Chenopodium quinoa Willd.) was known as the “golden grain” by the native Andean people in South America, and has been a source of valuable food over thousands of years. It can produce a variety of secondary metabolites with broad spectra of bioactivities. At least 193 secondary metabolites from quinoa have been identified in the past 40 years. They mainly include phenolic acids, flavonoids, terpenoids, steroids, and nitrogen-containing compounds. These metabolites exhibit many physiological functions, such as insecticidal, molluscicidal and antimicrobial activities, as well as various kinds of biological activities such as antioxidant, cytotoxic, anti-diabetic and anti-inflammatory properties. This review focuses on our knowledge of the structures, biological activities and functions of quinoa secondary metabolites. Biosynthesis, development and utilization of the secondary metabolites especially from quinoa bran were prospected.Quinoa ( Chenopodium quinoa Willd.), a dicotyledonous plant belonging to Chenopodiaceae family, is one of the oldest native crops in the Andean region of South America, with approximately 7000 years of cultivation [Quinoa  and cinnamic acid analogues (17–29). Benzoic acid (1) was derived from cinnamic acid (19) in planta in the biosynthetic pathway of phenolic acids [About 29 phenolic acid analogues have been identified in quinoa. According to their structural features, they can be classified as benzoic acid analogues , gallic acid (8), protocatechuic acid (10), syringic acid (12), vanillic acid (13), and their analogues. They are rich in the leaves and seeds of quinoa [At least 16 benzoic acid analogues have been identified from quinoa. Their biological activities are listed in f quinoa ,27. Thouf quinoa ,29, allef quinoa , antioxif quinoa , and antf quinoa  activiti17), chlorogenic acid (18), cinnamic acid (19), coumaric acid (20/21), ferulic acid (24), rosmarinic acid (28), sinapinic acid (29), and their analogues. Ferulic acid (24) and its derivatives were the predominant phenolics in bound form to be present in quinoa seeds [Thirteen cinnamic acid analogues have been identified from quinoa. Their biological activities are listed in 24) and sinapic acid (29) had more phytotoxic effects on cucumber seedling as compared to the other tested phenolic acids [Both ferulic acid  and quercetin (46). Other aglycones in quinoa include acacetin (30), myricetin (45), daidzein (62), and genistein (63). According to the structural features, quinoa flavonoids can be classified as flavones (30–33), flavonols (34–54), flavanones , flavanols (58–60), and isoflavones (61–65). Flavonoids play important roles in plants against the feeding insects and herbivores [Flavonoids are based upon a fifteen-carbon skeleton consisting of two benzene rings linked via a heterocyclic pyrene ring . They corbivores . Flavonorbivores .30), isovitexin (31), orientin (32) and vitexin (33), have been identified from quinoa. Their biological activities are listed in 33) and substantial amounts of isovitexin (31), whereas those grown in daylight only contained isovitexin (31). It is remarkable that no isovitexin (31) was present in quinoa seeds [30), isovitexin (31), orientin (32) and vitexin (33) were also isolated from other plant species which showed various biological activities such as antioxidant [Four flavones, namely acacetin  and quercetin (46) are two main flavonols. They are in the form of glycosides present in quinoa. Structure-activity relationship of their antioxidant activity showed that the ability to quench free hydroxyl radicals increased with the amount of hydroxyl groups in the ring B. For example, myricetin (45) was a stronger antioxidant than kaempferol (35) [ortho-dihydroxy substitution in the ring B [46) was the strongest antioxidant among the flavonoids. Both isorhamnetin (34) and kaempferol (35) were the most abundant flavonoids in quinoa leaves, and it also contained large amounts of rutin (54) [38–41) exhibited moderate antioxidant activity while two quercetin 3-glycosides  showed strong antioxidant activity, suggesting that quinoa could represent an important source of free radical inhibitors [Both kaempferol (rol (35) . In addie ring B . Quercettin (54) . Four kahibitors .35) and its derivatives showed antibacterial activity against Gram-positive and Gram-negative bacteria, as well as against the fungus Candida glabrata [Many flavonoids are characterized by antibacterial, antifungal and antiviral activities, not only against plant pathogens, but also against the pathogens for humans and animals . Kaempfeglabrata ,122.46–53) have been identified in quinoa. Kaempferol (35), myricetin (45) and quercetin (46) acted as the deterrents against Radopholus similis and Meloidogyne incognita [47) and rutin (54) from Pinus banksiana inhibited the development of Lymantria dispar and increased its mortality [46), quercetin 3-O-glucoside (47) and its six derivatives exhibited inhibitory activity on the shoot growth of Arabidopsis thaliana as well as on the spore germination of the fungus Neurospora crassa [About eight quercetin derivatives , neohesperidin (56), and naringin (57) were identified in quinoa seeds (55) and neohesperidin (56) were found in the sprouts [Three flavanones hesperidin , epicatechin (59), and epigallocatechin (60) were found in quinoa seeds. Their biological activities are listed in Three flavanols namely catechin (61), daidzein (62), genistein (63), prunetin (64), and puerarin (65) were found in quinoa (Radopholus similis [62) and genistein (63) by gut microorganisms [Five isoflavanones, i.e., biochanin , and have a characteristic bitter or astringent taste to protect it from birds and insects, and possess detergent properties . The sap74) was an iridoid glycoside isolated from the flour of quinoa [78) was also isolated from rice to show antibacterial activity on Xanthomonas oryzae pv. oryzae (Xoo) [Quinoa monoterpenoids and their biological activities are listed in f quinoa . γ-Terpiae (Xoo) .81) was identified in quinoa [Only one sesquiterpene namely caryophyllene  and glycosides (saponins), are mainly present in the bran to protect quinoa from pests and herbivores  and pathogenic microorganisms . Quinoa Candida albicans at 50 μg/mL [Fusobacterium nucleatum, with a minimum inhibitory concentration (MIC) of 31.3 μg/mL. It could be used as an antibacterial agent to treat halitosis [Botrytis cinerea was treated with the saponin extracts, mycelial growth and conidial germination were significantly inhibited [The crude saponin fraction inhibited the growth of 50 μg/mL . The alkalitosis . When thnhibited .Pomacea canaliculata, GAS) were treated with the crude saponin under laboratory conditions in 24 h at approximately 33 μg/mL, they were completely killed [Pomacea maculata) were treated with saponins above 7 μg/mL after 72 h, they were also 100% killed. Quinoa saponin could be a viable product to safely control P. maculata in rice fields [Carassius auratus) and tilapia (Oreochromis mossambicus), while providing adequate protection from Pomacea snails to newly sprouted rice seeds under laboratory conditions [When golden apple snails , hederagenin (83), spergulagenic acid (84), serjanic acid (85), phytolaccagenic acid (86), gypsogenin (or named 3β-hydroxy-23-oxo-olean-12-en-28-oic acid) (87), 3β-hydroxy-27-oxo-olean-12-en-28-oic acid (88), and 3β,23,30-trihydroxy-olean-12-en-28-oic acid (89), and their glycosides are shown in β-amyrin in planta (134) [The quinoa triterpenoids contain either tetracycles or pentacycles in their core structures. Most of them are pentacyclic triterpenoids in the form of saponins. The saponins contain an aglycone (sapogenin) and one to three saccharide chains in their structures, and were classified according to the number of saccharide chains as mono-, di-, and tridesmosides. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed a complete preassignment and identification of the major saponins and aglycones . The maita (134) . Sugars,ta (134) .About 11 oleanolic acid analogues have been identified in quinoa. Their biological activities are listed in Oleanolic acid and its glycosides are mainly present in the bran (seeds) of quinoa. They showed a variety of biological activities such as antimicrobial ,221, ant83) was the main aglycone of saponins from quinoa leaves [50) >30 μM [Hedera helix) induced apoptosis of LoVo cells through the mitochondrial apoptotic pathway, which indicated that hederagenin might be a promising therapeutic candidate for the prevention and treatment of human colon cancer [About 10 hederagenin analogues have been identified in quinoa. Their biological activities are listed in a leaves . Hederaga leaves , cytotoxa leaves , antifuna leaves , leishmaa leaves , anti-ina leaves  activiti) >30 μM . Hederag) >30 μM , hence t) >30 μM . Hederagn cancer .84), a pentacyclic triterpene used in medicine, was found in diverse plant families [Spergulagenic acid  is the aglycone with only the bidesmosides to be found in quinoa [O-α-l-arabinopyranosyl serjanic acid 28-O-β-d-glucopyranosyl ester (113) and 3-O-β-d-glucuronopyranosyl serjanic acid 28-O-β-d-glucopyranosyl ester (116) had weaker hemolytic activity (IC50 > 100 μg/mL) than their sapogenin  [Serjanic acid (n quinoa . About 5n quinoa . Similaryl ester 3 and 3-O86) might be originated from serjanic acid (85) by subsequent oxidative enzymatic steps involving the formation of the corresponding alcohol substituted at C-23 in planta [Phytolaccagenic acid (n planta . It is oPhytolaccagenic acid saponins are highly concentrated in the bran (seed coats), which are more exposed to water during germination compared to oleanolic acid saponins . It was 87) and its glycoside 3-O-β-d-glucopyranosyl-(3)-α-l-arabinopyranosyl 23-oxo-olean-12-en-28-oic acid 28-O-β-d-gluco-pyranosyl ester (126) were isolated from quinoa (Gypsogenin (or named 3β-hydroxy-23-oxo-olean-12-en-28-oic acid) (m quinoa . They sh88) and its glycoside 3-O-β-d-glucopyranosyl-(1→3)-α-l-arabinopyranosyl 27-oxo-olean-12-en-28-oic acid 28-O-β-d-glucopyranosyl ester (127) were isolated from quinoa (88) showed same cytotoxic effect as 3β-hydroxy-23-oxo-olean-12-en-28-oic acid (87) with an IC50 value of 25.4 μg/mL. This suggests that the CHO groups at C-23 or C-27 are correlated with the increased cytotoxicity [3β-Hydroxy-27-oxo-olean-12-en-28-oic acid (m quinoa . 