NPM1 gene mutations is currently recognized as a distinct entity, due to its unique biological and clinical features. We summarize here the results of published studies investigating the clinical application of minimal/measurable residual disease (MRD) in patients with NPM1-mutated AML, receiving either intensive chemotherapy or hematopoietic stem cell transplantation. Several clinical trials have so far demonstrated a significant independent prognostic impact of molecular MRD monitoring in NPM1-mutated AML and, accordingly, the Consensus Document from the European Leukemia Net MRD Working Party has recently recommended that NPM1-mutated AML patients have MRD assessment at informative clinical timepoints during treatment and follow-up. However, several controversies remain, mainly with regard to the most clinically significant timepoints and the MRD thresholds to be considered, but also with respect to the optimal source to be analyzed, namely bone marrow or peripheral blood samples, and the correlation of MRD with other known prognostic indicators. Moreover, we discuss potential advantages, as well as drawbacks, of newer molecular technologies such as digital droplet PCR and next-generation sequencing in comparison to conventional RQ-PCR to quantify NPM1-mutated MRD. In conclusion, further prospective clinical trials are warranted to standardize MRD monitoring strategies and to optimize MRD-guided therapeutic interventions in NPM1-mutated AML patients.Acute myeloid leukemia (AML) with  How patient . The ris patient ,5,6. Intt [MRD−) . Moreovet [MRD−) ,8,9. Mort [MRD−) . Beyond t [MRD−) . RQ-PCR I1, ERG) . While MI1, ERG) ,10.NPM1) gene encodes for a protein which physiologically shuttles between nucleus and cytoplasm, acting as a molecular chaperone to establish multiple protein-protein interactions [Arf protein in the nucleolus, and regulation of centrosome duplication [NPM1 gene mutations, occurring in approximately 30% of adult AML cases, and in 50–60% of AML cases with normal karyotype, represent one of the most frequent molecular lesions observed in AML [NPM1 mutations in 2005 by Falini et al. [NPM1 gene mutations result in structural changes of the C-terminus of NPM1 protein, with subsequent aberrant cytoplasmic delocalization, leading to a unique immunohistochemical pattern detectable on BM trephine biopsy [Nucleophosmin  classification of myeloid neoplasms and acute leukemia [NPM1 mutations [FLT3 and DNMT3A gene mutations, documented in about 40% and 50% of NPM1-mutated AML cases, respectively [NPM1-mutated AML patients were generally considered limited to cases without concurrent FLT3-ITD mutations [FLT3-ITD have previously been found to be most clinically relevant when co-occurring with NPM1 and DNMT3A mutations, as opposed with either mutation alone [NPM1 mutation and FLT3-ITD with a low (<0.5) allelic ratio have a similar favorable outcome as patients with NPM1-mutated AML without FLT3-ITD [NPM1-mutated AML associated with FLT3-ITD with high allelic ratio, which is characterized by higher relapse rate and poorer overall survival (OS) [As expected for founder genetic lesions, pulation ,14,20. Nleukemia . While tutations ,22,23, pectively ,15,17. Iutations . Furtheron alone . HoweverFLT3-ITD ,27,28,29NPM1 mutations may be considered an ideal leukemia-specific target for MRD detection [NPM1-mutated gene copies [NPM1-mutated AML undergoing intensive therapeutic approaches , with levels of residual NPM1 transcripts as the most relevant factor affecting event-free survival (EFS) in multivariate analysis, also in the subset of patients undergoing hematopoietic stem cell transplantation (HSCT). Moreover, MRD thresholds of 0.01% and 0.1% NPM1-mutated/ABL1 obtained during first-line treatment and after HSCT or second-line therapy, respectively, discriminated between prognostic subgroups, but without influencing overall survival (OS) [NPM1-mutated AML [p = 0.001). Furthermore, according to Kronke et al., NPM1-mutated transcript levels were an independent prognostic factor also when analyzed after completion of consolidation therapy  and during follow-up, when serial MRD monitoring allowed early prediction of AML relapse in patients exceeding an arbitrary cut-off value of greater than 2% NPM1-mutated/ABL1 [NPM1-mutated MRD cut-off values, among 155 AML patients treated within intensive treatment protocols [NPM1-mutated/ABL1 [NPM1-mutated AML [NPM1-mutated ratios and a 3-log relative reduction, after induction therapy resulted the most appropriate checkpoint in order to identify patients in CR at high risk of relapse. Indeed, the authors observed 2-year CIR 77.8% and 26.4% for MRD-positive and MRD-negative after induction, respectively. In this series, early molecular response thus seemed to be more prognostically relevant than persistent MRD positivity after consolidation treatments [NPM1 mutation MRD monitoring in 346 AML patients enrolled in NCRI AML17 trial [NPM1-mutated transcripts in PB samples exceeding a value of 0.01% NPM1-mutated/ABL1 after the second chemotherapy cycle was associated with a greater risk of relapse at a 3-year follow-up than was the absence of such mutated transcripts . Notably, MRD positivity after two treatment cycles was observed in 15% of patients and also translated into lower OS rates in these subjects compared with MRD-negative patients . Additionally, the presence of MRD at this testing timepoint was the only independent prognostic factor for death in multivariate analysis and superseded other adverse baseline cytogenetic or molecular factors, such as FLT3-ITD or DNMT3A mutations [NPM1-mutated AML patients, showing that patients who did not achieve a 4-log reduction in NPM1-mutated transcripts in PB samples after induction therapy (45% of study cohort) had a higher 3-year CIR (65.8% versus 20.5%) and a shorter 3-year OS (40.8% versus 91.2%) compared with patients who obtained an adequate MRD reduction [FLT3-ITD, only age, white blood cell (WBC) count and <4-log reduction in PB NPM1-mutated MRD after induction, but not FLT3-ITD allelic ratio, were identified as prognostic factors. Among the 71 patients with non-favorable risk AML according to ELN risk stratification, DFS and OS were significantly improved by allogeneic HSCT only in those with a <4-log reduction in PB MRD, whereas this benefit was not documented in those with an MRD clearance >4-log, thereby suggesting the potential use of early MRD investigation in PB as a predictive factor for HSCT indication [As above mentioned, several clinical trials have so far validated the clinical relevance of molecular MRD monitoring in patients , trying R assays . A totalval (OS) . Anotherated AML . Achieveted/ABL1 . Shayegiated AML . The asseatments . Subsequ17 trial . The majutations . More reeduction . Interesdication .NPM1-mutated AML [NPM1-mutated transcripts [NPM1-mutated RQ-PCR MRD assays and analytical methods, any comparison between different studies remains extremely difficult, and evidence-based conclusions on the best MRD thresholds and timepoints cannot be definitely drawn [NPM1-mutated RQ-PCR sensitivity generally favor BM over PB on the order of 0.6–1.5 log10 [10 increment in sensitivity [NPM1-mutated transcripts, with a 0.01% absolute threshold to define positivity, in PB of patients after the second chemotherapy cycle was highly discriminatory and was the only significant prognostic factor [Collectively, the results of the studies presented above confirmed that RQ-PCR is a reliable molecular tool to assess MRD in most patients with ated AML ,47,54,57ated AML ,57,68 ornscripts ,66,67,69ly drawn ,75. Intely drawn . Absolut.5 log10 ,54,57,75c factor ,44,54,75NPM1-mutated transcript levels at diagnosis did not correlate with the type of NPM1 mutation or with other clinical characteristics, namely age, sex, WBC count, BM blast count, lactate dehydrogenase level, ELN risk stratification or FLT3 gene mutational status [NPM1-mutated baseline levels in cases with WBC count greater than 50 × 109/L or with FLT3-ITD positivity, whereas no significant correlation was documented with age, karyotype or other concurrent gene mutations [NPM1-mutated expression levels detected by RQ-PCR did not influence CR rate after induction chemotherapy [NPM1-mutated AML to retrospectively evaluate the potential significance of NPM1 variant allele frequency (VAF) at diagnosis, comutations and clinical features on patient outcomes [NPM1 VAF (≥0.44) observed on NGS correlated with shortened median OS  as well as median EFS . High NPM1-mutated allele burden at diagnosis was an independent predictor of unfavorable clinical outcomes, particularly in patients who received HSCT in first CR and in the subgroup of subjects with concomitant DNMT3A mutations. Because of BM multilineage involvement of NPM1 mutations not being restricted to cells with blast morphology [NPM1 mutant allele burden may be less amenable to eradication by intensive chemotherapy, potentially resulting in a higher likelihood of MRD persistence. Alternatively, high NPM1-mutated allele burden may indicate the presence of disease in different hematopoietic cell populations, belonging to the NPM1-mutated leukemic clone regardless of blast morphology, which could be intrinsically resistant to chemotherapy and could subsequently foster leukemia relapse, resulting in adverse clinical outcomes [In most series, l status ,12,19,26utations . Howeverotherapy ,12,19,26outcomes . Interesrphology ,20, the outcomes .NPM1-mutated MRD monitoring during follow-up of AML patients in CR after the end of intensive treatments could foresee hematologic relapse [NPM1-mutated transcripts again become detectable or, alternatively, when rising MRD levels are observed in patients with persistent low-level MRD positivity [NPM1 mutations or with rearrangements such as PML-RARA, RUNX1-RUNX1T1 or CBFb-MYH11, and subsequently developed a mathematical model to predict the time elapsing between molecular and hematologic relapse [CBFb-MYH11 displayed a slower clone regrowth than AML with other molecular signatures, including NPM1 mutations [NPM1-mutated transcripts may be detectable by molecular techniques over 2–3 months before overt morphologic relapse occurred [FLT3-ITD positivity or negativity  [NPM1-mutated/ABL1 cut-off value considered for MRD positivity, as shown in the study Shayegi et al. [NPM1-mutated AML patients, potentially allowing pre-emptive therapeutic approaches, preferably in the context of clinical trials [Several studies have investigated whether  relapse ,54,61,65sitivity . Ommen e relapse . Accordiutations ,77. In moccurred ,54,61,65ctively) . Furtherctively) , and mayi et al. . Finallyl trials ,77.NPM1-mutated AML is still controversial. In the retrospective study by Rollig et al., a significant improvement in RFS was documented in the donor group, indicating a beneficial effect of alloHSCT in NPM1-mutated AML patients in first CR, also observed in the favorable subgroup without FLT3-ITD [NPM1-mutated AML patients experiencing relapse often achieved response to salvage treatments, alloHSCT could represent a valuable consolidation option for NPM1-mutated AML patients in first CR if a sibling donor is available, especially in young patients, with a relatively low risk of non-relapse mortality [NPM1-mutated MRD levels  or second (20 cases) CR or with refractory disease (16 cases) [NPM1-mutated MRD positivity (>1%) was a significant predictor of poor outcome after alloHSCT, independently of other variables, such as FLT3-ITD mutational status, BM blast count at diagnosis and age [NPM1-mutated MRD in 51 patients [NPM1-mutated MRD levels, is currently questionable [NPM1-mutated MRD clearance, whereas it was maintained for those with insufficient MRD clearance, suggesting the relevant use of MRD evaluation in transplant selection, to be further validated in clinical studies [NPM1-mutated AML patients resulting MRD-positive prior to alloHSCT [The use of alloHSCT in patients with FLT3-ITD . Even inortality . Unfortuortality . NotablyD levels  [52,55,5patients . The 2-ypatients . Interestionable . Accordi studies ,75. HowealloHSCT .NPM1-mutated MRD monitoring has been shown to be predictive of relapse in several large retrospective series [NPM1 mutation levels, with relevant threshold 0.1%, for samples analyzed between days 61 and 120 and between 121 and 365 after alloHSCT [NPM1-mutated/ABL1 on RQ-PCR assay was strongly associated with an increased risk of relapse, therefore resulting in significantly worse outcomes in terms of DFS and OS [NPM1-mutated AML [NPM1-mutated around day 28 after alloHSCT predicted morphologic relapse in 15 of 18 cases (83%). Peri-transplant MRD evaluation, improving