Fosb transcription start site is enriched for E2F3 specifically over other E2F isoforms. We further conclude that ΔFosB expression is regulated specifically by E2F3a, not E2F3b, that E2f3a expression is specific to D1 receptor-expressing medium spiny neurons, and that E2F3a overexpression in NAc recapitulates the induction of Fosb and ΔFosb mRNA expression observed after chronic cocaine exposure. E2F3a knockdown in NAc does not abolish ΔFosb induction by cocaine, a result consistent with previously published data showing that singular knockdown of upstream regulators of ΔFosB is insufficient to block cocaine-induced expression. Finally, to elucidate potential combinatorial epigenetic mechanisms involved in E2F3a’s regulation of Fosb, we explore H3K4me3 enrichment at the Fosb promoter and find that it is not enhanced by E2F3a overexpression, suggesting that it may instead be a pre-existing permissive mark allowing for E2F3a to interact with Fosb. Together, these findings support a role for E2F3a as a novel, upstream regulator of the addiction-mediating transcription factor ΔFosB in NAc.The transcription factor ΔFosB has been proposed as a molecular switch for the transition from casual, volitional drug use into a chronically addicted state, but the upstream regulatory mechanisms governing ΔFosB expression are incompletely understood. In this study, we find a novel regulatory role for the transcription factor E2F3, recently implicated in transcriptional regulation by cocaine, in controlling ΔFosB induction in the mouse nucleus accumbens (NAc) following cocaine administration. We find that an E2F consensus sequence 500 bp upstream of the  A hallmark question in the neuroscience of addiction is how repeated drug use is translated into long-lasting changes in neural regulation. Long-lasting changes in behavior are mediated in part by intracellular transcriptional mechanisms which affect gene expression and neural circuitry. Therefore, pinpointing signaling pathways downstream of abused drugs is a high priority for understanding the addiction process. To this end, we identify and characterize E2F3a as a novel regulator of ΔFosB, a transcription factor widely studied as a molecular switch for the transition from casual drug use to addiction.Fosb gene, accumulates in nucleus accumbens (NAc) after repeated exposure to virtually all abused substances and thus has been proposed as a molecular switch for the transition from casual drug use into the chronically addicted state by reorganizing gene expression , cAMP response element binding protein (CREB), serum response factor (SRF), and early growth response factor 3  and that E2F3a, but not E2F3b, expression is increased in dopamine receptor type 1 (D1)-expressing medium spiny neurons (MSNs) following cocaine exposure. Further, overexpression of E2F3a, but not E2F3b, recapitulates induction of ΔFosB as seen with repeated cocaine administration. These data establish a novel role for E2F3a in the brain in regulating the molecular response to cocaine, specifically in regulating expression of the transcription factor ΔFosB in NAc.E2F transcription factors are involved in chromatin modification, gene regulation, and RNA processing via direct interaction with and recruitment of histone modifying enzymes and splicing machinery, as well as binding directly to splice sites and influencing the expression of splicing factors . E2F3 haTTCGCGC” . Here, wad libitum. All animal procedures were performed in accordance with guidelines of the Institutional Animal Care and Use Committee at the Icahn School of Medicine at Mount Sinai.C57BL/6J male mice (The Jackson Laboratory), 7–8 weeks old and weighing 25–30 g, were habituated to the animal facility 1 week before use and maintained at 22–25°C on a 12 h light/dark cycle. All animals had access to food and water For quantitative chromatin immunoprecipitation (qChIP) assays and gene expression measures following fluorescence activated cell sorting (FACS), mice were injected intraperitoneally with saline or cocaine (20 mg/kg) once daily for 7 d in their home cage. Mice were then euthanized 24 h following the final cocaine injection.For assaying the effects of viral-mediated E2F3a knockdown, mice were injected intraperitoneally with saline once daily for 4 d beginning 2 d post-viral infusion. On the fifth day, animals were either injected with a final dose of saline or a single, acute dose of cocaine (20 mg/kg) and euthanized 1 h later.E2f3a for knockdown experiments. In these systems, GFP expression is driven by a cytomegalovirus promoter, whereas the select gene of interest is driven by the IE4/5 promoter (Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) and prepared for stereotactic surgery. Thirty-three gauge syringe needles (Hamilton) were used to bilaterally infuse 0.5 µl virus in NAc at a rate of 0.1 µl/min at 1.6 mm anterior, 1.5 mm lateral, and 4.4 mm ventral from bregma at a 10° angle, or in PFC at 1.8 mm anterior, 0.75 mm lateral, and 2.7 mm ventral from bregma at a 15° angle. We used bicistronic p1005 HSV vectors expressing GFP alone or GFP plus E2f3a or E2f3b for overexpression experiments, or HSV vectors that expressed GFP as well as microRNAs targeted against LacZ or promoter . Viral epromoter  and replTissue was collected at the appropriate time point following pharmacological or viral manipulation via cervical dislocation followed by dissection of NAc with a 14-gauge blunt needle for cocaine studies, or a 15-gauge needle for viral studies to ensure only virally infected tissue was dissected. PFC was dissected with a single central 12-gauge punch for cocaine studies, or a 14-gauge punch for viral studies. Tissue was immediately frozen on dry ice. Single animal samples were used in all RNA-qPCR experiments. For qChIP experiments, animals were pooled as described below.m glycine to stop the fixing reaction for 5 min. Punches were then washed 5 times in cold PBS, and passed through a 21-gauge syringe 10 times to homogenize the tissue. The homogenized tissue was centrifuged at 1250 rcf, and then resuspended and incubated in a 0.5% NP-40 cellular lysis buffer for 15 min, centrifuged at 2700 rcf and resuspended in a 1% SDS nuclear lysis buffer for 10 min. The samples were then sheared using a Diagenode Bioruptor XL at high sonication intensity for 30 s on/30 s off for 10 min twice (to prevent overheating). Fragment size and chromatin DNA concentration was verified with an Agilent 2100 Bioanalyzer at 150–300 bp. Sheared chromatin was incubated overnight in a 0.1% SDS solution with the following antibodies previously bound to magnetic beads following the Dynabead protocol : antibody to E2F3 , antibody to Rabbit-IgG , antibody to H3K4me3 . 1 µg of DNA was used for E2F3 and IgG IPs, 250 ng for H3K4me3. After incubation, beads were washed four times: (1) low salt (150 mm NaCl), (2) high salt (500 mm NaCl), (3) 150 mm LiCl, and (4) TE + 50 mm NaCl. Chromatin was eluted off the beads using a 100 mm sodium bicarbonate/1% SDS solution at 65°C for 30 min. Then reverse crosslinking took place overnight at 65°C, and DNA purification was performed via Qiagen Spin Columns. Binding to the Fosb gene was determined by qRT-PCR using primers detailed in qChIP was performed on NAc or PFC punches, pooled from three mice, 72 h after viral infection, or from two mice, 24 h after the last drug treatment. qChIP methodology was modified from Male D1-Tomato and D2-GFP were used to tag D1-receptor expressing and dopamine 2 (D2)-receptor expressing MSNs, respectively, with tdTomato or GFP fluorophores. Whole, tagged neurons were isolated and collected as previously described . BrieflytC values of the treatment condition to the control condition with the ΔΔtC method . Student’s Fosb gene using MatInspector, three putative E2F binding sites . E2F2, E2F5, E2F6, E2F7, and E2F8 are sparsely expressed in the adult mouse brain  = 13.35, p < 0.0001; N = 4–5, 3 mice/sample for all qChIP assays; row a, t(8) = 9.904, p < 0.0001; We identified, through a detailed investigation of the ng sites . Two areosb mRNA A. To tesse brain  and wereFosb by E2F3 at this −500 bp site is specific to NAc, we repeated qChIP for E2F3 in three additional regions of the reward circuitry: PFC, VTA, and CPu. In PFC, significant binding over an IgG control was observed = 48.44, p < 0.0001; N = 5; row b, t(8) = 1.69, p = 0.2208; F = 7.024, p = 0.0243; t(5) = 0.275, p = 0.9999; N = 3–4; F = 25.113, p < 0.0001; N = 5; row d, t(8)= 1.451, p = 0.3324; Fosb after repeated cocaine is specific to NAc.Next, to determine whether regulation of E2f3’s two isoforms, E2F3a and E2F3b, we performed FACS on tagged D1 MSNs or D2 MSNs and analyzed the mRNA expression of each transcript. In isolated D1 MSNs, both E2f3a and E2f3b are expressed at baseline levels, but only E2f3a expression is increased by repeated cocaine exposure. Main effects of both drug treatment = 9.624, p = 0.0127; row e, F= 6.845, p = 0.028; N = 3–4; row e, E2f3a expression only was confirmed via post hoc analysis (E2f3a: t(5) = 4.186, p = 0.0047; E2f3b: t(4) = 0.3328, p = 0.9999;E2f3b is expressed minimally but no E2f3a expression was detected under control or cocaine conditions = 15.489, p = 0.0034; N = 3–4; E2f3a is induced by cocaine selectively in the