3β-Hydrtoxicity .89) and 3-O-β-d-glucopyranosyl-(1→3)-α-l-arabinopyranosyl 3β,23,30-trihydroxy olean-12-en-28-oic acid 28-O-β-d-glucopyranosyl ester (128) have been isolated from quinoa (83) was considered as the substrate for the production of 3β,23,30-trihydroxyolean-12-en-28-oic acid (89), following a mechanism involving a stereochemically specific enzyme able to insert one hydroxyl group into the C-30 position of the triterpene skeleton [3β,23,30-Trihydroxy-olean-12-en-28-oic acid  and gramisterol (130) have been isolated from quinoa seeds [129) showed anticomplementary activity [130) showed anti-cancer activity [Four tetracyclic triterpenoids including two nortriterpenoids citrostadienol  was considered as the precursors of other triterpenoids in their biosynthetic pathways [Among the other pentacyclic triterpenoids, β-amyrin (pathways .141–144) and tocotrienols . Their biological activities are listed in Mertoterpenoids are natural products of mixed biosynthetic origin which are partially derived from terpenoids. Meroterpenoids were also found in quinoa that include tocopherols (143) to be the most abundant followed by α-tocopherol (141), β-tocopherol (142) and δ-tocopherol (144) was the least [141) was considered a stronger antioxidant, whereas γ-tocopherol (143) was a stronger anti-inflammatory agent [143) was the main lipophilic tocopherol in quinoa [The total tocopherol content ranged from 37.49 to 59.82 μg/g . All fouhe least . Tocophehe least ,278. Amory agent ,280. γ-Tn quinoa .145) and β-tocotrienol (146) were also identified in quinoa seeds [Both α-tocotrienol , campesterol (160), Δ7-campesterol (161), Δ5-avenasterol (172), β-sitosterol (176), stigmasterol (181), and Δ7-stigmasterol (182) [7-avenasterol (173), β-sitosterol (176), and Δ7-stigmastenol (180) being the most abundant  [About 36 steroids have been identified in quinoa. Seven sterols were identified among the quinoa lipids, namely cholesterol (ol (182) . Eleven ctively) .27-steroids were identified in quinoa seeds which are listed in 148) possessing a 14α-hydroxy-7-en-6-one chromophore and A/B-cis ring fusion (5β-H) [Eleven Cn (5β-H) .Eating quinoa seeds or quinoa-derived products provides significant amounts of ecdysteroids that may be beneficial to animal or human health . Quinoa 28-steroids such as campesterol (160), makisterone A (166), and their derivatives have been identified from quinoa seeds [About 14 Coa seeds . Their boa seeds , antiangoa seeds , and inhoa seeds .29-steroids in quinoa included avenasterol (172/173), sitosterol (176), stigmasterol (181), and their derivatives. They were all identified in the lipid extract of quinoa seeds [The main Coa seeds . Their b176) has been reported to have a variety of biological activities such as anti-inflammatory [181) also exhibited various biological activities such as anti-inflammatory [β-Sitosterol , betanin (184) and isobetanin (185), were confirmed in red and black quinoa seeds, instead of anthocyanins [188), indicaxanthin (189), and miraxanthin V (190) in quinoa [Betalains are tyrosine-derived red-violet and yellow pigments found in quinoa . They arocyanins . Betaxann quinoa .188), whose dihydroxylated substructure demonstrated high antioxidant capacity [Betalains showed promising bioactive potential, such as high antioxidant and free radical scavenging activities . Betacyacapacity .186), trigonelline (191), and their derivatives. In mammals, betaine (186) acted as an osmolyte in the inner medulla of the kidney, preserving osmotic equilibrium, maintaining at the same time the tertiary structure of macromolecules [191) was considered to be an important multifunctional natural plant hormone with potential taxonomic value [Other nitrogen-containing metabolites in quinoa include betaine  greatly limits the use of quinoa as food . Approxi148), mainly present in the bran, has potential for development as an insect moulting hormone [With the increased demand for quinoa, the problem that comes with it is that the bran is discarded as an industrial production waste. In order to increase the added value of quinoa, the bran (seed coat) should be fully exploited and utilized . Quinoa  hormone . After tBiosynthesis research on quinoa secondary metabolites has rarely been reported. Methyl jasmonate was reported to induce accumulation of saponins in quinoa leaves and induce the expression of saponin biosynthetic genes in quinoa . Knowled


Caenorhabditis elegans has emerged as a genetically tractable animal host in which to study evolutionarily conserved mechanisms of innate immune signaling. We previously showed that the PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway regulates innate immunity of C. elegans through phosphorylation of the CREB/ATF bZIP transcription factor, ATF-7. Here, we have undertaken a genomic analysis of the transcriptional response of C. elegans to infection by Pseudomonas aeruginosa, combining genome-wide expression analysis by RNA-seq with ATF-7 chromatin immunoprecipitation followed by sequencing (ChIP-Seq). We observe that PMK-1-ATF-7 activity regulates a majority of all genes induced by pathogen infection, and observe ATF-7 occupancy in regulatory regions of pathogen-induced genes in a PMK-1-dependent manner. Moreover, functional analysis of a subset of these ATF-7-regulated pathogen-induced target genes supports a direct role for this transcriptional response in host defense. The genome-wide regulation through PMK-1– ATF-7 signaling reveals a striking level of control over the innate immune response to infection through a single transcriptional regulator.The nematode  Caenorhabditis elegans lacks adaptive immunity and is therefore particularly dependent on mounting an innate immune response against pathogens. A major component of this response is the conserved PMK-1/p38 MAPK signaling cascade, the activation of which results in phosphorylation of the bZIP transcription factor ATF-7. Signaling via PMK-1 and ATF-7 causes broad transcriptional changes including the induction of many genes that are predicted to have antimicrobial activity including C-type lectins and lysozymes. In this study, we show that ATF-7 directly regulates the majority of innate immune response genes upon pathogen infection of C. elegans, and demonstrate that many ATF-7 targets function to promote pathogen resistance.Innate immunity is the first line of defense against invading microbes across metazoans.  Drosophila melanogaster and mammalian innate immune signaling revealed a commonality in signaling pathways of innate immunity that has helped motivate the study of pathogen resistance mechanisms in genetically tractable host organisms such as Caenorhabditis elegans )) or the pmk-1 mutant background (pmk-1(km25);atf-7(qd328[atf-7::2xTY1::GFP])) after a four hour exposure to either E. coli OP50 or P. aeruginosa PA14, for a total of four conditions, similar to the treatment described in C. elegans genome (WS258)  .P. aeruginosa in wild-type animals according to our RNA-seq data, revealed that ATF-7 is preferentially located at the promoter regions of genes that are increased in expression by P. aeruginosa, and that this enrichment for ATF-7 is lessened by pmk-1 loss , the Unfolded Protein Response (xbp-1), and the oxidative stress response (skn-1), as well as several immunity regulators   . These o elegans . Howeveratf-7(qd22 qd130)), KU25 (pmk-1(km25)), ZD1807 (atf-7(qd328[atf-7::2xTY1::GFP])), ZD1976 (atf-7(qd328[atf-7::2xTY1::GFP]);pmk-1(km25)). C. elegans were maintained at 16°C on E. coli OP50 as described [atf-7(qd328) allele was generated by the CRISPR-Cas9 system as described [atf-7(qd328)) was verified by immunobloting, and pull-down was assessed by IP-IB. The atf-7(qd238) allele was confirmed to function as wild-type, as assayed by susceptibility to P. aeruginosa strain PA14 in a slow kill assay, and then crossed into the pmk-1(km25) mutant background.Strains used: N2, ZD386 (escribed  and veriP. aeruginosa strain PA14 or E. coli OP50 was grown overnight in Luria Broth (LB), seeded onto SKA media and then grown overnight at 37°C, followed by an additional day at room temperature as previously described [Slow Kill Assay (SKA) plates were prepared as previously described . P. aeruescribed . Large patf-7(qd328) on PA14, as one of the samples failed to pass quality control.After three washes in M9 buffer, animal pellets were resuspended in an equal volume of PBS + complete ULTRA protease inhibitor tablets (Roche), flash frozen in liquid nitrogen, and stored at -80°C until chromatin immunoprecipitation (ChIP). ChIP was preformed as described ,23 usingC. elegans WBPS9 assembly using bwa v. 0.7.12-r1039 [ChIP-seq reads were aligned against the 12-r1039  and the 12-r1039 . Sorted 12-r1039 , and in 12-r1039  was used12-r1039  on the satf-7(qd22 qd130) on PA14, as one of the samples failed to pass quality control.After three washes in M9 buffer, TRIzol Reagent (Invitrogen) was added to worm pellets and flash frozen in liquid nitrogen. Following an additional round of freeze-thaw, RNA was isolated using the Direct-zol RNA MiniPrep kit (Zymo Research). Libraries were prepared using the Kapa mRNA Hyperprep kit and paired end reads were sequenced on the Illumina NextSeq500 sequencer. Three biological replicates of at least 1,000 animals were prepared and sequenced for each condition, with the exception of only two replicates for C. elegans WBPS9 assembly/ WS258 annotation using STAR v. 2.5.3a [Reads were aligned against the . 