CCNB1 and CDC25B promoters. Taken together, our findings support a novel partnering role of EPS8 with FOXM1 in the regulation of cancer cell proliferation and provides interesting insight into future design of therapeutic strategy to inhibit cancer cell proliferation.One hallmark of cancer cells is sustaining proliferative signaling that leads to uncontrolled cell proliferation. Both the Forkhead box (FOX) M1 transcription factor and the Epidermal Growth Factor (EGF) receptor Pathway Substrate 8 (EPS8) are known to be activated by mitogenic signaling and their levels upregulated in cancer. Well-known to regulate Rac-mediated actin remodeling at the cell cortex, EPS8 carries a nuclear localization signal but its possible nuclear role remains unclear. Here, we demonstrated interaction of FOXM1 with EPS8 in yeast two-hybrid and immunoprecipitation assays. Immunostaining revealed co-localization of the two proteins during G2/M phase of the cell cycle. EPS8 became nuclear localized when CRM1/Exportin 1-dependent nuclear export was inhibited by Leptomycin B, and a functional nuclear export signal could be identified within EPS8 using EGFP-tagging and site-directed mutagenesis. Downregulation of EPS8 using shRNAs suppressed expression of FOXM1 and the FOXM1-target CCNB1, and slowed down G2/M transition in cervical cancer cells. Chromatin immunoprecipitation analysis indicated recruitment of EPS8 to the  CCNB1 and CDC25B, as direct targets of FOXM1  8. EPS8 is constitutively tyrosine-phosphorylated in human cancer cell lines and over-expression of EPS8 was able to transform NIH3T3 cells in focus forming assay . EPS8 lein vitro and tumorigenesis in vivo when injected into nude mice. Expression of cyclins and p53 were perturbed with an associated change in cell cycle kinetics although the underlying mechanism remains unclear. Wang et al.  , suggestCDNA library construction and screening for FOXM1-interacting proteins using a LexA-based yeast two-hybrid system were described previously . The MatCCNB1 and CDC25b promoters, Chromatin IP was performed as reported in Kwok et al.  were cultured at 37°C under 5% CO2 in DMEM supplemented with 5.9 g/L HEPES, 3.7 g/L Na2CO3, 292.4 mg/L L-glutamine, 100,000 units/L Penicillin G, 80,000 units/L streptomycin sulfate, and 10% (v/v) fetal bovine serum. Transient transfection was performed on cells seeded on coverslips in wells or plates using FuGENE ® HD reagent (Promega) according to the manufacturer's instructions. For suppression of EPS8 expression by RNA interference, cells were transiently transfected with the various shRNA plasmids (shRNAmirs carrying puromycin marker gene) for 72 h. For positive selection of shRNA-expressing cells, transiently transfected cells were subjected to treatment with 0.5 μg/ml puromycin for an additional 72 h before harvesting for immunoblot analysis.For immunocytochemical analysis, HeLa cells were synchronized at the G1/S boundary by double thymidine block as previously described . BrieflyImmunoblot analysis was conducted as previously described . To studTo gain further insight into the regulatory role of FOXM1, we conducted a yeast two-hybrid screen for FOXM1-interacting proteins using an insulinoma cDNA library . To avoiTo define the interaction domain(s) within EPS8, we made EPS8 constructs deleted of the N-terminus and/or the SH3 domain. Interestingly, deletion of the N-terminal 260 amino acids , but not the SH3 domain (in construct EPS8-ΔSH3), abolished the interaction. To determine the region within EPS8 that is sufficient to mediate interaction with FOXM1, we constructed EPS8 baits of increasing length starting from the N-terminus. As shown in Since FOXM1 is a transcription factor that acts in the nucleus, we studied whether EPS8 showed nuclear localization at the different phases of the cell cycle by synchronizing HeLa cells using double thymidine block. DNA analysis using flow cytometry indicated successful synchronization of HeLa cells at the G1/S boundary . Synchro2,3(F/I/L/V/M)x1,2,3Lx(I/V/L); x = any residue in , Pxx(B3x), B3(H/P); B = basic residue (K or R), x = any residue (CRM1/exportin 1-dependent nuclear export acts via leucine (L)/isoleucine (I)-rich NES in target proteins [Proposed consensus sequences reported as Lxsidue in ]. Sequensidue in . As prevsidue in , a nucle residue . To testCCNB1 and other M phase target genes, and EPS8 was previously shown to activate FOXM1 promoter activity. To test whether EPS8, which could interact and co-localize with FOXM1 in the nucleus, modulates expression of FOXM1 targets and cell cycle progression, we used EPS8-targeting shRNAs to deplete EPS8 in HeLa cells. Compared against the non-specific-targeting shRNA and non-transfected controls, transient transfection of three EPS8-targeting shRNA plasmids (carrying puromycin marker gene) into HeLa resulted in suppression of both EPS8 and FOXM1 expression  was performed using EPS8 and FOXM1 antibodies and the immunoprecipitates analyzed for enrichment of FOXM1 transcripts transcriptionally  cells both in vitro and in vivo  and EPS8  do interact physically and functionally. We argued that EPS8 is a novel partnering factor required for FOXM1 to exert its multiple roles in cancer cell proliferation and migration/invasion, and targeting the FOXM1-EPS8 interaction might provide an alternative anti-cancer strategy.K-MY designed the work, interpreted the data and critically revised the manuscript. AN performed most of the experiments as part of her PhD thesis work. MT and DS also took part in designing and conducting some of the experiments and revised the manuscript. WL made many of the constructs and helped with conducting some of the experiments. DC helped to design the work and advised us on some experiments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vibrio parahaemolyticus and mortality caused by other Vibrio species such as Vibrio harveyi are worldwide related to huge economic losses in industrial shrimp production. As a strategy to prevent disease outbreaks, a plant-based phenolic compound could be used as a biocontrol agent. Here, using the brine shrimp (Artemia franciscana) as a model system, we showed that phloroglucinol treatment of the parental animals at early life stages resulted in transgenerational inherited increased resistance in their progeny against biotic stress, i.e., bacteria (V. parahaemolyticus AHPND strain and V. harveyi) and abiotic stress, i.e., lethal heat shock. Increased resistance was recorded in three subsequent generations. Innate immune-related gene expression profiles and potential epigenetic mechanisms were studied to discover the underlying protective mechanisms. Our results showed that phloroglucinol treatment of the brine shrimp parents significantly (P < 0.05) enhanced the expression of a core set of innate immune genes  in subsequent generations. We also demonstrated that epigenetic mechanisms such as DNA methylation, m6A RNA methylation, and histone acetylation and methylation  might play a role in regulation of gene expression leading toward the observed transgenerational inheritance of the resistant brine shrimp progenies. To our knowledge, this is the first report on transgenerational inheritance of a compound-induced robust protected phenotype in brine shrimp, particularly protected against AHPND caused by V. parahaemolyticus and vibriosis caused by V. harveyi. Results showed that epigenetic reprogramming is likely to play a role in the underlying mechanism.Emerging, infectious diseases in shrimp like acute hepatopancreatic necrosis disease (AHPND) caused by  Vibrio parahaemolyticus infects various shrimp species and causes acute hepatopancreatic necrosis disease (AHPND), initially named early mortality syndrome (EMS). V. harveyi is another pathogen from the Vibrionaceae family that causes substantial mortality by vibriosis and economic loss in shrimp aquaculture  can be maintained under axenic conditions, which allows the control of host-associated microbial communities. Well-established vibrio infection models as well as abiotic stress models are available , resulting in enhanced disease resistance against Vibrio infections . NLHS has been successfully used in parthenogenetic brine shrimp for inducing transgenerational inherited resistance against vibriosis (V. parahaemolyticus AHPND infection in gnotobiotic brine shrimp larvae as well as in freshwater shrimp (Macrobrachium rosenbergii) larvae by inducing endogenous heat shock protein 70 (Hsp70).Exposure to non-lethal heat shock (NLHS) of shrimp vannamei , green m viridis , and brinciscana  induced ibriosis . Thus, eibriosis . Therefoibriosis , 25, 26.ibriosis , 27. Kumibriosis  showed tV. parahaemolyticus and V. harveyi) and abiotic stressors .In this study, using brine shrimp as an animal model, we investigated whether phloroglucinol exposure of the parental generation at early life stages could elicit a disease-resistant phenotype in the animals as well as transgenerational inheritance of the acquired disease-resistant phenotype. Hereto, the brine shrimp parental population (TF0) was exposed to phloroglucinol (2 μM) until day after hatching (DAH) 16. Phloroglucinol exposure of the parental generation could induce and transmit a disease-resistant phenotype in three subsequent unexposed generations. Subsequent generations were more resistant against biotic  and abiotic  stressors. Underlying molecular and epigenetic mechanisms of the observed transgenerational inheritance were examined by studying the expression of innate immune-related genes, measuring global DNA (5-mC) methylation, RNA (m6A) methylation, and histone modifications. To the best of our knowledge, this is the first description of transgenerational inheritance of compound-induced robustness, resulting in protection against both biotic (AHPND strain A. franciscana) cysts (dormant embryos) originating from the Great Salt Lake in Utah (USA)  were used for production of the parental generation (F0). Artemia cysts were hatched in axenic conditions. Briefly, 2 g of A. franciscana cysts were hydrated in 89 ml of distilled water for 1 h. Sterile larvae were obtained via decapsulation using 3.3 ml of NaOH (32%) and 50 ml of NaOCl (50%), providing 0.2-μm filtered aeration. Decapsulation was stopped after 2 min by adding 50 ml of Na2S2O3 at 10 g/L followed by washing the cysts with filtered autoclaved seawater (FASW) containing 35 g/L of instant ocean synthetic sea salt . The decapsulated cysts, suspended in 1-L glass bottles containing FASW, were incubated for hatching at 28°C with constant illumination of approximately 27 μE/m2 s. After 28 h of incubation, hatched larvae at developmental stage instar II (mouth was opened to ingest food) were collected and used for the experiments.Brine shrimp , a polyphenol derivative organic plant-derived compound, was dissolved in sterile distilled water at 0.4 g/L (3.17 mM). Before starting the current experiments, we had tested the protective effect of phloroglucinol in brine shrimp larvae who were continuously exposed to different doses (0.25 to 10 μM) of phloroglucinol and subsequently infected with 2/s). During the entire culture period, all animals were daily ad libitum fed with live green algae (Tetraselmis suecica). In the treated population, the parental brine shrimp (TF0) were treated with phloroglucinol (2 μM), starting from