same neuronal population in which cocaine induction of ΔFosB occurs. Thus, To explore the cell-type-specific expression of F = 9.332, p = 0.0007; N = 6; row g, Fosb and ΔFosb mRNA expression was confirmed via post hoc analysis (Fosb: t(10) = 3.138, p = 0.0078; ΔFosb: t(9) = 2.868, p = 0.0185; Fosb–ΔFosb mRNA was not affected by E2F3a overexpression (t(9) = 0.3142, p = 0.7605; row h, Fosb splice site in Exon IV. E2F3b overexpression did not affect mRNA expression of either Fosb isoform (Fosb: t(10) = 1.336, p = 0.5757; ΔFosb: t(10) = 0.575, p = 0.9999; To probe which E2F3 isoform regulates ΔFosB expression, we performed stereotactic surgery to infuse HSV-GFP, -E2F3a, or -E2F3b into NAc. A main effect of virus was observed  = 1.637, p = 0.2162; N = 6; Because significant binding of E2F3 was observed in PFC E, but waFosb to induce ΔFosb expression, we knocked down expression of E2F3a with a miRNA and assessed ΔFosb mRNA levels following an acute dose of experimenter-administered cocaine. We found a main effect of treatment  = 3.297, p = 0.0415; N = 5–7; row j, post hoc analysis that an acute dose of cocaine induces a near-significant increase in ΔFosb mRNA expression  = 2.492, p = 0.0508; ΔFosb  = 1.223, p = 0.6855; row j, t(10) = 1.3311, p = 0.9999; Fosb promoter is sufficient for induction of ΔFosb expression but is not solely required. This is consistent with published data on two other transcription factors that regulate ΔFosB: SRF and CREB. Ablation of both of these upstream regulators simultaneously blocks the cocaine-induced increase in ΔFosb, but knockdown of either molecule alone is insufficient  = 3.619, p = 0.0224; N = 5–6, 3 mice/sample for all qChIP assays; row k, post hoc analysis (t(7) = 3.285, p = 0.0093; To determine whether this increase in Fosb or if perhaps H3K4me3 is a pre-existing mark. No effect of E2F3a overexpression was observed on H3K4me3 enrichment  = 0.057, p = 0.8126; N = 5–6; row l, F = 37.61, p < 0.0001; row l, Fosb.In the same genome-wide study that implicated E2F3 as a regulator of molecular responses to cocaine in the NAc, H3K4me3 enrichment was highly correlated with predicted E2F3 binding motifs, suggesting that this mark may be important in E2F3 action . We therFosb promoter at a site 500 bp upstream of the TSS is increased after repeated cocaine exposure. E2F3a overexpression in this brain region increases E2F3 binding at this same site and induces transcription of both Fosb products, Fosb and ΔFosb mRNA. Interestingly, we observed significant baseline binding of E2F3 500 bp upstream of TSS in PFC, VTA, and CPu but not regulation by cocaine. This result could point to a role for E2F3 in regulating steady-state Fosb expression in these reward circuitry regions, but one that is not affected by drug exposure. Further, using FACS, we determine that E2F3a’s regulation of ΔFosB is restricted to D1 MSNs in NAc, based on E2f3a’s restricted baseline and cocaine-induced expression in this cell type. There was also no effect of cocaine on much lower levels of E2f3b in either D1 or D2 MSNs. Based on overexpression experiments, we deduced that E2F3 binding in NAc, which cannot be differentiated by qChIP because of antibody limitations, is likely E2F3a, given that overexpression of E2F3b has no effect on expression of either Fosb isoform, whereas E2F3a increases expression of both isoforms. By contrast, miRNA knockdown of E2F3a is not sufficient to abolish the induction of ΔFosB following cocaine. Together, these results implicate E2F3a as a novel isoform- and region-specific regulator of ΔFosB, opening a new avenue for targeting this upstream regulator of addiction-mediated behaviors and a previously unrecognized signal transduction pathway affected in NAc by cocaine exposure.Here, we demonstrate that a specific isoform of the E2F3 transcription factor, E2F3a, is a novel regulator of the cocaine-elicited expression of the addiction-associated transcription factor ΔFosB. We show that in NAc specifically, E2F3 binding at the Before this investigation, two studies implicated E2F3a expression in NAc as a mediator of the transcriptional, alternative splicing, and behavioral responses to cocaine . The firFosb, alternative splicing of Exon IV, and the cell-type specificity of these events. Our findings add E2F3 to a list of several other factors that influence ΔFosB expression at different points in its production. PTB (encoded by Ptbp1) has been shown to regulate Fosb splicing in vitro . We did not find any direct involvement of E2F3a in the alternative splicing of Fosb and ΔFosb, as there was no evidence of binding at the Exon IV alternative splice site and no effect on the ratio of the two isoforms (Ptbp1 itself (Ptbp1 via E2F3a, increasing expression of a specific isoform of PTB that leads to the alternative splicing event giving rise to ΔFosB. However, prior work has shown no effect of chronic cocaine on Fosb splicing (There are three critical points of regulation in the expression of ΔFosB: transcription of in vitro . Specifiisoforms B. Intere1 itself . Thus, isplicing .Fosb promoter at −250 bp in response to cocaine exposure (ΔFosb mRNA after an acute cocaine exposure (The transcription factors, CREB and SRF, have previously been shown to bind the exposure . Deletioexposure F.Fosb in NAc, PFC, VTA, and CPu. However, E2F3 binding to Fosb is specifically altered by cocaine exposure in NAc only (Fosb expression after cocaine exposure that is specific to NAc. Moreover, we show that expression of E2f3a is specific to D1 MSNs in NAc (E2F3a is expressed throughout the brain , and we NAc only , indicats in NAc A,B. Prevs in NAc . These pE2f3a in D2 MSNs in NAc at baseline, and a D1 MSN-specific increase in the transcript elicited by chronic cocaine exposure (E2f3a expression was detected at baseline in D2 MSNs, how ΔFosB is regulated in this cell type remains an outstanding question. Finally, our results support regional specificity for E2F3a’s function and suggest that it acts differently in distinct cell populations in different regions.In addition, another past study of ΔFosB’s function in D1 and D2 MSNs investigated the effects of overexpression in each cell type on cocaine-elicited reward. This investigation found that ΔFosB overexpression in D1 but not D2 MSNs in NAc was sufficient to cause cocaine conditioned place preference at a dose of 3.75 mg/kg . Here, wexposure A,B. TogeFosb in the CPu after cocaine exposure (Given this MSN-specific and region-specific expression, our data support a broader understanding that ubiquitously expressed transcription factors target specific loci in different cells and specific brain regions. Despite this principle, it is interesting that we observed a non-significant decrease in E2F3 binding at the −500 bp site on exposure F. NAc anexposure . ΔFosB iexposure . Given oThat ΔFosB expression is specifically regulated in NAc by E2F3a, and not E2F3b, is also of note. The bioinformatic prediction of E2F3 as one of the most prominent upstream regulators of cocaine-induced gene expression and alternative splicing changes suggested a novel role for the E2F family in cocaine action but did not discriminate among family members or between these two E2F3 isoforms . E2F3a aFosb promoter was unaffected. This result is consistent with the former hypothesis, that H3K4me3 is a pre-existing mark in NAc neuronal euchromatin that could allow E2F3a to bind its consensus sequence 500 bp upstream of the Fosb TSS and induce transcription. It is interesting to note that H3K4me3 is also enriched at baseline at −250 bp (Fosb (Fosb transcription, though this speculation deserves further investigation in follow-up studies of Fosb’s transcriptional activators.In addition to predicting E2F3 as a novel upstream regulator,  −250 bp B, the sibp (Fosb ; this imFosb in NAc, such as H3K4me1, H3K27me3, or H3K27ac, among many others. Likewise, we did not investigate the status of H3K4me3 enrichment at Fosb’s −500 bp site in other brain reward regions. To this point, however, histone deposition data from the Roadmap Epigenomics Consortium shows enrichment of H3K4me3 at the −500 bp site upstream of the Fosb promoter in adult mouse forebrain, hindbrain, and midbrain (Fosb in NAc but not in the other brain regions studied. More targeted investigations are therefore needed to identify the histone modifications and perhaps other chromatin-remodeling factors that are responsible for E2F3a’s unique action on Fosb expression within D1 MSNs of the NAc.We did not investigate any other permissive or repressive chromatin marks that might interact with E2F3a at midbrain . This obIn summary, we demonstrate a novel role for E2F3a, a specific isoform of the E2F family of transcription factors, in regulating the expression of ΔFosB downstream of cocaine action in NAc. ΔFosB is a critical regulator of the behavioral responses to diverse substances of abuse that can lead to addiction. Thus, understanding how ΔFosB is regulated by upstream transcription factors like E2F3a in the context of cocaine exposure will lead to a greater understanding of how this critical regulator might be exploited for potential therapeutic development in the treatment of addiction and other disorders related to the use of commonly abused substances.