2.5.3a  with the. 2.5.3a  with the. 2.5.3a  in the RDifferences in gene expression between conditions (expressed as log2-transformed fold-changes in expression levels) were estimated under a general linear model (GLM) framework fitted on the read counts. In this model, read counts of each gene in each sample were modeled under a negative binomial distribution, based on the fitted mean of the counts and aforementioned dispersion parameters. Differential expression significance was assessed using a Wald test on the fitted count data  . P-valueRaw data presented in this manuscript have been deposited in NCBI’s Gene Expression Omnibus  and are Metagene analyses of gene expression and ATF-7 binding enrichment were generated by ngs.plot as described , using CCorrelations between ATF-7 binding and regulation of gene expression were interrogated using the gene set enrichment analysis (GSEA) framework . BrieflyGenes with adjusted p-values <0.05 were considered for Gene Ontology enrichment analysis using the DAVID online webtool, considering as a background the union of all genes with a non-zero baseMean value across any of the DE comparison, based on unique WormBase IDs.E. coli HT115 expressing plasmids targeting the gene of interest or the empty L4440 vector backbone for two generations prior to each experiment. Animal populations were synchronized by egg lay. At the L4 larval stage, approximately 30 worms were transferred to prepared SKA plates and incubated at 25°C. Animals were scored for killing twice daily until the majority of animals had died. Within each experiment, three plates were prepared and scored per RNAi treatment. All clones were obtained from the Ahringer [PA14 plates were prepared as described as above. N2 animals were grown on NGM, supplemented with 25 ug/mL carbenicillin and 2mM isopropyl b-D-1 thiogalactopyranoside (IPTG), that was seeded with either the Ahringer  or VidalAhringer  RNAi libS1 Fig(A) Top GO terms and InterPRO classifications of transcripts that are significantly downregulated  in N2 animals exposed to PA14 versus OP50. (B) Average expression (RPM) across the gene body of genes that are two-fold downregulated by exposure to pathogenic PA14. (C) Venn diagram of decreased genes that are dependent upon pmk-1 or atf-7 for complete  downregulation by PA14.(TIF)Click here for additional data file.S2 Figpmk-1 (A) or atf-7 (B) (y-axis). Transcripts highlighted in purple correspond to genes that were significantly different compared to OP50 in both genotypes . Blue dots indicate genes that are significantly different in the N2 PA14/OP50 comparison only. Red dots represent genes that reach significance in only the mutant condition. Grey dots indicate genes with detected transcripts in at least one condition being compared, but that failed to reach significance cutoffs in either data set.2x2 comparison of genes differentially expressed by exposure to PA14 in N2 animals (x- axis) or upon loss of (TIF)Click here for additional data file.S3 Fig(A) Metagene analysis of ATF-7::GFP binding in all four ChIP conditions across all genes, genes that are two-fold upregulated, or two-fold downregulated by RNA-seq upon exposure to PA14 in a wild-type (N2) background. Shading represents standard error among replicates. (B) Motif analysis of ATF-7::GFP ChIP peaks. The top 400 peaks were considered for motif analysis. (C) Ranked ATF-7::GFP peaks called in animals in all four ChIP conditions. Double red arrow indicates the peaks that were retained for further analysis.(TIF)Click here for additional data file.S4 FigA,B) Overlap of genes whose promoters are bound by ATF-7::GFP , or P. aeruginosa exposure (B)) and genes whose expression is induced (green) or repressed (red) by P. aeruginosa exposure in an ATF-7-dependent manner. Percentages reflect the portion of ATF-7 dependent genes that are also bound by ATF-7. Bonferroni-adjusted hypergeometric p-value < 1E-4 for all overlaps.  Gene Set Enrichment Analysis (GSEA) of transcripts detected by RNA-seq  for association with ATF-7::GFP peaks in WT (C) or pmk-1(km25) mutant (D) animals exposed to OP50.((TIF)Click here for additional data file.S5 FigRepresentative survival curves of animals treated with RNAi against indicated genes that resulted in a significant  reduction in survival on PA14 compared to EV controls in 2/2 experiments. Animals were treated with RNAi for two generations prior to exposure to PA14. EV refers to HT115 carrying the Empty Vector control plasmid, L4440.(TIF)Click here for additional data file.S6 FigPercentages reflect the portion of ATF-7 dependent genes that are also bound by the transcription factor of interest. Target gene sets were identified using WormExp ,38–41. S(TIF)Click here for additional data file.S1 TableSee separate electronic (.xlsx) file.(XLSX)Click here for additional data file.S2 TableSee separate electronic (.xlsx) file.(XLSX)Click here for additional data file.S3 TableProtein domains classified using the David 6.8 Functional Annotation Tool. “Yes,” indicates a significant  reduction in survival on PA14 compared to Empty Vector control in 2/2 experiments.(TIF)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Caenorhabditis elegans immunoglobulin heavy chain-binding protein (BiP) homologue Heat-Shock Protein 4 (HSP-4), is selectively induced in alae-secreting daughter cells but is repressed in hypodermal daughter cells. Surprisingly, this lineage-dependent induction bypasses the requirement for UPR signaling. Instead, its induction in alae-secreting cells is controlled by a specific developmental program, while its repression in the hypodermal-fated cells requires a transcriptional regulator B-Lymphocyte–Induced Maturation Protein 1 (BLMP-1/BLIMP1), involved in differentiation of mammalian secretory cells. The HSP-4 induction is anticipatory and is required for the integrity of secreted alae. Thus, differentiation programs can directly control a broad-specificity chaperone that is normally stress dependent to ensure the integrity of secreted proteins.Differentiation of secretory cells leads to sharp increases in protein synthesis, challenging endoplasmic reticulum (ER) proteostasis. Anticipatory activation of the unfolded protein response (UPR) prepares cells for the onset of secretory function by expanding the ER size and folding capacity. How cells ensure that the repertoire of induced chaperones matches their postdifferentiation folding needs is not well understood. We find that during differentiation of stem-like seam cells, a typical UPR target, the  A study in the nematode Caenorhabditis elegans shows that dedicated developmental programs can bypass the requirements for the unfolded protein response during the differentiation of secretory cells, anticipating their future high folding needs. Caenorhabditis elegans, we find that a chaperone BiP/HSP-4, which is usually induced in most cells by stress, is selectively induced during differentiation of stem cells into the alae-secreting cells while being repressed in their sister lineage, the hypodermal cells. We find that induction of this chaperone is independent of the known ER stress pathways, while its repression requires a known regulator of development in mammals, BLIMP1/BLMP-1. The pre-emptive induction of BiP/HSP-4 is important for the integrity of secreted alae and cuticle, suggesting that a general molecular chaperone that is a canonical target of ER stress pathways can be selectively regulated by development to ensure the quality of secreted proteome and functionality of the cells postdifferentiation.During differentiation, cells that specialize in secretion of proteins, such as antibody-secreting B cells, prepare for the onset of secretory function by expanding the size of the major secretory organelle, the endoplasmic reticulum (ER), and by increasing the expression of molecular chaperones and folding enzymes. This pre-emptive expansion of the ER depends on activation of the ER stress response pathways and is required for the secretory phenotype. In addition, cells may also need to up-regulate a selected subset of chaperones because different secreted proteins may require different chaperones for their folding and secretion. Except in specialized cases, how this selective up-regulation is achieved, and whether it depends on the ER stress pathways, is not well understood. Using  Cellular identity is largely defined by the proteins expressed in the cell or cellular proteome, whose functionality depends on successful folding, localization, and functional maintenance of expressed proteins. During cellular differentiation, rapid onset of new protein synthesis challenges the proteostasis and may result in the production of dysfunctional proteins and folding stress if not matched by corresponding increases in required chaperones . This isIn addition to their expansion, proteostasis networks may need to be remodeled during differentiation since different secreted proteins may require different chaperones for their biogenesis ,7. An eaxbp1 gene in the mouse B-cell lineage prevents development of antibody-secreting plasma cells -like) element ), SJ30(ire-1(zc14) II; zcIs4[phsp-4::GFP]), SJ17(xbp-1(zc12) III), RB772(atf-6(ok551) X), RB545(pek-1(ok275) X), VC1099(hsp-4(gk514)II), JT191(daf-28(sa191)V), RW11606(unc-119(tm4063) III; stIs11606 [egl-18a::H1-wCherry + unc-119(+)]), SD1546]), PS3729(unc-119(ed4) III; syIs78[AJM-1::GFP + unc-119(+)]), CB1372(daf-7(e1372) III), CF1038(daf-16(mu86) I), CB1370(daf-2(e1370) III), PS3551(hsf-1(sy441) I), TP12(kaIs12[COL-19::GFP]), and CB6208(bus-8(e2887) X).The following strains were obtained from the dpy-5(e907) I; sEx10514 [rCesT05E11.3::GFP + pCeh361]) and BC10700(dpy-5(e907) I; sEx10700 [rCesZK632.6::GFP + pCeh361]) strains and a strain expressing phsp-3::YFP were a gift from the Morimoto lab . WT (N2) animals were a subclone of N2Bristol from the Morimoto Lab.BC10514(hsp-4(gk514): hsp-4_Ext_F:CCTCCGATTACTCCTGCTTG; hsp-4_Int_F:GTTTGATGCTGGGTTGACAAAG; and hsp-4_Ext_R:GAGTCTTCAAGAATGGGCGAG. For ire-1(zc14): ire-1(zc14)_F:ATCAGCCAACGACCAATCTGC and ire-1(zc14)_R:GAAGCTTTGGATGGGCGAATAG; the mutation was confirmed by digesting the PCR product with BstBI. For atf-6(ok551): atf-6_Ext_F:ATACCGCGTCAAGGAATCAC; atf-6_Int_R:TTAAATCTCACGCAGGCAAG; and atf-6_Ext_R:AATTGGCCAGTCCCTGTCAC. For pek-1(ok275): pek-1_Ext_F:TCGGAGCACACGATTTCTCG; pek-1_Int_R:CTTGTGGACCCGGAGATACG; and pek-1_Ext_R:CTGAGCACATCTGACGTAAG.Primers used for PCR or to sequence-verify crosses with UPR mutant alleles were as follows. For daf-28(sa191) genetic background, appropriate strains were grown at 20 °C under noncrowded/noncontaminated conditions on fresh plates seeded with OP50 Escherichia coli for at least 2 generations. 