DAH1 after hatching until DAH16. For this purpose, every 3rd day, water was exchanged, adding fresh phloroglucinol (2 μM). Treatment was stopped at DAH16, to ensure that the uterus, which normally develops by 3 weeks and carries the cysts/embryos, was not directly exposed to the compound. In the control population, parental shrimp (CF0) were cultured in the absence of phloroglucinol and maintained under the same culture and water exchange conditions.The transgenerational experiment  was perfAdult females of the F0 generation (after 30 days post-hatching) from both control and treatment groups produced larvae (F1 generation). All F1 larvae were further cultured to maturity under controlled environmental conditions as previously explained. F2 larvae were collected from adult F1 brine shrimp. The experiment continued till the F3 generation . F1, F2,Artemia) switches from an ovoviviparous to an oviparous mode of reproduction. Hence, to induce the production of F1, F2, and F3 cysts, the adult F0, F1, and F2 animals in both the control and treatment group were cultured by adding autoclaved artificial seawater with gradually increasing the salinity from 35 to 80 g/L over a 2-week period , brine shrimp (k period . The genk period . One dayVibrio clade were used: (i) V. parahaemolyticus AHPND strain M0904 (Vibrio harveyi strain BB120 (ATCC BAA-1116) (−1). Bacterial cell density was measured spectrophotometrically at 550 nm, taking the McFarland standard into account, assuming that an optical density of 1.0 corresponds to 1.2 × 109 cells/ml. Sterile LVS3 (Aeromonas hydrophila) were used as brine shrimp feed during the bacterial challenge assay. An LVS3 stock culture was prepared as previously described (7 cells/ml.Two pathogenic strains from the in M0904  and (ii)AA-1116) . The M09AA-1116) . BacteriAA-1116)  and 40% escribed , and the7 CFU/ml) with V. parahaemolyticus (M0904) or V. harveyi (BB120) as previously described (A. hydrophila). During the survival assay, the glass tubes with experimentally infected brine shrimp larvae from both the control and treatment group were placed randomly in a rotor and maintained at 6 rotations per minute (rpm). The rotor was placed in the climatized challenge room where the temperature was constantly maintained at 28°C and 2000 lux permanent illumination was present. The survival of the brine shrimp larvae was scored from 12 h post infection onwards till 60 h post infection.One-day-old brine shrimp larvae from F1 to F3, at each generation from both phloroglucinol treated and non-treated parents, were experimentally infected  with V. parahaemolyticus (M0904) or V. harveyi (BB120) and monitored for survival from 12 to 48 h post infection. Also, for each group, 5 replicates  of 20 one-day-old larvae were exposed to abiotic stress, namely, lethal heat shock (15 min at 42.5°C) and monitored for survival every 3 h post exposure from 0 to 12 h post exposure. During the survival assay as previously described, the glass tubes were placed randomly in a rotor (6 rpm) and placed in the climatized challenge room (temperature 28°C and illumination 2000 lux). In addition, from each group, three replicates  of 100 one-day-old larvae were sampled for (i) gene expression analyses, (ii) examination of DNA methylation, (iii) examination of RNA methylation, and (iv) studying histone modifications. The later analyses were always performed on larvae sampled before and after challenge (6 and 12 h post infection). Also, 100 remaining 1-day-old larvae from each group were allowed to grow out to juveniles. At the age of 16 days, for each group, 3 replicates of 5 juveniles were sampled for (i) gene expression analyses, (ii) examination of DNA methylation, (iii) examination of RNA methylation, and (iv) studying histone modifications. Sampled larvae and juveniles were frozen in liquid nitrogen and stored at −80°C until analyses. The remaining 40 juveniles per group (F1–F3) were grown to adults and 7 adult pairs were examined for reproduction performance (production of cysts and nauplii).CF1 to CF3 cysts and TF1 to TF3 cysts originating from non-treated and treated parents, respectively, were used for the common garden experiment , 31. AllA260/A280 ratios >2.0 and A260/A230 ratios >1.5 used for the analysis. Reverse transcription was done from 1 μg of total RNA samples with the RevertAid H Minus First Strand cDNA Synthesis Kit  according to the manufacturer's guidelines.Total RNA was extracted from brine shrimp samples (larvae and juveniles) in triplicate  with Qiagen RNeasy Plus Mini Kit (Cat No. 74136). In each replicate, 100 larvae and 5 juveniles were used for RNA extraction. The quality and quantity of RNA isolates were checked on NanoDrop spectrophotometer  and RNA samples with E”) was calculated for each gene by E = b(−1/m), where “b” is the base of the logarithm.For each set of gene specific primers, the efficiency and specificity were verified via melting curve analysis and standard curves was prepared. Briefly, after PCR amplification, the amplified PCR product was cloned into the plasmid pGEMT vector and then sequenced to confirm the qPCR primers of all genes. Dilution series (1:10) of all the cloned plasmid were prepared, and a standard curve was drawn by plotting the Ct (threshold cycle) values against the logarithm of the dilution factors. The slope of the regression line is related to the amplification efficiency (“−ΔΔCt method) following Livak and Schmittgen , heat shock protein 90 (hsp90), down syndrome cell adhesion molecule (DSCAM), lipopolysaccharide and β-1,3-glucan-binding protein (Lgbp), prophenoloxidase (proPO), high mobility group box 1 (HMGB1), peroxinectin (PXN), superoxide dismutase (SOD), transglutaminase 1 (TGase1), and transglutaminase 2 (TGase2), were measured by RT-qPCR with pair of specific primers  using Sthmittgen  was usedhmittgen . The logA260/A280 ratios between 1.8 and 2.0 and A260/A230 ratios >2.0 were used for further analysis.Genomic DNA was extracted from cysts (100 cysts/replicate), larvae (100 larvae/replicate), and juveniles (5 juvenile/replicate) of F0, F1, F2, and F3 generations of brine shrimp in triplicates using the Qiagen DNeasy Blood & Tissue Kit (Cat No. 69504) according to the manufacturer guidelines. The quality and quantity of purified DNA were checked using the NanoDrop spectrophotometer . DNA samples with Global DNA methylation was determined by using the MethylFlash Global DNA Methylation (5-mC) ELISA Easy Kit (Colorimetric) . The methylated fraction of DNA was quantified using 5-methylcytosine (5-mC)-specific antibodies and the amount of methylated DNA is proportional to the optical density (OD) measured at an absorbance of 450 nm in an ELISA microplate spectrophotometer (Tecan Infinite M200 Microplate Reader). In brief, a standard curve was generated by plotting the OD values of the positive control (PC) at each percentage point in defined dilutions, and slope of the standard curve was calculated. From all samples, i.e., brine shrimp cysts, larvae and juveniles of F0, F1, F2, and F3 generations, 100 ng DNA was used for analysis in triplicates.According to the manufacturer's guidelines, the following formula was used to calculate the percentage of methylated DNA (5-mC) in the total DNA:S) × 100%, where S is the amount of input sample DNA (100 ng), OD is the optical density at 450 nm, and NC stands for the negative control. Genomic DNA methylation levels were expressed as 5-mC%.5-mC% = (Sample OD – NC OD)/(Slope × N6-methyladenosine (m6A) in RNA was measured using the EpiQuick m6A RNA Methylation Quantification Kit (colorimetric)  following the manufacturer's instructions and also previous studies –39. In bS × 100%. In this equation, NC stands for negative control, PC stands for positive control, and S represents the amount of input RNA: 200 ng.m6A (ng) = (Sample OD – NC OD)/Slope and m6A % = m6A Amount (ng)/g for 5 min. Next, the samples were homogenized (until no clumps left in solution) and incubated overnight in 0.4 N HCl on a rotor at 4°C to promote the lysis of nuclei and solubilization of histones. The extract was centrifuged at 16,000 × g for 10 min, and then the supernatant was transferred to a new protein LoBind Eppendorf tube. Next, trichloroacetic acid (TCA) (Sigma-Aldrich) was added (drop by drop) to the supernatant, and the tubes were mixed for several times until it turns to a milky solution and afterwards incubated on ice for 2 h to promote the precipitation of histones. The precipitated histones were centrifuged at 16,000 × g for 10 min and pelleted histone was washed twice with ice-cold acetone to remove the TCA. Finally, histone pellet was air dried and resuspended in nuclease-free water. Histone concentration was determined following the Bradford method using bovine serum albumin as standard , larvae (3 replicates with 100 larvae/replicate), and juveniles (3 replicates with 5 juveniles/replicate). Briefly, the brine shrimp samples, i.e., cysts (after decapsulation), larvae, and juveniles were washed twice with PBS in 1.5 ml of protein LoBind Eppendorf tube at 1,000 × standard  and subsstandard .Histone H3 and H4 total acetylation were analyzed from 7- to 16-day brine shrimp parental generation and three subsequent F1, F2, F3 generations (cysts and juveniles). Briefly, 1 μg of histone samples both from control and treatment brine shrimp was used to examine the H3 and H4 total acetylation in an ELISA-based format using Histone H3 Total Acetylation Detection Fast Kit  and Histone H4 Total Acetylation Detection Fast Kit , respectively, in triplicate following the manufacturer's instructions.Next, the histone H3 and H4 multiple modifications in brine shrimp cysts of F1, F2, and F3 generation from both treatment and control were screened using EpiQuik™ Histone H3 Modification Multiplex Assay Kit  and EpiQuik™ Histone H4 Modification Multiplex Assay Kit  according to the manufacturer's instructions. In brief, 200 ng of histone extracts  was used to determine the histone H3 and H4 modification at specific sites by a capture antibody that was coated in strip wells that targets the appropriate histone modification pattern and detected with a detection antibody. Colorimetric data were obtained from Tecan Infinite M200 Microplate Reader and OD values were measured at 450 nm wavelength and 655 nm as reference wavelength for background subtraction. H3 and H4 modifications were calculated following the manufacturer's instructions, which also accounts for total histone protein amounts, and the final values for each modification were presented as percentage over control.S × 1000/(Assay Control OD – Blank OD) ÷ P, where S is the amount of input sample protein in ng and P is the amount of input assay control in ng.H3 or H4 Modification or total H3 or H4 (ng/μg protein) = (sample OD – blank OD) ÷ H3 or H4 Modification % = Amount of H3 or H4 modification (ng/μg protein)/Amount of total H3 or H4 (ng/μg protein) × 100%.Relative Change % = H3 or H4 modification % in treated sample/H3 or H4 modification % in control sample × 100%.2, 5 mM NaH2PO4, and 40 mM HEPES, pH 7.4) supplemented with a protease inhibitor cocktail . Afterwards, samples were centrifuged  and the protein concentrations in the supernatants were measured by the Bradford method (Stored (−80°C) parenteral brine shrimp  were homogenized in cold buffer K  were combined with