Symbiodiniaceae, the functional roles of coral-associated bacteria remain largely elusive. By manipulating the Symbiodiniaceae bacterial community, we demonstrated that a bacterium that produces an antioxidant carotenoid could mitigate thermal and light stresses in cultured Symbiodiniaceae isolated from a reef-building coral. Therefore, this study illuminates the unexplored roles of coral-associated bacteria under stressful conditions.Occupying less than 1% of the seas, coral reefs are estimated to harbor ∼25% of all marine species. However, the destruction of coral reefs has intensified in the face of global climate changes, such as rising seawater temperatures, which induce the overproduction of reactive oxygen species harmful to corals. Although reef-building corals form complex consortia with bacteria and photosynthetic endosymbiotic algae of the family  Symbiodiniaceae and bacteria, collectively termed the coral holobiont. These bacteria are hypothesized to be involved in the stress resistance of the coral holobiont, but their functional roles remain largely elusive. Here, we show that cultured Symbiodiniaceae algae isolated from the reef-building coral Galaxea fascicularis are associated with novel bacteria affiliated with the family Flavobacteriaceae. Antibiotic treatment eliminated the bacteria from cultured Symbiodiniaceae, resulting in a decreased maximum quantum yield of PSII (variable fluorescence divided by maximum fluorescence [Fv/Fm]) and an increased production of reactive oxygen species (ROS) under thermal and light stresses. We then isolated this bacterial strain, named GF1. GF1 inoculation in the antibiotic-treated Symbiodiniaceae cultures restored the Fv/Fm and reduced the ROS production. Furthermore, we found that GF1 produces the carotenoid zeaxanthin, which possesses potent antioxidant activity. Zeaxanthin supplementation to cultured Symbiodiniaceae ameliorated the Fv/Fm and ROS production, suggesting that GF1 mitigates thermal and light stresses in cultured Symbiodiniaceae via zeaxanthin production. These findings could advance our understanding of the roles of bacteria in Symbiodiniaceae and the coral holobiont, thereby contributing to the development of novel approaches toward coral protection through the use of symbiotic bacteria and their metabolites.Reef-building corals form a complex consortium with photosynthetic algae in the family  Symbiodiniaceae and other microorganisms, such as bacteria and fungi  was dominated by bacteria affiliated with the classes Alphaproteobacteria and Flavobacteriia (Flavobacteriia) sequences and an increase in the relative abundance of bacteria affiliated with the class Cytophagia  and light stress (at a temperature of 24°C and a high light intensity of 200 μmol photons m−2 s−1). Consequently, the Abx group showed a significantly lower maximum quantum yield of PSII (variable fluorescence divided by maximum fluorescence [Fv/Fm]) and increased ROS production under thermal and light stresses, whereas no significant differences in the Fv/Fm and ROS production were observed under nonstressful conditions between the control and Abx groups  with a 16S rRNA gene sequence identical to the representative sequence of OTU2 from the Symbiodiniaceae culture (Muricauda lutaonensis CC-HSB-11 (Flavobacteriaceae (Symbiodiniaceae with GF1 and cultured them for 3 months without antibiotics to stabilize the association with GF1 (Symbiodiniaceae (Abx+GF1) showed a similar bacterial community  in the cultured Symbiodiniaceae, we analyzed the metabolites produced in GF1, which accumulates orange pigments  with 85× coverage and an g domain , suggestg domain , 28. The3,345 bp g domain . The yieg domain .10.1128/mBio.01019-19.3TABLE S1Table S1, XLSX file, 0.1 MB.MRM parameters for zeaxanthin analysis. Download Copyright © 2020 Motone et al.2020Motone et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01019-19.6TABLE S4Table S4, XLSX file, 2.0 MB.Genome annotation of GF1 according to the RAST annotator. Download Copyright © 2020 Motone et al.2020Motone et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Symbiodiniaceae can mitigate the oxidative damage induced by thermal and light stresses. Incubating the Abx algal culture with 1 μg/ml (1.76 μM) zeaxanthin resulted in significantly higher Fv/Fm and lower ROS production under thermal stress  of zeaxanthin required to mitigate light stress (0.1 μg/ml). Conversely, the estimated zeaxanthin production (8.04 × 10−2 μg/ml) was an order of magnitude less than the effective amount (1 μg/ml) under the thermal stress condition (To investigate whether zeaxanthin dosages (0.1 and 1 μg/ml) are comparable to the zeaxanthin production by GF1, we performed colony-forming assays of GF1 in the Abx+GF1 algal culture and estimated the zeaxanthin production before and after stress challenges. The CFU of GF1 were 6.16 × 10ectively , and 5.9ectively . As 1 g l weight , the zeaexposure  and 9.72exposure . Given texposure , GF1 wasondition . This inondition .in situ hybridization (FISH) using a 6-carboxyfluorescein (FAM)-labeled oligonucleotide probe, CF319a (Flavobacteriaceae bacteria (GF1). FAM signals were detected from bacteria in the vicinity of autofluorescent Symbiodiniaceae cells in the control and Abx+GF1 cultures but were absent in the Abx algal culture (Flavobacteriaceae bacteria (GF1) and that Flavobacteriaceae bacteria were intimately associated with Symbiodiniaceae rather than present only in the medium.To determine whether there are close interactions between bacteria and algae, we performed fluorescence , CF319a , to spec culture . This reFlavobacteriaceae bacteria are associated with corals. To this end, we isolated bacteria from coral tissue homogenates and sequenced the 16S rRNA genes of the bacteria. Consequently, 14.7% of all of the isolates that formed colonies on marine agar plates (19/129 colonies) showed a 16S rRNA gene sequence identical to that of GF1  methanol at a flow rate of 0.4 ml/min. A 1-μg/ml zeaxanthin standard was shown for retention time comparison. Zeaxanthin was not detected from the methanol used for extraction. Download FIG S1, PDF file, 0.2 MB.LC-UV-visible chromatograms of the methanol extract of GF1. Zeaxanthin and other metabolites were detected in GF1 at a wavelength of 450 nm. Samples were separated by an InertSustain AQ-CCopyright © 2020 Motone et al.2020Motone et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Alphaproteobacteria (OTU1) were not eliminated from the Symbiodiniaceae culture by antibiotics, which is consistent with a previous report  that are most likely essential for the survival of cultured Symbiodiniaceae  are beneficial to cultured Symbiodiniaceae.We observed that s report . Since t1 groups , it is dshilonii  and Vibrilyticus  are the iniaceae –42. Inde1.05b.01 , which phway see . Furtherhway see , 44 for 10.1128/mBio.01019-19.2FIG S212 biosynthesis genes in the genome of alpha proteobacterium Mf 1.05b.01. The annotation of each gene, which starts with “peg,” is listed in FIG S2, PDF file, 0.1 MB.Putative vitamin BCopyright © 2020 Motone et al.2020Motone et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Symbiodiniaceae from corals, we used antibiotics to suppress the growth of bacteria; otherwise, the algal cells would not have been able to grow  and kept at 24°C in an aquarium (Shimizu Laboratory Supplies) containing ∼200 liters of artificial seawater prepared with Coral Pro Salt . The aquarium was equipped with a light-emitting diode (LED) lamp , and 10% of the seawater was replaced every 3 to 4 days.The Symbiodiniaceae, a G. fascicularis colony was fragmented to obtain a single polyp and centrifuged at 100 × g for 10 s in a 1.5-ml tube to remove surface-associated seawater. The tissue of the polyp was harvested by further centrifugation at 8,000 × g for 2 min and suspended in 1 ml of f/2 liquid medium. Then, the tissue suspension was serially diluted and incubated on f/2 agar plates supplemented with antibiotics   was used to measure light intensity. All of the Symbiodiniaceae isolates were identified as Durusdinium (formerly Symbiodinium clade D) (To isolate tomycin)  at 24°C clade D)  based onclade D) .Symbiodiniaceae was perturbed by incubating an algal culture for 2 months in f/2 liquid medium supplemented with the antibiotic cocktail described above. To isolate a bacterium from the cultured Symbiodiniaceae, the algal suspension was serially diluted and spread on marine agar plates . After incubation at 25°C for 5 days, a colony showing orange pigmentation was isolated, purified, and named GF1. The antibiotic-treated Symbiodiniaceae (∼105 cells) were inoculated with ∼106 CFU of GF1 and cultured in the absence of antibiotics for at least 3 months to stabilize the association with Symbiodiniaceae before being used in subsequent experiments.The bacterial community of Symbiodiniaceae cells were harvested via centrifugation at 8,000 × g for 5 min and immediately stored at −80°C until processing. The total DNAs were extracted using the PowerSoil DNA Isolation kit  according to the manufacturer’s instructions. The variable region V1/V2 of the 16S rRNA gene was amplified using the primers 8F-overhang (5ʹ-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGAGTTTGATCMTGGCTCAG-3ʹ) and 338R-overhang (5ʹ-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGCTGCCTCCCGTAGGAGT-3ʹ). PCR amplifications were performed in a final volume of 25 μl, containing 12.5 μl of KAPA HiFi HotStart ReadyMix, 0.75 μl of each primer (10 μM), and 3 ng of template DNA. The PCR cycling conditions were as follows: initial denaturing at 95°C for 4 min and then 23 cycles of 98°C for 20 s, 55°C for 15 s, and 72°C for 15 s, followed by a final extension at 72°C for 4 min. The amplicons were subsequently indexed using the Nextera XT Index kit  and sequenced on the Illumina Miseq platform using 2 × 250-bp paired-end v2 chemistry.For the taxonomic analysis, the forward reads were cropped to 240 bp using Trimmomatic  and subs10.1128/mBio.01019-19.5TABLE S3Table S3, XLSX file, 0.1 MB.OTU sequence counts, taxonomic classification, and 16S rRNA representative sequence. Download Copyright © 2020 Motone et al.2020Motone et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the http://www.clustal.org/).Genomic DNA was isolated from GF1 grown on marine agar plates using the DNeasy Blood & Tissue kit (Qiagen). The 16S rRNA gene of GF1 was