20–40 YA animals were then picked to fresh plates, and L2d stage larvae were picked among their progeny based on their morphology [E. coli at 20 °C, and plates were examined daily until there was no food left. Predauers were imaged 1–2 days later.To generate L2d stage animals in rphology ,36: L2d hsp-4::GFP-containing plasmid (#21896) was obtained from Addgene . A 54-bp vector-derived region between the end of the hsp-4 promoter and the start of the GFP, which incidentally contained a PQM-1/DAE-like element, was removed using restriction enzyme PpuMI. To construct the phsp-4-ER stress element-II(del)::GFP transgene, 171 bp of the ER stress element-II–like region was deleted using Q5 Site-Directed Mutagenesis Kit . To construct phsp-4-ER stress element(del)-xbp-1(mut)::GFP, the XBP-1/ATF-6 element in the ER stress element(del) promoter was mutated from GATGACGTGT to GAgGggGTGT. All constructs were verified by sequencing . The mutagenesis primers were as follows: ESERII_del_F:CGGGTCTCTAAGGAAAGGATTC; ESERII_del_R:CCCAGTTGGACATCGGGTC; XBP_1_F:CCTCTCCGATAAGTACACGTTGC; XBP_1_R:GGGTGTATTAGTGCTGGAGAAATC. Transgenes were injected as a mix of 20 ng/μL plasmid DNA and 80 ng/μL sonicated salmon sperm DNA.patf-6 RNAi, xbp-1(zc12);phsp-4::GFP animals were imaged at the L4 stage. For hsp-4 RNAi, COL-19::GFP deposition was examined in young adult animals. For blmp-1 RNAi, 15–20 L4 stage progeny of RNAi-treated daf-28(sa191);phsp-4::GFP parents were placed on fresh RNAi plates, gastrula-stage embryos were picked 1–2 days later, and L2d stage animals were scored 41–46 hours later. All experiments were repeated with a different population of animals 2–3 times.The RNAi clones were from the Ahringer library. For RNAi experiments, animals were grown for one or two generations on 0.4 mM-IPTG–containing plates, spotted with designated RNAi bacteria. For xbp-1 mutant animals on atf-6 RNAi, to ensure proper induction of the RNAi.Prior to the experiments, the RNAi plates were tested for the expected phenotypes, such as, for example, larval arrest of second generation of AAGAAAGCTCTCGAAAAGTC is located within the modENCODE BLMP-1 peak on the hsp-4 promoter, near the XBP-1/ATF-6 (CRE-like) element (G/CT/C found in the MHC class I promoter (underlined) and partial matches (in bold) to an interferon-stimulated response element, found in most interferon-inducible promoters (A/GNGAAANNGAAACT), and to the positive regulatory domain (PRD) element found in the INF-β promoter (G(A)AAAG/CT/CGAAAG/CT/C) [hsp-4 promoter contains this core sequence, we designated it as a putative BLMP-1-binding site (A sequence T element . This seAG/CT/C) . BecauseAG/CT/C)  and the ing site .Animals were mounted on 2% agar pads, immobilized with sodium azide, and imaged with Zeiss LSM700 microscope, using 1.4NA 63x oil objective. Where indicated, 12-bit confocal z-stacks were reconstructed in ImageJ as 3D projections.Animals were mounted as above or immobilized by chilling on plates. Imaging was performed with a Leica M205FA microscope  and Hamamatsu Orca R2 camera , keeping magnification and intensity of fluorescence source  constant within each experiment.S1 Data(XLSX)Click here for additional data file.S1 Figegl-18::mCherry transgene in L1–L2 stage daf-28(sa191) animals. Left panel, L1 animal (20 hours postgastrula) shows differential expression of egl-18 reporter in seam cells following the first asymmetric division. Anterior (a) and posterior (p) daughters are indicated. Right panel, early L2 animal (26 hours postgastrula) following the symmetric division, with similar egl-18 expression in anterior and posterior daughters; stars indicate anterior daughters from the previous round of division in the L1 stage. daf-28(sa191), an allele causing ectopic L2d entry; pegl-18, egl-18 promoter, active in the posterior seam cells after asymmetric divisions; L1, first larval stage; L2, second larval stage; L2d, predauer L2; L4, fourth larval stage; PD, postdauer.(A) Schematic diagram of cell divisions in V1–V4 and V6 seam lineages during postembryonic development. Positions and lineages of seam cells in L1 stage larvae are indicated in and above the worm outline. Left diagram corresponds to reproductive development, right to dauer development. After most asymmetric divisions, anterior daughter cells (a) fuse with the hyp7 hypodermal syncytial cell, while posterior (p) daughters retain their stem-like seam fate. After the last asymmetric division in the L4 stage, posterior daughters initiate terminal differentiation into the alae-secreting cell, fuse with each other, and begin to secrete alae-constituents and other cuticular proteins . Additionally, posterior daughters may undergo differentiation into alae-secreting cells at the end of the L2d stage, resulting in secretion of the dauer cuticle ; these cells do not fuse and resume asymmetric divisions in PD animals. (B) p(TIF)Click here for additional data file.S2 Fighsp-4::GFP. daf-7(e1372) mutant animals enter the L2d stage at 20 °C, similar to the daf-28(sa191) animals; in other strains, the predauer stage was induced by starvation/crowding. Imaging as in daf-28(sa191), an allele causing ectopic L2d entry; GFP, green fluorescent protein; HSP-4, Heat-Shock Protein 4; L2, second larval stage; L2d, predauer L2.Fluorescence micrographs of predauer animals of indicated mutant strains, expressing p(TIF)Click here for additional data file.S3 Figdaf-28(sa191) L2d animals carrying indicated transgenes. Upper panels are projections of confocal stacks through half of the animal, overlaid on a transmitted light image; middle and bottom panels show projections of confocal stacks through the middle of the body or through the hypodermal layer. AJM-1::GFP protein marks apical junctions and outlines seam-cell boundaries . Open arrows point to various cells showing induction of the transcriptional reporters for indicated UPR-target genes . Double-headed arrows indicate individual animals. Scale bars: 20 μm. (B) cnx-1 reporter is induced in V5 seam-lineage–derived neuroblast cells in early L2 animals. Small arrows point to the seam cells outlines. Scale bar: 5 μm. (C) ER stress is able to induce expression of the hsp-3 and enpl-1 transcriptional reporters in seam cells and in hypodermis. The hsp-3 reporter can be induced equally strongly in both anterior and posterior daughters of dividing seam cells in stressed animals. Small arrows point to seam-cell outlines. Animals were incubated on plates containing 10 μg/ml tunicamycin for 24 hours. DMSO (vehicle control)-treated animals were not different from untreated. Scale bars: 10 μm. AJM, Apical Junction Molecule; BiP, immunoglobulin heavy chain-binding protein; daf-28(sa191), an allele causing ectopic L2d entry; GRP94, Glucose Regulated Protein, 94 kDa; ER, endoplasmic reticulum; ex, excretory cell; GFP, green fluorescent protein; HSP, Heat-Shock Protein; int, intestinal cell; L2, second larval stage; L2d, predauer L2; n, head neuron; ph, pharynx; UPR, unfolded protein response; vc, ventral cord neuron.(A) Confocal micrographs of (TIF)Click here for additional data file.S4 Fighsp-4::GFP reporter (gray line). Previously identified and putative regulatory elements/transcription factor binding sites are indicated relative to the coding region. Corresponding sequences, their positions, and orientation relative to the sense strain are indicated. (B) hsp-4 reporter lacking either only the ERSE-II region (left panel) or both known ER stress elements (right panel) is still specifically induced in the differentiating alae-secreting cells. (C) Screenshot of the WormBase GBrowse image of BLMP-1 binding peak in hsp-4 promoter, based on ModeEncode CHIP data. CHIP, Chromatin precipitation; ER, endoplasmic reticulum; GFP, green fluorescent protein; HSP-4, Heat-Shock Protein 4.(A) Schematic representation of the promoter used in p(TIF)Click here for additional data file.S5 Figblmp-1 results in mild induction of hsp-3 expression in seam cells but not hypodermis of late L2d animals. RNAi and scoring as in blmp-1 did not result in induction in seam cells of two additional UPR target genes, enpl-1 and cnx-1, coding for C. elegans orthologues of GRP94 and calnexin, respectively. BiP, immunoglobulin heavy chain-binding protein; BLMP-1, a C. elegans orthologue of B-Lymphocyte-Induced Maturation Protein 1 BLIMP1; GRP94, Glucose Regulated Protein, 94 kDa; hsp-3, heat shock protein 3; L2, second larval stage; L2d, predauer L2; RNAi, RNA interference; UPR, unfolded protein response.(A) Down-regulation of (TIF)Click here for additional data file.


