In addition, xenin potentiates the biological actions of GIP. The present study assessed the actions and therapeutic utility of a (DAla2)GIP/xenin-8-Gln was assessed in BRIN-BD11 beta cells. Acute and persistent glucose-lowering and insulin-releasing effects were then examined in vivo. Finally, the metabolic benefits of twice daily injection of (DAla2)GIP/xenin-8-Gln was determined in high-fat-fed mice.Following confirmation of enzymatic stability, insulin secretory activity of (DAlap < 0.05 to p < 0.001) enhanced in vitro insulin secretion from pancreatic clonal BRIN-BD11 cells, with xenin (and particularly GIP)-related signalling pathways, being important for this action. Administration of (DAla2)GIP or (DAla2)GIP/xenin-8-Gln in combination with glucose significantly (p < 0.05) lowered blood glucose and increased plasma insulin in mice, with a protracted response of up to 4 h. All treatments elicited appetite-suppressive effects (p < 0.05), particularly (DAla2)GIP/xenin-8-Gln and xenin-8-Gln at elevated doses of 250 nmol/kg. Twice-daily administration of (DAla2)GIP/xenin-8-Gln or (DAla2)GIP for 21 days to high-fat-fed mice returned circulating blood glucose to lean control levels. In addition, (DAla2)GIP/xenin-8-Gln treatment significantly (p < 0.05) reduced glycaemic levels during a 24 h glucose profile assessment. Neither of the treatment regimens had an effect on body weight, energy intake or circulating insulin concentrations. However, insulin sensitivity was significantly (p < 0.001) improved by both treatments. Interestingly, GIP-mediated glucose-lowering (p < 0.05) and insulin-releasing (p < 0.05 to p < 0.01) effects were substantially improved by (DAla2)GIP and (DAla2)GIP/xenin-8-Gln treatment. Pancreatic islet and beta cell area (p < 0.001), as well as pancreatic insulin content (p < 0.05), were augmented in (DAla2)GIP/xenin-8-Gln-treated mice, related to enhanced proliferation and decreased apoptosis of beta cells, whereas (DAla2)GIP evoked increases (p < 0.05 to p < 0.01) in islet number.All peptides significantly  contains peer-reviewed but unedited supplementary material, which is available to authorised users. A defect in the postprandial insulin-secretory incretin response, mediated by the gut hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), is a specific pathophysiological characteristic of type 2 diabetes . In contNear normalisation of blood glucose levels has been shown to restore the insulin-secretory effect of GIP in both animal models of type 2 diabetes  and in h2)GIP/xenin-8-Gln, by linking GIP(1-14) to xenin-8-Gln, retaining the regions of each peptide known to be important for biological activity  DPP-4 degradation profiles  were assessed as described previously . Xenin-b2)GIP/xenin-8-Gln-induced insulin secretion in BRIN-BD11 cells. Cells were incubated in 5.6 mmol/l glucose with (DAla2)GIP/xenin-8-Gln (10−6 mol/l) alone or in combination with the GIP receptor antagonist GIP(6-30)Cex-K40[Pal]   , that were derived from a nucleus colony obtained from the National Institutes of Health, Bethesda, Maryland. Longer-term experiments were conducted with male high-fat-fed NIH Swiss mice. For further details of mice age and maintenance please see 2)GIP/xenin-8-Gln-induced glucose-lowering and insulin-secretory actions in mice, (DAla2)GIP/xenin-8-Gln was administered by i.p. injection (25 nmol/kg body weight) in combination with glucose (18 mmol/kg) in the absence and presence of GIP(6-30)Cex-K40[Pal] and SR142948A (both at 25 nmol/kg), and glucose and insulin were measured as described above. In a final series of experiments, test peptides (25 nmol/kg) or saline vehicle (0.9% wt/vol. NaCl) were injected 4 h before a glucose load (18 mmol/kg) in non-fasted mice and blood glucose and plasma insulin measured as above.Specific information on acute feeding, glucose homeostasis and insulin-secretory studies is given in ESM 2)GIP or (DAla2)GIP/xenin-8-Gln hybrid (both at 25 nmol/kg) were administered for 21 days in high-fat-fed mice. Energy intake and body weight were monitored daily and non-fasting blood glucose and plasma insulin concentrations were assessed at 3 day intervals. On day 21, a non-fasted 24 h glucose profile was conducted. At the end of the treatment period, i.p. glucose tolerance (18 mmol/kg), biological response to GIP  and insulin sensitivity  tests were performed. Terminal analysis included measurement of total body fat and lean mass by dual-energy X-ray absorptiometry (DEXA) scanning  and extraction of pancreatic tissue for analysis as detailed below.Twice-daily (09:30 and 17:30 hours) i.p. injections of saline vehicle, , as described previously , or procBlood samples were collected from the cut tip on the tail vein of conscious mice into chilled fluoride/heparin glucose micro-centrifuge tubes . Blood glucose was measured directly using a hand-held Ascencia Contour blood glucose meter . Plasma and pancreatic insulin was assayed by a modified dextran-coated charcoal radioimmunoassay .p < 0.05.Statistical analysis was performed using GraphPad PRISM . For in vitro, islet histology and pancreatic insulin content studies, all samples were numbered and blinded. For acute and subchronic metabolic tests, there was no blinding. There were no inclusion and exclusion criteria applied. Results are expressed as means ± SEM and data were compared using repeated measures ANOVA followed by the Student–Newman–Keuls post hoc test. Groups of data were considered to be significantly different if 2)GIP/xenin-8-Gln and (DAla2)GIP remained fully intact when incubated in the presence of DPP-4 for up to 12 h (ESM Table p < 0.05 to p < 0.001) stimulated insulin secretion from BRIN-BD11 cells at a concentration of 10−6 mol/l GIP evoked a significant (p < 0.01 to p < 0.001) increase in insulin release at 10−8 mol/l when compared with respective glucose controls GIP-mediated elevation of insulin secretion, while the neurotensin receptor inhibitor SR142948A had a similar, but less prominent, effect on xenin-8-Gln  following co-culture of xenin-8-Gln and SR142948A when compared with 5.6 mmol/l glucose control GIP/xenin-8-Gln was completely inhibited (p < 0.001) by GIP(6-30)Cex-K40[Pal] and partially reversed (p < 0.05) by SR142948A GIP, xenin-8-Gln nor (DAla2)GIP/xenin-8-Gln had any effect on intracellular Ca2+ concentrations GIP/xenin-8-Gln induced a significant (p < 0.05) reduction in food intake at 30 min post injection when compared with saline controls GIP induced significant (p < 0.05 to p < 0.01) reductions in food intake at 180 min post injection  superior to control mice at 90 min post injection, while xenin-8-Gln and (DAla2)GIP/xenin-8-Gln also evoked significant (p < 0.05) reductions in food intake at 120 min GIP or (DAla2)GIP/xenin-8-Gln concomitantly with glucose resulted in significantly (p < 0.05) lowered blood glucose values at 30 min post injection, culminating in significantly (p < 0.05) decreased overall AUC blood glucose values when compared with controls GIP also induced a significant (p < 0.05) reduction in blood glucose levels 15 min post injection  increased in all treatment groups compared with controls  acute glucose-lowering effect of (DAla2)GIP/xenin-8-Gln was blocked by both GIP(6-30)Cex-K40[Pal] and SR142948A GIP/xenin-8-Gln-induced insulinotropic actions GIP and (DAla2)GIP/xenin-8-Gln significantly reduced the overall AUC for blood glucose (p < 0.05) and increased plasma insulin AUC (p < 0.05) following a glucose challenge ion Fig. . Correspols Fig. . The ben48A Fig. , whereasons Fig. . When adons Fig. . Howevernge Fig. .Fig. 2Ac2)GIP or (DAla2)GIP/xenin-8-Gln for 21 days to high-fat-fed mice had no significant effect on cumulative energy intake or body weight compared with high-fat-fed controls  body weight and cumulative energy intake compared with lean controls GIP/xenin-8-Gln-treated high-fat fed mice over the 21 days, being significantly (p < 0.05 to p < 0.01) reduced on observation days 3, 6 and 15 compared with levels in control high-fat mice GIP- and (DAla2)GIP/xenin-8-Gln-treated mice had glucose levels that were not significantly different from lean controls from day 12 onwards GIP/xenin-8-Gln treated mice had similar blood glucose levels to lean controls at each observation point and significantly (p < 0.05) reduced levels compared with high-fat-fed controls at 17:00 hours GIP-treated mice were also reduced, but not to the same extent as in (DAla2)GIP/xenin-8-Gln mice GIP or (DAla2)GIP/xenin-8-Gln did have reduced plasma insulin levels compared with high-fat-fed controls on day 21 but the difference did not reach statistical significance GIP or (DAla2)GIP/xenin-8-Gln for 21 days reduced overall AUC for blood glucose levels of high-fat-fed mice following a glucose load but the difference failed to reach statistical significance (Table 2)GIP or (DAla2)GIP/xenin-8-Gln treatment compared with high-fat controls (Table p < 0.05 to p < 0.001) increased glycaemic excursion and overall insulin-secretory response compared with lean controls (Table 2)GIP and (DAla2)GIP/xenin-8-Gln treatment significantly (p < 0.05) improved the glucose-lowering and insulin-releasing actions of native GIP; overall blood glucose AUC values were significantly (p < 0.05) decreased by 36% and 30%, respectively, compared with saline-treated control. Moreover, AUCs for glycaemic values were not different when compared with lean controls in response to administration of GIP in combination with glucose (Table p < 0.05 to p < 0.01) augmented in (DAla2)GIP- and (DAla2)GIP/xenin-8-Gln-treated high-fat-fed mice compared with high-fat-fed controls (Table Treatment with (DAla2)GIP- and (DAla2)GIP/xenin-8-Gln-treated high-fat-fed mice following administration of exogenous insulin  improved in (DAla2)GIP- and (DAla2)GIP/xenin-8-Gln-treated mice compared with control high-fat mice GIP/xenin-8-Gln-treated mice was augmented (p < 0.05) compared with that of lean controls but was not significantly different from that of high-fat controls GIP-treated mice was similar to that of lean control mice  increased islet area compared with lean controls  increases in both beta and alpha cell areas GIP had no significant effect on overall pancreatic islet area or beta cell area but resulted in decreased (p < 0.001) alpha cell area compared with high-fat-fed controls GIP/xenin-8-Gln significantly (p < 0.001) reduced pancreatic alpha cell area compared with high-fat-fed controls  pancreatic islet and beta cell area compared with lean controls  augmented in (DAla2)GIP-treated mice compared with all other groups  decreased the number of small islets and increased the number of large islets, compared with lean controls GIP/xenin-8-Gln-treated high-fat-fed mice also had a decreased (p < 0.001) number of small- and medium-sized islets, and fewer (p < 0.001) large islets, when compared with lean controls GIP/xenin-8-Gln-treated high-fat-fed mice was significantly (p < 0.05 to p < 0.001) increased when compared with the rate in all other groups  decreased in (DAla2)GIP- and (DAla2)GIP/xenin-8-Gln-treated high-fat-fed mice compared with high-fat-fed controls, and was similar to levels in lean control mice ols Fig. . Represeups Fig. . In addiice Fig. .Fig. 5Ef2)GIP/xenin-8-Gln.Despite encouraging preclinical data and the success of DPP-4 inhibitors , 35, pro2)GIP and the hybrid peptide (DAla2)GIP/xenin-8-Gln were completely resistant to degradation by DPP-4, indicating that the N-terminal d-Ala2 modification employed effectively masked the DPP-4 binding site. As expected, the parent peptides, (DAla2)GIP and xenin-8-Gln, stimulated insulin release from clonal pancreatic BRIN-BD11 beta cells at physiological and elevated glucose concentrations  [2)GIP/xenin-8-Gln was equally effective in vitro as an insulin secretagogue