loading buffer, vortexed, heated for 5 min at 95°C, and then electrophoresed in 10% SDS-PAGE gel  with each lane receiving equivalent amounts of protein (5 μg). Protein extracted from a suspension of heat-shocked HeLa cells  (6 μg) served as positive control. Separated proteins were transferred to a polyvinylidene fluoride membrane (BioRad Immuno-BlotTM PVDF) for antibody probing. Membranes were incubated with blocking buffer [50 ml of 1 × phosphate-buffered saline containing 0.2% (v/v) Tween 20 and 5% (w/v) bovine serum albumin] at room temperature for 60 min. All antibody dilutions were made in Tris-saline and all washing steps were performed using Tris–saline buffer. For Hsp70 analysis, membranes were incubated with a mouse monoclonal anti-Hsp70 antibody (3A3)  and horseradish peroxidase-conjugated donkey anti-mouse IgG .For histone analysis, 5 μg of total histone extracts from F1, F2, and F3 cysts were electrophoresed on a 4–20% SDS-PAGE gel ; histone calf thymus  (2 μg) served as a positive control. Separated proteins were transferred to a polyvinylidene fluoride membrane (BioRad Immuno-BlotTM PVDF) for antibody probing using (i) rabbit anti-histone H3 trimethyl K4 immunoglobulin  in combination with goat anti-rabbit IgG (H + L) , (ii) rabbit anti-histone H3 trimethyl K9 immunoglobulin  in combination with goat anti-rabbit IgG (H + L) , and (iii) a mouse monoclonal antibody against histone H3 trimethyl K27  in combination with goat anti-mouse IgG (H + L) . The membranes were then incubated with clarity Western ECL substrate (chemiluminescence reagent) (BioRad Laboratories) for 5 min and the signals were detected by a ChemiDoc MP imaging system . The relative signal intensity was quantified using Bio-Rad Image lab 4.1 software.p levels. Survival differences between the control and treatment at each time point were tested for the significance based on LSD at different p levels. Results for the gene expressions were represented as fold-changes relative to the geometrical mean of two internal control genes (EF-1a and GAPDH). The expression level in the control was regarded as 1.0 and thereby the expression ratio of the treatments was expressed in relation to the control. Analysis for significant differences in expression levels between the control and treatment groups was performed with the single-tailed Student's t-tests using log-transformed data, and a two-way analysis of variance (ANOVA) was also performed for the interaction analysis using the Statistical Package for the Social Sciences (SPSS) 19.0 . Significant differences between the control and the treatment group for DNA and m6A RNA methylation, total histone H3 and H4 acetylation, and histone multiplex modifications at each generation were determined by the Student's t-test using SPSS version 20.0 . The significance level was set at P < 0.05.Survival data were analyzed by logistic regression analysis using GenStat 16  software to determine significant differences between the control and treatment group. For each time point, least significant difference (LSD) values were generated at different V. parahaemolyticus-induced mortality   . Next, wV. parahaemolyticus or V. harveyi as compared to their respective (CF1) controls  at an early life stage increased the resistance of their progeny for three subsequent unexposed generations  against a bacterial infection with V. parahaemolyticus or Vibrio harveyi at the end of the monitoring period.TF1 brine shrimp whose parents (TF0) were exposed to phloroglucinol showed significant higher survival rates following challenge test with controls i,Bi. Inte shrimp i–iii. ThWestern blotting was used to measure Hsp70 protein production in brine shrimp from parents , which were treated by continuous exposure to phloroglucinol (2 μM). Exposure to phloroglucinol significantly increased Hsp70 production (~2-fold) on DAH16 as compared to the control (CF0) . Next, rP > 0.05) in total offspring production (cysts and nauplii) by TF1 to TF3 generations in comparison to total offspring production by their respective controls (CF1 to CF3)  suffered no reproduction cost or changes in reproductive behavior. There were no significant differences ( to CF3) . Thus, pVibrio spp. infections) or abiotic  stressor in the axenic nauplii collected from TF1 to TF3 generations was associated with altered immune-related gene expression during the common garden experiment. Newly hatched larvae from both control and treatment groups of F1–F3 generations were sampled. Remaining animals from both control and treatment groups (F1–F3) were cultured under conventional control condition without any challenge or treatment and were sampled at the juvenile (16 days old) life stage  were significantly upregulated (2.07- and 2.31-fold rise in TF1 and TF2 juveniles and 2.11- and 1.89-fold rise in TF1 and TF2 larvae) as compared to animals of CF1 to CF2, whereas mRNA levels for the lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) were significantly downregulated (~0.6 fold decline in juveniles and ~ 0.1 fold in larvae) (peroxinectin mRNA levels were only significantly upregulated in TF1 (2.9-fold rise) and downregulated in TF2 (0.59-fold decline) and TF3 (0.72-fold decline) animals. Superoxide dismutase (SOD) mRNA levels were significantly upregulated in TF1 juveniles (2.2-fold rise) and Heat shock protein 90 (HSP90) mRNA levels were significantly upregulated in TF1 (1.58-fold rise) and TF2 (2-fold rise) juveniles, but in larvae, the relative expression was <1.5, whereas HSP70 was significantly upregulated (1.67-fold rise) only in the TF1 larvae. Moreover, there were no significant changes in the expression of HMGB1, ProPO, TGase I, and TGase II either in TF1 to TF3 larvae or juveniles. These results of “phloroglucinol treated, non-challenged” gene expression profile suggest that immune receptor DSCAM, LGBP, and peroxinectin were differentially induced in TF1 to TF3 generation of brine shrimp.In the phloroglucinol-treated, non-challenged TF1 to TF2 larvae and juveniles, mRNA levels for the pattern recognition receptor down syndrome cell adhesion molecule ( larvae) . TranscrV. parahaemolyticus or V. harveyi) brine shrimp larvae were upregulated in a time-dependent manner   in 5-mC% for TF1 animals compared to their respective controls. The 5-mC% was statistically the same between TF2 and CF2 and between TF3 and CF3  in the parental generation and subsequent generation (TF1–TF3) animals. The global DNA methylation level was measured in (i) the parental generations TF0 and CF0 both at DAH7 and at DAH16, (ii) TF1 to TF3 and CF1 to CF3 cysts, (iii) TF1 to TF3 and CF1 to CF3 juveniles, and (iv) larvae 12 h post challenge (harveyi) . At DAH1harveyi) . Interesharveyi) . Furtherharveyi) . DNA met and CF3 . DNA metV. parahaemolyticus or V. harveyi)  a significantly lower m6A RNA methylation level in TF1 larvae compared to CF1, and (ii) a significant increase in m6A RNA methylation level in TF2 and TF3 larvae as compared to their respective controls  methylation levels and higher disease resistance.To gain more insights related to phenotypic observation of phloroglucinol-induced transgenerational inherited increased resistance, next we aimed to identify the m6A (N6-methyladenosine) RNA methylation level in the brine shrimp. The m6A RNA methylation level was measured in (i) the parental generations TF0 and CF0 both at DAH7 and at DAH16, (ii) TF1 to TF3 and CF1 to CF3 cysts, (iii) TF1 to TF3 and CF1 to CF3 juveniles, and in (iv) larvae 12 h post challenge (harveyi) . At DAH7harveyi) . The m6Aharveyi) . In TF1 harveyi) . The m6Acontrols . RNA  to test whether histone modifications have also any role in phloroglucinol-induced observed phenotype of transgenerational inherited increased resistance in brine shrimp.At DAH7, total histone H3 acetylation levels in TF0 parents and CF0 controls were statistically the same. However, at DAH16, the total histone H3 acetylation level was significantly higher in phloroglucinol-treated TF0 parents as compared to CF0 controls. Total histone H3 acetylation was significantly higher in the TF1 to TF2 cysts vs. their respective controls. For juveniles, the total histone H3 acetylation levels in treated brine shrimp and controls were always statistically the same  since enNext, to gain insight into specific histone modifications, the occurrence of 21 histone H3 protein modifications  and 10 histone H4 protein modifications  was examined for the TF1 to TF3 and CF1 to CF3 cysts. Increased methylation was observed for H3K4 and H3K27 and increased acetylation for H3K14 and H3K56. Specifically, the H3K4me1, H3K4me2, H3K4me3, H3K27me1, H3K14ac, and H3K56ac contents were significantly higher for TF1 vs. CF1 cysts. For TF2 vs. CF2 cysts, H3K4me3, H3K27me1, H3K27me2, H3K27me3, H3K14ac, and H3K56ac were significantly higher. Data for TF3 vs. CF3 were statistically the same . In caseTo verify the reliability of the data obtained by the multiplex histone ELISA, H3K4me3, H3K27me3, and H3K9me3 contents for TF1–TF3 and CF1–CF3 cysts were also examined by Western blotting. ELISA results for H3K4me3, H3K27me3, and H3K9me3 were successfully confirmed by Western blotting . OverallArtemia  as model organism, we showed that frequent application of phloroglucinol at early life induces phenotypic traits  at the end of the monitoring period, and these acquired traits can be transmitted to three successive, unexposed generations , non-coding RNA (ncRNA) molecules, and RNA methylation. These acquired epigenetic marks can be stably passed through the gametes and persist for multiple generations , 45. TheDSCAM and LGBP. The pathogen recognition receptors (PRRs), e.g., DSCAM and LGBP, are germline-encoded host sensors of the innate immune system that detect pathogen-associated molecular patterns (PAMPs)  in both larvae and juveniles and in post-Vibrio exposed larvae , e.g.,  (PAMPs) . DSCAM ichanisms –51. Morecination , 52–60. bacteria , 62. LGBnd fungi , 64. In d larvae , 5. LGBPHSP70, HSP90, and HMGB1), genes responsible for humoral immune defense , and the antioxidant defense gene SOD. Our results suggested that the observed phenomenon could also be associated, at least in part, with the induction of the signaling molecules HSP70 and HSP90. A positive correlation between elevated HSP70 and HSP90 transcript levels and increased resistance phenotypes was observed in the TF1 and TF2 generation progenies (whose ancestors were treated with the compound) before challenge and in the TF1–TF3 generations after being challenged with the two different strains of Vibrio . This result is consistent with the known important roles of HSP70 and HSP90 signaling proteins in defining the resistance of organisms against stressor by performing multifaceted functions, such as acting as molecular chaperone for proteins, functioning as danger-associated molecular pattern (DAMP) during inflammation and various cellular processes  is another defense-related gene and is a cell adhesive protein known to be strongly associated with the proPO system  mRNA were not upregulated in TF1–TF3 generation (both nauplii and juvenile) in the absence of challenge. However, despite the absence of any significant change in the gene expression levels of proPO or TGase in these animals, a significant upregulation of PXN was observed at TF1 (both nauplii and juvenile) and TF2 generations (only nauplii stage). Surprisingly, PXN was downregulated at TF2 (juvenile) and TF3 (both nauplii and juveniles) generations. From this result, it also appears that compound application in parental generation induces the production of PXN. This priming effect is passed on to the first two subsequent generations, but in the absence of the compound, they fade away and are reversed. However, once nauplii were challenged with either V. parahaemolyticus or V. harveyi, an increase in levels of proPO mRNA expression was observed across TF1–TF3 generations. Interestingly, the proPO gene expression patterns were followed by PXN once the animals were challenged with either of the pathogens. These results show a strong link between the two genes. By comparing the gene expression analysis results of proPO and PXN before and during the challenge, the only possible conclusion is that the compound application in the parental generation induces some kind of modifications on these immune-related genes that could result in the faster and more efficient expression of the mRNA during the challenge. Significantly elevated proPO expression levels in treated shrimp were correlated with higher survival rates in subsequent generations. However, we did not measure the phenol oxidase enzyme activity in shrimp, nor did we analyze protein expression at that time. However, we did collect samples for a future LC-MS/MS study to identify differentially expressed and regulated proteins. Free radicals and reactive oxygen species (ROS) are produced in the metabolic pathways of aerobic cells. The activated innate immune system also engages in phagocytosis to eliminate ROS and invading microorganisms with the aid of the antioxidant enzymes such as SOD . The results highlight possible involvement of DNA methylation in the epigenetic modifications for creating a resistant phenotype and gene expression regulation in transgenerational brine shrimp. Global DNA methylation can be further varied depending on the genotype, different environmental stressors, and their interactions (V. parahaemolyticus), indicating that both phloroglucinol and pathogen might have impacted DNA methylation in brine shrimp. There are few reports showing that DNA methylation is affected by environmental stress such as heat shock in the red flour beetle Tribolium castaneum . Moreover, DNA methylation changes the way DNA binds and interacts with proteins to regulate the transcription, and recent reports also suggested that methylation was involved in defining exon recognition or exon–intron boundaries that affects splice variant production , 81. In ractions . Here, wastaneum  and acronciscana . Likewis methylation and the induced inherited bacterial resistant phenotype. Nevertheless, this does not completely rule out the possibility that m6A RNA methylations might have possibly been involved in developing resistance in brine shrimp after phloroglucinol treatment to the parental generation and in transgenerational inherited robustness in brine shrimp.Recent studies strongly suggest that RNAs also have dynamic regulatory roles in several biological processes, analogous to epigenetic DNA and histone modifications , 88. RNAd larvae . Castro-Artemia  displayed a differential expression pattern. Third, there is evidence that epigenetic mechanism such as DNA (5-mC) methylation, RNA (m6A) methylation, and histone modifications  might be involved in the acquisition of the resistant phenotype. To the best of our knowledge, this is the first demonstration of transgenerational inheritance of a compound-induced robustness, enhancing protection against both biotic  and abiotic stressors (thermotolerance). Therefore, parental conditioning of the brood stock could be a unique and powerful novel strategy for prophylaxis of infectious diseases for future shrimp farming applications. Brine shrimp (A. franciscana) is regarded as a model for crustacean shrimp. However, species differ, and further experiments are necessary to find out if our data can be extrapolated to other shrimp species.In conclusion, this study reports three major novel findings. At first, phloroglucinol treatment of brine shrimp parents generated transgenerational inheritance of increased resistance against biotic  and abiotic stressors (thermotolerance) across three subsequent generations without involving a fitness-related cost. Secondly, some of the innate immune-related genes under study (All datasets generated for this study are included in the article/SR, PN, PB, and DV conceived the study and designed research. SR and VK performed the experiments. SR analyzed the data and wrote the manuscript. All authors were involved in editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We report the complete genome characterization of a novel human papillomavirus (HPV) (ICB2) isolated from a skin swab. The L1 region of HPV ICB2 shares 87.9% nucleotide similarity with its closest relative, HPV37, and thus constitutes a novel human betapapillomavirus. We report the complete genome characterization of a novel human papillomavirus (HPV) (ICB2) isolated from a skin swab. The L1 region of HPV ICB2 shares 87.9% nucleotide similarity with its closest relative, HPV37, and thus constitutes a novel human betapapillomavirus. Papillomaviridae family. HPVs infect basal keratinocytes of the mucosal and cutaneous epithelia. Based on the nucleotide sequences of the major capsid protein L1, HPVs are classified into five major genera, Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Mupapillomavirus, and Nupapillomavirus  are double-stranded circular DNA viruses with a genome of approximately 8 kb and belong to the mavirus 13. The mu cancers . Moreoveuggested –7.Here, we report the complete genome sequence of a novel HPV type  isolated from a human forearm skin swab.A partial L1 region sequence of HPV ICB2 (99 bp) was previously obtained from DNA extracted from the skin swab using broad-spectrum primers in combination with next-generation sequencing . MultiplThe resulting amplicon of approximately 8 kb was then cloned in pCR-XL-2 TOPO vector using the TOPO XL-2 complete PCR cloning kit . The sequence of the whole genome was obtained by Sanger sequencing using a primer-walking strategy . This sequencing service uses cycle sequencing technology (dideoxy chain termination/cycle sequencing) on an ABI 3730XL sequencing machine. The viral genome was covered at least twice in order to identify and correct sequencing errors. Thirty-one sequences were generated and aligned to reconstruct the whole genome using the CAP3 sequence assembly program , with dewww.hpvcenter.se) for assignment of HPV type number.The clone has been submitted to the International Human Papillomavirus Reference Center in Stockholm (Betapapillomavirus. HPV ICB2 thus constitutes a novel human betapapillomavirus by sharing less than 90% nucleotide sequence identity with the closest HPV type in the L1 ORF (The L1 open reading frame (ORF) of HPV ICB2 showed 87.9% nucleotide identity with its closest relative, HPV37, which belongs to the species beta-2 of the genus The G+C content of ICB2 is 40.7%. The virus has the typical genome organization of other cutaneotrophic HPVs; it is composed of five early  and two late (L1 and L2) ORFs, and no E5 was identified.4-CGGT (n = 2), ACC-N5-GGT (n = 1), and ACC-N1-GGT (n = 1). A putative TATA box domain (TATAAGA) for the downstream early promoter was also identified.The long control region (LCR) is 382 bp. This region contains two polyadenylation sites (AATAAA) for L1 and L2 transcripts and four consensus palindromic E2-binding sites, as follows: ACCG-N29CxxC and CxxC(x)30CxxC] are present and are separated by 36 amino acids no acids .29CxxC] and one LxCxE motif are located in the E7 protein (Betapapillomavirus genus.A zinc-binding domain [CxxC(x) protein . An ATP-MK080568.The complete genome sequence of HPV ICB2 is available in GenBank under accession number


Comparative analysis reveals adaptive evolution of bat IFITMs and a naturally polymorphic site within the conserved CD225 domain that affects S-palmitoylation and antiviral function of bat IFITM3. Host interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral restriction factors. Of these, IFITM3 potently inhibits viruses that enter cells through acidic endosomes, many of which are zoonotic and emerging viruses with bats (order Chiroptera) as their natural hosts. We previously demonstrated that microbat IFITM3 is antiviral. Here, we show that bat IFITMs are characterized by strong adaptive evolution and identify a highly variable and functionally important site—codon 70—within the conserved CD225 domain of IFITMs. Mutation of this residue in microbat IFITM3 impairs restriction of representatives of four different virus families that enter cells via endosomes. This mutant shows altered subcellular localization and reduced S-palmitoylation, a phenotype copied by mutation of conserved cysteine residues in microbat IFITM3. Furthermore, we show that microbat IFITM3 is S-palmitoylated on cysteine residues C71, C72, and C105, mutation of each cysteine individually impairs virus restriction, and a triple C71A-C72A-C105A mutant loses all restriction activity, concomitant with subcellular re-localization of microbat IFITM3 to Golgi-associated sites. Thus, we propose that S-palmitoylation is critical for Chiropteran IFITM3 function and identify a key molecular determinant of IFITM3 S-palmitoylation. IFITM1, IFITM2, and IFITM3—encoding proteins with antiviral functions and two IFITM family members that lack antiviral function—IFITM5 and IFITM10. Mice have orthologs of all these IFITMs as well as two additional genes, Ifitm6 and Ifitm7. Phylogenetic analysis of vertebrate IFITMs indicates that IFITM1, IFITM2, and IFITM3 group with murine ifitm6 and ifitm7 in a clade of immunity-related IFITMs (IR-IFITMs), with IFITM5 and IFITM10 falling as separate lineages (http://pfam.xfam.org/family/PF04505) (Interferon-induced transmembrane proteins (IFITMs) are antiviral factors that act uniquely and early in viral replication cycles to restrict the entry of a diverse range of primarily enveloped viruses into cells . Humans 20YEML23 localize to the plasma membrane and lose their ability to inhibit influenza A virus (IAV), alphavirus, and coronavirus infection by endosomal routes  domain, transmembrane domain, and extracellular  C terminus , 5, althl routes , 17, 18.Studies focusing on IFITM3 restriction of IAV and Semliki Forest virus (SFV) indicate that virus internalization is unaffected by IFITM3 expression and, for SFV at least, the viral envelope glycoprotein undergoes low pH-induced conformational changes . HoweverIFITMs are heavily regulated by posttranslational modifications (PTMs). One major modification is S-palmitoylation, a reversible 16-carbon lipid PTM that increases protein hydrophobicity and influences the behavior of proteins in membrane environments . For humIfitm3 knockout mice show enhanced morbidity after infection with IAV, alphaviruses, and flaviviruses  are of particular interest because these animals have been increasingly recognized as reservoir hosts from which numerous viruses have ultimately emerged, with severe pathogenic and socioeconomic consequences in humans and livestock. Indeed, a recent analysis showed that bats host a significantly higher proportion