We showed previously that murine nociception and analgesic responses are regulated by the expression of this gene in dorsal root ganglion (DRG) associated with a cis-eQTL. In this report, we identify an exon-level cis-eQTL (rs6471859) that regulates human DRG CA8 alternative splicing, producing a truncated 1,697bp transcript . Our functional genomic studies show the “G” allele at rs6471859 produces a cryptic 3’UTR splice site regulating expression of CA8-204. We developed constructs to study the expression and function of the naturally occurring CA8-204G transcript , CA8-204C  and CA8-201 (full length transcript). CA8-204G transcript expression occurred predominantly in non-neuronal cells (HEK293), while CA8-204C expression was restricted to neuronal derived cells (NBL) in vitro. CA8-204G produced a stable truncated transcript in HEK293 cells that was barely detectable in NBL cells. We also show CA8-204 produces a stable peptide that inhibits pITPR1 and Ca++ release in HEK293 cells. These results imply homozygous G/G individuals at rs6471859, which are common in the general population, produce exclusively CA8-204G that is barely detectable in neuronal cells. CA8 null mutations that greatly impact neuronal functions are associated with severe forms of spinal cerebellar ataxia, and our data suggest G/G homozygotes should display a similar phenotype. To address this question, we show in vivo using AAV8-FLAG-CA8-204G and AAV8-V5-CA8-201 gene transfer delivered via intra-neural sciatic nerve injection (SN), that these viral constructs are able to transduce DRG cells and produce similar analgesic and anti-hyperalgesic responses to inflammatory pain. Immunohistochemistry (IHC) examinations of DRG tissues further show CA8-204G peptide is expressed in advillin expressing neuronal cells, but to a lesser extent compared to glial cells. These findings explain why G/G homozygotes that exclusively produce this truncated functional peptide in DRG evade a severe phenotype. These genomic studies significantly advance the literature regarding structure-function studies on CA8-ITPR1 critical to calcium signaling pathways, synaptic functioning, neuronal excitability and analgesic responses.Carbonic anhydrase-8 (CA8) is an intracellular protein that functions as an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1) critical to intracellular Ca cis-eQTL that regulates murine expression of this gene. This study focuses on a human DRG exon-level cis-eQTL (rs6471859) that regulates CA8-204 alternative splicing producing a truncated 1,697bp transcript. Herein, we demonstrate the “G” allele at rs6471859 produces a cryptic 3’UTR splice site regulating tissue-specific CA8-204 expression. In vitro studies show the “G” allele (CA8-204G) produces a stable peptide that inhibits ITPR1 activation and Ca++ release in non-neuronal cells, but not in neuronal cells. However, using AAV8 gene transfer in vivo we show CA8-204G peptide is expressed in both glial and to a lesser extent in neuronal cells, producing profound analgesia and anti-hyperalgesia using inflammatory pain models, similar to the full length CA8-201 positive control. These data significantly extend our understanding of CA8 structure-function, demonstrating the truncated peptide may represent a novel therapeutic candidate.Carbonic anhydrase-8 (CA8) inhibits IP3 binding to the inositol trisphosphate receptor-1(ITPR1), which regulates intracellular calcium signaling critical to neuronal functions. Recessive CA8-null mutants are associated with spinocerebellar ataxia and neurodegenerative disorders. We have previously demonstrated that nociception and analgesic responses are associated with a DRG  ITPR1 converts inositol trisphosphate IP3) signaling to intracellular calcium signaling . Previou signalincis-expression quantitative trait (eQTL) in DRG associated with variable Car8 gene expression. This Car8 DRG cis-eQTL was shown to regulate nociception and analgesic responses [Previously, we reported Car8/CA8 and its role in calcium signaling are critical to nociception, inflammatory and neuropathic pain , 6. Addicis-eQTL in human DRG. Through our functional genomic studies, we found the “G” allele to be associated with probe PSR0801622.hg1 (P = 0.0095) exclusively recognizing a truncated 1,697 bp transcript . CA8-204 comprises coding sequence for exons 1–3 with a retained intron with a self-contained polyadenylation signal and coding for an extended exon 3 associated with a new stop. It is believed that CA8-204 is non-coding in current public databases. Based on the work of Hirota et al., (2003) this truncated peptide is not expected to bind ITPR1 directly, impact IP3 binding, nor influence calcium signaling [in vitro. Additionally, 1000 Genomes Project Phase 3 data show G/G homozygous individuals at rs6471859 are prevalent in the general population, with their frequencies differing by ethnic background. While the G allele is on average more prevalent in populations of East Asian ancestry ; and less prevalent on average in populations of African ancestry ; there is a near equal average prevalence in populations of European ancestry . Thus, G/G homozygotes are common in the general population and are expected to exclusively express the no-coding CA8-204 transcript predominantly in non-neuronal DRG cells. Given CA8 deficiency is associated with severe spinal cerebellar ataxia and that we have reported CA8 is critical to nociception, inflammatory and neuropathic pain [cis-eQTL on DRG CA8 tissue-specific alternative splicing, and the role of CA8-204 in calcium signaling, nociception and analgesic responses.In this report, we identify a 3’UTR single nucleotide polymorphism (SNP)rs6471859) that functions as an exon-level 9 that fuin vitro we found that the ‘G’ allele produces a cryptic splice site regulating expression of this transcript primarily in non-neuronal HEK293 cells. We also determined that CA8-204 codes for a truncated protein product, which inhibits steady state pITPR1, much like wild type CA8 (CA8-201). Similarly, like CA8-201, this truncated CA8-204 peptide also negatively regulates intracellular calcium release in vitro. Finally, using gene transfer containing AAV8-CA8-204 viral particles in vivo we show this peptide is expressed in both neuronal and glial cells within transduced DRG producing analgesia and anti-hyperalgesia. Collectively, these studies explain why G/G homozygotes at rs6471859 that exclusively produce this truncated peptide in DRG evade a severe phenotype. These genomic studies significantly advance the literature regarding structure-function studies on CA8-ITPR1 critical to calcium signaling pathways, synaptic functioning, neuronal excitability and analgesic responses.Herein using QPCR, we show that rs6471859 regulates tissue-specific alternative splicing of CA8-204. Specifically, Table 1 shows the association between rs6471859 with exon-level probe PSR08016202.hg1 (P = 0.0095) regulating expression of alternative transcript CA8-204. CA8-204 is comprised of the first three exons of CA8-201 with an extended exon 3 due to a retained intron containing a new stop . These data suggest that rs6471859 represents an exon-level cis-eQTL that resides at nucleotide position 1,416 within the retained intron of the CA8-204 transcript regulating expression of this alternatively spliced transcript in DRG. The exact mechanism by which rs6471859 regulates the alternative splicing of CA8-204 remains unknown.G or CA8-204C) and transfected HEK293  and NBL  cell lines with pCMV-N-FLAG-tagged vectors coding for the naturally occurring CA8-204G transcript or CA8-204C produced by reversion mutation of CA8-204. We then ran qPCR (4 replicates from at least 3 separate cultures) to quantify the production of these transcripts. QPCR of the cDNA from CA8-204G and CA8-204C showed selective expression in HEK293 and NBL cells, respectively . Results from qPCR revealed enriched or unique expression of CA8-204G in non-neuronal cells (HEK293) compared with low levels of exogenous expression of CA8-204C. The reverse results occurred in NBL cells, where CA8-204C expression  was greatly enriched over CA8-204G expression .To better understand how rs6471859 regulates alternative splicing of CA8-204 we developed two constructs  suggested that like CA8-201, CA8-204G produced a peptide of the expected size of 153 amino acids (26-27kDa). However, this protein was not detected using similar immunoblotting methods with NBL cell lysates. IHC analyses in vitro using HEK293 and NBL cells, shows no detectable CA8-204G found in NBL cells .To determine whether CA8-204 produced a stable peptide, we transfected HEK293 cells with CA8-204G in the regulation of ITPR1 activation (pITPR1) by measuring forskolin-induced phosphorylation in transfected HEK293 cells followed by western blotting. Our results demonstrate CA8-204G, similar to CA8-201, inhibits the forskolin-induced phosphorylation in vitro .We then investigated the role of CA8-204G affects ATP-stimulated ITPR1-dependent cytosolic calcium release. Once again, HEK293 and NBL cells were harvested after transfection with CA8-204G, CA8-201 or empty vector. ATP (1μM) was used to induce free calcium release in vitro, and cells were monitored in real time by measuring calcium release (Fura2-AM dye). Empty vector had no effect on ATP-mediated free calcium release. HEK293 cells transfected with either CA8-201 or CA8-204G demonstrated reduced cytoplasmic free calcium after ATP-stimulation . In contrast, only CA8-201 inhibited ATP-stimulated calcium release in NBL cells . Collectively, these results demonstrate tissue-specific transcription, translation and functions of CA8-204G on calcium signaling in vitro is restricted to non-neuronal cells.We next investigated whether