PCR-amplified using KOD FX Neo  and the primers 8F (5ʹ-AGAGTTTGATCMTGGCTCAG-3ʹ) and 1492R (5ʹ-GGTTACCTTGTTACGACTT-3ʹ) according to the manufacturer’s instructions. The PCR cycling conditions were as follows: initial denaturing at 94°C for 2 min and then 30 cycles of 98°C for 10 s, 55°C for 30 s, and 68°C for 80 s, followed by a final extension at 68°C for 3 min. The 16S rRNA gene of GF1 was sequenced . Other bacterial sequences were obtained through EzBioCloud . The phySymbiodiniaceae cells were acclimatized in 5 ml f/2 medium in a 14-ml round-bottom tube  under the same culture conditions described above for at least 5 days before stress exposure. For the heat stress challenge, Symbiodiniaceae cells at an initial cell density of 106 cells/ml were exposed to thermal (31.5°C) or nonthermal (24°C) stress at a light intensity of 50 μmol photons m−2 s−1. For the light stress experiment, the Symbiodiniaceae cells were incubated at 24°C under light (200 μmol photons m−2 s−1) or nonlight (50 μmol photons m−2 s−1) stress. The Fv/Fm was measured from the bottom of the tubes using a pulse-amplitude-modulated fluorometer  after the algal cultures had been dark adapted for 30 min. To quantify the ROS production in the algal cultures, 195 μl of algal suspension was incubated with 5 μl of 2ʹ,7ʹ-dichlorofluorescin diacetate  in a 96-well plate for ∼30 min in darkness, followed by measurement of the fluorescence intensity (excitation at 485 nm and emission at 527 nm) using a microplate fluorometer . The data were represented as relative fluorescence normalized by algal cell numbers after subtracting the fluorescence intensity in f/2 medium without algal cells.de novo assembled using SPAdes (version 3.13.1) (A sequencing library was prepared from the genomic DNA of GF1 using the Nextera DNA Library Preparation kit (Illumina) according to the manufacturer’s protocol and sequenced on the Illumina Miseq platform using 2 × 150-bp paired-end v2 chemistry. The raw sequencing reads were filtered using fastp (version 0.20.0)  with the 3.13.1)  with the 3.13.1) . All of  3.13.1) .18 column  at a column temperature of 35°C. The mobile phase comprised 90% (vol/vol) methanol containing 0.1% (vol/vol) formic acid at a flow rate of 0.4 ml/min. Electrospray ionization was performed at 4 kV, with the positive mode at 250°C in the desolvation line and 300°C in the interface. Nebulizing and drying gases were set at flow rates of 2 and 10 liters/min, respectively. Data acquisition was performed based on the MRM mode  for 10 s. The LC-MS/MS analysis was performed using LC  triple quadrupole mass spectrometry . The samples were separated by an InertSustain AQ-CMRM mode , and the−2 s−1) for 5 days, the Abx algal culture was exposed to either thermal stress  for 6 days or light stress  for 1 day to induce oxidative damage. Subsequently, the Abx algal culture was supplemented with zeaxanthin to a final concentration of 0.1 or 1 μg/ml and incubated for 2 days under the same stressful conditions followed by Fv/Fm and ROS production measurements as described above.A stock solution of zeaxanthin  was prepared in dimethyl sulfoxide. After acclimation under nonstressful conditions . The coral tissue suspensions were serially diluted and spread on marine agar plates to form 30 to 100 bacterial colonies. Among the colonies formed during the 10-day incubation at 25°C, those showing orange pigmentation similar to that of GF1 were directly subjected to PCR amplifications as described above. The amplified 16S rRNA genes of the bacteria were then sequenced (Eurofins Genomics) and compared with that of GF1.The Symbiodiniaceae, the Abx algal culture was spread on a marine agar plate. After incubation at 25°C for 10 days, a white colony was isolated and incubated at 98°C for 5 min in ultrapure water to extract DNA. After centrifugation at 14,000 × g for 1 min, the supernatant was used as a DNA template for PCR using 8F and 1492R primers. The amplified 16S rRNA gene was sequenced (Eurofins Genomics). The sequence was identical to the representative sequence of OTU1    and anno00000.1) . All of notators .10.1128/mBio.01019-19.7TABLE S5Table S5, XLSX file, 1.5 MB.Genome annotation of alpha proteobacterium Mf 1.05b.01 according to the RAST annotator. Download Copyright © 2020 Motone et al.2020Motone et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the PRJNA514199. The 16S rRNA gene of GF1 was deposited in GenBank under accession number MK391175. The GF1 genome sequencing data were deposited in GenBank under accession number PRJNA588666. The 16S rRNA gene of the alphaproteobacterium isolated in the present study was deposited in GenBank under accession number MN658478.The 16S rRNA amplicon sequencing data were deposited in the NCBI Sequence Read Archive under accession number


G-quadruplexes (GQs) formed from four consecutive repeats have been shown to possess high-structural and mechanical diversity. In principle, a GQ can form from any four repeats that are not necessarily consecutive. To understand the dynamics of GQs with positional multiplicity, we studied five and six repeats human telomeric sequence using a combination of single molecule FRET and optical tweezers. Our results suggest preferential formation of GQs at the 3′ end both in K+ and Na+ solutions, with minor populations of 5′-GQ or long-loop GQs. A vectorial folding assay which mimics the directional nature of telomere extension showed that the 3′ preference holds even when folding is allowed to begin from the 5′ side. In 100 mM K+, the unassociated T2AG3 segment has a streamlining effect in that one or two mechanically distinct species was observed at a single position instead of six or more observed without an unassociated repeat. We did not observe such streamlining effect in 100 mM Na+. Location of GQ and reduction in conformational diversity in the presence of extra repeats have implications in telomerase inhibition, T-loop formation and telomere end protection.A human telomere ends in a single-stranded 3′ tail, composed of repeats of T This G-nditions ,9–10. Tenditions  and telonditions .2AG3 repeats , nuclear magnetic resonance and X-ray crystallography have revealed extreme polymorphism in human telomeric GQs composed of four T repeats . Such di studies .2AG3) repeats and stretches for several kilobases, it has been assumed that GQs can form anywhere along the G-rich strand . GQ locations on telomeric overhangs can modulate telomerase extension or mechanism of alternative lengthening of telomere (ALT) (2AG3 repeat at the 5′ end (2AG3 repeats have been observed using single molecule fluorescence resonance energy transfer (smFRET)  . An exone 5′ end . Anothere 5′ end . Ensembl(smFRET) . In addi(smFRET) . In a re(smFRET) . However2AG3 repeats. Using guanine to thymine (G to T) mutation at the central G of a T2AG3 repeat, which is known to restrict the participation of the repeat in GQ formation /Cy3/T17TCGGGAGCGGACGCACGG-3′ (hTel28) and 5′-TGGCGACGGCAGCGAGGC5TGGG(TTAGGG)/Cy3/T17TCGGGAGCGGACGCACGG-3′ (hTel34), where the telomeric repeat motif is bold-faced. For the repeat site selective mutation studies, five hTel28 mutants were designed such as GTG(TTAGGG)4T (hTel28.1), GGG(TTAGTG)(TTAGGG)3T (hTel28.2), GGG(TTAGGG)(TTAGTG)(TTAGGG)2T (hTel28.3), GGG(TTAGGG)2(TTAGTG)(TTAGGG)T (hTel28.4) and GGG(TTAGGG)3(TTAGTG)T (hTel28.5), where the underline denotes mutation form G to T. A truncated GQ strand hTel16, where GGG(TTAGGG)3T was replaced with GGG(TTAGGG)2T, was also used in our experiments. For further probing GQ formation in hTel34, an internal position was labeled with Cy3 and referred to as hTel34′: 5′-TGGCGACGGCAGCGAGGC3TGGG(TTAGGG)/Cy3/2T(TTAGGG)T17TCGGGAGCGGACGCACGG-3′. Another sequence designed to the same effect was designated as 6T.hTel22.6T: 5′TGGCGACGGCAGCGAGGC6GGG(TTAGGG)4T6T/Cy3/T17TCGGGAGCGGACGCACGG-3′. A complementary stem strand of sequence 3′/Biotin/ACCGCTGCCGTCGCTCCG/Cy5/5′ was used to immobilize the telomeric sequences of interest onto the slide surface. A second complementary strand (λ-bridge) of sequence 3′ AGCCCTCGCCTGCGTGCCTCCAGCGGCGGG 5′ was used to bridge the hTel strand with the 12 nt COS site of the λ-DNA. The other end of the λ-DNA could be further annealed with a digoxigenin labeled strand of sequence 3′AGGTCGCCGCCC/dig/5′. The hTel strands were first annealed with the complementary stem in 1.1:1 ratio in a buffer containing 50 mM NaCl and 10 mM Tris–HCl, pH 8, at 95°C for 5 min, followed by slow cooling to room temperature. The final construct for single molecule experiments by total internal reflection microscopy (TIRF) was generated by adding the λ-bridge DNA to the above mixture in the ratio of 1.5:1 to the complementary stem strand followed by incubation with rotation at room temperature for an hour.5′-TGGCGACGGCAGCGAGGC+ at 80°C for 10 min and then quenched on ice for 5 min. The hTel constructs and bovine serum albumin (BSA) were added to the λ-DNA at a final concentration of 8 nM and 0.1 mg/ml, respectively, and incubated/rotated at room temperature for 2–3 h. The dig-strand was next added to a final concentration of 200 nM and then incubated with rotation at room temperature for 1 h.For integrated smFRET-optical tweezers assay, λ-DNA  was first heated in presence of 120 mM NaIn order to eliminate non-specific surface binding, coverslips and quartz slides/glass slides used in our experiments were passivated with polyethylene-glycol (PEG)  . The PEGFor integrated smFRET-optical tweezers experiments, a similar imaging chamber was incubated in blocking buffer containing 10 mM Tris HCl pH 8, 50 mM NaCl, 1 mg/ml BSA [NEB] and 1 mg/ml tRNA [Ambion] for 1 h. The DNA constructs were immobilized on the surface at ∼10 pM concentration, via biotin-neutravidin interaction. Next, anti-digoxigenin coated polystyrene beads (Polysciences) were attached to the immobilized construct by incubation in a buffer containing 10 mM Tris HCl pH 8 and 50 mM NaCl for 30 min. At last, data were acquired in an imaging buffer consisting of 50 mM Tris–HCl pH 8, 0.8% w/v D-Glucose [Sigma], 0.5 mg/ml BSA [NEB], 165 U/ml glucose oxidase [Sigma], 2170 U/ml catalase [Roche], 3 mM Trolox [Sigma] and pre-determined amount of NaCl/KCl.An integrated single molecule fluorescence-optical trap instrument was recently developed in our lab to probe conformational changes of biomolecular systems under tension ,38. In sID and