as either of the parent peptides. Its insulinotropic effects appeared to be more dependent on activation of GIP receptor-related cell signalling pathways, when compared with xenin. In full agreement, the acute in vivo insulinotropic actions of (DAla2)GIP/xenin-8-Gln were also chiefly dependent upon activation of GIP receptor pathways. Interestingly, the acute glucose-lowering actions of (DAla2)GIP/xenin-8-Gln appeared to be associated with activation of both GIP and xenin pathways, suggesting possible insulin-independent glucose-lowering effects of xenin [2)GIP/xenin-8-Gln likely relates to enhancement of GIP-mediated biological actions, as demonstrated here and previously [2)GIP/xenin-8-Gln-induced insulin release is necessary, but similar (DAla2)GIP/xenin-8-Gln-induced acute insulin-secretory effects were also noted in lean control mice, confirming biological efficacy. Our previous studies using clonal beta cells have shown that (DAla2)GIP activates adenylate cyclase pathways, whereas xenin-based peptides likely operate through the action of phospholipase C [2)GIP/xenin-8-Gln nor its parent peptides significantly affected intracellular Ca2+ levels in BRIN-BD11 beta cells.In harmony with previous studies , 29, GIP/xenin-8-Gln even at the lowest dose examined and especially at elevated doses. This is in agreement with previous findings suggesting that elevated doses of xenin are required to impart significant appetite-suppressive activity, whereas lower doses have beneficial metabolic effects [2)GIP/xenin-8-Gln was then assessed. Even 4 h after a single injection of (DAla2)GIP/xenin-8-Gln or (DAla2)GIP, glucose-lowering and insulin-releasing actions were clearly evident. This presumably relates to enhanced enzymatic stability of (DAla2) modified GIP-forms and corresponds well with the findings of other studies [2)GIP/xenin-8-Gln in plasma would be useful to determine the pharmacokinetic profile of the hybrid peptide. Moreover, methodology such as peptide acylation or PEGylation may help to extend the biological half-life of (DAla2)GIP/xenin-8-Gln [2)GIP/xenin-8-Gln were equal, or indeed enhanced, when compared with either parent peptide administered at the same dose. This indicates that the hybrid peptide still fully retained the ability to activate GIP- and xenin-related cellular signalling pathways involved in glucose homeostasis, insulin secretion and appetite suppression [In accordance with preserved bioactivity of the hybrid peptide, we observed satiety effects of GIP resulted in sustained and significant reductions of circulating blood glucose levels, both in terms of individual observation points and during assessment of a 24 h blood glucose profile [2)GIP/xenin-8-Gln had equal or improved beneficial effects on glycaemic status in high-fat mice. Indeed, reductions in glucose levels were much more apparent in mice treated with (DAla2)GIP/xenin-8-Gln during assessment of the more-in-depth 24 h blood glucose profile. Interestingly, significantly greater reductions in insulin-induced blood glucose levels were observed in (DAla2)GIP/xenin-8-Gln- and (DAla2)GIP-treated mice. This clearly points towards beneficial effects of both treatments that are additional to effects on pancreatic beta cell function. This improvement of insulin action was not a consequence of reduced adipose tissue mass. Thus, it presumably reflects alleviation of glucotoxicity, or the actions of GIP to improve insulin resistance [2)GIP/xenin-8-Gln had no effect on energy intake, although similar observations have been reported previously with xenin-based drugs [Chronic treatment of high-fat-fed mice with GIP/xenin-8-Gln concerned the stimulation of beta cell function [2)GIP/xenin-8-Gln-treated high-fat-fed mice. In agreement, pancreatic islet and beta cell areas were increased in (DAla2)GIP/xenin-8-Gln-treated mice, in line with the proliferative and anti-apoptotic beta cell actions of (DAla2)GIP/xenin-8-Gln, as has previously been reported for GIP [2)GIP/xenin-8-Gln. Moreover, GIP is known to stimulate GLP-1 secretion from the gut [2)GIP/xenin-8-Gln-treated mice, the insulin-secretory and subsequent glucose-lowering effects of native GIP were substantially enhanced in these mice. As expected, the biological action of GIP was severely perturbed in high-fat-fed mice, akin to the situation in patients with type 2 diabetes [2)GIP/xenin-8-Gln directly to augmentation of GIP action, as previously observed with other xenin-related compounds [2)GIP- and (DAla2)GIP/xenin-8-Gln-treated mice, has also been shown to independently restore GIP insulin-secretory function in type 2 diabetes [2)GIP/xenin-8-Gln in restoring GIP action in type 2 diabetes.As expected, a key component of the beneficial action of GIP/xenin-8-Gln has an impressive profile of beneficial metabolic effects in high-fat-fed mice, including improvement of glucose tolerance, insulin resistance and pancreatic islet morphology. Importantly, there was a clear augmentation of the biological action of native GIP in high-fat-fed mice, suggestive of restored GIP effectiveness by (DAla2)GIP/xenin-8-Gln. Further studies are required to fully evaluate the molecular mechanisms and full range of actions of (DAla2)GIP/xenin-8-Gln. However, it is evident that this stable GIP/xenin hybrid may represent an attractive potential new therapeutic for type 2 diabetes.In conclusion, the present study has demonstrated that the novel hybrid peptide analogue (DAlaESM 1(PDF 95 kb)Below is the link to the electronic supplementary material.


Caenorhabditis elegansSER-2 tyramine receptor. We show that arginines within a putative methylation motif in the third intracellular loop of SER-2 are methylated by PRMT5 in vitro. Our data also suggest that this modification enhances SER-2 signaling in vivo to modulate animal behavior. The identification of a third G protein-coupled receptor to be functionally regulated by arginine methylation suggests that this post-translational modification may be utilized to regulate signaling through a broad array of G protein-coupled receptors.G protein-coupled receptors are 7-pass transmembrane receptors that couple to heterotrimeric G proteins to mediate cellular responses to a diverse array of stimuli. Understanding the mechanisms that regulate G protein-coupled receptors is crucial to manipulating their signaling for therapeutic benefit. One key regulatory mechanism that contributes to the functional diversity of many signaling proteins is post-translational modification. Whereas phosphorylation remains the best studied of such modifications, arginine methylation by protein arginine methyltransferases is emerging as a key regulator of protein function. We previously published the first functional evidence that arginine methylation of G protein-coupled receptors modulates their signaling. We report here a third receptor that is regulated by arginine methylation, the  Methylation of D2-like dopamine receptors (human D2 and C. elegansDOP-3) by protein arginine methyltransferase 5  promoted signaling in both cell culture and in vivo  signaling can be regulated is through post-translational modification of receptors. Although a large-scale proteomics analysis performed in 2003 identified several GPCRs as substrates of an anti-methylarginine antibody Likhite . MethylaPRMT5 is a type 2 protein arginine methyltransferase (PRMT) that transfers two methyl groups from S-adenosyl-L-methionine (SAM) to form symmetric dimethylarginines (SDMAs)  -induced immobilization (ser-2 loss-of-function (lof) animals were resistant to the paralytic effects of exogenous TA (ser-2(lof) animals  animals continued to display foraging behavior while reversing following nose touch, unlike the wild-type animals in their study, and expression of a wild-type ser-2 transgene rescued the behavioral phenotype (ser-2(lof) animals do not cease foraging while backing in response to nose touch.While exploring their environments during forward locomotion, -to-side . This fo-to-side . ExtensiC. elegansSER-2 tyramine receptor is also regulated by methylation. We show that human PRMT5 methylates a portion of the third intracellular loop of SER-2in vitro, and that the conserved arginines within the predicted methylation motif are required for methylation by PRMT5. Using C. elegans behavior as a readout for nervous system function, we show that PRMT-5 also promotes tyraminergic signaling through the C. elegansSER-2 receptor in vivo, and that changing the predicted arginine methylation target sites in SER-2 diminished its ability to regulate C. elegans behavior. Together, our data reveal a third receptor that appears to be functionally regulated by protein arginine methylation. This work suggests that arginine methylation may be a widespread post-translational modification used to regulate the activity of GPCRs.Following the bioinformatics analysis of OP50E. coli bacteria (Strains were maintained at 20° under standard conditions on nematode growth media (NGM) agar plates seeded with bacteria .N2 Bristol wild-type, OH313ser-2(pk1357), FG129 prmt-5(gk357), FG807 ser-2(pk1357);prmt-5(gk357), FG808 ser-2(pk1357);udEx460, FG809 ser-2(pk1357);udEx461, FG810 ser-2(pk1357);udEx462, FG811 ser-2(pk1357);udEx463, FG812 ser-2(pk1357);udEx464, FG813 ser-2(pk1357);udEx465, FG814 prmt-5(gk357);udEx466, FG815 prmt-5(gk357);udEx467 and FG816 prmt-5(gk357);udEx468.Strains used in this study include: prmt-5 and ser-2 rescue experiments, pJM67 elt-2::gfp plasmid (25 ng/μl) (prmt-5 or ser-2 rescuing plasmid (50 ng/μl).Germline transformations were performed as previously described .The ∼5.3 kb ser-2 cDNA (isoform e) was PCR amplified from ser-2 in pFLAG (gift from Rick Komunicki) .The ∼2.2 kb ser-2 (isoform e) was isolated from pFG102 with KpnI/SacI, and subcloned into these sites of pPD49.26 .The cDNA encoding ser-2 promoter  was used to incorporate the R245A and R247A substitutions into the prmt-5 was isolated from pFG66  was amplified by PCR from ser-2p::ser-2 (pFG290), incorporating sequence encoding an N- and C-terminal S-tag (amino acids KETAAAKFERQHMDS) as well as a 5′ BamHI and a 3′ XmaI site. This DNA was then inserted into the corresponding sites of pGEX-5X-3.A portion of the cDNA encoding the third intracellular loop (ICL) of SER-2(R245A/R247A) (amino acid residues 240 to 269 of isoform e) was amplified by PCR from ser-2p::ser-2(R245A/R247A) (pFG291), incorporating sequence encoding an N- and C-terminal S-tag (amino acids KETAAAKFERQHMDS) as well as a 5′ BamHI and a 3′ XmaI site. This DNA was then inserted into the corresponding sites of pGEX-5X-3.A portion of the cDNA encoding the third intracellular loop (ICL) of All constructs were verified by sequencing where appropriate.All behavioral assays were performed on at least three separate days, in parallel with controls. Assays were performed at room temperature using young adult animals aged 24 hr post the L4 larval stage. For all behavioral experiments the combined data of ≥ 3 transgenic lines is shown, and the number of transgenic animals assayed in each experiment is indicated within the figure legends. In all cases n ≥ 24 for non-transgenic animals. The Student’s two-tailed t-Test and one-way Anova with Tukey’s Honestly Significant Difference (HSD) were used for statistical analyses.To quantify resistance to tyramine-induced immobilization, young adult animals were transferred to agar plates supplemented with 12 mM tyramine. Approximately 8 animals were transferred to assay plates and scored for locomotion every minute for a 10 min period. Animals were scored as immobilized if there was no sustained forward or backward locomotion in a 5 sec interval  while reversing was recorded. Five trials per animal and ≥30 animals per genotype were scored as follows: no foraging (no head movement while reversing) and foraging (head movement while reversing). Occasionally, animals did not reverse upon nose touch and were scored as no response.The nose touch assay was performed essentially as described ] or GST-SER-2240-269(R245A/R247A) [GST-S-SER-2(R245A/R247A) 3ICL-S (30 amino acids)] were pelleted, resuspended in lysis buffer  and subjected to two rounds of French Press lysis. Lysates was centrifuged at 30,000 × g for 30 min at 4°. Supernatants were incubated with Glutathione Sepharose High Performance beads (Amersham Biosciences) that had been equilibrated with wash buffer . Unbound sample was allowed to flow through and the resin was washed 3x with 10 mL of wash buffer. GST-tagged proteins were eluted with elution buffer . Fractions containing the protein eluate were dialyzed in dialysis buffer  for storage at -80°.Overnight cultures of in vitro methylation assay was performed essentially as described (Likhite ) in a total volume of 20 μl with 6 μg of substrate  in 1x methylation buffer . Reactions were incubated at 37° for 4 hr, resolved by SDS-polyacrylamide gel electrophoresis, and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were sprayed with 6% PPO enhance reagent  three times at 10 min intervals before being exposed to Kodak BioMax MS film with a BioMax Transcreen LE Intensifying Screen at -80° for two weeks and subsequently developed. Band intensities were quantified with Bio-Rad ImageLab software and were normalized according to gel loading.The Following film exposure for quantification of methylation signal, the PVDF membrane was washed two times with 100% methanol and two times in PBS-T to remove the PPO. The washed membrane was then blocked with blocking solution (5% milk in PBS-T) for one hour at room temperature. Polyclonal anti-GST primary antibody  was used at a 1:20,000 dilution. The secondary antibody  was used at a 1:10,000 dilution. The chemiluminescent reaction was performed using Amersham ECL Prime Western Blotting Detecting Reagent .TMpred (Prediction of Transmembrane Regions and Orientation)  and TMHMhttps://doi.org/10.25387/g3.6193037.Strains and plasmids are available upon request. The authors affirm that all data necessary for confirming the conclusions of this article are represented fully within the article and its figures. Figure S1 shows the sequence alignments of the predicted arginine methylation motifs from related receptors, as included in the Discussion. Supplemental material available at Figshare: C. elegansDOP-3 receptors ] was methylated by PRMT5 (245 and Arg247) were necessary for SER-2 methylation, we generated a recombinant fragment in which these arginines were changed to alanines (R245A/R247A). SER-2 receptor methylation was markedly diminished when the two conserved arginine residues were replaced with alanines  was used as substrate  animals are resistant to this paralysis  animals displayed an intermediate level of immobilization when compared to wild-type and ser-2(lof) animals (SER-2 receptor and PRMT-5  displayed immobilization levels similar to those of the ser-2(lof) single mutants, suggesting that these two proteins function in the same pathway. The partial tyramine resistance observed in prmt-5(lof) animals is consistent with PRMT-5 playing a role in promoting SER-2-mediated tyramine signaling.Tyramine (TA) modulates  animals . AnimalsPRMT-5 regulates tyramine-modulated paralysis by acting in the same cells as SER-2, we used the ser-2 promoter  animals expressing the ser-2p::prmt-5 transgene displayed an immobilization phenotype similar to that of wild-type animals (ser-2 promoter to restore SER-2 (isoform e) receptor expression [ser-2p::ser-2(WT)] in ser-2(lof) animals also fully rescued TA-induced immobilization (PRMT-5 methylation motif (Arg245 and Arg247) contributed to SER-2 function in vivo, we generated a SER-2(R245A/R247A) mutant receptor using site-directed mutagenesis. ser-2(lof) animals expressing the ser-2p::ser-2(R245A/R247A) transgene phenocopied prmt-5(lof) animals  animals  can stimulate pharyngeal pumping by mimicking the presence of food, while the addition of exogenous TA antagonizes 5-HT-stimulated pumping (PRMT-5 function. Similar to the TA-induced immobilization experiments (prmt-5(lof) animals displayed an intermediate pharyngeal pumping phenotype when compared to wild-type and ser-2(lof) animals (prmt-5(lof) animals expressing the ser-2p::prmt-5 transgene to restore PRMT-5 function in ser-2-expressing cells showed a pharyngeal pumping rate similar to wild-type animals in the presence of 5-HT and TA, consistent with PRMT-5 regulating pharyngeal pumping by acting in the same cells as SER-2. To assess the contribution of Arg245 and Arg247 to SER-2 function, we compared TA inhibition of 5-HT-stimulated pharyngeal pumping in ser-2(lof) animals expressing the ser-2p::ser-2(WT)vs.ser-2p::ser-2(R245A/R247A) transgene. While ser-2(lof) animals expressing wild-type SER-2 displayed a pumping rate similar to wild-type animals, ser-2(lof) animals expressing SER-2(R245A/R247A) were again similar to animals lacking PRMT-5  animals fail to suppress foraging behavior while reversing in response to nose touch  animals suppress foraging in response to nose touch. While only 16% of wild-type animals continued foraging while reversing, 51% of prmt-5(lof) animals continued foraging as they reversed following nose touch (ser-2(lof) animals did not suppress foraging while reversing. Restoration of PRMT-5 function in ser-2-expressing cells of prmt-5(lof) animals (by expressing the ser-2p::prmt-5 transgene) suppressed foraging to the extent seen in wild-type animals. Furthermore, while expression of wild-type SER-2 in ser-2(lof) animals suppressed foraging to wild-type levels, ser-2(lof) animals expressing SER-2(R245A/R247A) did not fully suppress foraging, similar to prmt-5(lof) animals. Taken together, these data are consistent with a role for arginine methylation promoting SER-2 signaling to dampen foraging behavior in response to nose touch.Consistent with se touch . By compC. elegansDOP-3), is regulated by protein arginine methylation  of the putative methylation motif reduced this methylation  is encountered, the ASH nociceptors signal to the AIBs which, in turn, inhibit the RIMs  . The RIMSER-2 receptors found on C. elegans head muscles , suggesting that methylation may regulate tyraminergic signaling across multiple phyla. Interestingly, these tyramine receptors, along with the human D2 and C. elegansDOP-3 receptors, all signal through Gαi/o, which traditionally functions to inhibit adenylyl cyclase and decrease cAMP production  receptor as the closest human homolog of the tyraminergic SER-2 receptor , suggesting that even though the receptor binds a different biogenic amine, its regulation by methylation has likely been conserved through evolution.BLAST  analysis identified the serotonin 1A  is necessary for retrieval of the olfactory memory. However, an additional tyraminergic GPCR, TYRA-2, is also required for imprinted olfactory aversion , suggesting that methylation of these receptors may contribute to the formation and/or retrieval of imprinted memories. For example, the introduction of pathogenic bacteria may alter the methylation status of these receptors, perhaps in a cell-specific manner.Although not examined here, the most recently reported SER-2 receptor (Arg245 and Arg247) lies between two predicted sites of phosphorylation in the third intracellular loop (Ser243 and Thr250) (SER-2 methylation by PRMT-5 could regulate the local phosphorylation state of these residues to regulate receptor signaling. Finally, since GPCR phosphorylation can lead to receptor desensitization and subsequent downregulation (The addition of a methyl group to an arginine residue removes a hydrogen bond donor and decreases the electrostatic surface potential at the residue, resulting in a change in size and hydrophobicity that can affect its interaction with binding partners . Thus, pgulation , methylaThe work described here provides evidence of a third GPCR that is functionally regulated by arginine methylation. In humans, GPCRs are the largest family of tractable drug targets  and are


SMARCA4/BRG1 and SMARCA2/BRM, the two mutually exclusive catalytic subunits of the BAF complex, display a well-established synthetic lethal relationship in SMARCA4-deficient cancers. Using CRISPR-Cas9 screening, we identify SMARCA4 as a novel dependency in SMARCA2-deficient esophageal squamous cell carcinoma (ESCC) models, reciprocal to the known synthetic lethal interaction. Restoration of SMARCA2 expression alleviates the dependency on SMARCA4, while engineered loss of SMARCA2 renders ESCC models vulnerable to concomitant depletion of SMARCA4. Dependency on SMARCA4 is linked to its ATPase activity, but not to bromodomain function. We highlight the relevance of SMARCA4 as a drug target in esophageal cancer using an engineered ESCC cell model harboring a SMARCA4 allele amenable to targeted proteolysis and identify SMARCA4-dependent cell models with low or absent SMARCA2 expression from additional tumor types. These findings expand the concept of SMARCA2/SMARCA4 paralog dependency and suggest that pharmacological inhibition of SMARCA4 represents a novel therapeutic opportunity for SMARCA2-deficient cancers. Selective targeting of tumors harboring loss-of-function alterations of a synthetic lethal gene by pharmacological inhibition of the functional synthetic lethal partner has been proposed for cancer therapy. The response of patients with BRCA1/BRCA2-deficient tumors to Polymerase (PARP) inhibitors has demonstrated how this approach can be successfully exploited in the clinic7. Several other synthetic lethal interactions highlighting potential cancer drug targets have been described in pre-clinical models9. Most notably, synthetic lethal interactions of paralog genes, such as ENO1/ENO2 or STAG1/STAG212 with redundant - but cell essential - functions result in acute and strong dependencies on the functional paralog in tumor cells deficient for the corresponding paralog gene.Synthetic lethality is a well-established concept developed from genetic studies in fruit flies and yeast13. BAF complexes, also referred to as Switch/Sucrose Non-Fermenting (SWI/SNF) complexes, are large, multi-subunit complexes that catalyze chromatin remodeling via ATP-dependent mobilization of nucleosomes, thereby impacting on chromatin accessibility of the transcriptional machinery and global gene expression14. The core BAF complex consists of a DNA-dependent ATPase, as the sole known enzymatic activity, and three subunits  that are critical for structural integrity and chromatin recruitment. BAF complexes are further variably composed of accessory components in a cell-type and context specific manner15. Multiple BAF components are represented by paralogous subunits that are incorporated into the complex in a mutual exclusive fashion. Most notably, the BAF ATPase function may be exerted by either SMARCA4 or its homologous paralog SMARCA217.Cancer-relevant paralog dependencies are prominently observed for BRG1-Associated Factor (BAF) chromatin remodeling complexes19, are known oncogenic driver events, indicating a tumor suppressive role for BAF complexes20. Cancer-associated BAF alterations do not result in complete abrogation of BAF activity, which would be detrimental for viability of most cells, implying that tumors are dependent on the aberrant function of the residual BAF complex13. This is exemplified by the ATPase subunit SMARCA4, which is frequently inactivated by loss-of-function mutations or epigenetic silencing in non-small cell lung cancer (NSCLC) and other tumor types24. Consequently, SMARCA4-deficient cell models are rendered highly dependent on