the risk assessment for relapse, may identify high-risk patients who could benefit from pre-emptive treatments after alloHSCT [NPM1-mutated AML patients [p < 0.001) for MRD-positive and MRD-negative patients, respectively [NPM1-mutated AML patients undergoing alloHSCT should be carefully monitored by MRD assays after transplant in order to early detect imminent morphologic relapse [NPM1-mutated MRD positivity after alloHSCT, are not currently standardized [NPM1 mutations), in CR after either conventional chemotherapy only (58 cases) or consecutive alloHSCT (147 cases) [NPM1-mutated AML patients in first or second CR after intensive chemotherapy or autologous or allogeneic HSCT (3 cases), who experienced either molecular relapse or persistently detectable MRD positivity in sequential RQ-PCR analyses [NPM1-mutated MRD [Furthermore, since the first observation in a small patient cohort by Bacher et al. , post-tre series ,44,63,65alloHSCT . Similar relapse ,61. Howedardized ,61,75,79dardized , Platzbe7 cases) . In 53 p7 cases) . Moreoveanalyses . Moleculated MRD . AlthougNPM1 mutations are found in only 20–25% of older AML patients, and in 35–40% of those with normal karyotype [NPM1-mutated AML patients [NPM1-mutated AML in younger adults is generally associated with favorable clinical outcomes, especially in the absence of FLT3-ITD, in older patients the risk of relapse is markedly higher (1-year CIR 71% in patients aged >65 years) and survival significantly lower (2-year OS 19%) [NPM1-mutated MRD monitoring may potentially be useful for patients who obtain morphologic CR in order to define the best personalized post-remissional treatment approach and to early detect relapse [NPM1-mutated MRD also enrolled some patients aged >60 years , IDH2 (92%) and NPM1 (91%) genes, whereas FLT3-ITD (75%) and IDH1 (75%) were less stable. Interestingly, DNMT3A mutations were consistently documented in primary and relapse samples from all the 5 cases with NPM1 mutation loss at relapse, suggesting that DNMT3A mutation most likely preceded NPM1 mutation in the pathogenesis of the disease and a common ancestral DNMT3A-mutated clone with NPM1 wild-type gave rise to both primary and relapsed AML [NPM1 mutations may be hampered in the rare cases where relapse develops from an ancestral NPM1-negative clone, probably arising from pre-existing non-malignant clonal hematopoiesis, therefore resulting in false-negative MRD results [NPM1 mutation subtype from D to A has been recently described in a patient with late NPM1-mutated AML relapse, 8 years after the first diagnosis [NPM1 mutation subtype may be misinterpreted by follow-up strategies only focusing on rising MRD levels alone, which assume recurrence of the NPM1 mutation subtype previously documented at initial diagnosis [As expected for founder genetic lesions,  relapse ,90,91. N relapse ,92,93. Hcurrence , many gry et al.  to 25% dy et al. ,94. Notaic clone ,32,40. I relapse . High stpsed AML . It shou results ,94,95. Fiagnosis . Clinicaiagnosis . These rNPM1-mutated AML patients. As discussed above, at least 55 frameshift insertion NPM1 mutations have so far been recognized, and several other different gene lesions are theoretically possible, rendering relative quantification of rare NPM1 mutations by conventional RQ-PCR highly challenging [NPM1 mutation types [NPM1-mutated AML patient series, ddPCR demonstrated excellent sensitivity and agreement with RQ-PCR, also allowing for the detection of a variety of rare NPM1 mutation subtypes , SRSF2 (64%), TET2 (55%) and ASXL1 (46%), whereas NPM1 mutation was rarely persisting  [NPM1 mutation, there was a non-significant trend towards higher molecular MRD levels by RQ-PCR in patients with compared to those without persisting pre-leukemic lesions. A correlation was observed between persistence of pre-leukemic clones during first remission and inferior OS and higher incidence of relapse, which was abrogated, in this series, by alloHSCT, suggesting that mutation persistence may guide post-remission treatments [DNMT3A, TET2, SRSF2, ASXL1 and TP53, whereas mutations in NPM1 gene, transcription factors or receptor tyrosine kinase genes were often cleared [DNMT3A, TET2 and ASXL1 genes (DTA mutations) was not correlated with an increased relapse rate. Only after exclusion of DTA mutations from the analysis, the documentation of molecular MRD display significantly higher relapse rates and lower survival rates, thereby confirming that persistent mutations in genes associated with age-related CHIP did not have prognostic value in adult patients within a 4-year time frame [NPM1 transcripts generally absent in pre-leukemic clones, could potentially be integrated with evaluation of persisting pre-leukemic molecular lesions at remission. The combination of these two variables may ultimately provide complimentary prognostic information and it could potentially be argued that monitoring both leukemic and pre-leukemic clones after therapy could, in perspective, complement or even supersede pre-treatment genetic factors for AML prognostic stratification [In the seminal analysis of comprehensive genomic data on samples collected from 50 patients at AML presentation and remission after induction chemotherapy, Klco et al. documented persistent leukemia-associated mutations in at least 5% of BM cells at remission in 48% of cases, which were associated with increased relapse risk and reduced OS. Interestingly, contrary to other genes, ed cases . Accordied cases . Furthernts, 2%) . Intereseatments . More reeatments ,109. Speme frame . Overallme frame ,108,109.NPM1-mutated AML patients, and the Consensus Document from ELN MRD Working Party clearly indicates that patients showing NPM1 mutations should have molecular assessment of MRD, we have discussed that several controversies have been raised, and further prospective clinical trials are warranted to standardize the optimal material, timepoints, molecular tools and cut-offs for a meaningful application of MRD monitoring to inform treatment decisions [Even though several published clinical studies  support ecisions ,74,110. ecisions . Interesecisions ,112,113.


Drosophila melanogaster. Initial EGFR activation occurs in the center of leg discs by expression of the EGFR ligand Vn and the EGFR ligand-processing protease Rho, each through single enhancers, vnE and rhoE, that integrate inputs from Wg, Dpp, Dll and Sp1. Deletion of vnE and rhoE eliminates vn and rho expression in the center of the leg imaginal discs, respectively. Animals with deletions of both vnE and rhoE  show distal but not medial leg truncations, suggesting that the distal source of EGFR ligands acts at short-range to only specify distal-most fates, and that multiple additional ‘ring’ enhancers are responsible for medial fates. Further, based on the cis-regulatory logic of vnE and rhoE we identified many additional leg enhancers, suggesting that this logic is broadly used by many genes during Drosophila limb development.We characterized the establishment of an Epidermal Growth Factor Receptor (EGFR) organizing center (EOC) during leg development in  The EGFR signaling pathway plays a major role in innumerable developmental processes in all animals and its deregulation leads to different types of cancer, as well as many other developmental diseases in humans. Here we explored the integration of inputs from the Wnt- and TGF-beta signaling pathways and the leg-specifying transcription factors Distal-less and Sp1 at enhancer elements of EGFR ligands. These enhancers trigger a specific EGFR-dependent developmental output in the fly leg that is limited to specifying distal-most fates. Our findings suggest that activation of the EGFR pathway during fly leg development occurs through the activation of multiple EGFR ligand enhancers that are active at different positions along the proximo-distal axis. Similar enhancer elements are likely to control EGFR activation in humans as well. Such DNA elements might be ‘hot spots’ that cause formation of EGFR-dependent tumors if mutations in them occur. Thus, understanding the molecular characteristics of such DNA elements could facilitate the detection and treatment of cancer. CRMs integrate the information that a single cell or group of cells receives and, in response, trigger changes in cellular and tissue fate specification . S–9. Scis-acRE CRM , 10. DurPD) axis  Fig 1A)cis-regulDll, the EGFR pathway is initially triggered by Wg and Dpp, which activate two types of EGFR ligands in the center of the leg disc . Using this approach, we discovered at least one leg disc enhancer element with a PD bias for each of the genes we tested  multiple fragments generated leg disc expression patterns. Interestingly, we uncovered two leg disc enhancers for the EGFR ligand Spi. Overall, 18 of the 23 tested fragments (78%) are leg enhancers, suggesting that there is a battery of leg disc gene CRMs that drive expression differentially along the leg disc PD axis and are regulated by the direct input of Wg, Dpp, Dll and Sp1.To validate the larger set of predicted CRMs, we picked 20 additional genomic fragments , making it difficult to validate experimentally. Nevertheless, it also seems to predict enhancer loci because, in addition to rhoE, some of the identified regions corresponded to previously identified leg CRMs such as Dll DKO 08482, dacp7d23 , DllSA1, Dllem212 , Egfrtsla, Egfrf24, grkΔFRT, Krn27-7-B, Mad1-2, pntΔ88, rho7M43, ru1, ruinga, Sp1HR Exel8041, vnL6, vnGAL4. Transgenic alleles used for in vivo clonal ectopic expression of genes were: UAS-armΔN, UAS-tkvQD, UAS-Dll.The following mutant alleles and enhancer trap alleles were used in this study: vein and spitz the following strains were used: UAS-vnRNAi(TRiP.HMC04390)/CyO, Dfd:EYFP; UAS-spiRNAi(TRiP.HMS01120) crossed to either Dll-GAL4 (Dllem212), spiDf(2L)Exel8041/ CyO, Dfd-EYFP; vnL6/TM6B, or spiDf(2L)Exel8041/CyO, Dfd-EYFP; vnGAL4/TM6B . CrosseDll-expressing leg disc region a yw; Dll-Gal4 (Dllem212), UAS-Flp; Ubi-GFP M- y+ FRT80B/CTb strain was crossed to a corresponding FRT80B-containg mutant strain (ruingarhorhoE-DfvnL6 or ru1rho7M43vnvnE-Df). For Flp-FRT inducible mitotic recombination and subsequent mosaic clonal analysis fly larvae were heat-shocked at 48h post egg laying (PEL), 72h PEL or 90h PEL and dissected for staining as crawling stage larvae at around 120h PEL. For generation of Flp-FRT mitotic recombination clones, larvae were heat-shocked for 40 minutes at 37°C. Mitotic recombination clones were generated using the following strains: w hs-Flp1.22 Ubi-RFP FRT19A, yw hs-Flp1.22; Ubi-GFP FRT40A /CyO; E/TM6B, yw hs-Flp1.22; FRT42D Ubi-GFP/CyO; E/TM6B, yw hs-Flp1.22; E/CyO; Ubi-GFP FRT80B /TM6B, yw hs-Flp1.22; E/CyO; FRT82B Ubi-GFP/TM6B, yw hs-Flp1.22; FRT42D M- hs-GFP/CyO; E/TM6B, yw hs-Flp1.22; E/CyO; Ubi-GFP M- FRT80B/TM6B. The corresponding strains carrying mutant alleles were used in crosses for generation of mutant clones in the resulting progeny. E in these genotypes designates either vnE-lacZ or rhoE-lacZ inserted in landing sites 51D or 86Fa on chromosome II and III, respectively. To induce GFP expression in larvae marked with hs-GFP, an additional heat-shock was given 1 h before dissection for 20 min to 1 hour at 37°C.For generation of mutant clones that encompass the entire E designates either vnE-lacZ or rhoE-lacZ inserted in site 86Fa: yw hs-Flp1.22tub-Gal4 UAS-GFP; tub-Gal80 FRT40A/CyO; E/TM2, yw hs-Flp1.22tub-Gal4 UAS-GFP; FRT42D tub-Gal80/CyO; E/TM2, yw; Mad1-2FRT40A; UAS-Dll/CTb, yw; FRT42D arr2; UAS-Dll/CTb, yw; FRT42D DllSA1; UAS-armΔN/CTb, yw; FRT42D DllSA1; UAS-tkvQD/ CTb, yw; y+ FRT40A; UAS-Dll/CTb, yw; FRT42D y+; UAS-Dll/CTb, yw; FRT42D y+; UAS-armΔN/CTb, yw; FRT42D y+; UAS-tkvQD/ CTb.The following strains were used for MARCM experiments where in vivo clonal experiments, at least 20 examples of discs with clones of the correct genotype were examined, which is typical for experiments of this type, and more than one independent experiment was carried out for each tested genotype.For all enhancer-reporter transgenic constructs were made using the lacZ reporter vector pRVV54 as an acceptor vector [All wildtype and mutagenized r vector . Coordinr vector .vnE and rhoE enhancers was performed according to the QuikChange II protocol (Agilent Technologies). vnE and rhoE enhancers were first introduced in pBluescript SK+ vector for site-directed mutagenesis and the resulting mutated enhancers were consequently transferred to pRVV54 for in vivo analysis in the fruit fly. Primers used for mutagenizing of putative binding site are listed in Site-directed mutagenesis of the Zn-finger-His , Sp1424AA-His (used to examine cooperativity