude and latency showing that A8 increased the N1 amplitude in WT but not in GPR110 KO injured mice without affecting N1 latency. No significant difference in the N1 amplitude or latency in sham animals was observed between two genotypes. The data are expressed as mean ± SEM (n=8-10). Each dot symbol represents each animal per group. ***p<0.001, ****p<0.0001 vs. Sham-WT. C. Average tracts of ERG at 2 weeks post injury obtained using a light-adapted (photopic) protocol. D, E. Quantitative analysis of a and b amplitude and latency showing that A8 or injury did not change a and b amplitude and latency for both WT and GPR110 KO mice. No significant difference in these ERG parameters was observed between two genotypes.