the paralog ATPase SMARCA2 to maintain residual BAF activity27.Loss-of-function alterations of individual BAF complex components, such as bi-allelic inactivation of the BAF core component SMARCB1 in malignant rhabdoid tumors28, is one of the most common cancers worldwide with a five-year-survival rate ranging from 15 to 25%29. Esophageal squamous cell carcinoma (ESCC) is the predominant subtype of esophageal carcinoma, with particular high incidence in Eastern and Central Asia31. Treatment of ESCC is currently largely limited to surgery and chemotherapy/radiotherapy approaches32. With the exception of a few actionable genomic alterations, such as EGFR amplification, targeted therapy options for ESCC are limited36.Esophageal carcinoma, which is histologically classified as adenocarcinoma or squamous cell carcinomaIn this study, using a pooled single guide RNA (sgRNA) viability screen targeting epigenetic factors in a panel of ESCC cell models, we identify the BAF chromatin remodeling ATPase subunit SMARCA4 as a novel dependency in a subset of ESCC cell lines with low or absent expression of SMARCA2. By applying SMARCA2 reconstitution and knock-out strategies in ESCC cell models, we define a novel synthetic lethal relationship reciprocal to the SMARCA2 dependency observed in SMARCA4-deficient cancer cells. This concept is further extended to non-ESCC cancer cell models, indicating SMARCA4 as a potential therapeutic target in tumors with low or absent expression of SMARCA2.37 across ten ESCC cell lines. Stable Cas9-expressing cell lines were transduced with a sgRNA library consisting of >1300 sgRNAs targeting 179 epigenetic regulators38 and negative selection of sgRNA-expressing cells was monitored by next-generation sequencing after 18 population doublings  showed no or less dependency (α-RRA scores ≥ −0.5) on SMARCA4. Of note, none of the ESCC lines showed dependency on the paralogous BAF subunit SMARCA2  displayed low or non-detectable levels of SMARCA2 mRNA and protein expression  expression constructs harboring loss-of-function mutations within the DEXDc- (K785A) and bromodomains (N1540A)42  SMARCA4res also protected KYSE-510 cells from SMARCA4 knock-out, the ATP-binding deficient form (K785A) of SMARCA4res did not rescue the depletion of SMARCA4 sgRNA-expressing KYSE-510 cells 42 Fig. . Wild-tynts Fig. . In linents Fig. , SMARCA4lls Fig. .Figure 3res were also stably expressed in the SMARCA2-deficient cell line KYSE-30  in KYSE-510 cells lacking endogenous SMARCA2 expression  and bromodomain-mutant (N1482A) forms of SMARCA2 , HuP-T4 (pancreas carcinoma) and OV-90 (ovarian carcinoma) - with low or non-detectable SMARCA2 protein expression suitable for CRISPR-Cas9 depletion studies  was designed to determine whether acute degradation of SMARCA4 represents a therapeutic approach against SMARCA2-deficient cancers . Treatment with dBRD9 for four hours led to a decrease in SMARCA4 protein levels in the KYSE-30-SMARCA4res-BDBRD9 cell line, but not parental KYSE-30 cells  of 322 ± 122 nM. In contrast, parental KYSE-30 cells were not affected by treatment with dBRD9 59. Missense mutations or in-frame deletions affecting the ATPase function of SMARCA2 are causative for Nicolaides-Baraitser syndrome, a rare condition characterized by sparse hair, facial and limb abnormalities and intellectual disabilities61. In cancer, mutations of SMARCA2 are rarely observed14. The lack of SMARCA2 expression in cancer cells might therefore be related to cell-specific and epigenetic regulation mechanisms62. A notable exception is adenoid cystic carcinoma (ACC), a rare type of cancer arising from salivary glands where mutations within the ATPase domain of SMARCA2 are found in approximately 5% of cases63, indicating a potential tumor suppressive function of SMARCA2 in this context.In contrast, loss-of-function mutations of SMARCA2 are primarily linked to developmental/neurological disorders64. While it is difficult to define a threshold for expression of SMARCA2 that maintains sufficient BAF functionality in the absence of SMARCA4, the fact that homozygous loss of SMARCA4 is embryonic lethal, indicates that SMARCA2 cannot universally compensate for SMARCA4 function56. In adult tissues, dual inactivation of SMARCA2 and SMARCA4 primarily affects viability of vascular endothelial cells65. Pharmacological targeting of SMARCA4 would therefore require a thorough analysis of potential toxicities in normal cell and tissues.Using a PROTAC approach in an engineered cell model, we demonstrate that pharmacological targeting of SMARCA4 effectively impairs proliferation of a SMARCA2-deficient cell line. Our data suggest that SMARCA4 inhibition would allow for a selective, cancer-cell directed therapy sparing normal, SMARCA2-expressing cells and tissues. Expression of SMARCA2 is predominantly detected in brain, liver, muscle and endothelial cells and has been linked to slow-cycling and differentiated cell states68. In small-cell carcinomas of the ovary, hypercalcemic type (SCCOHTs), characterized by bi-allelic inactivating mutations of SMARCA4, loss of SMARCA2 expression is frequently observed71. These examples depict a striking exemption of the concept of hard-wired SMARCA2/SMARCA4 paralog dependency and constitute a potential mechanism for resistance to BAF targeted cancer therapies72. Further studies are required to decipher whether alternative ATP-dependent nucleosome-remodeling complexes, such as ISWI and CHD complexes73, can fulfill a compensatory function in the absence of BAF activity.In human cancer also concomitant loss of SMARCA2 and SMARCA4 expression has been described75 and tolerability of systemic SMARCA4 inactivation, the results of this study suggest that targeting of SMARCA4 might be a valuable strategy for the treatment of tumors characterized by low or absent expression of SMARCA2.Our study highlights the use of functional genomic screening for identification of novel vulnerabilities in cancer cell lines. Given the possibility to develop selective SMARCA4 ATPase inhibitors or PROTACs selectively inducing SMARCA4 proteolysisKYSE-30 and KYSE-450 (ESCC) were cultured in 45% RPMI 1640 (Gibco) + 45% Ham’s F12 + 10% fetal calf serum (FCS). For cultivation of KYSE-70 and KYSE-410 (ESCC) RPMI 1640 (Gibco) supplied with 10% FCS, whereas for KYSE-140, KYSE-150, KYSE-180, KYSE-510 and COLO-680N (ESCC) RPMI1640 (ATCC #30-2001) + 10% FCS was used. T.T (ESCC) cell line was cultured in DMEM:F12 (ATCC: 30-2006) containing 10% FCS. KYSE-270 (ESCC) was cultured in RPMI1640 (Gibco) + HAM’s F12  (1:1) including 2 mM Glutamine and 2% FCS. HCT 116 (colon carcinoma) cells were cultured in McCoy’s 5 A medium  supplemented with GlutaMAX  and 10% FCS, SK-CO-1 (colon carcinoma) was cultured in EMEM  with GlutaMAX and 10% FCS supplemented with Sodium-Pyruvate, and OV-90 (ovarian carcinoma) was cultured in a 1:1 mixture of MCDB 105 medium  with glutamine and HEPES  containing a final concentration of 1.5 g/l sodium bicarbonate and Medium 199  containing a final concentration of 2.2 g/l sodium bicarbonate. HuP-T4 (pancreas carcinoma) was cultured in MEM + Earl´s Salt  + 20% FCS and GlutaMAX.CDK1, POLR2A or RPA3). For Cas9-expressing cell lines the following concentrations of Puromycin were added to the standard medium: T.T, and KYSE-270: 4 µg/ml; KYSE-70, KYSE-140, HCT 116, and HuP-T4: 2 µg/ml; KYSE-450, KYSE-510, SK-CO-1, and OV-90: 1 µg/ml; COLO-680N: 0.5 µg/ml; KYSE-30, KYSE-150, and KYSE-410: 0.25 µg/ml. The medium for the cell lines expressing SMARCA4res, SMARCA2ect, or SMARCA4-BDBRD9 was supplied with 25 µg/ml Hygromycin B. All supplements were obtained from Gibco, FCS (SH30071.03) from GE Healthcare Life Sciences, Puromycin from Sigma (P9620), and Hygromycin B from Invitrogen (10687010). Sources and information for authentication of cell lines  used in this study are attached in Supplementary Table Lentiviral particles were generated via usage of the Lenti-X Single Shot protocol . Low passage cell lines were transduced with lentiviral expression vector encoding Cas9 and puromycin and selected over two weeks. Cas9 editing efficacy was monitored via depletion of sgRNAs targeting positive control genes  such that the mean of these control genes was −1 for all cell lines.Statistical analysis of depletion signals was performed with the MAGeCK tool (V 0.5.6)et al. (http://crispr.mit.edu/) and cloned into GFP encoding vectors. Cas9-expressing cells were transduced and the fraction of GFP-positive cells was determined on day 3 post-transduction. Cells were split for 21-28 days. Fold changes of initially transduced cell populations were calculated from day 3 to the respective time points. For the heatmap representation, depletion of GFP-positive cell populations on day 28 was related to the fraction  of GFP-positive cells  of the POLR2A positive control on day 28.The sgRNAs were designed using a tool provided by Zhang All sgRNA sequences used for depletion experiments and generation of knock-out cell lines are listed in Supplementary Dataset File ™ VILO™ kit (Thermo Scientific). For qPCR analysis, QuantiTect® Multiplex PCR kit  and StepOne Real-Time PCR Sytem™ (Applied Biosytems) was used. The respective primers were all ordered from Applied Biosystems, including house-keeping genes: 18 S rRNA , ACTB , GAPDH  and primers for SMARCA2 (Hs01030858_m1 MGB/FAM) and SMARCA4 (Hs00231324 MGB/FAM). SMARCA2/4 expression was calculated from duplicates in relation to three different house-keeping genes listed above.RNA was isolated using RNeasy Mini Kit  and reversly transcribed using SuperScriptLysates were generated using MSD Tris Lysis Buffer  supplied with Protease & Phosphatase Inhibitor Cocktail . Capillary Western immunoassay  was conducted according to the manufacturer’s protocol. Dilutions of protein were prepared to obtain a final protein concentration of 0.4 µg/µl.Antibodies used were anti-BRG1/SMARCA4 , anti-SMARCA2  and anti-GAPDH . Respective antibodies were used in a multiplex assay together with anti-GAPDH for relative quantification of protein levels.https://ordino.caleydoapp.org/ 43. SMARCA4 sensitivity measurements were obtained from McDonald et al.39 (RSA scores) and Meyers et al.40 (Ceres scores). The visualizations and statistical tests were performed using R version 3.5.0 (R Core Team (2018) R: A language and environment for statistical computing, R Foundation for Statistical Computing, Vienna, Austria; available online at https://www.R-project.org/.), including the R package ggplot2 version 3.0.0 (H. Wickham (2016) ggplot2: Elegant Graphics for Data Analysis; 10.1007/978-3-319-24277-4).SMARCA2 gene expression values (TPM – transcripts per million) were obtained from res and SMARCA2ect the following constructs were generated by gene synthesis  based on the SMARCA4 cDNA sequence NCBI NM_ 001128844.1 and SMARCA2 cDNA sequence NCBI NM_003070.5 and cloning into the parental pLVX vector : pLVX-empty-IRES-Hygro; pLVX-SMARCA4-wt-IRES-Hygro; pLVX-SMARCA4-ATP-binding deficient (K785A)-IRES-Hygro; pLVX-SMARCA4-BD-dead(N1540A)-IRES-Hygro; pLVX-SMARCA2-wt-IRES-Hygro; pLVX-SMARCA2-ATP-binding-deficient(K755A)-IRES-Hygro; pLVX-SMARCA2-BD-dead(N1482A)-IRES-Hygro; pLVX-SMARCA4res-BDBRD9-IRES-Hygro. ATP-binding deficient and BD-dead sites were selected similar to known inactivating mutations in SMARCA2/442. The sequence of SMARCA4res-BDBRD9 is shown in Supplementary Table For expression of SMARCA4For siRNA-mediated gene knock-down, cells were reversely transfected using Lipofectamine RNAiMAX reagent according to the manufacturer’s protocol . The final concentration of the respective siRNAs was 25 nM. The siRNA duplexes were purchased from Thermo Scientific/ Dharmacon: Non-targeting pool (D-001810-10-20), SMARCA4_1 (J-010431-06), SMARCA4_2 (J-010431-07).All-in-one pSpCas9 BB-2A-GFP (PX458) vectors were ordered from Genscript, China with sgRNAs targeting the N-terminus of SMARCA2/4 as listed in Supplementary Table res-BDBRD9 and parental KYSE-30 cells were cultured for approximately 18 passages post-thawing and plated at low densities (100 cells/well) in a 96-well plate. The next day, cells were treated with varying doses of dBRD944 as indicated. Viability was determined using CellTiter-Glo . Ten days post-treatment, 100 μl CellTiter-Glo solution were added directly to the cell medium, mixed and incubated for 10 min prior to determination of the luminescence signal.KYSE-30-SMARCA4Supplementary InformationDataset 1Dataset 2Dataset 3