with Dll), MadMH1-His (only the MH1 domain) and PanHMG-His (only the HMG domain) vectors are listed in Plasmids for recombinant protein production were made by introducing cDNA sequences into pET21 series vectors (Novagen-EMD Millipore) and their derivatives, resulting in C-terminally tagged His proteins. Primers used to generate Dll-His (full-length Dll), Sp1vnE and rhoE CRISPR/Cas9 alleles were generated by using pCFD4 vector for driving gRNA expression [vnEDf allele: CGATTTTAATGCGAAAGCTA and TTTGGCTTTCAACGCTTAAT. The following sequences were used as gRNAs for generation of the rhoEDf allele: GAGCCGAGGGCACAAATTGA and ATGATGATGATGTATTGCCC. We created a vector containing a cassette with P3-RFP [FRT(F5)-hs-neo-FRT(F5) selectable markers flanked by minimal inverted ΦC31 [vnE and rhoE pCFD4-based gRNA vectors (250ng/μl) were co-injected with the corresponding vnE and rhoE homologous arm donor cassette vectors (500ng/μl) and resulting flies were screened for P3-RFP expression. To generate rhoE deletion allele in the background of ruinga, injections to generate the rhoEDf were repeated in a nos-Cas9/CyO; ruinga/TM3 strain. Positive fly lines were verified by PCR for correct insertion of the donor cassettes. Deletion alleles without P3-RFP were generated through RMCE by injection with an empty multiple cloning site vector containing inverted ΦC31 attB sites (pRVV578) [P3-RFP-containing and -non-containing enhancer deletion alleles exhibited identical expression patterns and phenotypes. The WT vnE, rhoE and the D. virilis vnE enhancers were cloned into pRVV578 and resupplied by RMCE in a similar manner (primers are listed in The pression  and a gepression . The folh P3-RFP  and FRT(ted ΦC31  attP sitted ΦC31 . This vepRVV578) . The P3-Recombinant proteins were expressed in BL21 (DE3) cells (Agilent Technologies) through IPTG induction for 4h. Proteins were subsequently purified through Cobalt chromatography with TALON Metal Affinity Resin . EMSA gels were performed as previously described .Immunostainings of fly imaginal discs was performed by standard protocol. The following antibodies were used in this study: rabbit anti-β-galactosidase (Cappel), mouse anti-β-galactosidase , guinea pig anti-Dll , rat antAdult legs were dissected, mounted, and analyzed by light microscopy. All adults of the relevant genotype that eclosed within an 8-hour period were scored. Roman numerals in the figure legends indicate the tarsal segments present in each phenotypic class . For example, a truncation designated as I-III means that tarsal segments I, II and III were present, with segment III partially defective e.g. . n referin situ probes vn, ru, spi, Krn, and grk DNA sequences were amplified from genomic DNA and rho DNA sequence was amplified from cDNA clone  using primers listed in To generate vectors for rho probes were then hydrolized for 30 minutes at 60°C as previously described [In situ hybridization was then performed as previously described [RNA antisense probes were transcribed with either T3 or T7 RNA polymerase  and reported as the ratio of β-gal:Dll (staining control) in arbitrary units (AU). Ordinary one-way ANOVA adjusted for multiple comparisons (Dunnett's test) were performed and graphed in Prism software (graphpad.com) to compare wild-type fluorescence to mutant enhancer genotypes where ns = not significant, * = p≤ 0.0332, ** = p≤ 0.0021, *** = p≤ 0.0002 and **** = p<0.0001 . n refers to the number of individual leg discs scored. The number of leg discs scored for each genotype is reported in the figure legends.Mid-third instar larvae carrying wild-type or mutant yw and 100 Sp1-GFPBAC L3 wandering larvae were used to perform independent chromatin IPs as previously described [Sp1-GFPBAC is a GFP-tagged Sp1 in BAC clone CH321-64M02 inserted in landing site VK00033 (gift from Dr. Rebecca Spokony). All 6 leg discs from each larva were used as material for each IP. Chromatin from the yw larvae pools was immuno-precipitated with goat anti-Dll antibody  while chromatin from the Sp1-GFPBAC larvae pools was immuno-precipitated with rabbit anti-GFP antibody . DNA from non-immunoprecipitated 10% chromatin input was isolated from each pool as reference control. Both control and immunoprecipitated DNA samples were prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Preparation Kit and sequenced on an Illumina HiSeq 2000 according to the manufacturer's specifications. Experiments were performed in duplicate and peak calling was based on merged reads for duplicate ChIPs. Sequences were aligned to the Drosophila genome using the Burrows-Wheeler Aligner and ChIP-seq peaks were called using MACSv2 [Triplicate pools of 100 escribed . The Sp1g MACSv2 , 61. Peagrep within the MotifDb Bioconductor/R package. To generate BED files containing position information for each of the above PWMs, the matchPWM command from the Biostrings Bioconductor/R package was used. In-house code was used to run the command iteratively through the chromosomes (using DM3 build). Only hits above a minimum score of 80% were retained. IGVtools within the Integrative Genomics Viewer (IGV) was used to sort and index the BED files prior to intersection. Intersections of all BED files  were done using Bedtools2 run locally from the command line. ChIP-seq peaks for Dll and Sp1 were first intersected with the FAIRE peaks. The product of this intersection was then sequentially intersected with each of the PWM files, always returning the peak coordinates from the initial file. The command intersectBed was used with options: -wa, -F 1.0, -u. To determine the gene nearest to each of the intersected ChIP peaks, packages within R/Bioconductor were used. The annotation package TxDb.Dmelanogaster.UCSC.dm3.ensGene was downloaded and annotated transcripts extracted. The distanceToNearest function was used to find the nearest annotated transcript to each of the ChIP Peaks. In-house R script was then used to generate the table containing the coordinates of the ChIP peaks, as well as the nearest annotated gene  and rhoE (B) in third instar eye-antennae discs. (C-D) Expression pattern of (C) rhoLLE1 and (D) rhoLLE2 throughout leg disc development. (E) Schematic representation of ru genomic locus with enhancer bashing results. Fragments represented in tan did not drive expression in leg discs. (F-H) Expression pattern of ru from in situ (F), ruinga (G) and ruLLE (H). (I) Schematic representation of spi genomic locus with enhancer bashing results. (J-K) Expression pattern of spi from in situ (J) and spi-lacZ reporter construct (K). (L) Schematic representation of Krn genomic locus with enhancer bashing results and Krn27-7-B mutant. Fragments represented in tan did not drive expression in leg discs. (M) Expression pattern of Krn from in situ. (N) Schematic representation of grk genomic locus with grkΔFRT mutant. (O) Expression pattern of grk from in situ.(A-B) Expression pattern of (TIF)Click here for additional data file.S2 FigvnvnE-Df mutant. (B) Adult leg of vnvnE-Df mutant. Filled arrowhead indicates intact pretarsal claw. (C) Expression pattern of C15 and Al in rhorhoE-Df mutant. (D) Adult leg of rhorhoE-Df mutant. Filled arrowhead indicates intact pretarsal claw. (E) Expression pattern of Al and BarH1 in WT (rhorhoE-DfvnvnE-Df/+) and (F) rhorhoE-DfvnvnE-Df double mutant. (G) Expression pattern of C15/Bar/Dac in WT (rhorhoE-DfvnvnE-DfKrn27-7-B/+) and (H) rhorhoE-DfvnvnE-DfKrn27-7-B triple mutants. (I-J) spi vn double RNAi driven by vn-GAL4. Expression pattern of C15 and BarH1 in third instar leg discs (I) and adult leg (J). Open arrowhead indicates absent pretarsal claw). (K-L) Expression pattern of ruinga-lacZ in ruingarhorhoE-Df/+ (K) and ruingarhorhoE-Df/ rhorhoE-Df (L) leg imaginal discs.(A) Expression pattern of C15 and Al in (TIF)Click here for additional data file.S3 FigvnE- or (B) rhoE-driven lacZ expression in DllSA1 mutant clones generated at 90h PEL. (C-E) vnE-driven lacZ expression in wing discs in Dll ectopic overexpression clones in WT background (C); Dll ectopic overexpression clones in arr2 mutant background (D); Dll overexpression clones in Mad1-2 mutant background (E); (C-E) clones were generated at 48h PEL. (F-G) vnE-driven lacZ expression in leg discs of Sp1HR mutant animals (F); Sp1HR mutant clones generated at 72h PEL (G). (H) rhoE-driven lacZ expression in leg discs of Sp1HR mutant animals. (I-J) vnE- (I) or rhoE- (J) driven lacZ expression in btdXA mutant clones generated at 48h PEL. (K) cic-lacZ expression in leg discs. (L-M) vnE- (L) or rhoE- (M) driven lacZ expression in leg discs with cicQ474X mutant clones generated at 48h PEL.(A) (TIF)Click here for additional data file.S4 FigvnE- (A) and rhoE-  driven expression of WT and intermediately mutant CRMs. (B and D) schematic representation of binding sites in vnE and rhoE, respectively. Mutated sites for the CRM-reporter genes shown in A, C, and E are indicated by the *, † and ‡. (F) Quantification of expression levels; fluorescence was calculated as a ratio of β-gal:Dll intensity in the center of the discs  (TIF)Click here for additional data file.S5 FigDll_LE2 (A); spi_LE2 (B); noc_LE2 (C); Antp_LE1 (D); Antp_LE2 (E); Ote/fj_LE (F).Schematic representation of identified genomic loci and the expression patterns they drive in reporter genes from (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Increasing evidence suggests that dysregulation of long non-coding RNAs (lncRNAs) is implicated in chemoresistance in cancers. However, the function and molecular mechanisms of lncRNAs in gastric cancer chemoresistance are still not well understood. In this study, we aimed to investigate the functional role and the underlying molecular mechanisms of lncRNA HOXD cluster antisense RNA 1 (HOXD-AS1) in cisplatin (DDP) resistance in gastric cancer. Our results revealed that HOXD-AS1 was upregulated in DDP-resistant gastric cancer tissues and cells. Patients with gastric cancer with high HOXD-AS1 expression levels had a poor prognosis. Knockdown of HOXD-AS1 facilitated the sensitivity of DDP-resistant gastric cancer cells to DDP. Additionally, HOXD-AS1 epigenetically silenced PDCD4 through binding to the histone methyltransferase enhancer of zeste homologue 2 (EZH2) on the promoter of PDCD4, thus increasing H3K27me3. More importantly, PDCD4 silencing counteracted HOXD-AS1 knockdown-mediated enhancement of DDP sensitivity in DDP-resistant gastric cancer cells. In summary, HOXD-AS1 led to DDP resistance in gastric cancer by epigenetically suppressing PDCD4 expression, providing a novel therapeutic strategy for patients with gastric cancer with chemoresistance. DespiteLong non-coding RNAs (lncRNAs) are a class of endogenous non-coding RNAs greater than 200 bp in length. LncRNAs have been reported to exert oncogenic or tumour suppressor roles and have been elucidated to regulate the biological processes and cell functions in many malignancies –8. AdditIn the present study, we aimed to explore the functional role and molecular mechanism of HOXD-AS1 in gastric cancer DDP resistance. We found that HOXD-AS1 levels were extremely elevated in DDP-resistant gastric cancer tissues and cells. In addition, HOXD-AS1 silencing enhanced the sensitivity of DDP-resistant gastric cancer cells to DDP. In a mechanistic manner, HOXD-AS1 rendered DDP resistance of gastric cancer cells through epigenetically silencing PDCD4 via recruiting enhancer of zeste homologue 2 (EZH2). Our study revealed a novel HOXD-AS1/PDCD4 regulatory axis conferring DDP resistance in gastric cancer.2.2.1.n = 42) and adjacent normal (n = 42) tissues were collected from patients with gastric cancer undergoing surgery at the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China. All patients were treated with DDP and divided into two groups: DDP-sensitive  DDP-resistant (stable or deteriorating) patients. Additionally, the patients were further divided into high and low HOXD-AS1 expression groups based on the median of HOXD-AS1 levels. Our research was approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University. All patients gave signed informed consent. To identify functional lncRNAs in gastric cancer, we downloaded three sets of gene expression profiling data for gastric cancer  from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). Moreover, the normalized RNA-seq data from the Stomach Adenocarcinoma (STAD) dataset were downloaded from The Cancer Genome Atlas (TCGA) data portal website (https://cancergenome.nih.gov/). All of the data from the databases were analysed using R language.Tumour  and human gastric cancer cell lines (BGC823 and SGC7901) were purchased from the American Type Culture Collection . The DDP-resistant strains (BGC823/DDP and SGC7901/DDP) were derived from BGC823 and SGC7901 by cisplatin (DDP) exposure. All cells were cultured in RPMI-1640 medium  containing 10% fetal bovine serum at 37°C in a humidified incubator under 5% CO2.2.The empty vector pcDNA3.1 (vector) or HOXD-AS1 overexpressing vector pcDNA3.1-HOXD-AS1 (HOXD-AS1) and small interfering RNAs against HOXD-AS1 (si-HOXD-AS1), EZH2 (si-EZH2) or PDCD4 (si-PDCD4) or their scramble negative siRNA (si-con) were obtained from Genepharma . Cell transfections were carried out using Lipofectamine 2000 .2.3.