CA8-204G in non-neuronal cells, we investigated the impact of this alternative transcript using a pain model in vivo. AAV-mediated gene transfer to DRG cells including glial and sensory neurons through SN injections is an effective route in the treatment of chronic pain [MT (negative control) and AAV8-FLAG-CA8-204G. Thermal behaviors were monitored daily and maximum latencies were recorded through day 15 for mice receiving SN injections. Mice receiving CA8-201 or CA8-204G, but not those injected with CA8-201MT, demonstrated elevations in their thermal latencies starting at Day 13 reaching a maximum by Day 15  of approximately 3.0 seconds above baseline. This increase in thermal withdrawal latencies in C57BL/6J mice exceeds a dose of 80 mg of oral morphine in a 60 kg adult, after allometric conversion [G, but not CA8-201MT showed rapid recovery through Day 22  and once again demonstrating profound analgesia with significant increases over baseline in thermal withdrawal latencies after Day 20 .Due to the tissue-specific transcription, translation and functions of CA8-204nic pain , 6. We cMT, V5-CA8-201 and FLAG-CA8-204G. While overexpression calculated for total population of cells (neurons and glia) in CA8-201 and CA8-204G showed a very similar pattern ), surprisingly, and in contrast to our in vitro data, significant CA8-204G, like CA8-201, was present in both neuronal and glial cells. However, while CA8-204G expression was not significantly different from CA8-201 in glial cells, expression was reduced (about 50%) when compared with CA8-201 in neuronal cells ). Nonetheless, this level of neuronal expression in vivo appears to be enough to produce analgesia and anti-hyperalgesia in this animal model. Thus, moderate levels of the functional CA8-204 peptide appear to be protective in vivo. Thus, the “all” or “none” expression based on cell type in vitro was not replicated in vivo. Presumably, even this moderate level of expression of CA8-204 in neuronal cells explains how G/G homozygotes evade a deleterious phenotype.Lumbar DRG sections (L4 and L5) were collected from each group (N = 8) after perfusion and immunohistochemistry (IHC) was performed using primary anti-FLAG and -V5 antibodies, with fluorescent  secondary antibody. Because DRG are comprised of both neurons and glial cells, to measure the overexpression of CA8 peptides after SN, we used anti-S100 (glia) and anti-advillin (sensory neurons) antibodies with fluorescent secondary antibody  to define the tissue-specific expression of each CA8 peptide. Total cells were counted using DAPI staining. The percentage of overexpression in neuronal and glial cells was measured separately for V5-CA8-201cis-acting regulatory element within the intronic or exonic sequences of CA8-204 [http://www.umd.be/HSF3/HSF.shtml) predicts splice sites (donor or acceptor) by calculating maximum entropy for the target nucleotide sequence. Consensus values range from 0 to 100 for HSF (Human Splicing Factor). The threshold is defined at 65 for HSF, which means that every signal with a score above this threshold is considered to be a splice site (donor or acceptor). The webserver also predicts the nature of the splice site.We performed a bioinformatic analysis using Human Splicing Factor (HSF 3.1) web server to understand the role of rs6471859 as a G including the naturally occurring “G” allele at position 1,416 in the 3’UTR; and on CA8-204C (reversion mutation) by replacing the “G” with a “C” nucleotide at rs6471859. Table 2 provides results of HSF3.1 analyses. The “G” allele at rs6471859 is predicted to create a donor site within the sequence “CAGGTCTTG” with CAG as potential new splice site (71.92% consensus). This splice site is lost with the “C” allele (CACGTCTTG) at rs6471859. These findings suggest a potential mechanism associated with the observed alternative splicing of the CA8 gene.The program was run for sequence CA8-204cis-eQTL (rs6471859) that regulates alternative splicing of CA8; (2) a cryptic splice site is produced by the “G” allele at position 1,416 (rs6471859) within the 3’UTR of the CA8-204 transcript; (3) CA8-204G  and CA8-204C (reversion mutation) expression constructs demonstrate tissue-specific splicing in vitro; where CA8-204G is transcribed predominantly in non-neuronal cells (HEK293), CA8-204C is transcribed predominantly in neuronal-derived cells (NBL); (4) Surprisingly, we also show that CA8-204G is translated into a stable truncated peptide in HEK293 cells; (5) and in contrast to the work of Hirota et al., (2003), CA8-204 inhibits forskolin-induced ITPR1 activation (pITPR1) and ATP-stimulated calcium release [in vitro data demonstrate tissue-specific transcription; translation and functions of CA8-204G are restricted predominantly to non-neuronal cells. Therefore, it appears that homozygous G/G individuals exclusively produce CA8-204, predominantly in non-neuronal DRG cells. These results raise numerous important questions because according to 1000 Genomes Project Phase 3 allele frequencies for rs6471859; G/G homozygotes are common across in almost all populations. To further address the presumed discordance between these in vitro results with what is known about the deleterious effects of CA8 deficiency, we show: (6) AAV8-mediated gene transfer of CA8-204 via direct sciatic nerve injection to transduce DRG in vivo showing CA8-204 overexpression of CA8-204, like CA8-201, produces profound analgesia after about 13 days; (7) overexpression of CA8-201 and CA8-204G showed very similar patterns in DRG neuronal and glial cells ( and (8) surprisingly, while CA8-204G expression was seen in neuronal cells, but to a lesser extent (about 50% compared to CA8-201)). Importantly, this level of neuronal expression in vivo appears to be enough to produce anti-hyperalgesia and profound analgesia in the inflammatory pain model tested. Thus, it is critical to appreciate that despite its moderate levels, the functional CA8-204 peptide in somatosensory cells appears to be protective in vivo. This is consistent with the described CA8-S100P null mutation that only produces a phenotype in recessive homozygotes [In this report we identify the following for the first time: (1) a human DRG exon-level -eQTL rs671859 thahttps://gtexportal.org/home/snp/rs6471859). It is also important to note that our in vitro data could have been misleading if we wrongly concluded that there is an “all” or “none” expression of CA8-204 based eQTL data by cell type. Only the in vivo pain model data presented enabled us to explain the discordance between the population genetic data for rs6471859 and the lack of a severe phenotype in G/G homozygotes due to the splice variants reported. Dorsal root ganglia (DRG) comprises both of neuronal and neuroglia population of cells. Markers such as S100 and advillin were used to identify glial and neuronal cell population. Glial cells in form of satellite glial cells and sensory ganglia surely regulate the afferent signaling and neuronal excitability, with a possible role in chronic pain formation as shown in literature [in vivo explains how G/G homozygotes evade a deleterious phenotype. Our overall findings are consistent with reports suggesting that CA8 null mutations producing severe spinal cerebellar ataxia are recessive, and heterozygotes are phenotypically normal. Thus, heterozygotes producing approximately 50% of normal cellular CA8 evade a severe phenotype [in vitro. This may likely occur because of this retained intron harboring a polyadenylation signal. Retained introns with self-contained 3’UTR polyadenylation signals are prevalent in animals [in vitro .GTEX data reported for rs6471859 show it represents a multi-tissue eQTL including both neuronal and non-neuronal tissues as follows: lung 4.0e-23); whole blood (8.8e-6); brain-hippocampus (2.7e-4), -cortex (3.5e-3), -cerebellum (2.3e-3), and -pituitary (3.2e-7). However, no splice eQTL data are reported , which showed that virtually the entire CA8 peptide (exons 1–8) is required for the negative regulation of IP3 binding to ITPR1 [+-K+-ATPase and protein 4.1N, which are shown to dynamically impact channel activation [Remarkably, our results demonstrated that CA8-204 also produces a stable functional peptide, which contradicts the earlier findings of Hirota t al. 200, which stivation –34.in vivo, suggesting that this peptide is fully functional and may serve as a validated therapeutic candidate as a long acting local anesthetic using gene therapy.Our results clearly demonstrated that CA8-204 overexpression is protective of inflammatory pain Director of Compliance, IACUC/IBC/ESCRO, Office of Research at University of Miami, Miller School of Medicine approved the protocol for the use of animal subjects in research study (IBC #17–075). Before SN injections all mice were anesthetized using intraperitoneal injection of a cocktail of ketamine, xylazine and acepromazine . Perfusion was carried out only once mice were anesthetized with isoflurane. The tests for assessing neuropathic pain were performed using IACUC approved protocols and the thermal beam projected for 15 seconds taken as cut-off threshold to prevent potential injury.CA8 exon-level DRG analyses were run essentially as described elsewhere . BrieflyG) was generated from in pCMV-Sport6 vector (Harvard Facility) and cloned using PCR with forward primer 5’-GGGGACAACTTTGTACAAAAAAGTTGGCATG GCGGACCTGAGCTTC-3’ and the reverse primer: 5’-GGGGACAACTTTGTACAAAAAAGTTGGCATGGCGGACCTGAGCTTC-3’ and cloned into pCMV-FLAG(DYKDDDDK)-N-terminal vector (Takara), with forward primer 5’-TTTGTCGACAACGCACGCCTGCTTGCAC-3’ and reverse primer 5’-TTTTGGTACCTTACAGTAATGCTGTCAAACACTTCAACAG-3’. The restriction enzymes SalI and KpnI (NEB) were used for restriction digestion on PCR