of zoonotic viruses than other mammalian orders . MoreoveHere, we performed evolutionary analyses of mammalian IFITMs, including those identified from bats, to shed light on IFITM function and the nature of past selection pressures, and to identify key amino acids for experimental studies of IFITM function.Myotis myotis, Eptesicus serotinus, and Sus scrofa and proteomics informed by transcriptomics . The resulting phylogeny revealed relatively high levels of sequence divergence (nucleotide substitutions per site) between the IFITM genes  to synonymous (dS) nucleotide substitutions per site, with dN > dS indicative of positive selection , performed using the adaptive Branch-Site Random Effects Likelihood method .The IFITM gene family is notable for gene duplications, variation in copy number, and the presence of pseudogenes , 55, andd method  that is IFITM genes used for selection pressure analysis.Table S1 To visualize amino acid diversity, a logo diagram was constructed for the alignment of 31 “functional” IFITMs. This revealed notable amino acid diversity at codon 70, located within the otherwise highly conserved CD225 domain and directly upstream of the double cysteine motif C71/C72 . The amiP < 0.1; mean dN-dS value of 2.64). Although there was no statistically significant evidence for positive selection on codon 70 (P = 0.192), it had the second highest dN-dS value of any site (1.91), with 14 non-synonymous nucleotide changes (compared with 13.5 at codon 121) and only two synonymous changes across the phylogeny. Similar results  were obtained using the fast unconstrained Bayesian approximation method.We next performed a site-specific selection analysis using the phylogeny of the 31 functional IFITMs shown in M. myotis IFITM3 (mbIFITM3) and a mbIFITM3 P70W mutant: W is a possible ancestral codon for the Laurasiatherian and primate IFITM clades  human IFITM3  and human IFITM3 encoding each of the other six amino acids that occur naturally in mammals at position 70 . In addition, we made C-terminally HA-tagged wt microbat M clades  and occuThe mutant IFITM3 proteins were tested for their ability to restrict cell entry by Zika virus (ZIKV), SFV, and IAV . Flow cyTo assess the impact of P70 mutations on the subcellular localization of huIFITM3 and mbIFITM3, confocal immunofluorescence imaging was performed on A549 cells stably expressing either wt or P70W mutants of C-terminally HA-tagged huIFITM3 and mbIFITM3 . In fixecis Golgi marker Giantin . A549 cells stably expressing single mutants C71A, C72A, and C105A, the double C71A-C72A mutant and the triple C71A-C72A-C105A mutant were generated. Two methods were used to assess S-palmitoylation: (i) the acyl exchange gel shift method described above , and ii. For thiImportantly, the results of both methods for the different cysteine mutants of mbIFITM3 showed that the single cysteine mutants  have reduced palmitoylation compared with wt mbIFITM3, with a greater reduction for C72A than C71A. A double C71A–C72A mutant still showed some acylation, indicating that C105 can be acylated, whereas the triple mutant abrogated palmitoylation . In summWe next studied the ability of the mbIFITM3 S-palmitoylation–defective cysteine mutants to restrict viral entry. The infectivity of ZIKV, SFV, and virus pseudotypes expressing the envelope proteins of the lyssaviruses West Caucasian Bat virus (WCBV) and Lagos Bat virus (LBV) was tested in cells expressing either wt mbIFITM3, mbIFITM3 P70W, or the cysteine mutants of mbIFITM3 . All cyscis Golgi marker Giantin . None of these mutants differed from wt in restricting three different viruses IAV, SFV, and ZIKV. An earlier report also showed that substituting P70 of human IFITM3 with T, the residue present in human IFITM2, did not alter IAV restriction or subcellular localization (M. myotis because this is the only Chiropteran IFITM reported to act as a restriction factor (Alphaviridae), ZIKV (Flaviviridae), and two lyssaviruses, concomitant with re-localization to perinuclear Golgi-associated sites and reduced S-palmitoylation. As for mbIFITM3 P70W, S-palmitoylation–defective cysteine mutants of microbat IFITM3 showed markedly different localization from wt, with reduced expression at the cell surface and retention at Golgi-associated sites. Based on the evidence that restriction relies on the localization of IFITM proteins at sites of viral entry , perhaps suggesting a threshold level of S-palmitoylated IFITM3 is needed for restriction.69NPCC72 in human and microbat IFITM3, which separates two alpha helices , and the pig NPTr cell line, using RACE and RT-PCR methods as previously described  and synonymous (dS) nucleotide substitutions per site  among the 31 functional IFITM genes using both the single-likelihood ancestor counting and fast unconstrained Bayesian approximation programs as implemented in the Datamonkey web server (http://datamonkey.org), using default settings (To assess selection pressures acting on IFITM genes (Table S1), we computed the relative numbers of non-synonymous , as previously (RACE was conducted as described previously  on primaeviously .A549 cells (ATCC CCL-185) were cultured in Ham’s F-12 Nutrient Mixture with GlutaMAX , supplemented with 10% (vol/vol) FBS (Gibco). SFV_Zs-Green  according to the manufacturer’s instructions using the following primers : mbIFITM3 P70W: CGAAGCCCAGGCAGCACCAGTTGAAGAACAGGGTGTTG and CAACACCCTGTTCTTCAACTGGTGCTGCCTGGGCTTCG; huIFITM3 P70A: TTCAACACCCTGTTCATGAATGCCTGCTGCCTG and CAGGCAGCAGGCATTCATGAACAGGGTGTTGAA; huIFITM3 P70T: TGTTCAACACCCTGTTCATGAATACCTGCTGCCTGG and CCAGGCAGCAGGTATTCATGAACAGGGTGTTGAACA; huIFITM3 P70G: GTTCAACACCCTGTTCATGAATGGCTGCTGCCTGGG and CCCAGGCAGCAGCCATTCATGAACAGGGTGTTGAAC; huIFITM3 P70W: CCTGTTCAACACCCTGTTCATGAATTGGTGCTGCCTGGGCT and AGCCCAGGCAGCACCAATTCATGAACAGGGTGTTGAACAGG; huIFITM3 P70F: CTGTTCAACACCCTGTTCATGAATTTCTGCTGCCTGGGC and GCCCAGGCAGCAGAAATTCATGAACAGGGTGTTGAACAG; huIFITM3 P70V: GTTCAACACCCTGTTCATGAATGTCTGCTGCCTGGG and CCCAGGCAGCAGACATTCATGAACAGGGTGTTGAAC. To generate cysteine mutants, C-terminally HA-tagged wt mbIFITM3 was subcloned from pHR-SIN CSGW PGK Puro into pcDNA3.1 by BamHI/NotI digest. Cysteine-to-alanine mutations were then introduced using the Q5 Site-Directed Mutagenesis Kit (NEB) according to the manufacturer’s instructions. Primers were as follows : For C71A – CTTCAACCCCGCCTGCCTGGGCTTC and AACAGGGTGTTGAACAGG; for C72A – CAACCCCTGCGCCCTGGGCTTCG and AAGAACAGGGTGTTGAACAG; for C105A CACCGCCAAGGCCCTGAACATCTGGGC and CTGGCGTAGCTCTGGGCG; for C71A-C72A – CTTCAACCCCGCCGCCCTGGGCTTCG and AACAGGGTGTTGAACAGG. The C71A-C72A-C105A construct was cloned using the C105A primers with the C71A-C72A construct as a template. Each of the five mutant sequences were then subcloned back into pHR-SIN CSGW PGK Puro by BamHI/NotI digest. All constructs were confirmed by Sanger sequencing.A549 cell lines stably expressing variants of IFITM3 were generated by lentiviral transduction as previously described , but usiCells were infected in F12 with 2% FBS (infection media) and analyzed after 8 h for SFV and 28 h for Zika virus. For flow cytometric analysis, cells were trypsinized and fixed in 4% (wt/vol) formaldehyde (TAAB) for 20 min. For Zs-Green–expressing SFV infections, fixed cells were washed in PBS, resuspended in FACS resuspension buffer , and stored at 4°C until analyzed. For ZIKV infections, fixed cells were washed in PBS, resuspended in FACS wash buffer  for 25 min at RT, and then stained with mouse anti-flavivirus antibody  diluted 1:500 in FACS wash buffer for 90 min at RT. After washing twice with FACS wash buffer, the cells were incubated with Alexa Fluor (AF) 488–conjugated antimouse secondary antibody (diluted 1:800 in FACS wash buffer) for 1 h at RT. The cells were then washed twice in FACS wash buffer and stored in FACS resuspension buffer at 4°C until analyzed. For flow cytometry  cells were gated on forward and side scatter and analyzed for FITC fluorescence. The data were processed using FlowJo vX.0.7 software (Tree Star). For SFV and ZIKV infections analyzed by high-throughput confocal imaging  and analysis of multi-cycle influenza replication was performed as described previously .AAR03484) or LBV  were described previously  was detected with either goat antirat conjugated to AF 488 (1:500) or with donkey anti-rat AF 488 (1:500). Rabbit anti-giantin primary antibody  was detected with goat antirabbit AF 647, sheep anti-TGN46 primary antibody  was detected with donkey antigoat AF 647 (1:500) and mouse anti-CD63 primary antibody  were incubated for 16 h with the alkyne palmitate (C16) analogue YnPalm (at 50 μM from 50 mM stock dissolved in DMSO) in F12 media with 3% FBS. A no-YnPalm control was incubated in DMSO only in F12 media with 3% FBS. The Cells were washed twice with PBS before lysis in 0.1 ml lysis buffer . Total protein concentration of lysates was quantitated using the Pierce BCA assay (Cat. no. 23225) and then adjusted to 1 mg/ml with lysis buffer. For each click reaction, 25 μg total protein was incubated with click-mix comprising  100 μM capture reagent AzTB (azido-TAMRA-biotin), 1 mM CuSO4, 1 mM Tris(2-carboxyethyl)phosphine (TCEP), and 100 μM Tris[methyl]amine (TBTA) for 60 min at room temperature. Proteins were then precipitated with two volumes methanol, 0.5 volume chloroform, and one volume water. The protein pellet was washed twice with methanol before boiling in 1× non-reducing loading buffer for 5 min and loading onto a 4–15% TGX stain-free gel . After SDS–PAGE, the gels were visualised using a Typhoon FLA 9500 imager  to detect TAMRA  and Cy5 (for MW markers). Proteins were then transferred onto PVDF for Western blotting using anti-HA  and anti-HSP90 . Densitometric quantification of TAMRA fluorescence (from Typhoon images) and HA (following ECL) was performed on 16-bit images using Image J 1.47v.A549 cells (3 × 105 cells/10 cm dish) were washed twice in PBS and then lysed in 0.5 ml lysis buffer . The total protein concentration of lysates was quantitated using the Pierce BCA assay. 75 μg of total protein in 100 μl of lysis buffer was subjected to acyl–biotin exchange using a modified protocol (2OH) cleavage, alkylation was performed with biotin–maleimide  in TEA buffer at 1 mM final concentration for 2 h at room temperature. After methanol–chloroform–H2O precipitation (2:0.5:1), protein pellets were resuspended in 4× non-reducing SDS–PAGE buffer , heated for 5 min at 95°C and allowed to cool for 30 min 6 μl (=10 μg) total protein was then reacted with streptavidin  in PBS  for 5 min at RT. Assuming that IFITM3 comprised all of the total protein, streptavidin was used at a 0.5× molar excess and titrations showed streptavidin was non-limiting at this amount . Protein concentrations were determined using the Pierce BCA assay. Proteins were separated by 15% SDS–PAGE and transferred to PVDF membranes. Membranes were blocked in 5% milk in PBST (PBS with 0.1% [vol/vol] Tween 20) and incubated with antibodies diluted in PBST, either for 1 h at RT or overnight at 4°C. The following primary antibodies were used: rat anti-HA , mouse anti-HA , rabbit anti-calreticulin , and anti-calnexin (CANX) . For blots imaged using an Odyssey system (Li-COR), goat antirat IRDye 800 secondary  or goat antirabbit IRDye 680  was used. For ECL detection, HRP-conjugated secondary antibodies and Luminata HRP substrate  were used.