−ΔΔCt method was used to analyse the data.Trizol reagent  was used for total RNA extraction. Then, cDNA samples were obtained by reverse transcription using a PrimeScript RT Reagent Kit (TaKaRa). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using an SYBR green qRT-PCR assay. GAPDH was used as an internal control for HOXD-AS1 and PDCD4. The primers were as follows: HOXD-AS1 forward: 5′-GGC TCT TCC CTA ATG TGT GG-3′, reverse: 5′-CTC TGG TTG GGT GAC TGG TT-3′; PDCD4 forward: 5′-GGC CTC CAA GGA GTA AGA CC-3′, reverse: 5′-AGG GGT CTA CAT GGC AAC TG-3′. The 22.4.The sensitivity of gastric cancer cells to DDP was evaluated by the MTT  assay. The IC50 value  of BGC823/DDP and SGC7901/DDP cells to DDP was estimated using relative survival curves.2.5.Cell apoptosis was determined using an Annexin V–FITC/PI Apoptosis Detection Kit . In brief, BGC823/DDP and SGC7901/DDP cells were harvested after treatment and then suspended in annexin-binding buffer. Afterwards, annexin V/FITC and PI solution were used to stain cells for flow cytometry analysis. FACSan flow cytometry  was used to evaluate cell apoptosis.2.6.The separation of nuclear and cytoplasmic RNA from BGC823/DDP cells was performed using a Cytoplasmic and Nuclear RNA Purification Kit  in accordance with the manufacturer's instructions.2.7.Biotinylated HOXD-AS1 and antisense-HOXD-AS1 RNAs were mixed with BGC823/DDP cell lysates, followed by incubation with Streptavidin Magnetic Beads . Then, the bound protein was detected by western blot.2.8.RNA immunoprecipitation (RIP) was carried out using the Magna RIP RNA-binding protein immunoprecipitation kit  and antibodies against EZH2 and immunoglobulin G (IgG)  following the manufacturer's instructions. The RNA levels in the immunoprecipitates were determined by qRT-PCR analysis.2.9.Chromatin immunoprecipitation (ChIP) assays were conducted using an EZ-ChIP kit (Millipore) with anti-EZH2 , anti-H3K27me3 (Millipore) or anti-IgG (Millipore) antibodies. The precipitated DNA was analysed by qRT-PCR.2.10.The PDCD4 promoter reporter vector was obtained from Genechem . Then, BGC823/DDP cells were co-transfected with (HOXD-AS1 or vector) or (si-HOXD-AS1 or si-con) and the reporter promoter. Finally, luciferase activity in BGC823/DDP cells was detected using the Luciferase Reporter assay system .2.11.Western blotting was performed as described previously . The ant2.12.−1 DDP or the same volume of phosphate-buffered saline (PBS) every 7 days. Tumour size was monitored every 7 days, and tumour volumes were calculated based on the following formula: volume = 0.5 × length × width2. On the 35th day post treatment, the mice were killed, and xenograft tumours were detected. HOXD-AS1 expression and PDCD4 protein levels were measured by qRT-PCR and western blot assays.The animal study was approved by the research ethics committee of the First Affiliated Hospital of Zhengzhou University. Lentivirus carrying shRNA to HOXD-AS1 (sh-HOXD-AS1) was commercially designed and synthesized by Ribobio . BGC823/DDP cells stably infected with sh-HOXD-AS1 or sh-con were transplanted into BALB/c-nude mice (six weeks old) from Slac Laboratory , and then the mice were injected intraperitoneally with 6 mg kg2.13.t-test and one-way ANOVA. p < 0.05 was considered significant.All data are presented as the means ± s.d. from at least three independent experiments. The statistical differences between groups were evaluated by Student's 3.3.1.a–c). To confirm the result from GEO DataSets, the expression of HOXD-AS1 was further analysed in gastric cancer tissues from the TCGA dataset. The results indicated that HOXD-AS1 expression was significantly elevated in gastric cancer tumour tissues compared with normal tissues from the STAD dataset (d). To verify the results from the databases, qRT-PCR analysis was conducted to measure HOXD-AS1 expression in 42 pairs of tumour and adjacent normal tissues. HOXD-AS1 was dramatically elevated in gastric cancer tumour tissues (e). Moreover, HOXD-AS1 expression in DDP-resistant gastric cancer tissues was much higher than that in DDP-sensitive gastric cancer tissues (f). Furthermore, HOXD-AS1 expression was notably upregulated in gastric cancer cell lines (BGC823 and SGC7901) compared with GES-1 cells . Prominently, DDP-resistant gastric cancer cells (BGC823/DDP and SGC7901/DDP) exhibited a higher HOXD-AS1 expression level than their parental cells . Patients with high HOXD-AS1 expression levels had a poor survival (p = 0.0083) (i). The relationship between HOXD-AS1 expression and clinical characteristics is shown in p = 0.005) and tumour–node–metastasis (TNM) staging (p = 0.021) in patients with gastric cancer. Collectively, HOXD-AS1 upregulation may be implicated in gastric cancer DDP resistance.To analyse the different lncRNA expressions in gastric cancer tumour and normal tissues, we downloaded gastric cancer gene expression data from GEO DataSets . Among the differentially expressed lncRNAs, HOXD-AS1 was highly expressed in the gastric cancer tumour tissues compared with normal tissues in all three above-mentioned datasets a–c. To c dataset d. To ver tissues e. Moreov tissues f. Furthe-1 cells g,h. Promal cells g,h. Pati 0.0083) i. The re3.2.a,b). To further explore the role of HOXD-AS1 in DDP-resistant gastric cancer cells, HOXD-AS1 siRNAs or si-con was transfected into BGC823/DDP and SGC7901/DDP cells. The qRT-PCR analysis revealed that HOXD-AS1 was remarkedly reduced in BGC823/DDP and SGC7901/DDP cells with si-HOXD-AS1 or si-HOXD-AS1 no. 1 transfection . In addition, HOXD-AS1 knockdown enhanced the DDP sensitivity of BGC823/DDP and SGC7901/DDP cells . To further explore the effect of HOXD-AS1 on DDP-induced apoptosis, flow cytometry analysis was carried out in BGC823/DDP and SGC7901/DDP cells exposed to 20 µM DDP. As expected, HOXD-AS1 silencing distinctly enhanced DDP-induced apoptosis in BGC823/DDP and SGC7901/DDP cells . Together, the downregulation of HOXD-AS1 could overcome DDP resistance of DDP-resistant gastric cancer cells.To evaluate the DDP resistance of gastric cancer cells, the IC50 of DDP was determined using MTT assay of gastric cancer cells. The drug sensitivity assay indicated that the IC50 of DDP in DDP-resistant gastric cancer cells (BGC823/DDP and SGC7901/DDP) was markedly increased compared with their parental cells a,b. To f3.3.a). HOXD-AS1 knockdown pointedly elevated PDCD4 expression in BGC823/DDP cells (figure 3b). Moreover, the introduction of si-EZH2 lowered EZH2 and increased the protein level of PDCD4 in BGC823/DDP cells (figure 3c). To further explore whether HOXD-AS1 could bind with EZH2 in BGC823/DDP cells, we conducted RNA pull-down and RIP assays in BGC823/DDP cells. The results showed that HOXD-AS1 could pull down EZH2 protein (figure 3d) and HOXD-AS1 was markedly enriched by EZH2 antibody (figure 3e). All these results revealed that HOXD-AS1 could bind with EZH2 in gastric cancer cells. To further investigate whether HOXD-AS1 transcriptionally suppressed PDCD4 expression via the enrichment of EZH2 to the PDCD4 promoter, ChIP assays were conducted in BGC823/DDP cells. The results showed that HOXD-AS1 silencing remarkably reduced EZH2 binding and H3K27me3 occupancy of the PDCD4 promoter (figure 3f). Also, the luciferase reporter assay revealed that HOXD-AS1 inhibition improved the activity of the PDCD4 promoter; in contrast, upregulation of HOXD-AS1 suppressed the promoter activity (figure 3g). All these data demonstrated that HOXD-AS1 epigenetically silenced PDCD4 through recruiting EZH2 and increasing the H3K27me3 level on the promoter region of PDCD4 in gastric cancer cells.HOXD-AS1 has been reported to silence gene expression by recruiting EZH2, a vital catalytic subunit of polycomb repressive complex 2 (PRC2), to the specific genome sites of target genes ,17. Morefigure 33.4.a,b), which could be abolished by PDCD4 knockdown. Moreover, HOXD-AS1 silencing could overcome DDP resistance in BGC823/DDP and SGC7901/DDP cells . Furthermore, PDCD4 inhibition reversed the improved sensitivity of BGC823/DDP and SGC7901/DDP cells to DDP triggered by HOXD-AS1 knockdown . To further determine the effect of HOXD-AS1 and PDCD4 on apoptosis, flow cytometry analysis was conducted in BGC823/DDP and SGC7901/DDP cells with 20 µM DDP exposure. As expected, the introduction of si-HOXD-AS1 markedly increased apoptosis in BGC823/DDP and SGC7901/DDP cells . However, si-HOXD-AS1-mediated apoptosis enhancement was eliminated by PDCD4 knockdown . Together, HOXD-AS1 silencing overcame DDP resistance in DDP-resistant gastric cancer cells through PDCD4 inhibition.To further investigate whether HOXD-AS1 enhanced the response of gastric cancer cells to DDP via regulating PDCD4 expression, si-con, si-HOXD-AS1 or si-HOXD-AS1 + si-PDCD4 were transfected into BGC823/DDP and SGC7901/DDP cells. Introduction of si-HOXD-AS1 elevated PDCD4 expression in BGC823/DDP and SGC7901/DDP cells a,b, whicDP cells c,d. Furtnockdown c,d. To fDP cells e,f. Howenockdown e,f. Toge3.5.in vivo, a mouse xenograft model was established. sh-con or sh-HOXD-AS1 stably infected BGC823/DDP cells were subcutaneously injected into nude mice and treated with DDP or PBS. The data indicated that HOXD-AS1 silencing or DDP exposure blocked tumour growth, as demonstrated by the reduced tumour volume (a) and tumour weight (b). HOXD-AS1 silencing combined with DDP exposure contributed to a more evident decrease in tumour growth . Moreover, qRT-PCR and western blot assays revealed that HOXD-AS1 expression was lowered, while PDCD4 expression was elevated in tumours after the introduction of sh-HOXD-AS1 or DDP treatment , especially after the combination of sh-HOXD-AS1 and DDP exposure. All these data demonstrated that HOXD-AS1 silencing enhanced DDP sensitivity of gastric cancer cells in vivo. Moreover, the schematic diagram of the mechanism of HOXD-AS1 epigenetically regulating PDCD4 is shown in To validate the functional role of HOXD-AS1 in DDP resistance r volume a and tumr weight b. HOXD-Ar growth a,b. Morereatment c,d, espe4.Chemoresistance has severely restricted therapeutic outcome for patients with gastric cancer. Therefore, identifying the underlying mechanism and discovering new therapeutic strategies for chemoresistance is very important. In the present study, we revealed that HOXD-AS1 expression was remarkably upregulated in DDP-resistant gastric cancer tissues and cells. Additionally, HOXD-AS1 knockdown could improve DDP sensitivity of gastric cancer cells. More importantly, HOXD-AS1 silencing facilitated the DDP sensitivity of gastric cancer cells through epigenetically silencing PDCD4 via recruiting EZH2. Collectively, HOXD-AS1 is a positive regulator in gastric cancer DDP resistance and targeting HOXD-AS1 may be an effective treatment for gastric cancer chemoresistance.Elucidating the underlying mechanism of chemoresistance could contribute to developing reasonable and effective therapeutic targets to overcome chemoresistance. Our results revealed that HOXD-AS1 was increased in DDP-resistant gastric cancer tissues and cells, and HOXD-AS1 silencing facilitated the DDP sensitivity of DDP-resistant BGC823/DDP and SGC7901/DDP cells. All these findings confirmed that HOXD-AS1 could confer DDP resistance in gastric cancer cells. In addition, abnormal HOXD-AS1 expression was reported to be implicated with chemoresistance in various cancers. For instance, HOXD-AS1 expression was elevated in glioma, and HOXD-AS1 knockdown could suppress proliferation, migration and invasion and sensitize glioma cells U87 and U251 to DDP by acting as an miR-204 sponge . MoreoveHow elevated HOXD-AS1 led to DDP resistance in gastric cancer has not been determined. Hence, the functional mechanism of HOXD-AS1 was further explored in this work. An increasing number of studies have reported that about 20% of lncRNAs could silence genes by binding to EZH2, a subunit of PRC2 . AdditioIn conclusion, our study demonstrated that HOXD-AS1 knockdown improved DDP sensitivity of gastric cancer cells by epigenetically silencing PDCD4, providing a promising therapeutic target to overcome DDP resistance in gastric cancer.