While VZV DNA and antigen have been detected in acute and chronic VZV keratitis, it is unclear whether productive infection of corneal cells is ongoing or whether residual, noninfectious VZV antigens elicit inflammation. Herein, we examined VZV-infected primary human corneal epithelial cells (HCECs) and keratocytes (HKs) to elucidate the pathogenesis of VZV keratitis.HCECs and HKs were mock- or VZV infected. Seven days later, cells were examined for morphology, proinflammatory cytokine and matrix metalloproteinase (MMP) release, ability to recruit peripheral blood mononuclear cells (PBMCs) and neutrophils, and MMP substrate cleavage.Both cell types synthesized infectious virus. VZV-infected HCECs proliferated, whereas VZV-infected HKs died. Compared to mock-infected cells, VZV-infected HCECs secreted significantly more IL-6, IL-8, IL-10, and IL-12p70 that were confirmed at the transcript level, and MMP-1 and MMP-9; conditioned supernatant attracted PBMCs and neutrophils and cleaved MMP substrates. In contrast, VZV-infected HKs suppressed cytokine secretion except for IL-8, which attracted neutrophils, and suppressed MMP release and substrate cleavage.in vitro model for VZV infection of corneal epithelial cells. Furthermore, the proliferation and persistence of VZV-infected HCECs suggest that these cells may serve as viral reservoirs if immune clearance is incomplete. Finally, the finding that VZV-infected HKs die and suppress most proinflammatory cytokines and MMPs may explain the widespread death of these cells with unchecked viral spread due to ineffective recruitment of PBMCs.Overall, VZV-infected HCECs recapitulate findings of VZV keratitis with respect to epithelial cell proliferation, pseudodendrite formation and creation of a proinflammatory environment, providing an  Varicella zoster virus (VZV) produces varicella then establishes latency in ganglionic neurons, including cranial nerve ganglia.4Epithelial and stromal keratitis are corneal complications of VZV and can lead to vision loss after acute disease and during recurrence due to long-term corneal scarring and haze.614Based on its rapid resolution upon corticosteroid treatment, stromal keratitis was thought to be an immune response to viral antigens within the corneal stroma, comprised of stromal fibroblasts underlying the epithelial cell layer.Since there is no animal model for VZV keratitis, we infected primary human corneal epithelial cells (HCECs) and stromal fibroblasts  with VZV and analyzed the cytopathology and inflammatory responses. Understanding the role of ongoing virus replication and inflammation in VZV keratitis can guide antiviral and corticosteroid therapy, mitigating ocular damage and vision loss produced by VZV.https://fiji.sc/).The VZV Gilden strain (GenBank #MH379685) and primary HCECs and HKs from adult human cornea  were used. Cells were confirmed by immunofluorescence antibody assay (IFA) and quantified by Fiji image processing software . Cells were co-cultured with lysates from uninfected or VZV-infected human fetal lung fibroblast  for 24 hours (200 plaque-forming units [PFU]/cm2),HCECs were seeded in corneal epithelial cell medium containing HCEC growth supplement/1% penicillin-streptomycin  and neutrophils purified from whole blood  using the direct human neutrophil isolation kit  were used.At 7DPI, HCECs and HKs were analyzed by IFA using primary mouse anti-cytokeratin 18 , rabbit anti-fibronectin , mouse anti-VZV glycoprotein B , and rabbit anti-GAPdH  antibodies then secondary donkey anti-mouse AlexaFluor 594 IgG and donkey and anti-rabbit AlexaFluor 488 antibodies  as described.VZV-infected HCECs were harvested at 3, 5, and 7 DPI; each sample was serially diluted onto 70% confluent monolayers of uninfected HFLs in DMEM F12 medium with 10% FBS (ATCC) to demonstrate virus transmission from HCECs to HFLs. Similarly, VZV-infected HKs at 3, 5, and 7 DPI were diluted onto HFLs. Three days later, HFLs were stained with 0.1% crystal violet and PFUs/mL were calculated.Proinflammatory cytokines  and MMPs in supernatants (MMP-1/2/3/9/10) were measured using commercial assays  as described.21RNA was extracted, cDNA transcribed then analyzed by RT-qPCR using primers to cytokines above and glyceraldehyde-3-phosphate-dehydrogenase , as described.http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). Positive control for PBMC and neutrophil migration was chemokine (C-C motif) ligand 2  diluted in either HCEC or HK medium (20 pg/mL) and 50 ng/mL IL-8 (Biolegend), respectively. Negative controls for PBMC and migration assays were HCEC and HKs medium only. To determine specificity for IL8-induced neutrophil migration in infected supernatant, mouse monoclonal IL-8 blocking antibody was added at 1 μg/mL . Fold differences of VZV-infected cells and positive and negative controls were relative to mock-infected cells.At 7 DPI, mock- and VZV-infected supernatants were assayed for their ability to attract PBMCs and neutrophils. Supernatants were loaded in a 96-well chemotaxis plate , 5-μm pore size filters placed on top, immune cells pipetted onto filters , and plate incubated at 37°C for 4 hours. Filters were removed; migrating immune cells were visualized by microscopy and quantified using ImageJ 3D Objects Counter . MMP-1 enzyme (500 pg/mL) served as positive control and MMP-1 enzyme plus MMP inhibitor  served as negative control . Relative fluorescence units were measured using a fluorescent plate reader (excitation/emission: 485 nm/520 nm).q-value ≤ 0.05) using the two-stage step-up method of Benjamini et al.P < 0.05, **P < 0.01, ***P < 0.001).Statistical analysis was performed using graphing software . Cytokine and MMP differences among mock- and VZV-infected cells were determined by multiple unpaired t-tests with a False Discovery Rate , consistent with proliferating VZV-infected HCECs over time. In contrast, input VZV-infected HKs at 3, 5, and 7 DPI significantly decreased PFU/mL in HFLs at 14,666 ± 1171, 8333 ± 1353 and 5400 ± 493 (n = 3), consistent with dying VZV-infected HKs over time. Supernatant from infected cells were unable to transmit virus to HFLs demonstrating the absence of cell-free virus (data not shown).Aside from VZV-infected corneal cells having the capacity to spread VZV to adjacent cells of the same type, VZV-infected HCECs and HKs were tested for their ability to transmit VZV to another cell type by cell-to-cell spread. VZV-infected HCECs and HKs at 3, 5, and 7 DPI were cocultured with uninfected HFLs; PFUs were counted at 3 DPI. The PFUs observed were due to VZV-infected HFLs since input VZV-infected HCECs perish in DMEM F12 medium, and input infected HKs were present in low amounts and are morphologically distinguishable. VZV-infected HKs die and form cell clearings, whereas VZV-infected HFLs swell and form syncytia. Input VZV-infected HCECs at 3, 5, and 7 DPI significantly increased the PFU/mL in HFLs at 367 ± 219, 2300 ± 82, and 5250 ± 204, respectively . VZV-infected HKs significantly decreased cell counts at 5 and 7 DPI relative to 1 DPI: 3 DPI (0.99 ± 0.07), 5 DPI (0.64 ± 0.07), 7 DPI .Phase-contrast imaging indicated that VZV-infected HCECs proliferate; VZV-infected HKs sloughed off. Compared to mock- (n = 5). To verify cytokine expression, transcripts were analyzed by RT-qPCR and fold differences between VZV- to mock-infected HCECs determined . To verify cytokine expression, transcripts from mock- and VZV-infected HKs were analyzed by RT-qPCR and fold differences between VZV- to mock-infected HKs determined . In contrast, VZV-infected HK supernatant did not increase PBMC migration (0.99 ± 0.16), consistent with the suppression of proinflammatory cytokines. Media only control had less migrating PBMCs than CCL2 diluted in HK medium .Relative to mock-, VZV-infected HCEC supernatant significantly increased PBMC infiltration (1.73 ± 0.15), consistent with the elevated proinflammatory cytokine levels detected. Media only control had less migrating PBMCs than CCL2 diluted in HCEC medium .Relative to mock-, VZV-infected HCEC supernatant significantly increased neutrophil migration (1.29 ± 0.04). Anti-IL-8 in VZV-infected supernatant, αIL-8 with IL-8 cytokine diluted in media and media only did not significantly increase migration . IL-8 cytokine diluted in HCEC medium significantly increased neutrophil infiltration . The ability of αIL-8 to block migration of neutrophils indicates that IL-8 in the infected conditioned media was responsible for neutrophil chemotaxis.Compared to mock-, VZV-infected HK supernatant significantly increased neutrophil migration (3.01 ± 0.68), consistent with the elevated levels of IL-8 (a neutrophil chemoattractant). Anti-IL-8 in VZV-infected HK supernatant, αIL-8 with IL-8 cytokine in media and media only did not significantly increase migration . IL-8 diluted in HK medium significantly increased PBMC infiltration . In contrast, VZV-infected HK supernatant significantly decreased MMP-1 (0.28 ± 0.01), MMP-2 , MMP-3 (0.66 ± 0.06) and MMP-9 (0.56 ± 0.04); MMP-10 was not detected (n = 3).Compared to mock-, VZV-infected HCEC supernatant significantly increased MMP-1 (1.70 ± 0.29) and MMP-9 (1.61 ± 0.07), but not MMP-3 or MMP-10; MMP-2 was not detected (n = 3). Positive control MMP-1 enzyme had increased activity (1.78 ± 0.03) compared to the negative control that was not detected.Compared to mock-, VZV-infected HCECs significantly increased MMP activity (1.44 ± 0.06), whereas VZV-infected HKs significantly decreased MMP activity  for the disparities in transcript and secreted cytokine levels remain to be determined. Consistent with the increased proinflammatory cytokine levels, VZV-infected HCEC supernatant recruited PBMCs and neutrophils in a migration assay, consistent with inflammatory infiltrates observed in VZV-infected corneal epithelium of patients. The number of proinflammatory cytokines secreted by VZV-infected HCECs is greater than that previously reported in VZV-infected human brain vascular adventitial fibroblasts (HBVAFs), vascular smooth muscle cells (HBVSMCs), perineurial cells (HPNCs) and HFLs.In VZV-infected HCECs, levels of secreted MMP-1 and MMP-9 were significantly increased, unlike the HSV-1-induced increases in MMP-2 and MMP-9 in HCECs.Compared to mock-infected HKs, VZV-infected HKs only secreted significantly higher levels of IL-8 that was confirmed at the transcript level, similar to HSV-1-induced IL-8 secretion in keratocytes;33Overall, our data shows that VZV-infected HCECs recapitulate the clinical findings seen in patients with VZV keratitis, including epithelial cell proliferation, pseudodendrite formation and increased proinflammatory cytokines and MMPs, providing an in vitro model for VZV infection of HCECs. The proliferation and persistence of VZV-infected HCECs suggest that these cells may serve as viral reservoirs if immune clearance is incomplete. Finally, the VZV-induced death of HKs and suppression of most proinflammatory cytokines and MMPs may explain the widespread death of these cells with unchecked viral spread due to ineffective recruitment of PBMCs.