IA, respectively, were corrected for background signals and crosstalk and used to estimate smFRET efficiency E using IA/(IA+ID). E histograms were constructed by averaging the first ten data points of each molecule's time trace.In the absence of force, a prism-type total internal reflection microscope, with 532 nm laser excitation and back-illuminated electron-multiplying charge-coupled device camera  was used to detect smFRET signals . The donet al. . These derived parameters , and ΔG‡) were then used to reconstruct the force profiles , using a quartz cell of 1 mm optical path length. The oligonucleotides were diluted to 20 μM in a buffer containing 20 mM Tris pH 8 and appropriate concentration of K4TGGG(TTAGGG)/Cy3/GGACGCACGG-3′. The hTel28 was then annealed to its complementary sequence in the ratio of 1:1.1 at 95°C for 5 mins, in a buffer containing 50 mM NaCl and 10 mM Tris–HCl, pH 8, followed by slow cooling to room temperature. The duplexes were then immobilized on the PEG-passivated surface. A total of 50 nM Rep-X was then incubated in a loading buffer  for 2 min. The unbound Rep-X was washed off simultaneously and the unwinding reaction was initiated by adding the unwinding buffer  [ThermoFisher Scientific], 10% glycerol, 1% BSA). For characterization of the maiden folded GQ conformation, the unwinding reaction was quenched after a minute and images were acquired for smFRET analysis. For the real-time observations, imaging was started a few seconds before addition of the unwinding buffer. A detailed protocol has been described by Hua et al. , flanked by two duplex handles at the 5′ and 3′ ends. CD spectrum of hTel28 in 100 mM K+ showed a positive peak at ∼289 nm with shoulders at 265 and 255 nm and a negative peak at 235 nm, suggestive of hybrid GQs s Figure  41). Th. Th4T), e Figure .+ concentration , or with an inner long loop bearing the second, third or fourth T2AG3 segment (long-loop GQs). Therefore, at least five different GQ structures can be conceived depending on which repeat is excluded . Ho. Ho+ sho, Figure . Previou00 mM K+ ,32, simif a loop . On the f a loop , consistE-values . hTel28.E-values . HoweverE-values . As the E-values , the difE distributions of the other four hTel28 variants and hTel28, 5′ end GQs and long-loop GQ may exist as minor populations , 64% 3′ GQ (128 events) and 18% long-loop GQ (35 events) of the recorded events, respectively  and refolding forces (frefold) were defined as the force(s) corresponding to midpoint(s) of the E transitions during abrupt unfolding and refolding. Diverse funfold values observed across different pulling cycles could be fit to two force clusters centered at ∼7.5 and 17 pN  of the pulling cycles . Combining these results with zero-force smFRET studies, we propose that in 100 mM KE decreases as the ssDNA tail is stretched. Thus, the reversible E changes between ∼0.68 and ∼0.39 under applied forces shown by 5′ GQs and classified as Type II partial unfolding of hTel28 may be due to an ultrastable GQ population. On the other hand, a greater degree of unraveling observed for Type I partial unfolding (2AG3 segment at the 5′ end with (dT)6 (hTel28.1T). Thus, hTel28.1T serves as a 22 nt long GQ forming sequence with a pendant (dT)6 at its 5′ end.We previously classified diverse mechanical behavior of hTel22 that contain four telomeric repeats into (i) abrupt and complete unfolding, (ii) partial unfolding and (iii) an ultrastable population that could not be perturbed by forces up to ∼28 pN . GQs fornfolding  can be dnfolding . Interesnfolding . Hence, + concentration-dependent manner  can be grouped into three force clusters centered at ∼3.5, 10 and 20 pN  and reminiscent of Type I partial unfolding of hTel28 . Th. Th+ cong Figure . Refoldig Figure  and D. Tg Figure , similarg Figure . The funN Figure . We alsof hTel28  30). In. In+ con+ shows signatures of hybrid GQs  major populations, centered at E ∼ 0.54 and 0.71, that we designate as E1 and E2 states . Se. Se+ shoFigure + , and in s Figure .1,Ewhich is the dominant population, unraveled gradually and reversibly  that could be fitted to two clusters centered at ∼7.5 and 16 pN  is similar in mechanical response to (dT)34, an unstructured ssDNA of the same length  populations centered at E ∼ 0.47 (0E′), 0.68 (1E′) and 0.86 (2E′)  or hTel34′ (1E′) can be likened with the non-cooperative unraveling via mutual strand slippage, observed with 3′ GQs in hTel28. This further hints at preferential GQ formation at the 3′ end in hTel34, similar to hTel28.With two additional Te chosen . In ordee chosen  so that e chosen . At 100 ) Figure . E0′ shoresponse  30,46) ,46 2AG3 response  (30). Li2AG3 repeats, as we studied here using five repeats and six repeats using fluorescence-force spectroscopy, GQ polymorphism in tandem with their positional multiplicity along the DNA add to the complexity. Indeed, previous studies of four to seven repeats using smFRET only (hence at zero force)  based on strand symmetry, strand orientation and glycosidic conformation . Recentlo force)  or optico force)  found ev2AG3 segments, on application of force, the 3′ GQ former unravels gradually whereas the 5′ GQ withstands forces as high as 28 pN  and ΔG‡s (apparent free energy of activation) as ∼6.6 nm and ∼7 kBT, respectively  of hTel22 under identical buffer conditions .,40.funfoectively . A Δx‡ onditions , attribunditions . BecauseAt applied forces of up to ∼28 pN, because the 3′ GQs in hTel28 are only partially unraveled, they rarely convert into long-loop GQs (∼16%) upon relaxation. In contrast, a long-loop GQ, when unfolded cooperatively, loses memory of its initial state and can relax into a mechanically different long-loop GQ or even a 3′ GQ (∼33%).2AG3 overhang were mechanically distinct from GQs formed in hTel22 , we also designed the internally labeled construct hTel34′ whose FRET response is sensitive to the conformation of the four 5′ most repeats. By comparative analysis of responses of hTel28 and hTel34, we assigned the major population, 1E (or 1E′) of hTel34 (or hTel34′) to 3′GQ with a 5′ 12 nt ssDNA overhang harboring two T2AG3 segments. To test this assignment further, we next evaluated the possibility that E1 (or E1′) instead represents an internal GQ formed from the second, third, fourth and fifth T2AG3 segments, leaving 6 nt overhangs at both the 3′ and 5′ ends , which is clearly not the case as shown in hTel34E′,20 pN ∼ 0.07 versus hTel28,20 pNE ∼ 0.28). Likewise, Type II unfolding would occur if an internal GQ is dominated by the 3′ extra T2AG3 segment but 20 pNE for Type II unfolding of 3′ GQ of hTel28 is ∼0.43, very different from hTel34E′,20 pN ∼0.07. Thus, the mechanical response of an internal GQ is inconsistent with that of 1E′-hTel34′. This further substantiates our assignment of 1E of hTel34 to 3′GQs.From hTel34 which contains six human telomeric repeats, we observed two major populations  5′ ends . Indeed,ose to E1. Therefo 5′ tail , left. T2E (hTel34) and 2E′ (hTel34′) show abrupt, cooperative unfolding under tension similar to long-loop GQs in hTel28. The funfold of 2E reveals two force clusters at ∼8 and 16 pN with underlying Δx‡s of ∼3.7 and 4 nm, respectively  and ΔG‡ (10 990 versus 59.83 s and 11.6 versus 5.7 kBT), indicating the presence of long and short-lived species that are structurally similar .GQs formed at the 5′ end of hTel28 were ultrastable. Analogous behavior might be overshadowed by ssDNA stretching in hTel34, but should be reflected as stable high FRET over the complete pulling cycle in hTel34′. Although +, CD spectra of hTel28 in 100 mM Na+ showed signatures of antiparallel GQs  and ΔG‡ as ∼4.3 nm, ∼50 s and 6.5 kBT, respectively. Additionally, Type II partial unfolding was observed in ∼30% (31 out of 104 events) of pulling cycles . In. In+, CDE ∼ 0.72 . Under tat ∼8 pN  and refoat ∼8 pN . We estio in Na+ . Other ved to K+ .+   ∼ 1190 s, funfold ∼ 14.8 pN) and short-lived (uτ(0) ∼ 68 s, funfold ∼ 6.8 pN) isostructural species with Δx‡ of ∼4 nm, which is close to a previously reported value for hTel22   with thend 19 pN . Upon sur hTel22  30). Th. Th+ . A long-loop GQ in hTel34′, if it exists, would harbor a 15 nt long loop or two 9 nt long loops and have E ∼ 0.85 of hTel22 instead of the observed E ∼ 0.72 . Th. Th+ and00 mM K+ . hTel34′E ∼ 0.72  and shownd 19 pN  and refond 19 pN . Prior t hTel34′ . We noteE ∼ 0.85 . BecauseE ∼ 0.72 . Moreove+. Thus, although GQ formation is polarized at the 3′ end in Na+ and K+, the regulatory effect of an associated telomeric repeat on GQ folding appears unique to K+ conditions.Taken together, our study suggests a complex, sequence dependent interplay between GQ and the unpaired Gs in five and six-repeat telomeric DNA. We note that, the predominant Type I partial unfolding observed in hTel28 and hTel34 is exclusive to 100 mM Kin vitro. In cells, telomeric DNA spans over 100–200 nt, beyond the double-stranded region, in the form of multiple G-rich hexanucleotide repeats. Using single molecule fluorescence-force spectroscopy, we demonstrated preferential GQ formation at the 3′ end of telomeres under physiologically relevant conditions. Although the sequences used in this study can harbor only one GQ, our results might imply a general scheme of polarized GQ formation being initiated from the 3′ end of long telomeric DNA, spanning multiple repeats of GQ-forming segments. The relative position of GQ(s) with respect to the 3′ end, i.e. the size of 3′ ssDNA tail downstream of the 3′ most GQ, has important implications in processes such as extension by telomerase and alternate lengthening of telomeres (ALT). Wang et al. showed that a minimum 3′ ssDNA tail of ∼6, 8 and 12 nt in length is essential for helicase unwinding of telomeric GQ, extension by telomerase, and lengthening by ALT, respectively (G-rich telomeric sequences are well known to form stable GQs ectively ,53–55. Tectively . Telomerectively . Stable 2AG3 repeats (2AG3 repeats in vivo. Our data also showed that the long telomeric repeats cannot be fully unwound at average stall forces of motor proteins and polymerases (In the cellular milieu, processes such as transcription and replication generate tension. Although folding properties under tension have been studied quite extensively telomeric DNA containing four T repeats ,58–60, c repeats . In thisymerases ,62. Thusgkz906_Supplemental_FileClick here for additional data file.