FGFR4 in a human gastric tumour that leads to constitutive activation of its product, FGFR4. The G636C-FGFR4 tyrosine kinase domain mutation was found in 1 of 83 primary human gastric tumours. The G636C mutation increased FGFR4 autophosphorylation, and activated FGFR4 downstream signalling molecules and enhanced anchorage-independent cell growth when expressed in NIH/3T3 cells. 3D-structural analysis and modelling of FGFR4 suggest that G636C destabilizes an auto-inhibitory conformation and stabilizes an active conformation, leading to increased kinase activation. Ba/F3 cell lines expressing the G636C-FGFR4 mutant were significantly more sensitive to ASP5878, a selective FGFR inhibitor, than the control. Oral administration of ASP5878 significantly inhibited the growth of tumours in mice engrafted with G636C-FGFR4/3T3 cells. Together, our results demonstrate that mutationally activated FGFR4 acts as an oncoprotein. These findings support the therapeutic targeting of FGFR4 in gastric cancer.Gastric cancer remains one of the leading causes of cancer death worldwide. Despite intensive investigations of treatments over the past three decades, the poor prognosis of patients with unresectable advanced or recurrent gastric cancer has not significantly changed, and improved therapies are required. Here, we report the identification of an oncogenic mutation in  The number of survivors is low, because diagnosis is often determined during the late stages when symptoms first appear, which are accompanied by metastasis and chemoresistance1. Earlier treatment would be aided by detailed knowledge of the molecular characteristics of gastric cancer and the identification of new biomarkers. Most gastric cancers are adenocarcinomas, and arise and progress as a result of complex genetic and environmental interactions. The Cancer Genome Atlas (TCGA) identifies mutations that classify gastric cancer into the following subtypes: EBV-positive, microsatellite unstable/instability, genomically stable and chromosomal instability. The development of trastuzumab targeting ERBB2 is a successful example of translational genetic profiling and precision medicine applied to gastric cancer2. Such novel findings help uncover molecular mechanisms and identify effective therapeutic targets for gastric cancer.Gastric cancer is an aggressive cancer with poor prognosis and the fifth-most common cause of cancer-related death worldwide4. FGFs and FGFRs are encoded by 18 and four genes, respectively, which are expressed by diverse cell types4. Upon ligand binding, FGFRs dimerize, autophosphorylate, and recruit adaptor proteins such as FGFR substrate 24. These activate intracellular signalling pathways involved in cell growth, differentiation, and survival3. In gastric tissue, FGF10-triggered FGFR2 signalling controls stomach progenitor maintenance, morphogenesis and cellular differentiation during early epithelial growth before differentiation5. Approximately 1.2–9% of patients with gastric cancer harbour FGFR2 amplifications associated with increased tumour cell proliferation6. Oncogenic gene alternations are present in all FGFR family members in human cancers. For example, FGFR1 amplification occurs in breast, lung, gastric and bladder cancers10. FGFR2 amplification, mutations and fusions occur in breast, liver, uterine, lung, gastric cancer13. FGFR3 mutations and fusions occur in bladder and lung cancers15. In contrast, FGFR4 is infrequently mutated in cancers16. The oncogenic mutations of FGFR4, N535K/D and V550E/L, occur in rhabdomyosarcoma (RMS), although other oncogenic mutations are unknown16. The G388C mutation may be involved in tumour progression, although it is a polymorphism17.The signalling pathways activated by fibroblast growth factor receptors (FGFRs) and their cognate ligands such as fibroblast growth factors (FGFs) play important roles in development, from early embryogenesis to the formation of organsHere, we describe the identification of a novel oncogenic mutation of FGFR4 (G636C) in gastric cancer. We show that G636C is a driver mutation that leads to enhanced sensitivity to a selective FGFR inhibitor. These results provide a rational basis for designing therapies that target FGFR4 in gastric cancer.FGFR4 occur in RMS, in which FGFR4 is highly expressed16. High FGFR4 expression correlates with tumour progression and survival in patients with gastric cancer18. We therefore hypothesized that FGFR4 plays an important role in gastric cancer and that mutations that activate the protein tyrosine kinase activity of FGFR4 promote an aggressive phenotype. Accordingly, we searched for activating FGFR4 mutations of the tyrosine kinase domain in 83 gastric cancer tissue specimens. We identified a mutation in one sample (St041) at nucleotide position 1906 (NM_213647.1), which substitutes Gly with Cys at amino acid residue 636 , the Catalogue Of Somatic Mutations In Cancer , dbSNP (http://www.ncbi.nlm.nih.gov/SNP) and the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php). G636C mutation was identified in oesophagogastric junctional adenocarcinoma and registered in the COSMIC database (COSMIC v89)19. This report did not include experimental analysis19. Further, human gastric cancer cell lines harbouring the G636C mutation are not listed in COSMIC (GRCh38 CELL_LINES v89).Oncogenic mutations of 20. G636 resides in the activation loop of the FGFR4 TK domain ain Fig. . Specifiain Fig. . ThroughIn contrast, we were unable to identify X-ray structures of phosphorylated catalytically active conformations of FGFR4. Homology modelling of phosphorylated G636C-FGFR4 in a catalytically active conformation indicated that C636 is located near the αC helix and engaged in hydrophobic interactions with V523 and L526 Fig. . FurtherTo examine whether G636C mutation confers constitutive activation of FGFR4, we transduced NIH/3T3 cells with the wild-type human FGFR4 or G636C expression vector or empty vector . Western blot analysis revealed that wt-R4/3T3 and G636C-R4/3T3 cells expressed comparable levels of FGFR4. Further, the levels of autophosphorylated FGFR4 and phosphorylated AKT and ERK in G636C-R4/3T3 cells were significantly higher compared with those of wt-R4/3T3 cells Fig. . These dWe hypothesized that constitutive FGFR4 activation is associated with increased cell division. In support of this hypothesis, we found that in spheroid cell cultures, G636C-R4/3T3 underwent anchorage-independent proliferation at a significantly higher rate compared with that of wild-type FGFR4 Fig. . Consist24 with the FGFR4 expression vectors. Ectopic expression of G636C-FGFR4 conferred IL-3-independent survival and proliferation of Ba/F3 cells. Together, these data strongly support the conclusion that the G636C mutation converted FGFR4 into an oncoprotein.We next transduced IL-3-dependent Ba/F3 cellsFGFR4-G636C. To support this prediction, we treated G636C-R4/BaF3 and G636C-R4/3T3 cells with the selective FGFR inhibitor ASP587826. G636C-R4/BaF3 cells were sensitive to ASP5878  in the absence of IL-3 . In addition, G636C-R4/3T3 cells were sensitive to ASP5878   Fig. . This ef28.We next evaluated the anti-tumour activity of ASP5878 in a mouse subcutaneous xenograft model employing G636C-R4/3T3 cells. In this model, once-daily oral administration of ASP5878 (3 mg/kg) inhibited tumour growth by 60% Fig.  without 30. These discoveries led to the development of specific inhibitors having efficacious anti-tumour activities and reduced adverse effects compared to standard cytotoxic drugs. Examples include erlotinib and crizotinib, which are specific for mutant EGFRs and ALK/ROS-driven lung cancers, respectively30. Although the number of therapies targeted to driver oncogenes of gastric cancer is limited, a humanized monoclonal antibody against HER2 (trastuzumab) was approved by the United States Food and Drug Administration (USFDA) for the treatment of patients with metastatic gastric cancer that overexpress HER22. Moreover, nine selective FGFR inhibitors are now in development for the treatment for gastric cancer with FGFR2 amplification6.Recent success in molecularly-targeted therapies for cancer was made possible, in part, by identification of oncogenic gene alterationsFGFR4 in a primary gastric cancer. Computational analysis shows that G636C activated FGFR4 by disrupting the auto-inhibitory conformation and stabilizing the catalytically active conformation of its A-loop  of oop Fig. . Functio6C of oop19. Further, mutations corresponding to G636-FGFR4 include D647N-FGFR1 in lung cancer, D650H-FGFR2 in stomach cancer, D650Y-FGFR2 in endometrial cancer and D641N-FGFR3 in bladder cancer34. In particular, the tyrosine kinase activity of FGFR3-D641N is increased compared with that of the wild-type isoform35. Analysis by COSIC using Hidden Markov Models (FATHMM) software predicted that these mutations are pathogenic (FATHMM score >0.95). Although further investigation is required to confirm the oncogenicity of these mutations, these observations suggest that G636C-FGFR4 and its corresponding mutations in other members of the FGFR family may serve as common therapeutic targets across tumour type. For example, the USFDA approved a TRK inhibitor for adult and paediatric patients with cancers that harbour oncogenic NTRK fusions involving NTRK1, NTRK2 or NTRK3, which are present at low frequencies (commonly <1%) in diverse tumour types36.We identified G636C-FGFR4 in only one of 83 gastric tumour specimens. In the absence of studies of larger numbers of gastric tumours from diverse sources, this finding indicates that G636C-FGFR4 is a minor mutation in gastric cancer. G636C-FGFR4 occurs in oesophagogastric junctional adenocarcinomaFGFR1 amplification, gastric cancer with FGFR2 amplification, cholangiocarcinomas with an FGFR2 fusion, and urothelial cancers with FGFR3 alterations28. The FGFR4-specific kinase inhibitors BLU9931 and FGF401 are now in clinical trials that include patients with HCC27. The development of these FGFR inhibitors may provide effective therapies for G636C-positive gastric cancer. For example, JNJ-42756493, a potent inhibitor of the tyrosine kinase activities of the four FGFR family members, may be beneficial for treating gastric cancer with FGFR2 amplification and those that harbour the G636C-FGFR4 mutation37.The FGFR-specific inhibitors NPV-BGJ398, AZD4547 and JNJ-42756493 are under development for treating lung and breast cancers with 38. Therefore, we evaluated potential mechanisms that may explain how G636C increases the kinase activity of FGFR4  purified from primary gastric tumours were obtained from Asterand Bioscience. Written informed consent was obtained from all patients. Patient anonymity was ensured, and the study was approved by the Institutional Review Committees at Astellas Pharma, Inc. (Astellas). Experiments were performed in accordance with Astellas’ guidelines.FGFR4 was amplified from cDNA templates using PCR with the forward primer (5′-CACTGTGGCCGTCAAGATGC-3′) and reverse primer (5′-TGCTGGTTTTCTTATAGTAGTCAA-3′). Nucleotide sequencing of PCR products was performed using Big Dye Terminator chemistry (Applied Biosystems) and the forward primer (5′-ATGCTCAAAGACAACGCCTCTGAC-3′). Sequences were analysed using SeqMan Pro software . Experiments were carried out in accordance with Astellas’ guidelines.Total RNAs were reverse-transcribed using SuperScript III reverse transcriptase (Thermo Fisher Scientific). The tyrosine kinase (TK) domain of FGFR4  and G636C-FGFR4 (G636C-R4) were cloned into the pMXs-Puro retroviral vector (Cell Biolabs). NIH/3T3 cells and Ba/F3 cells were obtained from the ATCC and RIKEN Bioresource Center, respectively. Although the cell lines used in this study were not authenticated in our laboratory, they were purchased from providers of authenticated cell lines and stored at early passages in a central cell bank at Astellas. Mycoplasma testing was performed using PCR. The experiments were conducted using low-passage cultures of these stocks. NIH/3T3 cells were cultured in DMEM medium supplemented with 10% heat-inactivated FBS. Ba/F3 cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and mouse IL-3 (10 ng/mL). Wt-R4- or G636C-R4- or mock-infected NIH/3T3 cells and G636C-R4-Ba/F3 cells were generated by Astellas. The pMXs-Puro retroviral vector containing the wt-R4 or G636C-R4 gene or empty vector was transfected into Platinum-E cells using FuGENE HD transfection reagent (Roche Diagnostics) to produce virus stocks. NIH/3T3 and Ba/F3 cells were then infected with viruses with genomes harbouring each FGFR4 construct or control virus. Stable transfectants were obtained and maintained under selection pressure using puromycin at 1.5 µg/mL. G636C-R4-Ba/F3 cells were