Activation of death receptor 5 (DR5) to induce apoptosis in cancer cells is an attractive strategy for cancer therapy. However, many tumor cell lines and primary tumors are resistant to DR5 targeted agents including recombinant tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and anti-DR5 agonistic antibodies. Here we identify tubulin proteins - primarily consisting of α and β subunits folded into microtubule polymers - as a crucial modulator of DR5 mediated apoptosis. Using affinity purification coupled with mass spectrometry, we found that DR5 interacts with both α- and β-tubulin proteins in cancer cells. Pharmacological disruption of microtubules increased DR5 protein expression and subsequently sensitized the cells to TRAIL-induced apoptosis. Similar results were observed by selectively silencing tubulin transcript using small RNA interference. We also demonstrate that tubulin/microtubule blockade augments TRAIL induced apoptosis by stabilizing DR5 protein. Together, our results link the tubulin/microtubule network to the stringent regulation of DR5 mediated apoptosis, which could lead to potential therapeutic strategies to enhance cancer therapy efficacy. Death receptor-5 (DR5), also known as TRAIL receptor 2 (TRAIL-R2), is a cell surface receptor of the TNF-receptor superfamily that contains a cytoplasmic death domain . This reIn a general scheme, apoptotic cell death is characterized by distinct morphological and biochemical changes: cell shrinkage, plasma membrane “blebbing”, chromatin condensation, and DNA and cells fragmentation . These dTo identify DR5 interacting proteins, we transfected 293T cells with a cDNA plasmid expressing Strep-tagged DR5 fusion protein or an empty plasmid Figure . Strep-DGiven the stable tubulin-DR5 complex formation, we tested the role of tubulin in regulating TRAIL induced apoptosis by manipulating cellular tubulin polymerization. To this end, we treated the cells with colchicine – a microtubule inhibitor that is known to selectively bind tubulin and disrupt microtubule polymerization or taxol- a microtubule stabilizer which inhibits the disassembly of microtubules , 21. NotWe tested whether tubulin plays a role in regulating DR5 protein stability. To this end, cells were left untreated or treated with colchicine to disrupt tubulin function, followed by treatment with cycloheximide (CHX) – a protein synthesis inhibitor that is widely used to shut down protein synthesis machinery in targeted cells. The levels of pre-existing cellular DR5 protein in MB-231 cells were monitored by immunoblotting at the indicated time points Figure . DR5 wasDespite significant progress in understanding how DR5 triggers apoptosis, the molecular mechanisms of tumor resistance to DR5-targeted therapies remain incompletely defined. DR5 is subject to post-translational processing, and its proper expression on the cell surface is a prerequisite for ligand binding and caspase activation . Here weTubulin proteins are essential components of microtubules that are featured by cylindrical structures being assembled from α- and β-tubulin heterodimers. The tubulin/microtubule network undergoes dynamic rearrangement in the course of apoptotic execution . In the Consistent with our data, taxol has been shown to enhance TRAIL killing activity in several cancer cell lines , 16-19. These data provide a novel mechanism by which therapeutic agents targeting tubulin/microtubules kill cancer cells. Our findings suggest that these drugs may function at least partly through upregulation of DR5 expression, leading to DR5 self-clustering and spontaneous caspase activation in targeted cells. Importantly, we showed synergistic effects of TRAIL and tubulin inhibitors to kill cancer cells. Such combinations may be able to overcome tumor resistance mechanisms, leading to better clinical outcomes in cancer patients.2. The cell line authentication was proved by growth rate, morphology, isoenzymology, short tandem repeat profiling, and mycoplasma testing (https://www.atcc.org/). Monoclonal antibodies specific to DR5 , Phospho-Histone H3 (ser10) (3377), β-tubulin (2128), Poly ADP ribose polymerase (PARP) (9542), caspase 8 (9746), and caspase 3 (9662) were from Cell Signaling Technology . Antibody to GAPDH (NB300-328) was from Novus Biologicals . Horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG-HRP (sc-2054) and anti-mouse IgG1-HRP (sc-2969) were from Santa Cruz Biotechnology . Anti-DR5 monoclonal antibody (CDM237) and IgG2b isotope control (CSI11889) used in co-immunoprecipitation (co-IP) assays were from Cell Sciences . Antibodies against beta-tubulin (T0198) and α-tubulin (T6199), cycloheximide  (C4859), poly-L-lysine hydrobromide (P9155), paclitaxel (T1912), and colchicine (C9754) were from Sigma-Aldrich Corporation . Anti-DR5 antibody used for immunocytochemistry was purchased from ProSci Inc . Goat anti-mouse-AF488 (A10667) and goat anti-rabbit-AF594 (A11012) fluorescence-conjugated secondary antibodies were from Life Technologies . DAPI-slow fade mounting solution (H-1200) was purchased from Vector Laboratories. Twin-Strep Purification kit (2-1121-011) was purchased from IBA Solutions For Life Sciences (Germany). Pierce Silver Stain for Mass Spectrometry kit (24600) and mouse IgG1 isotype control (MA5-14453) were from ThermoFisher Scientific . Recombinant human TRAIL/TNFSF10 (rhTRAIL) (375-TEC), phycoerythrin (PE)-conjugated monoclonal antibody DR5 (FAB6311P) and its corresponding IgG2b (IC0041P) isotope controls were from R&D Systems . The synthetic small interference RNA (siRNA) oligos specific to tubulin (L-013150-00-0005) and a scramble siRNA (D-001810-10-20) were purchased from Dharmacon. Transfections of siRNAs and cDNA plasmids were performed using Lipofectamine RNAiMAX Reagent from ThermoFisher.Human cancer cell lines MDA-MB-231 (MB-231), H460, HCT116, and 293T were purchased from the American Type Culture Collection (ATCC) and were cultured per the vendor’s protocols at 37°C in a humidified atmosphere with 5% COStrep-tag affinity purification was conducted using one-Strep starter Kit (IBA Solutions For Life Sciences) per the vendor’s protocol . Brieflyueous One Solution Cell Proliferation Assay kit from Promega . Briefly, 20 μl of CellTiter 96 AQueous One Solution reagent was added into each well of the 96-well plate containing 100 μl of culture medium. After incubation for 1-4 hours, absorbance was recorded at 490 nm using a 96-well plate reader . For colchicine and TRAIL synergy analysis, cells were treated with a single drug dose range of colchicine (50-200 ng/mL) or TRAIL (5-20 ng/mL) as well as in combination.Cell viability was determined using CellTiter 96 AQ6 cells/mL. After 20 min incubation on ice, 30 μl of cell suspension was mixed with 10 μl of 10 μg/ml anti-DR5-PE or corresponding IgG2b-PE as control and incubated for 45 min on ice in the dark. Cells were washed with PBS and re-suspended in 500 μl of PBS and analyzed on an Accuri C6 flow cytometer (BD Biosciences).DR5 expression on the cell surface was analyzed using flow cytometry. Cells at 70-80% confluence were washed with 1× PBS and harvested by incubation with enzyme-free dissociation buffer. Harvested cells were counted using Cellometer and spun down at 1,350 rpm. The cell pellets were re-suspended in a blocking buffer  to reach 5 × 10http://imagej.nih.gov).Whole cell lysates were prepared in RIPA buffer (Sigma-Aldrich). Protein concentrations were measured using BCA Protein Assay Kit (ThermoFisher Scientific). Equal amounts of total proteins were resolved onto SDS-PAGE using the 4-12% NuPAGE Bis-Tris gels (ThermoFisher Scientific) and transferred to PVDF membranes. Membranes were blocked, washed and incubated with primary antibody followed by secondary antibody and visualized by ECL reagents. Densitometry analysis was performed with the LAS-4000 Luminescent Image Analyzer (Fujifilm). Bands density was quantified with Image J software , and then dissolved in 3% acetonitrile in 0.1% formic acid. The peptides were separated on a 43-mm HPLC C18 ProtID-Chip (Agilent Technologies), followed by data-dependent auto-MS/MS on an nano-electrospray-ionization 6520 Q-TOF tandem MS spectrometer (Agilent Technologies). Protein identifications were achieved by searching Swiss-Prot Homo sapiens protein database using Spectrum Mill MS Proteomics Workbench software  with a global false discovery rate of 1%.Immunocytochemistry and confocal microscopy visualization was performed using a Zeiss LSM880 (Zeiss). Glass microscopy slides were coated with Poly-L-lysine before cells were seeded overnight. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.01% Triton-X. Slides were blocked with 3% bovine serum albumin in PBS before labeling. Immunolabeling for DR5 and alpha tubulin was visualized with fluorescence-tagged antibodies and counterstained with DAPI. Images were obtained at 40x magnification and analyzed using Zen software (Zeiss). Negative controls were unlabeled samples and secondary antibody only stained samples.t-test with a Welch’s correction. All assays were completed with an N≥ 3. Statistical significance is shown as either *p<0.05 or **p<0.01 as indicated in the results. The effects of drug combination were determined using the CompuSyn software (http://www.combosyn.com). Combination Index (CI) values were then calculated based on cell viability data. CI values of <1, =1 and >1 represent synergism, additive effect and antagonism, respectively.Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software). Statistical comparisons for cell viability, surface protein and total protein expression were determined using one-way ANOVA and un-paired


Increased inflammation associated with leaky gut is a major risk factor for morbidity and mortality in older adults; however, successful preventive and therapeutic strategies are not available to ameliorate these conditions. In this study, we demonstrate that a human-origin Lactobacillus paracasei D3-5 strain (D3-5), even when dead, extended life span of C. elegans. In addition, feeding D3-5 to older mice (>79 weeks) prevented high fat diet (HFD)-induced metabolic dysfunction and decreased leaky gut and inflammation, which were associated with improved physical and cognitive function. D3-5 feeding significantly increased mucin production and proportionately, the abundance of mucin-degrading bacteria Akkermansia muciniphila was also increased. Mechanistically, we show that the cell wall of D3-5 contains lipoteichoic acid (LTA), which enhanced mucin (Muc2) expression via TLR-2/p38-MAPK pathway, which in turn reduced age-related leaky gut and inflammation. This study indicates that the D3-5 and its LTA can prevent/treat age-related leaky gut and inflammation.