Despite the recent progress in unraveling the role of STIM2 in Ca2+ signaling, the mechanistic underpinnings of its activation remain underexplored. We use an engineering approach to direct ER-resident STIMs to the plasma membrane (PM) while maintaining their correct membrane topology, as well as Förster resonance energy transfer (FRET) sensors that enabled in cellulo real-time monitoring of STIM activities. This allowed us to determine the calcium affinities of STIM1 and STIM2 both in cellulo and in situ, explaining the current discrepancies in the literature. We also identified the key structural determinants, especially the corresponding G residue in STIM1, which define the distinct activation dynamics of STIM2. The chimeric E470G mutation could switch STIM2 from a slow and weak Orai channel activator into a fast and potent one like STIM1 and vice versa. The systemic dissection of STIM2 activation by protein engineering sets the stage for the elucidation of the regulation and function of STIM2-mediated signaling in mammals.The endoplasmic reticulum (ER) Ca Calcium ions play a major regulatory role in the physiology and biochemistry of the cell, and thus their levels and activities should be tightly regulated. The stromal interaction molecules (STIMs) are sensors of the calcium levels within the endoplasmic reticulum (ER)—which serves as a major intracellular calcium store—to mediate communication between the ER and the plasma membrane and are regarded as ubiquitous central players of calcium signaling in mammalian cells. STIM2 acts as a slow and weak activator of Orai1 calcium channels on the plasma membrane by direct binding; however, the affinity of STIMs for calcium or how Orai1 channels are activated remain unclear. In this study, we systematically analyzed the molecular determinants that govern the activation of STIM proteins. Adopting protein engineering approaches that enable the relocation of ER-resident STIM proteins at the plasma membrane, we determined the calcium affinities of STIMs under physiological conditions in mammalian cells. We identified a critical position within STIMs, which defines their distinct resting states and activation kinetics, as well as the efficacy to activate Orai1 channels. These findings shed new light on how STIM2 can efficiently respond to small changes within the ER lumen to regulate calcium homeostasis and signaling in mammalian cells. Usitivation , the in round 4) , 25, 30. readout , resultsof STIM1 .2+ with a stoichiometry of 1 in vitro f2+ levels were calculated using the following equation EThe obtained in situ values of CEPIA1er indicators were used in further calculations .All experiments were carried out at room temperature. Traces shown are representative of at least three independent repeats, with 15–60 single cells analyzed per each repeat.2+ affinities of full-length STIM1 using STIM1 puncta as readouts, confocal microscopy (described below) was used to collect corresponding YFP and R-CEPIA1er signals. ER Ca2+ stores were gradually depleted with 1 μM thapsigargin (TG); the resulting decrease in FRET signal or increases in punctate area were then plotted against the corresponding ER Ca2+ levels indicated by R-CEPIA1er fluorescence. Similar to previous reports [2+ affinities of the STIM constructs were then calculated by fitting the obtained puncta/FRET-Ca2+ relationship to the Hill equation using Prism 7 software.The FRET signals between the YFP-SOAR1L-SC1111/1121 pairs and R-CEPIA1er signals were acquired simultaneously from HEK293 cells or HeLa SK cells transiently coexpressing R-CEPIA1er, YFP-SOARL, and SC1111-CFP or SC1211-CFP. The same filters as described in the above sections were used. For measurements of Ca reports , 30, eitSubcellular distribution of fluorescently tagged STIM and Orai1 constructs was monitored using a Zeiss LSM 880 confocal system equipped with a 100× oil lens . The acquired raw images were analyzed using Image J software (NIH). Studies of the PM targeting of STIM- and STIM-CC1–binding SOAR were conducted using the Nikon Eclipse Ti-E microscope  equipped with an A1R-A1 confocal module with LU-N4 laser sources and CFI Plan Apochromat VC series Objective Lenses (60× or 40×). All acquired confocal images were analyzed by using the NIS-Elements AR microscope imaging software .2 for 1 h. The cells were then washed twice with fresh culture medium. Secondary goat anti-mouse IgG Alexa Fluor 568 antibody  was then incubated with the cells in the growth medium for 1 h. After extensive washing, images were acquired using a Nikon A1R confocal microscope at 40× magnification.To confirm the orientation of either end of the engineered PM-STIM protein, live-cell immunofluorescence staining without cell permeabilization was performed. HeLa cells were grown on 35 mm glass-bottomed dishes  and transfected with PM-STIM constructs using Lipofectamine 3000 . Next, 1CRAC in HEK293 Orai1-CFP stable cells transiently expressing STIM1-ΔK-YFP or corresponding 1121 or G379E variants was measured with conventional whole-cell recordings [2, 10 mM EGTA, and 10 mM Cs-HEPES (pH 7.2). The extracellular solution contained 130 mM NaCl, 4.5 mM KCl, 20 mM CaCl2, 10 mM TEA-Cl, 10 mM d-glucose, and 5 mM Na-HEPES (pH 7.4). A 10 mV junction potential compensation was applied to correct the liquid junction potential of the pipette solution relative to the extracellular solution. Currents from at least six cells for each condition were collected and averaged.Data were collected using an HEKA EPC 10 USB double patch amplifier controlled by Patchmaster software . The Icordings . After t- ions were introduced to neutralize the system. The total number of atoms was approximately 84,000. The CHARMM 36 force field [To determine the dynamics of SOAR1, its initial dimeric structure was taken from the X-ray structure (PDB: 3TEQ). The other structures  were built by homology modeling with SOAR1 as the template; the modeling was conducted using the Swiss-model server , 64. Force field  was usedce field .Molecular dynamics (MD) simulations were performed using the NAMD package suite . The simS1 Fig1-CC1 construct; SOAR, STIM-Orai–activating region; SOAR1L, STIM1(343–491); STIM, stromal interaction molecule; sα1–4: α helix 1 to 4 of SOAR region; TM, transmembrane region; Y, YFP tag; YFP, yellow fluorescent protein.(A–B) Full-length STIM chimeras or constructs with C-terminal deletions. (C–D) SCs and chimeras; (E–F) PM-anchoring SCs and chimeras; (G–H) SOAR constructs and chimeras. C, CFP tag; CC1, coiled-coil 1; CFP, cyan fluorescent protein; EF-SAM, EF-hand and sterile alpha motif domain; K, lysine-rich region; NT, N terminus; PM, plasma membrane; SC, STIM(TIF)Click here for additional data file.S2 Fig1-21. To facilitate the ER export of STIM1 and trafficking in the cytosol, PM-trafficking TP (Kir2.1233−252) and ER-exporting TP (Kir2.1374−380) were inserted upstream and downstream of the C-terminal CFP, YFP, or mCh fluorescent tag, respectively. (B) Live-cell immunofluorescence staining of HeLa cells expressing the designed YFP-tagged PM-targeting constructs. Alexa-Fluor-568–conjugated secondary antibody was used to determine the extracellular localization of the Myc tag in nonpermeabilized HeLa cells. (C) Confocal imaging of HeLa cells coexpressing PM-S2222-YFP and mCh-CAD cultured in the 2 mM Ca2+ medium or Ca2+-free medium. Scale bar, 10 μm. CAD, CRAC-activating domain; CFP, cyan fluorescent protein; CRAC, Ca2+-release-activated Ca2+ current; EF-SAM, EF-hand and sterile alpha motif domain; ER, endoplasmic reticulum; mCh, mCherry PM, plasma membrane; SP, signal peptide; STIM, stromal interaction molecule; TP, target peptide; YFP, yellow fluorescent protein.(A) Schematic illustration of the strategies used to force the ER-to-PM trafficking of engineered STIMs. First, a Myc tag was introduced into STIM between SP and EF-SAM to aid the determination of the orientation of the N terminus of STIM. The original ER SP of STIM was replaced by an extracellularly targeting peptide derived from CD8A(TIF)Click here for additional data file.S3 Fig2+ influx (blue and green traces); no such constitutive Ca2+ influx was observed in cells expressing constructs harboring the STIM1 EF-SAM (red and purple traces). (B) Statistics showing Ca2+ affinity (mM) of the various PM-anchoring SCs. (C) Some unengineered SCs show some PM-like distribution in approximately 25% of transfected cells. FRET signals between YFP-SOAR1L and PM-localized SC-CFP constructs in response to increases in extracellular Ca2+ concentration in these cells. Left, typical traces; right, statistical analysis of the apparent Kd . (D) Calibration of the ER Ca2+ levels using R-CEPIA1er and a Ca2+-insensitive ER marker, CFP-Sec61β in HeLa SK cells. Left, a typical trace used for calibration; right, statistics of the ER Ca2+ concentration. (E) In HeLa SK cells coexpressing R-CEPIA1er, YFP-SOAR1L, and SC1111-CFP or SC1211-CFP, ER Ca2+ levels and FRET signals between SCs and SOARL were monitored simultaneously. Typical traces of the rest state and TG-induced responses for R-CEPIA1er signals. Individual numerical values underlying (A)–(E) may be found in 1-CC1 construct; SK, STIM1 and STIM 2 double knockout; SOAR, STIM-Orai–activating region; STIM, stromal interaction molecule; TG, thapsigargin; WT, wild type; YFP, yellow fluorescent protein.(A) In HEK293-Orai1 stable cells transiently expressing WT STIM or corresponding STIM chimeras with swapped EF-SAM regions, only cells expressing constructs that contain the STIM2 EF-SAM (STIM1211 or STIM2) facilitate a high constitutive Ca(TIF)Click here for additional data file.S4 Fig2+ entry into the Orai1- and STIM-coexpressing cells. (E–G) Comparative analysis of interactions between STIM1-CC1-CFP and YFP-SOAR molecules coexpressed in HEK293 tsA cells, the tsA201 variant of HEK293 cells expressing a temperature sensitive mutant of the SV40 large T antigen. (E) SC1111-CFP+YFP-SOAR1, (F) SC2222-CFP+YFP-SOAR2, (G) SC1122-CFP+YFP-SOAR2, and (H) SC2211-CFP+YFP-SOAR1. The top diagrams show the two coexpressed STIM fragments. Top panel: representative traces of typical FRET signals between WT or chimeric STIM1-CC1-CFP and YFP-SOAR molecules; Bottom panel: confocal images of the typical colocalization of STIM1-CC1-CFP and YFP-SOAR molecules . All results are typical of at least three independent repeats, and at least 36 cells were examined for each condition. Individual numerical values underlying (A)–(H) may be found in 1-CC1 construct; SOAR, STIM-Orai–activating region; SOCE, store-operated Ca2+ entry; STIM, stromal interaction molecule; tsA, the tsA201 variant of HEK293 cells expressing a temperature sensitive mutant of the SV40 large T antigen; WT, wild type; YFP, yellow fluorescent protein.Panels with light yellow background are cells expressing constructs containing the STIM1 cytosolic region; panels with light cyan background are cells expressing molecules containing the STIM2 cytosolic region. (A–D) Comparison of the function of STIM1-YFP (A), STIM2-YFP (B), and the luminal-region–exchanged chimeras, STIM1122-CFP (C) or STIM2211-CFP (D), expressed in HEK293-Orai1-CFP cells or coexpressed with Orai1-YFP in HEK293 WT cells. Left, a diagram of the two coexpressed SOCE components. Top panel: confocal images of the typical cellular distribution of STIM1, STIM2, STIM1122, and STIM2211 at rest . Bottom panel: representative traces for a constitutive Ca(TIF)Click here for additional data file.S5 FigP < 0.001; *P < 0.05, t test, n = 3). (B) Statistics of mean constitutive Ca2+ influxes mediated by Orai1 and different SOAR2 (left) or SOAR1 (right) constructs. . Note: all signals were compared with and normalized to those mediated by SOAR1. Individual numerical values underlying (A) and (B) may be found in 2+ store depletion, SOAR detaches from CC1 and relocates to the vicinity of the PM, where it binds and activates the Orai1 channels. CC1, coiled-coil 1; ER, endoplasmic reticulum; FRET, Förster resonance energy transfer; PM, plasma membrane; SC, STIM1-CC1 construct; SOAR, STIM-Orai–activating region; STIM, stromal interaction molecule.