cultured in RPMI-1640 as described above but without IL-3. Experiments were carried out in accordance with institutional guidelines approved by Astellas.Human wild-type 2. The number of viable cells was determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) on days 1, 4 and 7. Experiments were carried out in accordance with Astellas’ guidelines.Mock/3T3, wt-R4/3T3 and G636C-R4/3T3 cells (1000 cells per well) were plated in DMEM supplemented with 10% heat-inactivated FBS in 96-well Sumilon Celltight spheroid plates (Sumitomo Bakelite) and then incubated at 37 °C in an atmosphere containing 5% CO50 value of ASP5878 was calculated using nonlinear regression analysis with the Sigmoid–Emax model, and geometric mean IC50 values and 95% confidence intervals (CIs) were calculated from three individual experiments. Microsoft Excel (Microsoft) and GraphPad Prism (GraphPad Software) were used for data analysis. Experiments were performed in accordance with Astellas’ guidelines.G636C-R4/BaF cells (1000 cells per well) were plated in RPMI-1640 supplemented with 10% heat-inactivated FBS with or without IL-3 (10 ng/mL) in 96-well plates. G636C-R4/3T3 cells (2000 cells per well) were plated in DMEM supplemented with 10% heat-inactivated FBS in PrimeSurface96U plates (Sumitomo Bakelite). The cells were treated with ASP5878 at 0–10,000 nM  for 4 or 5 days, and the number of viable cells was determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). The ICNIH/3T3 cells were lysed with cell lysis buffer containing phosphatase and protease inhibitors, and the levels of FGFR4, phospho-FGFR4, phospho-ERK1/2, phospho-AKT and β-actin were determined using immunoblotting. The antibodies were as follows: anti-FGFR4 from Santa Cruz Biotechnology, Inc. ; and anti-phospho-FGFR (Tyr653/654), anti-phospho-ERK (Thr202/Tyr204), anti-phospho-AKT, anti-β-actin and HRP-conjugated anti-rabbit IgG from Cell Signaling Technology . All antibodies were diluted 1/2000 for use. Proteins of interest were visualized by enhanced chemiluminescence using the Western Lightning Plus-ECL (PerkinElmer) and detected with LAS-4000 . Cell lysis buffer was purchased from Cell Signaling Technology, phosphatase inhibitor cocktail from Thermo Scientific  and protease inhibitor cocktail from Roche Diagnostics . Experiments were performed in accordance with Astellas’ guidelines.20. An X-ray structure of the phosphorylated catalytically active form of FGFR4 with ATP or ATP analogues is not available, to our knowledge. Therefore, the coordinates of the active conformation of G636C-FGFR4 were modelled according to the phosphorylated- and ATP analogue-bound active form of FGFR1 (PDB ID: 3GQI)40. The coordinates of 3GQI were used as a template for homology modelling of the active form of FGFR4 using the modelling software MOE . Phosphate moieties of Y642 and Y643 were added after homology modelling, and energy minimization of the coordinates of these residues was performed to relax the coordinates. Further energy minimization including R635 and G636C was performed, because an Arg residue corresponding to R635 forms hydrogen bonds with a phosphorylated Tyr residue corresponding to Y643 of other FGFR isoforms42.The amino acid sequence of the kinase domain of wild-type FGFR4 was obtained from the UniProt database . Substituting G636 with Cys generated the mutant G636C-FGFR4. The coordinates of the auto-inhibitory conformation of FGFR4 are registered in the protein database (PDB) (PDB ID: 4TYE)22, whose chemical structure is similar to that of ASP5878. Sequentially substituting or removing atoms and bonds of BLU9931 in 4XCU from the atoms binding to the back of the binding pocket generated the initial coordinates of FGFR4 bound to ASP5878. Repetitive optimization calculations of the coordinates were performed using energy minimization, and the atoms of the proteins were fixed using MOE. Finally, the coordinates of the residues within 4.5 angstroms from ASP5878 were repeatedly optimized until the energies converged. The coordinates of ASP5878-G636C-FGFR4 were introduced using homology modelling by MOE that were based on the coordinates of wild-type ASP5878-FGFR4. During the homology modelling, the coordinates of the atoms were initially assigned according to the template structure and finally optimized to minimize the energy level, which reflected the effect of the mutated residue on the coordinates near the residue as well as those of the binding pocket. To visualise the molecules, hydrogen atoms were added using Protonate3D included in MOE.The coordinates of ASP5878-bound wild-type FGFR4 were modelled according to the coordinates of wild-type FGFR4 bound to the FGFR kinase inhibitor BLU9931 (PDB ID: 4XCU)43) was synthesized in our laboratory. Experiments were performed in accordance with Astellas’ guidelines. ASP5878 was dissolved in dimethyl sulfoxide or 0.5% methylcellulose (MC) for in vitro or in vivo experiments, respectively.ASP5878  were obtained from Charles River Japan.6 cells mixed with Matrigel/PBS ([Matrigel:PBS = 1:1]) was subcutaneously inoculated into the flanks of mice. Tumour volume was calculated as follows: V = length × width2 × 0.5. Matrigel was purchased from Corning Incorporated (Life Sciences).Mock/3T3, wt-R4/3T3 and G636C-R4/3T3 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, and 0.1 ml of 3 × 106 cells/0.1 mL [matrigel:PBS = 1:1]/mouse). Seven days after inoculation, mice with tumours were divided such that the mean tumour volume was similar among the groups on the first day (day 0) of treatment. ASP5878 (3 mg/kg) was orally administered once daily for 8 days. Tumour diameters were measured using a caliper on days 0, 2, 4, and 7, and tumour volume was determined by calculating the volume of an ellipsoid as follows: length × width2 × 0.5. Body weight was measured using a standard balance.G636C-R4/3T3 cells were subcutaneously inoculated into the flanks of mice  and GraphPad Prism (GraphPad Software) were used for data analysis.The values for the mouse xenograft model are expressed as the mean ± SEM. Differences between groups were analysed using the Student Supplementary information


A simple and sustainable production of nanoplatelet graphite at low cost is presented using carbon-based materials, including the recycled lead-graphite pencils. In this work, exfoliated graphite nanoplatelets (EGNs), ball-milled exfoliated graphite nanoplatelets (BMEGNs) and recycled lead-graphite pencils (recycled 2B), as well as thermally cured polydimethylsiloxane (PDMS), are used to fabricate highly stretchable thermal-interface materials (TIMs) with good thermally conductive and mechanically robust properties. Several characterization techniques including scanning electron microscopy (SEM) and thermogravimetric analysis (TGA) showed that recycled nanoplatelet graphite with lateral size of tens of micrometers can be reliably produced. Experimentally, the thermal conductivity was measured for EGNs, BMEGNs and recycled 2B fillers with/without the effect of ball milling. The in-plane thermal conductivities of 12.97 W/mK (EGN), 13.53 W/mK (recycled 2B) and 14.56 W/mK (BMEGN) and through-plane thermal conductivities of 0.76 W/mK (EGN), 0.84 W/mK (recycled 2B) and 0.95 W/mK (BMEGN) were experimentally measured. Anisotropies were calculated as 15.31, 15.98 and 16.95 for EGN, recycled 2B and BMEGN, respectively. In addition, the mechanical robustness of the developed TIMs is such that they are capable of repeatedly bending at 180 degrees with outstanding flexibility, including the low-cost renewable material of recycled lead-graphite pencils. For heat dissipating application in high-power electronics, the TIMs of recycled 2B are capable of effectively reducing temperatures to approximately 6.2 °C as favorably compared with thermal grease alone. A previous study  indicateA schematic representation and the optical image of recycled nanoplatelet graphite by ball-milling of recycled 2B is shown in Experimentally, recycled 2B was selected in this study for the filler material, together with commercially available nanoplatelet graphite. Initially, the ball milling of recycled 2B to produce recycled nanoplatelet graphite was carried out in a ball-mill machine . 2.0 g of recycled 2B was typically charged into a stainless steel capsule (25 mL) with zirconia beads of 3 mm in diameter. The container was vibrated at a frequency of 20 Hz for durations of approximately 4 h, then carefully collected and washed with the solution of water and ethanol. The final products were dried in vacuum oven at 60 °C under a reduced pressure (~93 kPa) for 4 h to yield 1.08 g of recycled 2B. The ball-milling process is performed at dry ambient environment.The schematic of the screen-printing technique to functionally facilitate the spreading and bonding process of TIMs onto an aluminum heat sink in high-power applications is presented in The morphologies of EGN, recycled 2B and BMEGN were analyzed by scanning electron microscopy  at room temperature and atmospheric conditions. Thermogravimetric analysis (TGA) was measured in the temperature range from 50 to 1000 °C with a 200 °C/min ramp rate and a 4 °C/min resolution using TG–DTA . The through-plane thermal conductivity was measured by a plane heat source (hot plate) method as described by ASTM D5470-06  with theThe recycled 2B composite (10 vol % recycled 2B loading) is black a and mec−1, 1580 cm−1 and 2700 cm−1, respectively). The spectral feature of graphene can be identified as the G-band (1580 cm−1) [−1) are calculated as 27.2, 30.3 and 28.2 cm−1, respectively, for EGN, recycled 2B and BMEGN samples, indicating that the degree of graphitization is characteristically similar. Moreover, the previous work demonstrated that G and 2D Raman peaks could be affected by the number of graphene layers due to the evolution of the electronic structure and electron–phonon interactions [80 cm−1) . From thractions .T20% (temperature at 20 wt % loss) are 527.6 °C, 516.4 °C and 515.9 °C for BMEGN, recycled 2B and EGN, respectively. This suggests that the BMEGN composite has the highest thermal stability of all tested samples. The recycled 2B composites pose a relatively higher heat capacity as compared to their EGN counterpart.The thermal stability of the recycled 2B composite is shown in K┴) and in-plane (K//) directions are experimentally measured. K┴) [K//) measurement setup [K// increases from 12.97 W/mK (EGN) to 13.53 W/mK (recycled 2B), as compared with the BMEGN samples with the highest in-plane thermal conductivity of 14.56 W/mK. The relatively lower thermal conductivity of recycled 2B samples  is primarily attributed to impurities existing in the original 2B pencils: C (51.4%), Si (13.9%), O (20.3%), Fe (5.5%), Al (5.0%) and Ca (1.0%) [K┴) is comparatively small while the trend is the same, that is, the K┴ increases from 0.76 W/mK (EGN) to 0.84 W/mK (recycled 2B) and 0.95 W/mK (BMEGN) for ball-milled composites. Anisotropy in the thermal conductivities was experimentally observed  ,38. Figunt setup . Figure a (1.0%) . On the observed d as measGE voltage transmits across an IGBT inverter module at the pulse frequency of 20 kHz. The current loading and the pulsing frequency are mimicking the operating conditions in renewable energy sources such as wind power stations. The designed IGBT specification of electrical output has a current loading of a maximum up to 100 A at 600 V. In the actual experiments of standalone operation, VAC) and current (IAC), that is, the switching frequency/power at 60 Hz/2.0 kW, the output voltage is 270 V (Vp-p ~ 550 V) and output current is 9.8 A (Ip-p ~ 19.7 A). The output voltage and current perform a well-regulated sinusoidal waveform. As shown in RC1 and RC2, respectively  increases from 12.97 W/mK (EGN) to 13.53 W/mK  after four hours of ball milling. In summary, ball milling offers the potential of preparing TIM composites at low cost and with an easily scalable method. For the IGBT tests, the results indicate that the composite of recycled 2B is capable of effectively reducing temperatures by approximately 6.2 °C as favorably compared with thermal grease. This work provides new insights into the relationship between sample preparation methods and the formation of highly efficient conductive paths of polymer composites.Graphite-based materials, such as lead-graphite pencils, have been widely used and discarded easily due to their low cost. The present work shows the commercial potential of thermally conductive recycled 2B composites that can be ball milled with homogenously dispersed fillers into the matrix and directly applied to TIMs. The thermal conductivities of composites with recycled 2B filler were characterized by SEM and TGA. The mechanical (bending test) and thermal performance of three types of carbon filler  dispersed in epoxy resin were experimentally investigated. The recycled 2B is a renewable low-cost source, and ball milling can transform the initial bulk morphology into a spherical form. Therefore, the thermal conductivity of recycled 2B can be enhanced via uniformly dispersed particles (increasing the specific surface area). Fundamentally, ball milling can be effective in enhancing the mechanical mixing processes between a polymer matrix and embedded recycled 2B, such that filler agglomerates can be fragmented segments with a homogeneous dispersion inside the matrix. The measured in-plane thermal conductivity (