products and for the pCMV-N-FLAG (Takara) vector having similar restriction sites. CA8-204C was produced using GENEART site-directed mutagenesis system  and specific primers  were employed to create one nucleotide substitution at position 1416 in the CA8-204 transcript. This substitution was in the CDS sequence using the forward primer: 5’-ATGCAGATAGAAGAATTTCGACACATGTCAAGGGGGCAGA-3’ and the reverse primer: 5’-TCTGCCCCCTTGACATGTGTCGAAATTCTTCTATCTGCAT-3’. The pCMV-N-FLAG constructs containing CA8-204G and CA8-204C sequence was confirmed using Sanger sequencing. Human wildtype CA8 cDNA (CA8-201) was purchased from Origene (NCBI accession: NM_004056). The plasmid was digested with enzymes BamH1 and Not1 (NEB). The purified cDNA was ligated with linearized pAAV-MCS vector using quickstep ligation (Invitrogen) and transformed. The sequence of the insert was confirmed through sequencing. The CA8-201 cDNA was mutagenized to produce the S100P mutation (CA8-201MT), as described previously [G (CA8-204G) using primers specific for 1,697 bp long alternative variant of CA8 containing FLAG tag carrying BamHI and XhoI sites; forward primer: 5’-TTTGGATCCGCCACCATGGACTACAAGGACGACGATGA-3’ and reverse primer: 5’-TTTTCTCGAGTTACAGTAATGCTGTCAAACAC-3’. PCR fragments were restriction digested with BamHI and XhoI (NEB), purified and ligated with linearized pAAV-MCS (ITR) vector between BamHI and XhoI sites. The constructs were confirmed using Sanger sequencing. The recombinant AAV8-V5-CA8-201, AAV8-V5–CA8-201MT, AAV8-FLAG-CA8-204G viral particles were produced by the Miami Project Viral Vector Core at the University of Miami Miller School of Medicine, essentially as described previously [13 genome copies per mL.An expression construct containing the naturally occurring CA8-204 transcript (NCBI accession: NM_001321837.1) including the G allele at nucleotide position 1416 . All transfections were performed in Opti-MEM I reduced serum medium (Invitrogen) with 2 μg DNA and 6 μl Lipofectamine 2000 for 6-well plates or 0.5 μg-1 μg for 12-wells. Cells were maintained for 4–6 h (minimum of 2h) in the transfection media at 37o C/ 5% CO2 followed by replacing it with DMEM/F-12 (1:1) plus FBS and penicillin-streptomycin. A 48 h incubation of cells in these conditions is sufficient for their use in measurements of mRNA and protein expression using RNA extraction followed by RT (reverse transcriptase)- PCR and/or real-time PCR using SYBR-green PCR master mix on the Step One Plus real time PCR machine (Applied Biosystems), western blot and immunoprecipitation studies.HEK293 and NBL  cells, passed at least three times with 0.5% trypsin in DMEM/F12 (GIBCO) complemented with penicillin/streptomycin and 10% fetal bovine serum  were allowed to grow at the rate of 4 x 10G, CA8- 204C, CA8-201 and CA8-201MT expression constructs using the RNeasy RNA extraction kit (Qiagen) following the manufacturer’s protocol. Total RNA was quantified by an Epoch spectrophotometer (BIOTEK). Two-step RT-PCR was performed using the Access-quick RT-PCR system (Promega) and Faststart DNA Polymerase (Roche) according to the supplier’s protocols. RT-PCR was used to amplify exogenous CA8-204G and CA8-204C with the forward primer: 5’-ATGGACTACAAGGACGACGATG-3’ and the reverse primer: 5’-GGGCTATTTTCTGGGGTAAA-3’. For the RT-PCR, the PCR products were loaded on 1.2% agarose gel, and were visualized with ethidium bromide. No template was used as a negative control. Quantitative PCR was used to amplify exogenous CA8-204G and CA8-204C, flanking the 3’UTR region, with the forward primer: 5’-ATGGC GCTGGCCCCATGGGTT-3’ and the reverse primer: 5’-GGGC TATTTTCTGGGGTAAA-3’. ACTB (ß-actin) was used as internal control with primers forward: 5’-AAATCTGGCACCACACCTTC-3’ and reverse: 5’-CACCTTCACCGTTCCAGTTT-3’ . qPCR and analysis was carried out on a Step One Plus system  using Power SYBR-green PCR master mix (Applied Biosystems). Primers  were designed across the beginning and end of the 3’UTR of the CA8-204 alternative variants with retained intron. No endogenous CA8-204G or CA8-204C were detected at baseline in either cell line. Quantitation of CA8-204G or CA8-204C was normalized to the expression of ß-actin (ACTB). Primer efficiency was determined using the melting curve analyses after qPCR.Total RNA was extracted from cultured HEK293 and NBL cells transfected with CA8-204Transfected and non-transfected HEK293 and NBL cell cultures were lysed in RIPA buffer complementing with appropriate concentrations (1μM) of proteinase and phosphatase inhibitors (Sigma). Cell lysates containing proteins were separated on 4–15% or 10% SDS polyacrylamide gels and transferred to a PVDF membrane (Biorad) using transfer buffer containing 20% methanol in 25mM Tris-HCl and 192mM glycine. For transfer of large proteins such as pITPR1 western blot, 5–6% Tris-HCl/SDS polyacrylamide gel and 10% methanol in transfer buffer were used to increase the transfer efficiency. Blocking of the membrane was done with 5% skimmed milk in Tris-buffered saline (TBS) with 0.1% tween-20 for 1 h at room temperature followed by wash with TBS containing tween-20 and incubation with primary antibodies at 4°C. The blots were then incubated for 1 h at HRP-conjugated secondary antibodies at room temperature (Santa Cruz laboratories). Pierce Super Signal substrate , was used to visualize bands. Primary antibodies used were as follows: anti-CA8 , anti-V5 (Invitrogen), anti-pITPR1  and anti-ITPR1 , anti-FLAG (Sigma Aldrich), anti-FLAG (Aves), anti-Vinculin (Abcam) and anti-β-actin .G 24 hours after the seeding cells on the 12 well plates. Cells (1x 105) were split onto 12 mm glass coverslips , previously coated with poly-lysine (12h) and laminin for 2 h. Transfections were done as described above. Cell media was replaced with Fura-2 dye from the Fluo-4 Calcium Assay Kit (Life Technologies) on the day of the assay followed by the incubation at 37°C for 30 minutes. Cells were moved to the room temperature, washed briefly twice with calcium containing buffer . Coverslips were loaded in the upright position on the microscope washed and perfused with Ca2+-free media containing EGTA . Each coverslip was allow to equilibration for 5 minutes before imaging. Cells were visualized at every 2 seconds for 10 minutes while Ca2+ free buffer alone or media containing ATP were perfused onto the coverslips with cells. Individual cells were analyzed using the LAX system on Leica microscope .HEK293 and NBL (ATCC) cell lines, were passaged at least three times and were transfected with vectors vehicle (empty vector with FLAG tag), CA8-201 and CA8-204ad libitum. All animals were allowed to acclimatize for at least 10 days and were housed in a 12–12 h light–dark cycle in a sterile facility under controlled humidity and temperature.All experiments and procedures performed on animals were conducted in compliance with the guidelines from the Institutional Animal Care and Use Committee (IACUC) at University of Miami for the care and use of laboratory animals and the current guidelines for experimental pain in conscious animals, following an IACUC approved protocol , 37. MalMT (CA8-201MT) [G (CA8-204G) was used as the test group. SN (sciatic nerve) injections of viral particles was followed using a previously prescribed procedure [https://gtexportal.org/home/snp/rs6471859). Therefore, to minimize the number of animals used in these studies, we selected male C57BL/6J mice only. This represents a limitation of the current study.Viral particles from AAV8-V5-CA8-201 (CA8-201) were used in this study as positive control group. As previously shown, analgesic and anti-hyperalgesic activity is abolished in the (S100P) null mutant AAV8-V5-CA8-2018-201MT) , that warocedure . We haverocedure , 38–40. rocedure , 42. We o C) and cooled further, was subcutaneously injected in the mice through plantar surface of left hind paw.Total volume of 25–30 μl of 0.1% λ-carrageenan  dissolved in saline  were assessed as previously described . Mice we5 cells/ml. Culture volume was 2ml per well in 6-well plates, 1ml per well in 12-well plates. The cultures were incubated at 37°C in a water saturated atmosphere containing 5% CO2/95% air and maintained in Gibco DMEM+F12 (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1X penicillin-streptomycin . Transfections were performed using AAV8 vectors containing tagged genes of interest .HEK293 (ATCC) and NBL  cells were plated in 12-well plates with cover slips coated prior with poly-D-lysine, laminin-coated glass coverslips for immunofluorescence. Cells were seeded at a density of 1x 10Mice were anesthetized with isoflurane and perfused by a saline injection through the left ventricle with saline (30ml) followed by a thorough perfusion with a fixative of 4% paraformaldehyde+15% saturated picric acid solution in 0.16M PBS . Dorsal horn (DH) and DRG from the fourth and fifth lumbar regions (L4-L5) were dissected, and placed in the same fixative for 2–4 h post fix, then transferred to 20% sucrose overnight, ideally when the tissue touched the bottom. The tissues were embedded with OCT  on dry ice. From the embedded tissues, 16–20μm sections were cut using Leica 1900 Cryostat , and mounted on the slides for immunofluorescence staining.The immunostaining of DRG sections was performed essentially as previously described procedure , 6. BrieMT, CA8-201 and CA8-204G in DRG and the percentage of overexpression of positive neurons and glial cells in L4 and L5 DRG sections from nonadjacent slices (N = 8) from each group of animals were determined as described previously [G) and V5 (for CA8-201MT and CA8-201) cells and for each occurrence. The investigator was blinded to the group and arrangements of ganglia on the slides while making a quantitative assessment. Data were