Scientific Reports 10.1038/s41598-018-23187-8, published online 27 March 2018Correction to: This Article contains an error in the X axis of Figure 6g. The correct Figure 6 appears below as Figure


Zeta-potential value of PEI-rGO coated with PK5E7 polymer (PK5E7(PEI-rGO)) was −10.9 mV at pH 7.4 and converted to 29.2 mV at pH 6.0, showing pH-responsive charge-conversional property. Sharp-edged plate morphology of PEI-rGO was transformed to spherical nanostructures with vague edges by PK5E7 coating. Size of PK5E7(PEI-rGO) was found to be smaller than that of PEI-rGO in the serum condition, showing its increased serum stability. Loaded doxorubicin (DOX) in PK5E7(PEI-rGO) could be released rapidly in lysosomal condition . Furthermore, DOX-loaded PK5E7(PEI-rGO) showed enhanced anticancer activity in HeLa and A549 cells in the tumor microenvironment-mimicking condition , which would be mediated by non-specific cellular interaction with decorated serum proteins. These results indicate that the pH-responsive charge-conversional PKE polymer coating strategy of cationic rGO nanostructures possesses a potential for acidic tumor microenvironment-targeted drug delivery systems.Tumor tissue represents a slightly acidic pH condition compared to normal tissue due to the accumulation of lactic acids via anaerobic metabolism. In this work, pH-responsive charge-conversional polymer (poly(ethylene imine)-poly( The tumor microenvironment does not simply refer to cancer cell clusters but also to the cancer-associated fibroblasts, myoblasts, epithelial cells, and immune cells along with the extracellular matrix (ECM), oxygen level, pH, and so on ,2,3,4. CThe main component that constitutes the serum protein is negatively-charged albumin . TherefoGraphene is a carbon-based material having a flat two-dimensional monolayer of the hexagonal honeycomb structure . To obtaTherefore, we assumed that this PEI-rGO-based nano drug delivery system would acquire the serum-resistance and the cancer cell killing ability, responding to acidic tumor microenvironment condition by coating with pH-responsive charge-conversional polymers.l-lysine)-poly(l-glutamic acid)s (PKE polymers) with various composition ratios of cationic amino acids and anionic amino acids as pH-responsive charge-conversional polymers [Streptomyces peucetius caesius [Here, we used poly(ethylene imine)-poly nanostructures. Then, physicochemical properties, serum stability, DOX release profile, and anticancer activity of PKE polymer-coated PEI-rGO/DOX nanostructures were examined based on pH-responsive charge-conversion ability.1.8k) and l-glutathione (GSH) were purchased from Alfa Aesar . H-Lys(Z)-OH (Lys(Z)), l-Glutamic acid γ-benzyl ester (BGlu), trifluoroacetic acid, triphosgene, hydrobromic acid solution (33 wt% in acetic acid), agarose, and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich . Dimethylsulfoxide (DMSO), sodium chloride, and tris base were purchased Merck . D2O and trifluoroacetic acid-D were purchased Cambridge Isotope Laboratories . Tetrahydrofuran was purchased from Tokyo Chemical Industry . Doxorubicin (DOX) was purchased from Tocris Bioscience . Diethyl ether and n-hexane were purchased from Daejung . Thiazolyl blue tetrazolium bromide was purchased from Gold Bio . Dulbecco’s Modified Eagles’ Medium (1X) + GlutaMAX-1 (DMEM), Dulbecco’s Phosphate Buffered Saline (DPBS), 0.25% Trypsin-EDTA, fetal bovine serum (FBS), and Penicillin-Streptomycin  were purchased from Invitrogen-Gibco . All other chemicals were purchased and used without any further purification.Poly(ethylene imine) 1.8 kDa  as previously described [1.8k to synthesize PEI1.8k-polyLys(Z) and subsequently, BGlu-NCA  was reacted with PEI1.8k-polyLys(Z) to synthesize PEI1.8k-polyLys(Z)-polyBGlu. After removal of protecting groups, poly(ethylene imine)-poly(l-lysine)-poly(l-glutamic acid)s (PKE polymers) were obtained. The synthesized PKE polymers were named as PK5E5, PK5E7, PK5E9, and PK5E13, respectively, based on the ratios of conjugated l-lysine and l-glutamic acid moieties to PEI1.8k.PKE polymers were synthesized using PEIescribed . BrieflyPEI-conjugated reduced graphene oxide (PEI-rGO)  was kindThe DOX loading in PEI-rGO was confirmed by measuring absorbance (300–700 nm) of PEI-rGO and PEI-rGO/DOX respectively, using a microplate reader . The DOX loading efficiency was examined by measuring DOX fluorescence  of PEI-rGO/DOX in water or 90% DMSO. Ten microliters (10 μL) of PEI-rGO/DOX solution (1 mg/mL PEI-rGO) was added to 90 μL of water or DMSO. The DOX loading amount was calculated by comparison of DOX fluorescence values measured in water and 90% DMSO. DOX loading efficiency was calculated as below:w/w) by dropwise, respectively. The mixtures were incubated for 1 h with orbital shaking at room temperature. Then, they were centrifuged  and re-dispersed in solutions after removal of supernatants.To form PKE(PEI-rGO) or PKE(PEI-rGO/DOX) nanostructures, PEI-rGO or PEI-rGO/DOX solution (deionized water) were added to PKE polymer solutions (deionized water) having pre-determined weight ratios  and PK5E9(PEI-rGO/DOX) (weight ratio = 30) were measured at pH 7.4 and pH 6.0 to examine the property change after DOX loading to the PEI-rGO.The average sizes and zeta-potential values of the prepared PKE(PEI-rGO) and PKE(PEI-rGO/DOX) nanostructures were measured by using a Zeta-sizer Nano ZS90 . First, the average sizes and zeta-potential values of PKE polymer-coated PEI-rGOs (PKE(PEI-rGO)) with various PKE polymer weight ratios  were measured. Additionally, the average size and zeta-potential value of each PKE(PEI-rGO) nanostructure (weight ratio = 30) were measured at pH 7.4 and pH 6.0. Additionally, the average sizes and zeta-potential values of PK5E7 polymer coating (weight ratio = 30). The samples were absorbed onto TEM copper grid. After drying for 12 h and the images were visualized by EF-TEM  under the voltage of 80 kV.The energy filtered transmission electron microscope (EF-TEM) was used to observe the morphology of PEI-rGO and PEI-rGO/DOX nanostructures with and without PK5E7(PEI-rGO) was centrifuged  and dispersed in PBS or PBS containing 10% FBS at pH 7.4 or 6.0. Each sample was incubated at 37 °C with gentle shaking. After pre-determined incubation time , the average size of each sample was measured using Zeta-sizer.The nanostructure stability in serum condition was investigated by measuring the size changes of PEI-rGO based nanostructures. PEI-rGO or PK5E7 coated PEI-rGO/DOX was examined. One-hundred micrograms (100 μg) of PK5E7(PEI-rGO/DOX) (weight ratio = 30) was prepared for measuring DOX release. The total volume of PK5E7(PEI-rGO/DOX) solution was adjusted to 2 mL and the solution was placed in a dialysis membrane (MWCO: 3000) in 50 mL of PBS . The solutions were incubated at 37 °C with gentle shaking in dark condition. After pre-determined incubation time , released DOX amount was analyzed by measuring the DOX fluorescence outside the membrane  using a microplate reader.DOX release profile of PK5E7(PEI-rGO/DOX) was examined by MTT assay in human cervical adenocarcinoma cells (HeLa cells) and human lung adenocarcinoma epithelial cells (A549 cells). HeLa and A549 cells were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin (P/S) . Cells were seeded at 1 × 104 cells/well on 96-well cell culture plates and cultured for 24 h. After reaching 70–80% of cell confluency, the pH of the medium was adjusted to pH 7.4 or 6.0. One-hundred microliters (100 μL) of PK5E7(PEI-rGO/DOX) solution  was treated to each well and incubated for 24 h. DOX only, PK5E7, and PK5E7(PEI-rGO) with various concentration (pH 7.4) were also treated to HeLa cells or A549 cells as controls. Then, each medium was replaced with fresh medium containing 10% FBS and 1% P/S. After further incubation for 24 h, 25 μL of MTT/DPBS solution (2 mg/mL) was added to each well and incubated for 2 h. The medium was carefully removed and 150 μL of DMSO was added to the formazan crystal formed by proliferating cells. The absorbance at 570 nm was measured using a microplate reader. The anticancer activity was expressed as the relative cell viability (%) by comparing the absorbance values with cell only value.Anticancer activity of PK5E7(PEI-rGO/DOX) (weight ratio = 30) was evaluated by flow cytometry analysis in HeLa and A549 cells. Cells were seeded at a density of 2 × 105 cells/well on six-well cell culture plates. Having achieved 70–80% of confluency, the cells were treated with PK5E7(PEI-rGO/DOX) solutions in serum-free or serum-containing medium (pH 7.4 or 6.0) for 4 h, respectively. Then, the cells were washed twice with ice-cold DPBS and detached using trypsin-EDTA. The cells were resuspended in DPBS and analyzed by FACS using BD Accuri C6 flow cytometer  at a minimum of 1 × 104 cells gated per sample. The flow cytometry results were processed by Flowing software .The cellular uptake efficiency of PK5 cells/well on six-well cell culture plates. Having achieved 70–80% of confluency, the cells were treated with PK5E7(PEI-rGO/DOX) solutions (10 μg/mL DOX) in serum-free or serum-containing medium (pH 7.4 or 6.0) for 24 h, respectively. Then, the cells were washed twice with ice-cold DPBS and stained with Hoechst 33342 (10 μg/mL) for 15 min. After washing with DPBS twice, fluorescence images were observed by fluorescence microscope .A549 cells were seeded at a density of 2 × 10l-lysine residues were conjugated to one PEI1.8k molecule and conjugated l-glutamic acid residues in PKE polymers were 53.4, 75.9, 93.7, and 140.5 molecules per one PEI1.8k molecule, respectively. According to the ratios of conjugated l-lysine and l-glutamic acid moieties to PEI1.8k, PKE polymers were named as PK5E5, PK5E7, PK5E9, and PK5E13, respectively. PKE polymers showed pH-responsive charge-conversional ability, according to the protonation/deprotonation behavior of their lysine and glutamic acid residues responding to environmental pH change. For example, the zeta-potential value of PK5E9 polymer particle was increased from −35.0 mV to 27.5 mV as pH was decreased from 10.0 to 3.0. This change could induce the increase of both cellular uptake and serum stability in terms of gene delivery systems. The inherent characteristics of the pH-responsive charge-conversional ability of PKE polymers could be regulated via controlling the ratios between l-lysine and l-glutamic acid residues.According to the previous study , it was Therefore, it was assumed that we would modulate the physicochemical properties of PEI-rGO by coating with PKE polymers via electrostatic interaction for improvement of serum stability and anticancer efficacy responding to environmental pH.First, Z-average sizes and zeta-potential values of PKE(PEI-rGO) nanostructures with various weight ratios between PKE and PEI-rGO were measured to examine the PKE coating effect to PEI-rGO. It was found that intact PEI-rGO without coating had about 200 nm size and 50 mV zeta-potential value.5E5(PEI-rGO) were gradually increased from 204.7 nm to 681.3 nm with the increase of weight ratios from 10 to 30 and then decreased to 272.5 nm at a weight ratio of 50. Zeta-potential values of PK5E5(PEI-rGO) were gradually decreased from 38.7 mV to 22.7 mV with the increase of the weight ratios from 10 to 50. In the case of PK5E7 coating to PEI-rGO (5E7(PEI-rGO) showed homogeneous size distributions, regardless of weight ratios. Their zeta-potential values were reduced from 27.4 to 20.5 mV with the increase of weight ratios from 10 to 50. The size of PK5E9(PEI-rGO) was the largest (718.5 nm) at a weight ratio of 20 (5E9(PEI-rGO) showed zeta potential value of −35.0 at a weight ratio of 10, which remained unchanged significantly (−32.8~−31.8 mV) regardless of weight ratios. PK5E13(PEI-rGO) displayed sizes less than 250 nm (113.03~246.6 nm) and zeta-potential values maintaining