We found that high dose 25-hydroxyvitamin D3 (25(OH)D3) to weaned pigs would alleviate porcine epidemic diarrhea virus (PEDV) infection and immune response. Forty-two weaned pigs were allotted to 1 of 6 dietary 25(OH)D3 treatments D3/kg diet) for 26 days. On day 22 of the trial, all the treatments were orally administrated with PEDV except for one of the 5.5 μg 25(OH)D3/kg treatments, which was challenged with the same volume of sterile saline and served as control. Another 5.5 μg 25(OH)D3/kg group for PEDV challenge was named CON-PEDV. Average daily gain (p < 0.05) was reduced by PEDV infection. PEDV administration also induced severe diarrhea (p < 0.05), reduction of villous height and the ratio of villous height to crypt depth, and increase of crypt depth and serum diamine oxidase activity (p < 0.05). Serum IgM and complement component 4 levels were increased by PEDV challenge. However, 155.5 μg 25(OH)D3/kg supplementation alleviated intestinal damage (p < 0.05) compared with CON-PEDV. Furthermore, 155.5 μg 25(OH)D3/kg supplementation downregulated the mRNA abundance of inflammatory cytokines and interferon signal pathway-related genes (p < 0.05) compared with CON-PEDV. These results suggested that dietary supplementation of 155.5 μg 25(OH)D3/kg could alleviate intestinal damage and protect against PEDV-induced inflammatory status.We conducted this experiment to determine if feeding 25-hydroxyvitamin D Porcine epidemic diarrhea virus (PEDV) infection causes severe damage to the intestinal function and barrier integrity of pigs , leading3 might ease the infection of PEDV in pigs. However, studies on the feasibility of vitamin D3 as anti-PEDV infection agent in piglets are scarce.Vitamin D not only enhances calcium and phosphate absorption but also regulates immune function . Previou3 is higher than vitamin D3 [3 was used to investigate whether it could alleviate PEVD-infected diarrhea and intestinal injury.It is generally believed that the bioavailability of 25(OH)Dtamin D3 ,12,13. IThe experimental protocol involved in the present study was approved by the Animal Care Advisory Committee of Sichuan Agricultural University .3/kg. Each treatment consisted of four gilts and three barrows. At day 22 of this study, all the treatments were orally administrated with 35 mL of PEDV (5.6 × 103 TCID50/mL) except for one of the 5.5 μg 25(OH)D3/kg  treatment, which was served as control (CON) and administrated with the same volume of sterile saline. Another 5.5 μg 25(OH)D3/kg treatment for PEDV challenge was named by CON-PEDV. In order to prevent infection, the CON groups were housed in the next room of challenge groups. The settings for the two rooms were the same. Pigs in the CON group were negative for PEDV throughout the trial period. As shown in 3, which was not prepared in the vitamin premix. Each treatment was formed by supplementing with indicated 25(OH)D3 levels in the basal diet. All pigs had ad libitum access to water and experimental diets throughout the trial. 25(OH)D3 (Hy-D) was kindly provided by DSM Nutritional Products Ltd. Shanghai, China. The PEDV was kindly presented by professor Zhiwen Xu, College of Veterinary Medicine, Sichuan Agricultural University. Average daily gain (ADG) and average daily feed intake (ADFI) were determined via weighing to determine body weight and recording of feed intake.Forty-two crossbred healthy weaned pigs  with an initially body weight (BW) of 6.61 ± 0.41 kg were used in the 26 days trial. On the first day of the trial, all pigs were allotted on the basis of BW to six groups and each fed diets supplemented with either 5.5, 5.5, 43.0, 80.5, 118.0, or 155.5 μg 25(OH)DAfter the PEDV challenge, the fecal consistency and diarrhea incidence were assessed every day according to Hu et al. : 1 = harDiarrhea rate (%) = (A/5d) × 100, in which A = total days per pig with diarrhea after PEDV challenge.Mean cumulative score = ∑A/B, in which A = the sum of daily scores, and B = pigs per treatment.g for 15 min, and the serum samples were stored at −20 °C until analysis. All pigs were then euthanized by intramuscular injection of Shumianning . About 2 cm jejunal tissue sample was stored in 4% paraformaldehyde solution for histological analysis. Mucosal samples from the middle jejunum were scraped and rapidly frozen in liquid nitrogen, and then stored at −80 °C for further analysis.All pigs were bled via anterior vein on 27 day. The blood was used for extracting serum via centrifugation at 3000 The concentration of IgG, IgM, and complement component 3 (C3) and C4  in serum were detected by automatic biochemical analyzer . Immunology multiple control were performed before sample determination to ensure the outcomes were correct.After being embedded in paraffin, the jejunal samples were stained with hematoxylin and eosin for intestinal morphology measurement. A minimum of 20 well-orientated villi and crypts from each intestinal sample of pigs were measured using Image-Pro Plus 6.0 software. As a measurement of intestinal permeability, serum diamine oxidase activity (DAO) was detected using commercial assay kits  following the protocols of the manufacturer.β-actin.Total RNA was extracted from the mucosa of jejunum tissue using TRIzol reagent . Reverse transcription was performed with RNA using a PrimeScript RT reagent kit . The mRNA expression of genes of interest were quantified using an ABI 7900HT detection system  and the SYBR Premix Ex Taq II with ROX reagents . The primer sequences used for RT-PCR are listed in Protein sample processing was performed according to Zhang et al. . Brieflyt-test. On the condition of PEDV challenge, the data were performed using the PROC MIXED SAS 9.4 procedure according to the following model:p < 0.05 and trends at p < 0.10.In this study, each pig was used as the experimental unit. The differences between CON and CON-PEDV group were assayed by Student’s p < 0.05) and ADFI (p = 0.08) compared with CON. However, the performance was not influenced among the different 25(OH)D3 supplementation groups. PEDV infection induced severe diarrhea in piglets (p < 0.05). However, the dietary supplementation of 25(OH)D3 decrease diarrhea scores (p < 0.05) and diarrhea rate (p < 0.1).As shown in hea rate , p < 0.1p < 0.05, 3 supplementation had no effect on serum IgM, IgG, and C4 levels on the condition of PEDV challenge. In addition, dietary supplementation of 155.5 μg 25(OH)D3/kg decreased serum C3 levels compared with the 118.0 μg 25(OH)D3/kg group. PEDV infection increased serum IgM and C4 concentrations compared with CON (p < 0.05). However, 118.0 and 155.5 μg 25(OH)D3/kg treatments significantly increased villous height and VCR and decreased crypt depth compared with the CON-PEDV group (p < 0.05). Furthermore, dietary supplementation of 25(OH)D3 increased villous height and VCR and decreased crypt depth in a linear way (p < 0.05).Compared with CON, PEDV challenge decreased villous height and the ratio of villous height to crypt depth (VCR), and increased the crypt depth of jejunum in the CON-PEDV group (p < 0.05). However, the dietary supplementation of 25(OH)D3 tended to decrease serum DAO activity (p < 0.1).In addition, serum DAO activity of pigs in the CON-PEDV group was significantly increased compared with CON . 25(OH)D3 also tended to increase MUC2 and mRNA expression (p < 0.1). Interestingly, PEDV increased sucrase-isomaltase (SI) and occludin protein levels, but high doses 25(OH)D3 (155.5 μg 25(OH)D3/kg) inhibited these increase . However, a high dose of dietary 25(OH)D3 supplementation decreased TLR2, TLR9, MyD88, IL6, IL8, IFNλ1, STAT1, MxA, IFNAR1, and TRIF mRNA expression compared with CON-PEDV (p < 0.05).PEDV induced a significant increase in the mRNA expression of 3 could alleviate PEDV infection.Early studies suggested that vitamin D exerts broad-spectrum antiviral effects, including inhibiting the replication of dengue virus , hepatit3 levels linearly increased serum 25(OH)D3 concentrations, but no treatment effects were observed in the growth performance of weaned pigs [3 supplementation decreased diarrhea scores, and 155.5 μg 25(OH)D3/kg treatment showed the minimum diarrhea scores and diarrhea rate, which indicated that supplementation with a high dose of 25(OH)D3 might alleviate the symptoms of PEDV infection.We have previously demonstrated that increasing dietary 25(OH)Dned pigs . In the ned pigs ,22,23. W3 supplementation had no effect on serum C4 and IgM levels under the conditions of PEDV challenge. We inferred that 25(OH)D3 might exert an antiviral effect on intestinal mucosa, which is the main target of PEDV replication [The complement system is a part of the innate immune system, and not only participates in inflammation but also enhances the adaptive immune response . In thislication .3/kg 25(OH)D3 treatment was shown to be optimal in alleviating intestinal injury induced by PEDV. Therefore, this may be the reason why the 155.5 μg 25(OH)D3/kg treatment showed the minimum diarrhea scores and diarrhea rate among the treatments. Moreover, we found that claudin2 gene expression showed a linear response with increasing dietary 25(OH)D3 concentration, and 25(OH)D3 tended to increase MUC2 expression under the conditions of PEDV challenge. It suggested that high dose 25(OH)D3 supplementation could improve tight junction protein expression to maintain intestinal barrier integrity. Interestingly, levels of SI, an intestinal absorptive cell marker, were significantly increased by PEDV challenge. A previous study has shown that PEDV infection decreased goblet cells in intestinal villous [3 supplementation significantly reduced the SI expression induced by PEDV, which might be beneficial for maintaining normal intestinal function. We also found occludin expression was increased with PEDV challenge compared with CON, but 118.0 and 155.5 μg 25(OH)D3/kg groups inhibited this increase. Luo et al. [3/kg supplementation might ease PEDV infection through decreasing occludin expression.PEDV infection always leads to morphological changes of the small intestine with a reduction in villus height and damage to intestinal integrity ,27. In t villous . Hence, o et al.  demonstrIFNλ expression in the jejunum mucosa. This indicates that the IFNs signaling pathway was activated by PEDV in the intestine of piglets. Since the high dose of 25(OH)D3 supplementation showed a better protective effect than the low dose groups, we investigated whether high dose supplementation could alleviate PEDV infection by regulating immunity. We found that a high dose of 25(OH)D3 inhibited the PRR, IFNλ, STAT1, and MxA expression. It was suggested that dietary 25(OH)D3 supplementation inhibited the activation of intestinal immunity induced by PEDV. Previous studies have shown that vitamin D attenuated rotavirus infection and reduced the viability of Mycobacterium tuberculosis through regulating autophagy and cathelicidin [3 might be due to decreased PEDV replication. In addition, we also found that high doses of 25(OH)D3 inhibited jejunal mucosa IL6 and IL8 mRNA expression compared with CON-PEDV. This suggested that high dose 25(OH)D3 supplementation might inhibit intestinal inflammation induced by PEDV. Reducing the expression of intestinal inflammatory cytokines is also beneficial in maintaining normal intestinal function.Innate immune response plays an important role in defense against viral infections in mammalian cells. During viral infection, the virus is recognized by pattern-recognition receptors (PRRs) including toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIGI) or melanoma differentiation gene 5 (MDA5), then INFs and proinflammatory cytokines are produced for initiation of the inherent antiviral immune response ,32. Diffelicidin ,34. Ther3 alleviated the severity of diarrhea of piglets infected with PEDV by improving the intestinal structure and immune response, and maintaining regular intestinal function.In summary, the results of the current study indicate that dietary supplementation of 155.5 μg/kg 25(OH)D