summarized using mean ± SEM. Differences between groups were assessed using one-way / two-way ANOVA depending on the condition analyzed. The criterion for statistical significance was P<0.05 applying Bonferroni’s correction for multiple tests.Analyses of data from immune staining of CA8-201eviously . PercentQuantitation of data in all the other experiments was performed using one-way ANOVA followed by Bonferroni correction / Tukey’s multiple group comparison as post-hoc test for experiments with triplicates or more, with experiments repeated 3 times or more. Quantitation of data on thermal pain behavior tests was performed using two-way ANOVA repeated measures for each time-point (day) the measurements were recorded, followed by Bonferroni’s post-hoc correction.S1 Fig(I) (A) Immunostaining (IF) using anti-V5  antibodies. (B) IF using antibodies anti-V5 . (C) IF using anti-FLAG . IF from the HEK-293 cells transfected with AAV8-viral vectors containing CA8-201MT, CA8-201 and CA8-204G shows an increase in positive overexpression in CA8-201 and CA8-204G. This expression was not detected in the CA8-201MT in HEK-293 cells. (II) (A) IF using anti-V5  antibodies. (B) IF using anti-V5 antibodies . (C) IF using anti-FLAG antibodies . IF from NBL cells transfected with AAV8 virus containing CA8-201MT, CA8-201, or CA8-204G showed an increase CA8-201 overexpression only. V5 expression was barely detectable after transfection with CA8-201MT or CA8-204G in NBL cells. (III) The histogram represents the comparison of overexpression of CA8-204 with CA8-201, where percentages of overexpression were quantified using one-way ANOVA with Bonferroni's post-hoc test . Comparisons reveal the ubiquitous nature of CA8-201 overexpression in both HEK and NBL cells. Exogenous expression of CA8-204G was higher in HEK cells than NBL cells. There was virtually no V5-CA8 expression observed after transfection with CA8-201MT in either cell line. .(TIF)Click here for additional data file.S2 FigG. Western blotting suggests that while inhibition of pITPR1 expression through CA8-201 is observed, CA8-204G was unable to inhibit pITPR1expression in NBL cells due to absence of any CA8-204G expression. Quantitation of pITPR1 expression was performed using ImageJ software. Data were normalized with vinculin. N = 3, ***P<0.001, **P<0.01 Quantitative analysis was performed using the one-way ANOVA followed by Bonferroni post–hoc test for each possible comparison (GraphPad software).Immunoblot of pITPR1  in NBL cells, transfected with an empty vector, CA8-201 (WT) or CA8-204(TIF)Click here for additional data file.S3 FigMT, used in this study as a negative control, were stained with (A) glial (S100) or neuronal (advillin) markers. (A) IF done with anti-V5 against V5-CA8-201MT and anti-S100  antibodies individually in DRG sections, , and shown as merged (Merge). (B) IF done with anti-V5 and anti-advillin antibodies individually in DRG sections, , and shown as merged (Merge). V5-CA8-201MT failed to express in either glial or neuronal DRG cells. .Immunostaining of cells extracted from DRG of mice that received sciatic nerve injections of AAV8-V5-CA8-201(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.


EVI1 has potent oncogenic properties and confers poor prognosis and chemo-resistance in leukemia and solid tumors. To investigate to what extent EVI1 function might be regulated by post-translational modifications we carried out mass spectrometry- and antibody-based analyses and uncovered an ATM-mediated double phosphorylation of EVI1 at the carboxy-terminal S858/S860 SQS motif. In the presence of genotoxic stress EVI1-WT (SQS), but not site mutated EVI1-AQA was able to maintain transcriptional patterns and transformation potency, while under standard conditions carboxy-terminal mutation had no effect. Maintenance of hematopoietic progenitor cell clonogenic potential was profoundly impaired with EVI1-AQA compared with EVI1-WT, in particular in the presence of genotoxic stress. Exploring mechanistic events underlying these observations, we showed that after genotoxic stress EVI1-WT, but not EVI1-AQA increased its level of association with its functionally essential interaction partner CtBP1, implying a role for ATM in regulating EVI1 protein interactions via phosphorylation. This aspect of EVI1 regulation is therapeutically relevant, as chemotherapy-induced genotoxicity might detrimentally sustain EVI1 function via stress response mediated phosphorylation, and ATM-inhibition might be of specific targeted benefit in EVI1-overexpressing malignancies.The transcriptional regulator EVI1 has an essential role in early hematopoiesis and development. However, aberrantly high expression of  EVI1, which frequently results from chromosomal aberrations involving MECOM (MDS-EVI1 complex) locus on 3q26, where EVI1 is encoded, has potent oncogenic properties with transformation capabilities in vivo and in vitro , and the EVI1 antibodies #2265 and #2593  were used. For detection of phosphorylated EVI1 we used phospho-specific, affinity purified rabbit polyclonal antibodies raised against EVI1-phospho-peptides NSNHGSQpSPRNVEC for serine 860 (S860) phosphorylation (anti-pS860-EVI1), and NSNHGpSQpSPRNVEC for serine 858 and serine 860 (S858/S860) double phosphorylation (anti-pS858/pS860-EVI1) (Eurogentec)   . Followi® II XL Kit (Agilent). The Codon-optimized mouse Evi1 lentiviral vector pRRL.PPT.SF.EVI1mCo.IRES_EGFP.pre (The human EVI1 coding region was excised from pBABE-puro-flag-EVI1 (gift from Aubrey Thompson)  using SaEGFP.pre ,33,34 waEGFP.pre . Primer EVI1-expression (seq) analysis of EVI1-mediated modulation of gene expression RNA was extracted from HEK293 cells, which were subjected to transient transfection using half confluent cultures with pCMV-flag, pCMV-EVI1-WT-flag or pCMV-EVI1-AQA-flag, exposed to 150 μM H2O2 or left untreated for 8 h. Equal levels of EVI1 levels were monitored by western blot (ftp://ftp.ensembl.org/pub/current_gtf) . Biological pathways associated with differential gene expression were determined using a right-sided Fisher's exact test . The effect on levels of expression detected by RNAseq were confirmed by RT-PCR for selected transcripts using taqman technology with housekeeper transcripts (YWHAZ and β-ACTIN) chosen from a tested pool of housekeeper genes for minimal variation between samples and conditions (primer sequences and detailed methodology in Reporter gene assays were carried out in HEK293T cells as described before (details in pression . For Polern blot . Librarient_gtf) . Data an+ cells (104) were seeded in untreated methylcellulose medium , or supplemented with 30 μM of H2O2. Alternatively, 1 × 105 cells in 100 μl (96-well plate) were left untreated or irradiated (0.5 or 2 Gy). After 14 days, colony number and size were quantified and documented using a DMIL inverted microscope fitted with a MC170 HD camera (Leica)  . Inducti (Leica) .+ progenitor cells were isolated from bone marrow of 8–10 week old C57/BL6 mice as previously described (g at 32°C), and cultured for two days in pre-stimulation serum free cytokine supplemented medium (StemCell Technologies), prior to FACS selection of GFP+ cells. Equal transduction efficiency for EVI1-WT and EVI1-AQA was monitored by RT-PCR and quantitation of fluorescent signal , with induction of genotoxic stress monitored by the induction of γH2AX foci  peptide (NCBI accession: NP_001098548.2) Figure . This pen Figure . Mass spn Figure . Westernd Figure . Mass spFOS and PLZF , EVI1-WT and EVI1-AQA transfected HEK293 cells, with and without DNA damage mediated by H2O2. We applied a group ANOVA with P < 0.01 to the entire dataset and identified 1306 significantly differentially expressed transcripts (P < 0.05) upregulated transcripts with EVI1-WT transfection compared to empty vector, 22 are also significantly up-regulated in EVI1-AQA transfected cells. Likewise, of 90 significantly repressed transcripts with EVI1-WT transfection, 30 are also significantly repressed in EVI1-AQA transfected cells. However, in the presence of damage, 10 of 41 upregulated transcripts in EVI1-WT transfected cells were also significantly upregulated in EVI1-AQA transfected cells, and of 47 repressed transcripts in EVI1-WT transfected cells, only 7 were also significantly repressed by EVI1-AQA transfection. Pathways affected by EVI1-WT transfection in the presence of genotoxic stress in this model included fucose-carbohydrate biosynthesis, death-receptor- and MAPK signaling , for comparison with the wild type EVI1-SQS (EVI1- WT) constructs. We first assessed target-gene promoter repression of EVI1-AQA compared with EVI1-WT and measured EVI1-mediated repression of and PLZF ,42. EVI10 Figure . We nextnscripts , Sheet 1nscripts , Sheet 2s Figure . In untrA Figure , arrow. n = 4) increase in colony numbers after 14 days compared with vector only transduction, which under standard conditions was not significantly different from transduction with mutated EVI1-AQA . Transduction with EVI1-AQA did not affect replating efficiency compared to EVI1-WT in the first round of plating , and of 22.7% ± 2.4 SEM after radiation (P < 0.001)  after H2O2 treatment, and 24.9% ± 4.8 SEM after radiation  . As the interaction with the co-repressor CtBP1 is essential for EVI1-mediated transformation ,27, and P < 0.05) after treatment with H2O2 compared with EVI1-WT . Carboxy-terminal phosphorylation of EVI1 has previously been listed in global phosphoproteomic and targeted studies .Gene expression data from RNASupplementary DataClick here for additional data file.


However, non-hyperaemic 3‑dimensional quantitative coronary angiography-based vessel fractional flow reserve (vFFR) was 0.74  for which further physiological assessment was performed (Fig.