(A) Statistics showing resting FRET signals of SOAR constructs with SC1112 (left) or SC1111 (right). (***(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Trypanosoma cruzi, the causative agent of Chagas' disease. However, the involvement of Arg-1 in promoting intracellular replication of T. cruzi in AAM has not been proven so far in vivo. Because Arg-1 is only moderately expressed in T. cruzi-infected wildtype mice, we elucidated the role of Arg-1 and AAM during infection in IL-13-overexpressing (IL-13tg) mice, which are characterized by an inflammation-induced development of AAM and an accompanied elevated expression of Arg-1. In comparison to wildtype littermates, IL-13tg mice were highly susceptible to T. cruzi infection with enhanced parasitemia and impaired survival. Importantly, T. cruzi-infected IL-13tg mice developed an elevated alternative macrophage activation with increased arginase activity. To proof the hypothesis, that Arg-1 accounts for the increased susceptibility of IL-13tg mice, we blocked arginase activity in infected IL-13tg mice. Because this arginase inhibition resulted in a decreased susceptibility to experimental Chagas disease our study supports in summary the conclusion that IL-13/IL-4Rα-driven Arg-1 expression contributes to the permissiveness of the host to T. cruzi infection.Arginase-1 (Arg-1) is a marker for alternatively activated macrophages (AAM) and is mainly induced by the type 2 cytokines interleukin (IL)-4 and IL-13 through the common IL-4 receptor-alpha (Rα) subunit. Both, Arg-1 and AAM undermine macrophage effector functions against intracellular parasites and are therefore implicated in the susceptibility to infection with  Trypanosoma cruzi, a protozoan parasite that belongs to the family of Trypanosomatidae, is the causative agent of Chagas disease, a neglected tropical disease that is a major public health problem in Latin America -4 and IL-13 are only associated with an increased susceptibility to T. cruzi (T. cruzi are not clearly established.A T helper type 1 (Th1) immune response characterized by the production of interferon-gamma (IFN-γ) is protective against experimental infection with T. cruzi –5 througT. cruzi –10. HoweIn general, the Th2-cytokines IL-4 and IL-13 mediate the development of alternatively activated macrophages (AAM) through binding to the IL-4 receptor-alpha (Rα) chain . ParticuLeishmania major infection the IL-4Rα-mediated development of AAM and the associated strong Arg-1 induction account for the enhanced susceptibility of BALB/c mice  mice  mice  and analT. cruzi infection IL-13tg mice and wildtype littermates were infected i.p. with 50 blood trypomastigotes of the Tulahuen strain and the survival rate and parasite load during the acute phase of infection was determined  , 26. To  Figures , spleen  Figures , and liv Figures  were pres Figure . To quanT Figure  and AST T Figure  were quaT. cruzi-infected mice  and monitored survival and determined parasitemia  and STAT6−/− mice or neutralization of IL-4 by administration of monoclonal antibodies  and significantly ameliorated the outcome of experimental Chagas disease when compared to untreated IL-13tg mice. Hence, Arg-1 appears to be a key molecule that mediates the IL-13-dependent susceptibility to T. cruzi infection.Whereas classical macrophage activation was rather similar in tivation , 33, 34.rophages , 36. Becrophages , 18, we T. cruzi infection   , which re (ADMA) . Becauseactivity  the IL-1T. cruzi IL-13 induces an increased arginase activity and subsequently elevated amounts of polyamines. This excess supply of host polyamines may also account for the arginase-dependent uncontrolled parasite replication in T. cruzi-infected IL-13tg mice.Additionally, arginase activity results in the production of polyamines, which appear to be essential for intracellular parasite replication . ParasitIn summary, we here give evidence that IL-4Rα-induced Arg-1 mediates susceptibility to acute experimental Chagas disease by several - mutually not exclusive - mechanisms downstream of Arg-1 including L-arginine depletion in MDSCs and AAM and polyamine synthesis. A correlation between Arg-1, arginase activity and susceptibility was already shown in patients with visceral leishmaniasis , 50. Howtg mice on a BABL/c background were kindly provided by Andrew McKenzie  and bred under specific-pathogen-free conditions in the animal facility of the Research Center Borstel. For experiments, female transgenic mice and transgene-negative littermates aged 6 to 8 weeks were infected intraperitoneally (i.p.) with the T. cruzi strain Tulahuen (WHO reference strain M/HOM/CH/00/Tulahuen C2). To obtain high numbers of T. cruzi blood form trypomastigotes for infection experiments and to prevent the transfer of lymphocytes and antibodies, SCID mice mice  were i.p. infected with 1 × 107 parasites in cryopreserved T. cruzi stabilates. At day 12 post infection, blood was collected from infected SCID, mixed with heparin and parasites were enriched in the plasma by differential centrifugation. Parasites were resuspended in PBS/0.5% glucose and used for infection. An infection dose of 500 blood trypomastigotes was used to induce detectable inflammatory cytokine responses and pathology. For determining parasitemia and mortality, a sublethal dose of 50 parasites was used . Viable parasites were counted in a Neubauer chamber.Blood parasitemia was determined in 3 μl of tail vein blood that was lyzed in 27 μl NHhprt) gene, used for quantification of host DNA, was amplified with the forward primer 5-GTG GCC CTC TGT GTG CTC-3, the reverse primer 5-TCT ACA GTC ATA GGA ATG GAT CTA TCA-3, and the internal probe 5-ACC TGC TG-3. Quantitative PCR was performed on a Light Cycler 480 . Data were analyzed employing the “Second Derivative Maximum” and “Standard Curve Method.”Tissue parasite burdens were analyzed by quantitative real-time PCR of genomic DNA isolated from the spleen. A 70 bp sequence of the 140/116-kDa antigen gene of T. cruzi (accession no. U15616) was amplified with the forward primer 5-ACT CAT CGG GTT TGA AGC AT-3, the reverse primer 5-GCC AGG GTC TAG TAC TCT TTG CT-3, and the internal probe 5-CAG CAG GC-3_ A 107 bp stretch of the murine hypoxanthine–guanine phosphoribosyltransferase . Histopathological analyses were performed using standard protocols for hematoxylin/eosin staining. For quantification of heart inflammation, serial sections of heart tissue were analyzed for the degree of cellular infiltaraion using the ImageJ software  as described . For NOSSerum was prepared using the BD Microtainer SST Tubes (BD Pharmingen) and the aspartate transaminase (AST), and alanine transaminase (ALT) activity was determined by using the Reflotron System of Diagnosis (Roche Diagnostics).hprt) as a housekeeping gene to calculate the level of gene expression in relation to hprt. The following primer and probe sets were employed: Arg1: sense 5′-CCT GGA ACT GAA AGG AAA G-3′, antisense 5′-TTG GCA GAT ATG CAG GGA GT-3′, probe 2; Chi3l3: 5′-GAA CAC TGA GCT AAA AAC TCT CCT G-3′, antisense 5′- GAG ACC ATG GCA CTG AAC G-3′, probe 88; Hprt: sense 5′-TCC TCC TCA GAC CGC TTT T-3′, antisense 5′-CCT GGT TCA TCA TCG CTA ATC-3′, probe 95; Ifng: sense 5′-ATC TGG AGG AAC TGG CAA AA-3′, antisense 5′-TTC AAG ACT TCA AAG AGT CTG AGG TA-3′, probe 21; Il4: sense 5′-CAT CGG CAT TTT GAA CGA G-3′, antisense 5′-CGA GCT CAC TCT CTG TGG TG-3′, probe 2; Il6: sense 5′- CT ACC AAA CTG GAT ATA ATC AGG A-3′, antisense 5′-CCA GGT AGC TAT GGT ACT CCA-′, probe 6; Il12b: sense 5′-CCA TCA GCA GAT CAT TCT AGA CAA-3′, antisense 5′-CGC CAT TAT GAT TCA GAG ACT G-3′, probe 78; Il13: sense 5′-CCT CTG ACC CTT AAG GAG CTT AT-3′, antisense 5′-CGT TGC ACA GGG GAG TCT-3′, probe 17; Irgm: sens 5′-AAG GCC ACT AAC ATC GAA TCA-3′, antisense 5′-TGC CTT ATC TCA CTT AAT ACT CCT-3′, probe 82; Nos2: sense 5′-CTT TGC CAC GGA CGA GAC-3′, antisense 5′-TCA TTG TAC TCT GAG GGC TGA C-3′, probe 13; Retnla: sense 5′-TAT GAA CAG ATG GGC CTC CT-3′, antisense 5′-GGC AGT TGC AAG TAT CTC CT-3′, probe 3; Tnf : sense 5′-TGC CTA TGT CTC AGC CTC TTC-3′, antisense 5′-GAG GCC ATT TGG GAA CTT CT-3′, probe 49 .Organs were homogenized in 4M guanidinium-isothiocyanate buffer and total RNA was extracted by acid phenol extraction. cDNA was obtained using murine moloney leukemia virus reverse transcriptase  and oligo-dT  as a primer. Quantitative PCR was performed on a Light Cycler 480 Instrument (Roche Diagnostics). Data were analyzed employing the “Second Derivative Maximum” and “Standard Curve Method” using hypoxanthine-guanine phosphoribosyltransferase . After adding Griess reagents, the content of NO2 was determined by photometric measurement reading the absorbance at 540 nm on a microplate reader  as previously described (2SO4 (96%)/H3PO4 (85%)/H2O . As a degree of arginase activity, the urea concentration was measured at 540 nm after addition of 25 μl α-isonitrosopropiophenone  followed by heating at 95°C for 45 min. One unit of arginase activity is defined as the amount of enzyme that catalyzes the formation of 1 μmol urea/min.To detect reactive nitrogen intermediates (RNI) in uninfected and infected mice, spleen homogenates were collected at different time points. After deproteination of homogenates using Micron YM-30 centrifugal filters , NOt-test, the Mann-Whitney U test, the Kruskal-Wallis test or the Log-Rank test defining different error probabilities .Quantifiable data are expressed as means of individual determinations and standard deviation. Statistical analysis was performed using the unpaired Student's MA performed the experiments, analyzed the data, and wrote the manuscript; CH conceived and designed the experiments and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PROPEP transcripts that give rise to AtPep peptides which also activate immune signaling. Phytohormones such as methyl-jasmonate also induce PROPEP expression, suggestive of additional hormone-mediated feedback loops that similarly amplify immune signaling. The current study aimed to determine if perception of jasmonate is genetically required for AtPep1-induced immune responses in Arabidopsis thaliana.Plant cells detect the presence of potentially pathogenic microorganisms in the apoplast via plasma membrane-localized receptors. Activated receptors trigger phosphorylation-mediated signaling cascades that protect the cell from infection. It is thought that signaling triggered by the detection of exogenous signals, such as bacterial flagellin, can be amplified by endogenous signals, such as hormones or debris caused by cell damage, to potentiate robust immune responses. For example, perception of flagellin and other microbial molecules results in increased expression of endogenous coi1-16 mutants are severely compromised in some AtPep1-induced immune responses, while other AtPep1-induced responses are maintained but reduced. Our findings build on previously published work and suggest that JA perception plays a role in immune responses triggered by AtPep1.We assessed several AtPep1-induced immune responses in plants expressing a non-functional variant of the jasmonate receptor CORONATINE-INSENSITIVE 1 (COI1). We found that The online version of this article (10.1186/s13104-018-3628-7) contains supplementary material, which is available to authorized users. Arabidopsis thaliana by receptor kinases FLAGELLIN SENSING 2 (FLS2) and EF-Tu RECEPTOR (EFR), respectively [2+, the activation of receptor-like cytoplasmic kinases (RLCKs), a rapid and transient apoplastic oxidative burst, the activation of mitogen-activated and calcium-dependent protein kinases (MAPK and CDPKs), and transcriptional reprogramming resulting in a basal immune response that is effective against most potential pathogens [Plants lack a humoral immune system and rely solely on the innate ability of each cell to detect potentially harmful pathogens and defend against disease. Plasma membrane-localized pattern recognition receptors (PRRs) bind ‘non-self’ molecules characteristic of entire classes of microbes known as pathogen-associated molecular patterns (PAMPs), which are typically integral to microbial lifestyles and are thus under strong selection pressure . Exampleectively . PRRs alectively , 5. For ectively –9. Many ectively , 10. Upoectively –16. PRR athogens .PATHOGENESIS RELATED-1 (PR-1) and PLANT DEFENSIN 1.2 (PDF1.2), and expression of AtPep precursor PROPEP genes is induced by treatment with methyl-salicylate (MeSA), methyl-jasmonate (MeJA), immunogenic peptides, as well as pathogen infection and herbivore feeding [Interplay between plant immune and hormone signaling has been observed in several systems . In part feeding , 8, 21.  