negative values (−40.3~−37.0 mV), regardless of weight ratios  nanostructures could form aggregates at certain coating weight ratios of PKE polymers, which were dependent on PKE polymer types. It is thought that PKE polymers with different cationic lysine and anionic glutamic acid ratios, which means the different net charges and chain lengths, would act as electrostatic glues between the nanostructures for aggregate formation during coating process .5E5(PEI-rGO) and PK5E7(PEI-rGO) with the increase of PKE weight ratios mean that anionic glutamic acid residues of PKE polymers would induce the negative surface charges to PEI-rGO by coating. In the case of PK5E9(PEI-rGO) and PK5E13(PEI-rGO), their negative zeta-potential values mean that negative charges from glutamic acid residues exceed positive charges from lysine residues due to their having more anionic moieties (carboxylic acid of glutamic acid) than cationic moieties (amine of PEI and lysine).Decreased zeta-potential values of PKIt was expected that PKE(PEI-rGO) nanostructures would possess pH-responsive charge-conversional property, which means the maintenance of negatively charged surfaces at normal physiological pH, conferring serum stability and conversion to a positively charged surfaces in mild acidic condition like tumor microenvironment, providing improved adsorption to negatively-charged cellular membrane and cellular uptake. Therefore, Z-average sizes and zeta-potential values of PKE(PEI-rGO) nanostructures were examined at pH 7.0 and 6.4.5E5(PEI-rGO) at pH 7.4 (548.7 nm) was slightly decreased to 459.0 nm at pH 6.0, while its zeta-potential at pH 7.4 (21.2 mV) was increased to 37.6 mV at pH 6.0. PK5E7(PEI-rGO) presented 1425.7 nm size probably due to the aggregates formation during coating process and −10.9 mV zeta-potential value at pH 7.4 (5E9(PEI-rGO) exhibited 235.2 nm size and −30.8 mV zeta-potential value at pH 7.4. At pH 6.0, its size was increased to 661.6 nm and the zeta-potential value was 6.0 mV. In the case of PK5E13(PEI-rGO), the size (about 150 nm) and the zeta-potential value (−35.6~−31.8 mV) were not significantly changed, regardless of pH.As shown in t pH 7.4 B. Howeve5E7(PEI-rGO) and PK5E9(PEI-rGO) nanostructures possessed pH-responsive charge-conversional properties, due to the charge balance between carboxylic acid groups of glutamic acid residues and amines of lysine residues and PEI. In these pH conditions (pH 7.4 and 6.0), ε-amines of lysine and primary amines of PEI (pKa = ~10) would be almost protonated, contributing to the positive zeta-potential values of PKE(PEI-rGO) nanostructures, while carboxylic acid groups of glutamic acid residues and other amines of PEI (secondary and tertiary) would modulate net zeta-potential values, displaying pH-dependent protonation/deprotonation behaviors. It was found that coating by PK5E5 polymer with the least glutamic acid portion and PK5E13 polymer with the most glutamic acid portion constructed positively-charged and negatively-charged nanostructures, respectively, at both pH 7.4 and pH 6.0, which means that they lack pH-responsive charge-conversional properties. Although the PK5E9(PEI-rGO) also exhibited the charge-conversional ability, it was presumed that the PK5E9(PEI-rGO) nanostructure would be difficult to access to the cell membrane due to the large particle size and the low surface charge value at pH 6.0.These results indicated that only PKFirst, the drug loading efficiency of PEI-rGO was assessed by measurement of absorbance and fluorescence of DOX-loaded PEI-rGO (PEI-rGO/DOX). As shown in 5E7(PEI-rGO/DOX) was 1570 nm at pH 7.4 and 762.0 nm at pH 6.0. Its zeta-potential value was −13.1 mV at pH 7.4 and increased to 7.1 mV at pH 6.0. PK5E9(PEI-rGO/DOX) was found to have 182.5 nm size at pH 7.4 and 889.9 nm at pH 6.0. Its zeta-potential was −17.7 mV at pH 7.4 and increased to −0.64 mV at pH 6.0. Considering the sizes and zeta-potential values of PKE(PEI-rGO) without DOX loading, a similar tendency responding to pH change was observed even after DOX loading. Since PK5E7(PEI-rGO/DOX) showed suitable charge-conversion behavior from pH 7.4 to 6.0, PK5E7(PEI-rGO/DOX) was focused on for further characterization in this work.To examine the DOX loading effect to PKE(PEI-rGO)s, the average sizes and zeta-potential values of PKE(PEI-rGO/DOX)s were measured at pH 7.4 and 6.0 . The siz5E7 polymer was coated on PEI-rGO and PEI-rGO/DOX, spherical particles were observed  nanostructure against serum proteins was examined by measuring its sizes under serum condition (10% FBS). As shown in 5E7(PEI-rGO) gradually were increased until 4–8 h of incubation, showing the interaction with serum proteins. However, in the case of PK5E7(PEI-rGO), the size of the nanostructures was much smaller than that of uncoated PEI-rGO nanostructure at both pH 7.4 and 6.0, meaning that PKE polymer coating could reduce the interaction with serum proteins. At pH 6.0, it was confirmed that the size of nanostructures was increased by increasing the electrostatic interaction of the positively-charged nanostructures with negatively-charged serum proteins, compared to the result at pH 7.4. The size of PK5E7(PEI-rGO) nanostructure was maintained as about 400 nm even after 24 h of incubation at pH 7.4, showing its high serum stability. In the absence of serum (5E7(PEI-rGO) showed similar sizes, although the sizes were increased in acidic condition probably due to the swelling by protonation of amines. Therefore, this result suggested that PEI-rGO nanostructure could be protected from non-specific interactions with anionic serum proteins at normal physiological pH via coating with charge-conversional PK5E7 polymer.In terms of drug or gene delivery systems, serum proteins in blood plasma are one of the most important obstacles to interrupt their therapeutic effects by the formation of large aggregates . Therefoof serum , PEI-rGO5E7(PEI-rGO/DOX) was investigated in three different conditions simulating the normal physiological condition (pH 7.4), the lysosomal condition after endocytosis (pH 5.0), and the lysosomal reducing condition after endocytosis  (5E7(PEI-rGO/DOX) nanostructure. In the acidic condition, DOX, PK5E7, and PEI-rGO were positively charged and their electrostatic repulsion would weaken the interaction between bound DOX and PEI-rGO. Additionally, hydrogen bonding between rGO and DOX would be strong in the neutral condition, which can suppress DOX release at pH 7.4 compared to pH 5.0 [5E7(PEI-rGO/DOX) under the acidic condition.The drug delivery systems should release their cargo drug molecules to the desired location of cells under various conditions. The DOX release behavior of PK mM GSH) . DOX waso pH 5.0 . Thus, D5E7(PEI-rGO/DOX) would release DOX efficiently after endocytosis, according to this result.DOX can be adsorbed on graphene materials via various mechanisms, such as π-π interactions, hydrogen bonds, and hydrophobic interactions ,40,41. I5E7(PEI-rGO/DOX) was validated by MTT assay in HeLa and A549 cells (5E7 (5E7(PEI-rGO) (5E7(PEI-rGO/DOX) showed DOX concentration-dependent decrease of cell viability in both serum-free and serum condition, regardless of cell types. Interestingly, much higher anticancer efficacy of PK5E7(PEI-rGO/DOX) was observed at pH 6.0 than pH 7.4 in serum condition, although no significant change of cell viability was found in serum-free condition at both pH 7.4 and 6.0 in HeLa cells (5E7(PEI-rGO/DOX) than DOX only was probably due to the slow release of DOX from the nanocarrier [5E7 polymers [Anticancer efficacy of PK49 cells . First, lls  D, and wePEI-rGO) C, were iLa cells D,E. The La cells F,G. Loweocarrier  and the polymers . In addipolymers  and multpolymers  and thatpolymers ,48.5E7(PEI-rGO/DOX) at pH 6.0 due to its pH-responsive charge-conversion, according to the former results. Previously, it was reported that the human serum albumin (HAS)-coated lipoplexes could bind non-specifically to cell surface receptors, analogous to scavenger receptors, which mediate their endocytosis [5E7(PEI-rGO/DOX) could bind to cell membranes non-specifically at pH 6.0 in serum condition, which would induce its facilitated endocytosis and DOX delivery to cancer cells, finally leading to the high anticancer activity. In addition, the increased DOX efficacy in acidic environment was probably due to the expedited DOX release from PK5E7(PEI-rGO) in acidic condition as shown in 5E7(PEI-rGO/DOX) nanostructure would show an enhanced cancer cell killing effect in the acidic tumor microenvironment condition via aid of decorated serum proteins, maintaining the intact status under normal physiological condition.Contrary to pH 7.4, anionic serum proteins such as albumin would bind to PKocytosis . Anotherocytosis . Therefo5E7(PEI-rGO/DOX) in HeLa and A549 cells (5E7(PEI-rGO/DOX) was 10.1% at pH 7.4 and it was decreased to 8.1% at pH 6.0. However, in the presence of serum (5E7(PEI-rGO/DOX) was observed. The uptake efficiency was 27.8% at pH 7.4 and decreased to 8.1% at pH 6.0 in serum-free condition (5E7(PEI-rGO/DOX).Flow cytometry analysis was performed to elucidate the pH and serum effect onto the cellular uptake of PK49 cells . In HeLa49 cells , the celof serum , the uptondition . In seruondition , it was 5E7(PEI-rGO/DOX) at pH 6.0 than pH 7.4 would be induced by decelerated overall cellular activity [5E7(PEI-rGO/DOX) in acidic condition.In the serum-free condition, it was thought that relatively lower cellular uptake efficiency of PKactivity  and by cactivity  and PK5E5E7(PEI-rGO/DOX) at pH 6.0 in serum condition in comparison with the efficiency at pH 7.4, suggested that serum-protein-decorated PK5E7(PEI-rGO/DOX) by electrostatic interaction via charge-conversion would be internalized into cells more efficiently through non-specific interaction of albumin with cell membrane, overcoming the decelerated overall cellular activity and charge repulsion between positively-charged cell surface and PK5E7(PEI-rGO/DOX) in acidic condition.Interestingly, 1.7~2.6 times increased cellular uptake efficiency of PK5E7(PEI-rGO/DOX) nanostructures were further observed by fluorescence microscopy , PK5E7(PEI-rGO/DOX) displayed the highest cellular uptake activity, as shown in flow cytometry analysis result.Cellular uptake behaviors of PKcroscopy . It was Overall, the scheme for the formation of PKE(PEI-rGO/DOX) nanostructures and their serum stability and decorated serum protein-mediated cellular uptake by PKE coating is presented in 5E7(PEI-rGO) was identified by zeta-potential value measurement. The sharp-edged sheet structure of PEI-rGO was transformed to spherical nanostructures with vague edges by PK5E7 coating. It was also found that PK5E7 coating could endow the stability against serum proteins to PK5E7(PEI-rGO) nanostructure. Loaded doxorubicin (DOX) in PK5E7(PEI-rGO) could be released rapidly in lysosomal condition . Furthermore, DOX-loaded PK5E7(PEI-rGO) showed enhanced anticancer activity in cancer cells in tumor microenvironment-mimicking condition, which would be facilitated by improved cellular uptake via non-specific cellular interaction with decorated serum proteins. Therefore, pH-responsive charge-conversional PKE polymer-coating strategy of cationic nanostructures is thought to possess a potential for tumor microenvironment-targeted drug delivery systems.In this work, DOX-loaded PEI-rGO was coated with pH-responsive charge-conversional polymer, PKE polymer, forming PKE(PEI-rGO/DOX) nanostructures and investigated for drug delivery systems. The pH-responsive charge-conversional property of PK


Scientific Reports 10.1038/s41598-019-45379-6, published online 28 June 2019Correction to: This Article contains low resolution versions of Figures 2 and 3. High resolution versions have now been added as Supplementary Information files that accompany the Article.