At 14 days, urinary pH, electrolytes, urinary metabolites and ion-exchanger gene expression were determined. The results revealed that MSG-treated rats had significantly more alkaline urine and higher levels of urinary sodium and bicarbonate similar to NaHCO3 controls. These changes correlated with a lower expression of ion-exchanger genes, namely, CAII, NBC1, and AE1, which are involved in bicarbonate kidney reabsorption. The urinary metabolic profiles also revealed similar patterns for the MSG and NaHCO3 groups. In conclusion, MSG exhibits similar properties to NaHCO3, an alkalinizing agent, with regard to inducing alkaline urine, reducing bicarbonate kidney reabsorption, and generating a specific urinary metabolic pattern. We believe that these observations will be useful to further study the MSG effects in humans.Monosodium glutamate (MSG) is widely used as a flavor enhancer and its effects on human health are still debated. We aimed to investigate whether MSG can act as alkalinizing agent in murine models and if its metabolites are biomarkers of MSG consumption. For this purpose, adult male Wistar rats were given water added with 1 g% MSG or three types of control water, including sodium chloride (NaCl) and sodium bicarbonate (NaHCO Monosodium glutamate (MSG) is commercially produced as a flavor enhancer in processed foods and home cooking and its use is exponentially increasing worldwide . Althoug3) -propionate (TSP), 100% D2O and 3 mM sodium azide. The sample was vortexed briefly, and then 600 µL of the mixture was transferred to NMR glass tubes  with an outer diameter of 5 mm pending NMR analysis.Urine samples were thawed at room temperature and centrifuged at 18,000× 1H frequency of 600.13 MHz with a temperature of 300 K. A standard one-dimensional (1-D) NMR pulse sequence (recycle delay [RD]-90°-t1-90°-tm-90°-acquire free induction decay) was used t1 was fixed to 3 µs. The suppression of the water peak was achieved by selective irradiation during recycle delay of 2 s and mixing time (tm) of 100 ms. The 90 degree-pulse was adjusted to approximately 10 µs. A total of 128 scans were recorded into 64 K data points with a spectral width of 20 ppm [Urinary samples were analyzed using a Bruker 600 MHz spectrometer  at a f 20 ppm .1H 0.00), phased and baseline corrected using TopSpin 3.0 . The pre-processed spectra were imported into MATLAB software  and digitized with a resolution of 0.0005 ppm. Spectral regions containing the TSP peak (δ1H −1.00–0.005), water (δ1H 4.70–4.90) and urea (δ1H 5.71–6.00) were removed. Normalization using the probabilistic quotient method [2X and Q2Y values, representing the fitness and predictivity of the model, respectively. A two-tailed heteroscedastic t-test with Benjamini-Hochberg correction was also used to determine the statistical significance of differences between the groups. Statistical Total Correlation Spectroscopy (STOCSY), the Human Metabolome Database  [The urinary spectra were calibrated to the TSP peak (δt method  and peakt method  were app Canada) ,27 and i® reagent method. Tissues weighing about 50–100 mg were preserved in TRIzol® and incubated at room temperature (RT) for 3 min, 200 µL of chloroform was added the mixture was shaken vigorously and incubated at RT for 3 min. The reaction was then centrifuged at 12,000× g for 15 min at 4 °C. The upper aqueous phase (60%) was transferred to a new 1.5 mL sterile tube, 500 μL of isopropanol was added, mixed well, and then incubated at RT for 10 min and centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was discarded while the pellet was washed by 70% ethanol and centrifuged at 7500× g for 5 min at 4 °C (twice). The pellet was air dried at RT, re-suspended in 30–50 μL of DEPC water and then the pellet was dissolved by keeping it at 55 °C for 15 min. Total RNA were stored at −80 °C until used.Total RNAs were extracted from cortex and medulla layers of kidney using TRIzol® 480 real-time PCR system . Each PCR condition containing 2.5 μM of primers (forward and reverse primers), cDNA 50 ng/μL, and 2X LightCycler® 480 SYBR green I master mix. The amplification was initiated by pre-incubation at 95 °C for 10 min, followed by 40 cycles at 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, and 79 °C for 0.1 s. Each sample was prepared in duplicate sample and the crossing point (Cp) cycle was calculated and presented as mean ± SEM values. The gene expression levels were determined and the 2−∆cp value; Δcp = Cptarget − Cpactin was calculated. Primers for all genes were designed using NCBI with a least one exon-exon junction in the target (RNA concentration was measured with OD at 260 nm and reverse-transcribed to complementary DNA (cDNA) using the high capacity reverse transcription Kit . Obtained cDNA was diluted to 20 ng/μL and stored at −20 °C until used. Gene expression analysis was determined by real-time PCR, using beta-actin (ACTB) as the internal control for normalization , in Lighe target .p-values < 0.05 were considered as statistically significant.The statistical analysis of the urinary analysis and gene expression were reported as mean ± SEM per group of animals and the differences between groups was compared for statistical significance by Student’s t-test. Moreover, all comparisons of gene expression with 3 treatment groups had significantly higher urine pH compared to controls , whereas no significant difference in urinary pH was observed between NaCl-treated rats (pH = 6.93) and controls . Urinary K+ excretion in MSG group (1.83 ± 0.06 mEq/day) was significantly higher, whereas the NaHCO3-treated group (1.04 ± 0.12 mEq/day) was the opposite to controls (1.50 ± 0.10 mEq/day). Significantly higher levels of HCO3− were also revealed in MSG (0.95 ± 0.16 mEq/day) and NaHCO3-treated rats (1.14 ± 0.20 mEq/day) but not in NaCl-treated rats (0.20 ± 0.06 mEq/day) compared to controls (0.19 ± 0.02 mEq/day). No significant differences in urinary Cl- were observed between MSG (3.70 ± 0.26 mEq/day) and NaCl-treated rats (3.85 ± 0.31 mEq/day), whereas significantly lower levels were found in NaHCO3-treated rats (1.88 ± 0.20 mEq/day) compared to control rats (4.32 ± 0.28 mEq/day)  B.3− reabsorption  were suppressed in MSG-treated rats , similar to that of the NaHCO3 group  when compared to controls   . No sign1H-NMR spectra of 24 h urine samples from control, MSG, NaCl and NaHCO3-treatment groups at day 14 (D14) are plotted in 3-treated rats compared to NaCl and control rats . The distinct urinary metabolites between MSG and NaHCO3 are methylamine and dimethylamine, which were only observed in MSG-treated animals. Methylamine and dimethylamine are metabolites from the gut microbiota [In fact, excessive renal metabolism of glutamate can be a source of ROS. Moreover, chronic MSG intake is associated with decreased levels of antioxidant enzymes and increased lipid peroxidation; leading to cellular and functional damage of the kidneys . Such efcrobiota ,51. Thiscrobiota . MSG may3-treated rats. MSG consumption exhibits similar features to alkaline loading, causing alkaline urine, leading to alteration of di- and tricarboxylic metabolites, i.e., glutamate, alpha-ketoglutarate, citrate, and malonate reabsorption in the kidney. We may also speculate that MSG itself may alter the gut microbiome to catabolize dietary molecules, such as amino acids and choline and urinary metabolites could be potential markers for MSG consumption in animals and should now be studied in humans. However, there are two limitations in this present study to be aware of: the effects of MSG consumption were only tested in male animals, and the frequency of obtaining MSG was every time the animals drunk water. This behavior differs from humans because humans receiving MSG only consume MSG at their regular meal times, which is usually 2-3 times a day.In summary, MSG consumption may resemble alkali loading , causing3, an alkalinizing agent; it induces alkaline urine and alters the ion-exchanger gene expression that relates to bicarbonate kidney reabsorption. This implies that MSG consumption induces metabolic changes with a pattern of urinary metabolites, which may be used for monitoring MSG exposure in humans.Short-term MSG consumption exhibits similar effects as consuming NaHCO


Synechocystis sp. PCC 6803 and the chemoheterotrophic P. taiwanensis VLB 120 enabled HCDs up to 51.8 gCDW L−1. Respective biofilms have been grown as a biofilm in capillary flow-reactors, and oxygen evolution, total biomass, as well as the ratio of the two strains, have been followed under various cultivation conditions. Furthermore, biofilm formation on a microscopic level was analyzed via confocal laser scanning microscopy using a custom made flow-cell setup. The concept of mixed trophies co-cultivation was coupled to biotransformation, namely the oxyfunctionalization of cyclohexane to cyclohexanol. For benchmarking, the performance of the phototrophic reaction was compared to the chemical process, and to a biotechnological approach using a heterotrophic organism only. The data presented refer to our research paper “Mixed-species biofilms for high-cell-density application of Synechocystis sp. PCC 6803 in capillary reactors for continuous cyclohexane oxidation to cyclohexanol” Hoschek et al., 2019.Photosynthetic microorganisms offer promising perspectives for the sustainable production of value-added compounds. Nevertheless, the cultivation of phototrophic organisms to high cell densities (HCDs) is hampered by limited reactor concepts. Co-cultivation of the photoautotrophic  Bacterial strains and plasmids used for biocatalyst development are listed in In 2Bacterial strains and plasmids used in this study are listed in 3, without citrate):Composition of YBG11 , 0.019 g L−1 Na2CO3, 50 mM HEPES (pH 7.2); YBG11 trace elements (100x): 3.6 g L−1 CaCl2 2H2O, 0.28 g L−1 boric acid, 0.11 g L−1 MnCl2 4H2O, 0.02 g L−1 ZnSO4 7H2O, 0.039 g L−1 Na2MoO4 2H2O, 0.007 g L−1 CuSO4 5H2O, 0.005 g L−1 Co(NO3)2 6H2O, 0.162 g L−1 FeCl3 6H2O, 0.6 g L−1 Na2EDTA 2H2O, 4.2 g L−1 NaHCO32.1P. taiwanensis VLB 120_egfp and Synechocystis sp. PCC 6803 (pAH050) was analyzed by confocal laser scanning microscopy (CLSM). The schematic representation of the experimental set-up is given in Pseudomonas sp. as well as the autofluorescence of Synechocystis sp. was recorded individually so that the volume could be calculated for each channel individually. The total volume of the biofilm equals the sum of the volume occupied by each species. Ps = P. taiwanensis VLB120_egfp, Syn = Synechocystis sp. PCC 6803 (pAH050). For the volume ratio of Ps/Syn, the calculated volume of Pseudomonas sp. was divided by the volume of Synechocystis sp. PCC 6803.The development of a mixed trophies biofilm consisting of 2.2Synechocystis sp. PCC 6803 and P. taiwanensis VLB120 both containing the plasmid pAH032. The schematic representation of the experimental set-up is given in Biofilms were cultivated as mixed and single species biofilms of The amount of oxygen produced as well as the biofilm dry weight and composition regarding bacterial species was determined for each cultivation condition . For fur2.3Synechocystis sp. PCC 6803 (pAH050) and P. taiwanensis VLB 120 (pAH050) by the addition of cyclohexane. The biotransformation substrate cyclohexane was supplied via saturation of the medium and air phase by a silicon membrane, before the reactor inlet. The productivity of 3.76 gCHXOH m−2 day−1 was reached after 1 day of adaptation and was stable for 30 days. After 31 days, the setup was actively terminated Synechocystis sp. PCC 6803 (pAH050) was no longer able to perform photosynthesis, and this resulted in to decrease of the productivity 1.0–1.3 gCHXOH m−2day−1CHXOH m−2 day−1 and 1.35 gCHXOH m−2 day−1, respectively (After 36 days of cultivation, the biotransformation was initiated for a mixed species biofilm of ectively .2.4The here presented biotransformation using a mixed-trophies biofilm consisting of a photoautotrophic and a chemoheterotrophic strain has been compared to the conventional chemical process and to a biotechnological approach using a heterotrophic organism only . Thereby