feeding , 23 sugg feeding  and suppArabidopsis thaliana ecotype Columbia (Col-0) and previously described mutants bak1-5 [, glabra1 (Col gl1) [, SA-induction deficient 2-2 (sid2-2) [coi1-16 (in the Col gl1 background) [18 primer and SuperScript III (Invitrogen) following the manufacturer’s directions. Quantitative real-time PCR was performed using SsoAdvanced Universal SYBR Green Supermix (BioRad) and measured on a CFX96 Touch Real-Time PCR Detection System (BioRad). Melting curve analysis confirmed that all primer pairs amplify a single product; primer sequences are listed in Additional file s bak1-5 , glabra1Col gl1) , SA-indu(sid2-2) , and coikground)  were usekground) . For genArabidopsis seedlings constantly exposed to immunogenic peptides display severe growth inhibition, presumably due to continual activation of immune signaling that diverts resources away from normal growth and development. Although cross-talk between immune and hormone pathways has been well-demonstrated [coi1-16 [bak1-5 [sid2-2, which cannot synthesize SA due to lack of functional isochorismate synthase [nstrated , 19, how[coi1-16  was almo [bak1-5  to AtPepsynthase , was notcoi1-16 compared to controls, AtPep1-induced ROS was as severely inhibited as in bak1-5 mutants  mutant which cannot synthesize JA [As seedling inhibition is considered a late immune response, we extended our analysis to test if JA perception via COI1 is also required for an earlier immune response such as the RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD)-mediated burst of reactive oxygen species (ROS). We found that while elf18- and flg22-induced ROS was unaffected in affected . Thus, ockground , corroboesize JA , suggestrbohD mutants, and flg22-induced oxidative burst is maintained in mpk3 mpk6 mutants [While some PTI responses are directly linked via phosphorylation cascades, genetic evidence supports parallel activation of other outputs downstream of PRR activation . For exa mutants .gl1 and coi1-16 plants with flg22, elf18, or AtPep1 for 10 min and compared the activation of MPK6, MPK3, and MPK4/11 using immunoblot analysis. While flg22-induced MAPK activation was comparable between coi1-16 and control Col gl1 plants, we observed slightly reduced MAPK activation in coi1-16 mutants following treatment with elf18 and AtPep1 . Three biological replicates were performed with similar results. Statistically significant groups (p < 0.05) are indicated with lower-case letters based on a one-way ANOVA followed by Tukey’s post-test.Additional file 3:  AtPep1-induced PEPR1 expression in coi1-16 mutants. Twelve 14-day-old Col gl1 and coi1-16 seedlings were treated with water (-) or 1 µM AtPep1 (+) for 120 minutes prior to RNA extraction. Quantitative real-time PCR was used to assess expression level of PEPR1. Values are means + standard deviations , normalized against the relative average expression of UBOX from the same sample. Three independent biological replicates were performed with similar results. Statistically significant groups (p < 0.05) are indicated with lower-case letters based on a one-way ANOVA followed by Tukey’s post-test.


Delirium is a common, morbid, and costly geriatric syndrome, yet its pathophysiology remains poorly understood. In a nested matched case-control study within the Successful Aging after Elective Surgery (SAGES) study, a cohort of adults age ≥70 without dementia undergoing major non-cardiac surgery, we previously identified inflammatory proteins to be associated with delirium. Using the entire SAGES cohort, the current study examines the independent associations of these inflammatory proteins with postoperative delirium. Plasma was collected preoperatively (PREOP) and on postoperative day 2 (POD2). Neuroinflammatory marker chitinase-3-like protein [CHI3l1 or YKL-40]; PREOP and POD2) and systemic inflammatory markers interleukin [IL]-6 (POD2 only) and C-reactive protein  were measured using enzyme-linked immunosorbent assays. Generalized linear models were used to determine the independent (multivariable) associations between the inflammatory markers, measured in sample-based quartiles (Q). All models adjusted for age, sex, baseline cognition, surgery type, Charlson comorbidity index, and medical complications. Among the 555 patients , 58% were female and 86% underwent orthopedic surgeries. Postoperative delirium occurred in 24%. High YKL-40 PREOP and IL-6 at POD2 (Q4 vs. Q1) were significantly associated with an increased risk of delirium: relative risk (RR) [95% confidence interval (CI)] 2.2[1.1-4.4] and 2.7[1.3-5.7], respectively. CRP (PREOP and POD2) was not significantly associated with delirium . This work underscores the importance of inflammation (YKL-40 and IL-6) in the pathophysiology of postoperative delirium.


The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle composed of smooth peripheral tubules and rough, ribosome-studded central ER sheets whose morphology is determined, in part, by the ER-shaping proteins, reticulon (RTN) and cytoskeleton-linking membrane protein 63 (CLIMP-63), respectively. Here, stimulated emission depletion (STED) super-resolution microscopy shows that reticulon4a (RTN4a) and CLIMP-63 also regulate the organization and dynamics of peripheral ER tubule nanodomains. STED imaging shows that lumenal ER monomeric oxidizing environment-optimized green fluorescent protein (ERmoxGFP), membrane Sec61βGFP, knock-in calreticulin-GFP, and antibody-labeled ER-resident proteins calnexin and derlin-1 are all localized to periodic puncta along the length of peripheral ER tubules that are not readily observable by diffraction limited confocal microscopy. RTN4a segregates away from and restricts lumenal blob length, while CLIMP-63 associates with and increases lumenal blob length. RTN4a and CLIMP-63 also regulate the nanodomain distribution of ER-resident proteins, being required for the preferential segregation of calnexin and derlin-1 puncta away from lumenal ERmoxGFP blobs. High-speed (40 ms/frame) live cell STED imaging shows that RTN4a and CLIMP-63 regulate dynamic nanoscale lumenal compartmentalization along peripheral ER tubules. RTN4a enhances and CLIMP-63 disrupts the local accumulation of lumenal ERmoxGFP at spatially defined sites along ER tubules. The ER-shaping proteins RTN and CLIMP-63 therefore regulate lumenal ER nanodomain heterogeneity, interaction with ER-resident proteins, and dynamics in peripheral ER tubules. Stimulated emission depletion (STED) super-resolution microscopy shows that endoplasmic reticulum–shaping proteins regulate not only the macroscale organization of the ER (sheets versus tubules) but also the nanodomain heterogeneity and dynamics of peripheral ER tubules. Since the initial Singer-Nicholson fluid mosaic model of free membrane diffusion in the 1980s, the role of membrane nanodomains in the control of protein and lipid dynamics in the plasma membrane, regulating signal transduction, endocytosis and exocytosis, and thereby cellular behavior has been extensively characterized [The endoplasmic reticulum (ER) is a continuous membrane network that is classically divided into central ribosome-studded rough ER sheets, the site of protein synthesis, and peripheral smooth ER tubules, implicated in lipid synthesis and detoxification –5. A famThe thickness of an ER sheet and the diameter of an ER tubule are typically 30–100 nm ,14, beloHowever, molecular mechanisms that regulate the organization of ER nanodomains remain to be defined. Here, we apply STED microscopy to study lumenal compartmentalization in peripheral ER tubules using the lumenal ER reporter ER monomeric oxidizing environment-optimized green fluorescent protein (ERmoxGFP). ERmoxGFP contains the bovine prolactin signal sequence and Lys-Asp-Glu-Leu (KDEL) ER retention sequence linked to monomeric, cysteine-less moxGFP, a modified inert GFP optimized for use in oxidizing environments that minimally perturbs the cell . We findThe peripheral ER imaged by diffraction limited confocal microscopy presents a highly reticular network of interconnected ER tubules. Super-resolution two-dimensional (2D) STED live cell imaging of HT-1080 fibrosarcoma and COS-7 cells transfected with the ER lumenal reporter ERmoxGFP  shows thLine scan analysis shows distinct maxima and minima corresponding to local enrichment and depletion, respectively, of the fluorescent signal from the lumenal ERmoxGFP reporter along peripheral ER tubules in fixed and live cells . ER tubuAs RTN4a promotes ER tubule formation ,10–12, wWe then tested whether overexpression of mCherry-RTN4a, mCherry-CLIMP-63, and, as a control, mCherry-ATL1, impacts lumenal nanodomain organization in peripheral ER tubules. ERmoxGFP-transfected HT-1080 cells show expansion of the central ER and reduction of peripheral tubules upon mCherry-CLIMP-63 overexpression, formation of an extended network of peripheral tubules upon mCherry-RTN4a overexpression, and increased branched peripheral ER structures upon mCherry-ATL1 overexpression , consistLine scan quantification of ERmoxGFP in peripheral ER tubules of HT-1080 cells shows that mCherry-CLIMP-63 transfection decreases lumenal periodicity. ERmoxGFP tubules present significantly increased maxima length, increased variation in maxima length, and decreased maxima-to-minima intensity differentials. In contrast, overexpressed mCherry-RTN4a enhances the periodicity of ERmoxGFP, reducing the variation of maxima length and increasing maxima-to-minima intensity differentials. mCherry-ATL1 overexpression does not impact maxima length, variation of maxima length, or maxima-to-minima differentials . To quanWe then undertook to determine whether other ER markers present a similar peripheral ER tubule periodicity. As observed for ERmoxGFP, the peripheral reticular network of Sec61βGFP-labeled tubules in live HT-1080 and COS-7 cells showed a highly periodic distribution by STED . SimilarWe then extended our analysis to examine the distribution of the endogenous ER-resident proteins, calnexin and derlin-1, involved in protein quality control and ER-associated protein degradation (ERAD), respectively ,26. AntiQuantitative line scan analysis of 2D STED imaged peripheral ER tubules shows the increased association of calnexin and derlin-1 puncta with ERmoxGFP minima and an equal distribution between minima and maxima of Sec61βGFP-labeled tubules . SimilarWe then tested if RTN4a and CLIMP-63 impact the distribution of ER-resident proteins to ER tubule nanodomains. Upon RTN4 or CLIMP-63 knockdown or RTN4a or CLIMP-63 overexpression, calnexin or derlin-1 puncta no longer showed a preferential distribution to minima of lumenal ERmoxGFP but rather a balanced distribution to maxima and minima . ATL1 ovTo determine whether CLIMP-63 and RTN impact the dynamics of ERmoxGFP lumenal nanodomains, we acquired high-speed (40 ms/frame) 2D STED time-lapse image series of small ROIs encompassing individual peripheral ER