Recently, S1P1 and S1P2 were identified as specific receptor subtypes that contribute to brain injury in cerebral ischemia; however, the possible involvement of other S1P receptors remains unknown. S1P3 can be the candidate because of its upregulation in the ischemic brain, which was addressed in this study, along with underlying pathogenic mechanisms.The pathogenic roles of receptor-mediated sphingosine 1-phosphate (S1P) signaling in cerebral ischemia have been evidenced mainly through the efficacy of FTY720 that binds non-selectively to four of the five S1P receptors , a mouse model of transient focal cerebral ischemia. To identify S1P3 immediately after reperfusion with CAY10444 significantly reduced tMCAO-induced brain infarction, neurological deficit, and neurodegeneration. When S1P3 activity was inhibited, the number of activated microglia was markedly decreased in both the periischemic and ischemic core regions in the ischemic brain 1 and 3 days following tMCAO. Moreover, inhibiting S1P3 significantly restored the microglial shape from amoeboid to ramified microglia in the ischemic core region 3 days after tMCAO, and it attenuated microglial proliferation in the ischemic brain. In addition to these changes, S1P3 signaling influenced the proinflammatory M1 polarization, but not M2. The S1P3-dependent regulation of M1 polarization was clearly shown in activated microglia, which was affirmed by determining the in vivo activation of microglial NF-κB signaling that is responsible for M1 and in vitro expression levels of proinflammatory cytokines in activated microglia. As downstream effector pathways in an ischemic brain, S1P3 influenced phosphorylation of ERK1/2, p38 MAPK, and Akt.Inhibiting S1P3 as a pathogenic mediator in an ischemic brain along with underlying mechanisms, involving its modulation of microglial activation and M1 polarization, further suggesting that S1P3 can be a therapeutic target for cerebral ischemia.This study identified S1PThe online version of this article (10.1186/s12974-018-1323-1) contains supplementary material, which is available to authorized users. In addi1–5) , strongl1 is the only identified receptor subtype to be associated with cerebral ischemia [1, S1P2 (which is not a target for FTY720) was also revealed to influence brain injury after ischemic challenge [1 in cerebral ischemia [1 [1, FTY720-phosphate may also antagonize S1P3 because it reduced cellular responses through S1P-S1P3 signaling axis [3 was reported to be upregulated at mRNA levels in the brain after ischemic challenge [3 could be an additional pathogenic factor for cerebral ischemia, and FTY720’s efficacy in cerebral ischemia can also be mediated via suppressing S1P3. However, whether S1P3 influences brain injury in focal cerebral ischemia and the role of S1P3, pathogenic or neuroprotective, has not been identified.Cerebral ischemia, which is caused by a sudden interruption of blood flow to the brain, is a disease type where S1P receptors become validated drug targets mainly due to the efficacy of FTY720. Numerous in vivo studies have been conducted to prove the neuroprotective effects of FTY720 in the brain against ischemic challenge –9. FTY72ischemia , indicathallenge . These tischemia  demonstrhemia [1 , 16. In ing axis . Further3 in transient focal cerebral ischemia with a mouse model of transient middle cerebral artery occlusion and reperfusion (tMCAO). To identify the role, we used a selective S1P3 antagonist, CAY10444, that was given to mice immediately after reperfusion. We then assessed brain damage such as brain infarction, neurological functional deficit, and neural cell death. We further assessed whether S1P3 influenced microglial activation and polarization, a core pathogenic event in cerebral ischemia, along with a clarification of S1P3-dependent effector pathways in the brain after tMCAO challenge.In this study, we aimed to address the pathogenic role of S1P2O∶O2 (3∶1) mixture, and the right common carotid artery was isolated through a ventral neck incision. A silicone-coated 5–0 monofilament was introduced to the internal carotid artery from carotid bifurcation and advanced to occlude the middle cerebral artery (MCA). After 90 min of MCAO, the filament was withdrawn to allow complete reperfusion of the cerebral area. During surgery, rectal temperature was maintained at 37.0 ± 0.5 °C with a homoeothermic blanket. Sham-operated mice received similar surgical procedure except for the occlusion of MCA. After surgery, three mice were kept in a single cage; wet food and soft bedding were provided to minimize the suffering from the operation until they were sacrificed for brain sampling.Male ICR mice  were bought from the Orient Bio company (Korea) and housed under controlled environmental conditions of diurnal lighting (light on 07:00–19:00), temperature (22 ± 2 °C), and relative humidity (60 ± 10%). All animal handling and surgical procedures were carried out in accordance with the approved animal protocols specified by the Institutional Animal Care and Use Committee at Gachon University  . Following 1 week of laboratory acclimatization, the mice were challenged with tMCAO as described previously . In brieCAY10444  was dissolved in 1:1 mixture of chremophore EL and 100% ethanol, diluted in water, and injected intraperitoneally to mice at 0.1, 0.2, and 0.5 mg/kg at the time of reperfusion. For the tMCAO group, equal volumes of the vehicle were injected.2 exposure; their brains were quickly removed and sliced in the mice brain matrix at 2 mm thickness. The obtained brain slices were incubated with 2% 2,3,5-triphenyltetrazolium chloride (TTC) in physiological saline for 20 min at 37 °C. The TTC-stained brain slices were photographed, and the infarct area was calculated using ImageJ software .Functional neurological deficit was assessed using modified neurological severity score (mNSS) scale to determine the motor, sensory, balance, and reflex disorder 24 h following MCAO, as described previously –20. FollBrain tissue samples for histological analysis were obtained 1 or 3 days after tMCAO. Mice were anesthetized with a mixture of Zoletil 50®  and Rompun® , and their brains were perfused with ice-cold phosphate-buffered saline  followed by 4% paraformaldehyde. The brains were incubated in the same fixative solution overnight, cryoprotected with 30% sucrose, and cut into 20-μm sections using a microtome cryostat. To ensure anatomical similarity of brain regions, two coronal brain sections obtained from the rostral to middle portion of the striatum and the cortex of each mouse brain were used for histological evaluation. In a different set of experiments, the mice brains were transcardially washed with ice-cold PBS and the ipsilateral brain hemisphere was used for RNA and protein extraction.w/v KMnO4. Then, sections were stained with 0.001% (w/v) FJB in 0.1% (v/v) acetic acid solution for 30 min, rinsed in deionized water, dried in a slide warmer, cleared in xylene, and then cover-slipped.In order to identify any degenerating neurons following the tMCAO challenge, Fluoro Jade B (FJB) histochemical staining was performed 1 day after tMCAO induction. Brain sections were sequentially immersed in ethanol series , rinsed in deionized water, and oxidized in 0.06% 3 activity on microglia or astrocyte activation, Iba1 or GFAP immunohistochemistry was performed 1 or 3 days after tMCAO. Brain sections were oxidized with 1% H2O2 in PBS for 15 min and blocked with 1% fetal bovine serum (FBS) in 0.3% Triton-X100 in PBS for 1 h to block non-specific protein binding. Then, the brain sections were incubated with primary antibody against Iba1  or GFAP  overnight at 4 °C followed by anti-rabbit secondary antibody (1:200). Sections were exposed to avidin and biotinylated horse-radish peroxidase macromolecular complex (ABC) kit  and visualized with 3, 3′-diaminobenzidine tetrahydrochloride (DAB) exposure (0.02% DAB and 0.01% H2O2 in 0.05 M TRIS solution), dehydrated with ethanol, cleared in xylene, and mounted using mounting media.To evaluate the effect of S1P2O2 for 2 min). The stained sections were then washed with PBS (3 × 5 min), blocked, and incubated with primary antibodies against Iba1 (1:500) overnight at 4 °C. Sections were then labeled with appropriate secondary antibodies conjugated with Cy3 (1:1000) and mounted with VECTA SHIELD mounting medium.In order to determine whether the NF-κB pathway is triggered in activated microglia after the tMCAO challenge, cryostat brain sections were processed for double immunolabeling using antibodies against NF-κB (p65) and Iba1. The sections were incubated with TRIS-EDTA solution at 100 °C for 30 min for antigen retrieval, blocked with 1% FBS in 0.3% Triton X-100, and labeled with rabbit NF-κB (p65) (1:100) antibody overnight at 4 °C. The sections were labeled with a biotinylated secondary antibody (1:200) followed by incubation with an ABC kit. The signals were visualized with DAB staining  was administered twice a day at 12-h intervals on the second and third day after tMCAO challenge. Brain sections were prepared for Iba1/BrdU immunofluorescence as described previously [The role of S1Peviously , 21.2).The brain sections after staining or immunolabeling were photographed using bright-field and fluorescence microscopy  equipped with a DP72 camera. Representative images were prepared using Adobe Photoshop CS3. For quantification, three photographs were taken from different area of each region and the number of immunopositive cells was counted. Then, the average number of immunopositive cells from each region was expressed in per unit area ; the obtained proteins was thus separated in a 10% SDS-PAGE system and transferred to the polyvinylidene difluoride membrane. The membrane was blocked with 5% skim milk to avoid non-specific protein bindings and incubated with primary antibodies against rabbit pAkt, Akt, pERK1/2, ERK1/2, pp38, p38 , and mouse β-actin  overnight at 4 °C followed by incubation with respective secondary antibodies  for 2 h at room temperature and visualized with enhanced chemiluminescence (ECL) solution. The band intensity of each protein was analyzed using ImageQuant (TM) TL software, normalized with β-actin, and then expressed as fold changes of the sham-operated group.5 cells/well. Microglial cells were starved overnight and treated with CAY10444 (1 μM) or vehicle (0.1% DMSO in DMEM). Thirty minutes later, microglia were stimulated with lipopolysaccharides (LPS) (100 ng/ml) for additional 24 h. Alternatively, the shRNA targeted with S1P3 (shS1P3) receptor or non-targeted control shRNA was transfected into the cells in serum and antibiotic-free medium. After 6 h of incubation, the media were replaced with serum and antibiotic containing media for an additional 42 h. S1P3-infected microglial cells were then challenged with serum starvation for 12 h and stimulated with LPS, and then harvested for qRT-PCR analysis.Primary microglial cells were obtained from the brain cortices of 1–2-day-old mouse pups as described previously . The micTotal RNA was extracted from the ipsilateral hemisphere of mice brain and cultured microglia cells using TRIzol Reagent (Invitrogen). One microgram of total RNA was reverse transcribed (RT) to synthesize cDNA. The gene expression levels of the different markers of M1- and M2-polarized microglia were determined using the StepOnePlus™ qRT-PCR system (Applied Biosystems) with the FG Power SYBR Green PCR master mix (Life Technologies) and primer sets , and the data are presented as mean ± S.E.M. One-way ANOVA followed by the Newman-Keuls post hoc test was used to compare the data among the multiple experimental groups, while comparisons between the two groups were performed using the Student’s 3 antagonist (CAY10444) showed significantly decreased brain infarction in a dose-dependent manner  were significantly upregulated 1 and 3 days following the tMCAO challenge  at both day 1  and Iba1 1 day after the ischemic challenge. The vehicle-administered tMCAO group showed an enhanced expression of NF-κB(p65) which are easily identified in Iba1-immunopositive cells in the ischemic core region  reduced p-selectin production and leucocyte rolling via S1P-S1P3 signaling axis, which was reaffirmed in S1P3 knockouts [q-mediated signaling pathway under S1P3 activation [1 and S1P3, our previous and current in vivo findings strongly indicate that the reported FTY720’s efficacy in cerebral ischemia may be through suppressing at least the S1P1 and S1P3 activities. Besides FTY720-relevant target receptors, S1P2 was also identified to mediate brain injury in cerebral ischemia through the disruption of vascular integrity in the ischemic brain [4 or S1P5.The use of receptor-mediated S1P signaling has been assumed as a possible therapeutic strategy to overcome cerebral ischemia because FTY720, which is a non-selective modulator of 4 of 5 S1P receptors after being phosphorylated, exerts neuroprotective effects in rodent models –8. Curreischemia . The curfor S1P1 , 16 and nockouts . FTY720-tivation . Consideic brain , even th3 in the brain, its roles in ischemic conditions seem to be tissue-specific. In fact, earlier studies reported controversial roles of S1P3 in non-neural ischemic models: protective or harmful. In the heart, the deletion of both S1P2 and S1P3 was shown to aggravate myocardial infarction in mice, which supported the cardioprotective role of S1P3 [3 was shown to be associated with tissue injury after ischemic challenge. Deletion of bone marrow S1P3 attenuated tissue damage following renal ischemia/reperfusion, in which its deletion reduced the expression levels of proinflammatory cytokines and increased the expression levels of anti-inflammatory cytokines [1. Renal injury after ischemic challenge was reduced or exacerbated by exposure to an S1P1 agonist [1 deletion [1 knockdown reduced brain injury after ischemic challenge [1, S1P2, and S1P3) mediate pathogenesis in ischemic brain.Despite the clear pathogenic role of S1P of S1P3 . In the ytokines , 36. The agonist  or endotdeletion . Howeverhallenge . Regardl3 in cerebral ischemia because CAY10444 has been widely used as a specific antagonist for S1P3 [2, P2 receptor, or α1A-adrenoceptor [2- and S1P3-mediated increase in the intracellular calcium levels in Chinese hamster ovary cells. This inhibitory effect of CAY10444 was also mediated through the stimulation of P2 receptor or α1A-adrenoceptor [2 and an agonist for P2 receptor or α1A-adrenoceptor. The latter agonistic property could be excluded in the protective effects of CAY10444 against cerebral ischemia: the association of α1A-adrenoceptor with cerebral ischemia is unclear and suppressing P2 receptor is neuroprotective in this disease [2 could mediate the neuroprotective effects of CAY10444 in cerebral ischemia because S1P2 was reported as a pathogenic factor in this disease [3 in cerebral ischemia. In renal ischemic injury, blocking S1P2 activity by JTE013 resulted in renoprotection, whereas CAY10444 did not [2 antagonist. It would be tempting to address these opposite notions using genetic tools such as knockout mice for S1P3 in future studies.In this study, we have used CAY10444 to address the role of S1Pfor S1P3 –41. But,noceptor . CAY1044noceptor . These f disease , 44. Unl disease . However did not . The lat3 in the ischemic brain appears to be associated with the activation of brain residence microglia, which is a common pathogenic event in several central nervous system (CNS) disorders, including stroke [1-mediated brain damage after focal cerebral ischemia was mainly mediated through microglial activation [3 was also associated with microglial activation: inhibiting S1P3 using its specific antagonist reduced the number of activated microglia in the ischemic brain, in both a time- and region-dependent manner. Furthermore, the suppression of S1P3 activity in the ischemic brain attenuated microglial proliferation. In addition to the increase in the population, S1P3 in the ischemic brain was closely associated with the morphological transformation of activated microglia. In the ischemic core regions 3 days or more after ischemic challenge, most of the activated microglia were amoeboid shaped and were mainly responsible for neuronal damage in ischemic brain by releasing several proinflammatory mediators [3 resulted in a significant attenuation of the transformation of activated microglia into an amoeboid shape.The neuroharmful role of S1Pg stroke , 47. Preg stroke , 48. Rectivation . In thisediators , 50. We 3 in the ischemic brain may also link into astrogliosis, a core pathogenesis associated with inflammatory responses in cerebral ischemia [3 was identified as the receptor subtype to regulate astrogliosis, in which a pharmacological antagonism or genetic deletion of S1P3 reduced S1P-triggered inflammatory responses in astrocytes [3-triggered astrogliosis may occur in an ischemic brain. Indeed, we demonstrated that inhibiting S1P3 after tMCAO challenge resulted in a significant attenuation of astrogliosis.S1Pischemia . In factischemia , 51. Rectrocytes . These p3 activity in the ischemic brain attenuated M1 polarization, as evidenced by the attenuated gene expression of relative markers following tMCAO. However, S1P3 suppression did not alter the expression levels of M2 polarization-relevant markers following tMCAO. These data demonstrate that S1P3 in the ischemic brain is selectively associated with the M1 polarization. This unique role of S1P3 in M1 polarization was obvious in activated microglia, which was confirmed by determining the expression levels of microglial NF-κB, a characteristic marker for M1 polarization [3 significantly reduced the number of Iba1/p65 NF-κB double-immunopositive cells. These in vivo findings of the link between S1P3 and M1 microglial polarization were further affirmed in LPS-stimulated mouse primary microglia, in which inhibiting S1P3 by both genetic and pharmacological tools ensured the attenuation of proinflammatory cytokines. Therefore, S1P3 may mediate brain injury following tMCAO by altering the microglial polarization states to M1, further suggesting that S1P3 is a novel and selective player in regulating M1 microglial polarization.The phenotypical shift of activated microglia has also been extensively considered to understand the pathogenesis of cerebral ischemia . Activatrization . Inhibit3 in cerebral ischemia were linked to PI3K/Akt and MAPK pathways, including ERK1/2 and p38 MAPK. Inhibiting S1P3 following tMCAO increased the Akt phosphorylation in the ischemic brain, whereas it attenuated the phosphorylation of ERK1/2 and p38 MAPK. These signaling molecules are, in particular, considered to regulate the phenotype shift between M1 and M2 polarization. Akt activation in microglia is a signaling molecule that drives activated microglia towards M2 polarization. Additionally, the activation of PI3K/Akt signaling is critical for restricting inflammatory activation of microglia/macrophages and negatively regulates NF-κB signaling, whereas its inhibition drives activated microglia/macrophages towards their M1 polarization [3 activity in ischemic brain increased Akt phosphorylation without altering the expression markers of M2 polarization, indicating that the increased Akt phosphorylation by S1P3 inhibition may be linked to the restriction of M1 polarization rather than to the enhancing of M2 polarization. Persistent activation of ERK1/2 signaling has been reported to trigger NF-κB transcriptional activity [3 activation is closely associated with the M1 polarization of activated microglia in the ischemic brain because the suppression of S1P3 activity attenuated ERK1/2 and p38 MAPK phosphorylation in the ischemic brain.The underlying signaling mechanisms for the pathogenic roles of S1Prization , 55. In activity , 29 simiactivity , both ofactivity –58. This3 as a novel pathogenic factor in cerebral ischemia and provided underlying mechanisms, particularly in view of microglial activation. The medically relevant roles of the S1P receptor subtypes in cerebral ischemia have emerged through translational studies. Now, at least three subtypes have been identified to mediate brain injury in cerebral ischemia, including S1P1 [2 [3 (the current study). Even though S1P3 may be limited as a therapeutic target because of its negative effects on the heart, it would be a good therapeutic strategy for cerebral ischemia if S1P3-specific antagonist can act inside the CNS. In addition to the identification of novel roles of S1P3, our findings also implicate that the neuroprotective effects exerted by FTY720 in cerebral ischemia in previous studies occur additionally via suppressing S1P3 activity [1 [This study identified S1Ping S1P1 , S1P2 [1 S1P1 [2 , and S1Pactivity , similarivity [1 , 59, 60.Additional file 1:Figure S1. CAY10444 (CAY) administration attenuates tMCAO-induced neurodegeneration in post-ischemic brain. Figure S2. CAY10444 (CAY) administration does not alter tMCAO-induced microglial M2 polarization in post-ischemic brain. Figure S3. CAY10444 (CAY) administration attenuates tMCAO-induced astrocytes activation in post-ischemic brain. Table S1. Primer sets used for qRT-PCR analysis. (DOCX 1425 kb)