tubules . KymograPeripheral ER tubules are heterogenous, periodic structures composed of discrete nanodomains. STED imaging of lumenal (ERmoxGFP) or membrane (Sec61βGFP) ER reporters, of CRISPR/Cas9 knock-in calreticulin-GFP expressed at endogenous levels, and of various antibody-labeled ER-resident proteins shows that ER proteins are localized to discrete puncta interspersed with lumenal domains along ER tubules. Periodic distribution of ER reporters along ER tubules can be observed in various publications studying ER using various super-resolution microscope approaches and EM ,27–32 anSingle particle tracking has identified active lumenal flow within ER tubules . By highThe RTNs cause local ER curvature , and CLIIndeed, by STED imaging, puncta of various ER proteins  along ER tubules were closely associated with RTN-associated lumenal ERmoxGFP minima and therefore segregated away from lumenal nanodomains. Overexpression or knockdown of either CLIMP-63 or RTN4a disrupted the enrichment of resident ER proteins in nanodomains depleted of the lumenal ERmoxGFP reporter. Simplistically, increased ER tubule lumenal filling due to CLIMP-63 overexpression or RTN4 knockdown could facilitate overlap between resident ER proteins and fluid components, while, conversely, localized accumulation of overexpressed RTN along ER tubules may sequester both protein and lumenal ER tubule components away from RTN-enriched nanodomains. The fact that CLIMP-63 knockdown had minimal effect on lumenal nanodomain stability and distribution argues that it is but one of multiple redundant mechanisms to define lumenal enriched ER nanodomains. Regulation of ER nanodomain organization by these ER-shaping proteins in peripheral ER tubules parallels their roles in the formation of ER tubules and ER sheets. RTN and CLIMP-63 also contribute to nanohole formation in ER sheets [This study suggests that ER tubules are composed of protein-enriched nanodomains interspersed with fluid-filled nanodomains that may facilitate diffusion and exchange of small molecules, such as metabolites and enzyme substrates, as well as folding intermediates between protein complexes. The minimal overlap between the two endogenous ER markers studied, calnexin and derlin-1, suggests that multiple, discrete protein complexes exist within the ER. Further study is required to define how nanodomain organization and heterogeneity within the highly dynamic ER tubular network control functional interaction between ER-resident proteins, cargo proteins, enzymatic substrates, and metabolites to enable ER quality control and cargo processing.ERmoxGFP was a gift from Dr. Erik Snapp  (Addgene plasmid #68072), mCherry-CLIMP-63 and Sec61βGFP from Dr. Gia Voeltz , Sec61βmRFP from Dr. Patrick Lajoie , and mCherry-RTN4a and mCherry-ATL1 from Dr. Tom Rapoport  . Anti-CLIMP-63 antibody (G1/296) was purchased from Enzo Life Sciences  (Cat# ALX-804-604-C100). Rabbit anti-calnexin (Cat# C4731), mouse anti-derlin-1 (Cat# SAB4200148), rabbit anti-BiP (Cat# G9043), mouse anti-actin (Cat# A2228), goat anti-mouse HRP conjugate (Cat# AP308P), goat anti-rabbit HRP conjugate (Cat# AP307P), and rabbit anti-Stx17 (Cat# HPA001204) antibodies were from Sigma . Rabbit anti-RTN4/NOGO (Cat# 10950-1-AP) and anti-Gp78 (Cat# 16675-1-AP) antibodies were from Proteintech . Goat serum (Cat# 16210–064), goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 532 (Cat# A-11009), and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 568 (Cat# A-11004) were from Thermo Fisher Scientific . siCLIMP-63 (Cat# L-012755-01-0005), siRTN4 (L-010721-00-0005) and nontargeting Control siRNA (Cat# D-001810-01-50) were from Dharmacon, GE Healthcare Life Sciences . Sixteen percent paraformaldehyde (Cat# 15710) and 25% glutaraldehyde (Cat# 16220) were from Electron Microscopy Sciences , USA. Other chemicals were from Sigma .2 in complete RPMI 1640  containing 10% fetal bovine serum (FBS)  and 1% L-glutamine  unless otherwise stated. The U2OS knock-in cell line expressing GFP-calreticulin at endogenous levels generated with CRISPR/Cas technology was provided by Dr. Tom Rapoport . COS-7 and U2OS cells were grown at 37°C with 5% CO2 in complete DMEM  containing 10% FBS and 1% L-glutamine. All cell lines were mycoplasma-free, tested routinely by PCR  and, as necessary, treated with BM Cyclin  to eliminate mycoplasma contamination.HT-1080 cells were grown at 37°C with 5% COPlasmids were transfected for 22 hours using Effectene  according to the manufacturer’s protocols. siRNA transfection was done with Lipofectamine 2000 Transfection Reagent  in Opti-MEM Reduced Serum Media , which was replaced with fresh complete RPMI 1640 after 5 hours; after a further 21 hours plasmids were transfected with Effectene and incubated for 22 hours before fixation. For live cell imaging, cells were grown in ibidi 8-well μ-slides with #1.5H (170 μm ±5 μm) D 263 M Schott glass and transfected as described above. For live cell imaging at 37°C, complete RPMI 1640 medium was replaced with warmed RPMI 1640 medium  without sodium bicarbonate, supplemented with 1% L-glutamine, 10% FBS, 10% HEPES  for HT-1080 cells. For COS-7 and U2OS cells, complete DMEM medium was replaced by FluoroBrite DMEM media  warmed to 37°C before imaging. Western blots were conducted as previously described .2 and 1 mM MgCl2; two quick washes and then two 5-minute washes); (2) permeabilized with 0.2% Triton X-100 for 5 minutes, then washed with PBS-CM as above; (3) quenched with 1 mg/mL of NaBH4  for 10 minutes and washed with PBS-CM; (4) blocked with 10% Goat Serum  and 1% bovine serum albumin  in PBS-CM for 1 hour; (5) incubated with primary antibodies in Antibody Buffer  overnight at 4°C, then washed with PBS-CM, then three times for 5 minutes with Antibody Wash Buffer ; (6) incubated with secondary antibodies in Antibody Buffer for 1 hour, then washed with PBS-CM, then six times for 10 minutes with Antibody Wash Buffer on a rocker; and (7) rinsed with Milli-Q H2O and mounted with ProLong Diamond  and cured for 24–48 hours at room temperature.Cells grown on #1.5H coverslips  were (1) fixed with 3% paraformaldehyde with 0.2% glutaraldehyde in phosphate-buffered saline (PBS) at room temperature for 15 minutes and washed with PBS-CM . Time-gated fluorescence detection was used for STED to further improve lateral resolution. Live cell time-lapse imaging of GFP-tagged reporters was performed at 37°C using the 592-nm depletion laser on a 15,360-nm square ROI (0.8-second frame rate over 40 seconds) or a 3,840-nm square ROI (40-ms frame rate over 4 seconds) in the periphery of the cell. For double- or triple-labeled fixed samples, acquisition was done at a scan speed of 600 Hz with a line average of 6. GFP was excited at 488 nm and depleted using the 592-nm depletion laser. Alexa Fluor 532 was excited at 528 nm, Alex Fluor 568 at 577 nm, and mRFP at 584 nm, and all three were depleted using the 660-nm depletion laser. Sequential acquisition (in the order of AF568/AF532/GFP or mRFP/GFP) between frames (2D) or between stacks (3D) was used to avoid cross talk. Three-dimensional STED images were acquired at a step size of 100 nm. STED images were deconvolved using Huygens Professional software  that was also used to determine the theoretical PSFs from 2D STED live and fixed images and from 3D STED fixed images. XY FWHM values obtained from the theoretical PSFs for STED GFP images were 70 nm for 2D fixed and 78 nm for 2D live. For 3D fixed analysis, FWHM was 126 nm for XY and 340 nm for Z.Confocal and STED imaging was performed with the 100×/1.4 Oil HC PL APO CS2 STED White objective of a Leica TCS SP8 3× STED microscope  equipped with a white light laser, HyD detectors, and Leica Application Suite X (LAS X) software  along the line was exported for the ER reporters (ERmoxGFP or Sec61βGFP) and either calnexin, derlin-1, mCherry-CLIMP-63, mCherry-RTN4a, or mCherry-ATL1, and displayed using GraphPad Prism . Maximum and minimum fluorescence values were identified with a Java script. Blobs were defined as local maxima in the fluorescence signal and minima as troughs in fluorescence intensity, at least 20% below both adjacent maxima. FWHM of maxima was used to determine blob length. The percent decrease in the fluorescence signal of all minima was calculated relative to the adjacent maximum of lower fluorescence. Protein  puncta were localized to either maxima or minima and deemed intermediate if the puncta center was greater than 20 nm from the peak of the maxima or minima. Quantification of protein puncta distribution to maxima and minima in 3D projections was scored manually in LAS-X. Three-dimensional STED volume rendering was done with LAS-X. Analysis of the live cell imaging (40 ms/frame) was done with ImageJ/FIJI . GeneralS1 FigRepresentative confocal and STED images of calnexin and ERmomxGFP in ERmoxGFP transfected and untransfected cells. The punctate distribution of calnexin is observed more readily by STED (raw and decon) compared with confocal imaging in both (A) transfected and (B) untransfected cells. Scale bar, 5 μm; zooms, 2 μm. decon, deconvolution; ER, endoplasmic reticulum; ERmoxGFP, ER monomeric oxidizing environment-optimized green fluorescent protein; STED, stimulated emission depletion.(TIF)Click here for additional data file.S2 Fig2 test from at least 25 ROIs (2.5 um × 2.5 um) from 10 three-dimensional stacks for each condition at 2 degrees of freedom in three independent experiments. *P < 0.05; **P < 0.01. Numerical values that underlie the graphs are shown in P < 0.05; **P < 0.01; ***P < 0.001. Numerical values that underlie the graphs are shown in (A) Quantification of 3D localization of calnexin and derlin-1 puncta to ERmoxGFP maxima and minima of peripheral ER tubules in 3D STED images of siCTL or siCLIMP-63 HT-1080 cells. Significance assessed by χ(TIF)Click here for additional data file.S3 FigWestern blots of RTN4 and CLIMP-63 siRNA knockdown in COS-7 cells. The blots were probed with anti-CLIMP-63, anti-RTN4, or anti-β-actin as a loading control. CLIMP-63, cytoskeleton-linking membrane protein 63; RTN4, reticulon4; siRNA, small interfering RNA.(TIF)Click here for additional data file.S1 VideoLive HT-1080 cells overexpressing ERmoxGFP or Sec61βGFP were imaged with confocal or STED at 37°C for 40 seconds at a temporal resolution of 0.8 seconds per frame. (a) Confocal ERmoxGFP; (b) STED ERmoxGFP; (c) confocal Sec61βGFP; (d) STED Sec61βGFP. Video frame rate: 10 frames per second. ER, endoplasmic reticulum; ERmoxGFP, ER monomeric oxidizing environment-optimized green fluorescent protein; STED, stimulated emission depletion.(MP4)Click here for additional data file.S2 VideoERmoxGFP expressing HT-1080 cells were fixed, labeled with calnexin and derlin-1, and imaged sequentially  with 3D STED (vortex donut enabled). Video frame rate: 10 frames per second. ER, endoplasmic reticulum; ERmoxGFP, ER monomeric oxidizing environment-optimized green fluorescent protein; STED, stimulated emission depletion; 3D, three-dimensional.(MP4)Click here for additional data file.S3 VideoLive COS-7 cells were transfected with ERmoxGFP (CTL) or cotransfected with ERmoxGFP and either mCherry-CLIMP-63 (CLIMP-63 OX) or mCherry-RTN4a (RTN OX). ERmoxGFP was imaged with STED at 37°C over 4 seconds at a temporal resolution of 40 ms per frame. Arrows show sites of stable distribution of ERmoxGFP at specific sites along ER tubules over time, as determined by kymogram analysis . Video f(MP4)Click here for additional data file.S4 VideoLive COS-7 cells were transfected with siCTL, siCLIMP-63, or siRTN4, as indicated, and then with ERmoxGFP. ERmoxGFP was imaged with STED at 37°C over 4 seconds at a temporal resolution of 40 ms per frame. Arrows show sites of stable distribution of ERmoxGFP at specific sites along ER tubules over time, as determined by kymogram analysis . Video f(MP4)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.