In conclusion, we showed for the first time that the presence of a dense mucosal cholinergic innervation is associated with decreased secretion of IEC-derived pro-inflammatory IL-8 in the rectosigmoid of HSCR patients likely dependent on a7nAChR activation. Owing to the association between IL-8 and enterocolitis-prone, fiber-low HSCR patients, targeted therapies against IL-8 might be a promising immunotherapy candidate for HAEC treatment.Infants affected by Hirschsprung disease (HSCR), a neurodevelopmental congenital disorder, lack ganglia of the intrinsic enteric nervous system (aganglionosis) in a variable length of the colon, and are prone to developing severe Hirschsprung-associated enterocolitis (HAEC). HSCR patients typically show abnormal dense innervation of extrinsic cholinergic nerve fibers throughout the aganglionic rectosigmoid. Cholinergic signaling has been reported to reduce inflammatory response. Consequently, a sparse extrinsic cholinergic innervation in the mucosa of the rectosigmoid correlates with increased inflammatory immune cell frequencies and higher incidence of HAEC in HSCR patients. However, whether cholinergic signals influence the pro-inflammatory immune response of intestinal epithelial cells (IEC) is unknown. Here, we analyzed colonic IEC isolated from 43 HSCR patients with either a low or high mucosal cholinergic innervation density (fiber-low versus fiber-high) as well as from control tissue. Compared to fiber-high samples, IEC purified from fiber-low rectosigmoid expressed significantly higher levels of IL-8 but not TNF-α, IL-10, TGF-β1, Muc-2 or tight junction proteins. IEC from fiber-low rectosigmoid showed higher IL-8 protein concentrations in cell lysates as well as prominent IL-8 immunoreactivity compared to IEC from fiber-high tissue. Using the human colonic IEC cell line SW480 we demonstrated that cholinergic signals suppress lipopolysaccharide-induced IL-8 secretion  Hirschsprung disease (HSCR) is a multi-genetic congenital malformation of the colon characterized by the absence of enteric ganglia (aganglionosis) –4. DefecIn aganglionic rectosigmoid tissue from HSCR patients, acetylcholinesterase (AChE) activity is increased due to the presence of extrinsic AChE-positive nerve fibers innervating the muscularis, submucosa and the mucosa , 18–20. via pattern-recognition receptors and coordinate the underlying immune system towards tolerance or activation  are exposed to pathogenic and non-pathogenic antigens derived from harmful invaders or harmless commensals or food. Towards the luminal side IEC protect the host by the secretion of mucus and antimicrobial peptides. To maintain mucosal homeostasis, IEC sense intestinal signals tivation . The rectivation , 32. IL-tivation –36. Cons+ nerve fibers (fiber-high) had decreased inflammatory cytokines and TH17 T cells as well as lower incidence of enterocolitis development compared to HSCR patients with only sparse mucosal innervation (fiber-low)  and muscarinic (CHRM3) cholinergic receptors might be involved in neuronal immune cell modulation  , 37. AChdulation .IEC have been reported to express cholinergic receptors and their essential role in maintaining barrier functions has been described . Howeverin vitro possibly involved cholinergic receptors and signaling pathways using the human SW480 IEC cell line.Here we investigated the association of mucosal cholinergic innervation with cytokine expression of HSCR and Non-HSCR derived IEC and identified www.clinicaltrials.gov under access number NCT03617640 and is performed according to the Declaration of Helsinki. Colonic tissue from consenting patients was collected freshly in the operating room. For histological analysis a longitudinal strip of resected colonic tissue was immediately snap-frozen. Tissue for epithelial cell isolation was kept on ice in transport medium  supplemented with 10% Foetal Calf Serum , 10mM Hepes , 1% Penicillin-Streptomycin, 0.5µg/ml amphotericin B, 80µg/ml gentamicin  and was processed in less than 24h. Demographic and clinical patient data were extracted from medical records. The Non-HSCR patient group included five patients with anal atresia and one misdiagnosed HSCR patient. Tissue was collected during ostomy and pull-through resection, respectively. The control tissue showed the presence of an ENS and the absence of inflammation as defined by the local pathologists. A detailed patients characteristic is available in Keck et al.  and the Medical Faculty of Heidelberg (S-388/2015). The study is registered at Colonic tissue was separated into anatomical segments according to rectosigmoid and descending colon  and muscle layers stripped from mucosa. Mucosa was minced and digested with collagenase D (1mg/ml) and DNase I  in complete medium  supplemented with 10% FCS, 1% Penicillin-Streptomycin, 0.5µg/ml amphotericin B, 80µg/ml gentamicin, 10mM Hepes) at 37°C for 1h under vigorous shaking. Digestion suspension was filtered through a 100µm-cell strainer, and cells were washed in complete medium. Suspension cells were resuspended in 20% Percoll  and underlaid with 40% Percoll (3ml of each). Epithelial cells were purified from the 20%/40% Percoll interface. Cells were washed and used freshly for Fluorescence-activated cell sorting (FACS) analysis or immediately resuspended in RLT Lysis buffer and cryo-stored until further analysis.Viability was determined using the LIVE/DEAD Fixable Near-IR Dead cell stain . Epithelial purity was determined using human specific antibodies against CD45  and EpCAM . Cell surface markers were stained extracellular in 50µl FACS buffer (PBS supplemented with 2% FCS) for 15min on ice. Unspecific Fcγ receptor binding was blocked by human Fc block . Cells were analyzed using a FACS Canto II  and FlowJo software (TreeStar Inc.).-ΔCT method, with β2-microglobulin as the housekeeping gene, and results were multiplied by 1000. Primer pairs were designed using the clone manager software (Sci-Ed Software). The following primer pairs were used : β2-microglobulin: Forward: 5’-CAG CGT ACT CCA AAG ATT CA-3’ Reverse: 5’-GAA TGC TCC ACT TTT TCA AT-3’; IL-8: Forward: 5’-GTG CAG TTT TGC CAA GGA GT-3’ Reverse: 5’-CTC TGC ACC CAG TTT TCC TT-3’; TNFα: Forward: 5’-GCT GCA CTT TGG AGT GAT CG-3’ Reverse: 5’-GGG CTA CAG GCT TGT CAC TC-3’; TGFβ1: Forward: 5’-ATT GAG GGC TTT CGC CTT AG-3’ Reverse: 5’-AAT GGT GGC CAG GTC ACC TC-3’; IL10: Forward: 5’-GCC TTC AGC AGA GTG AAG AC-3’ Reverse: 5’-GGC TTG GCA ACC CAG GTA AC-3’; MUC2: Forward: 5’-GCC CTT GCG TCC ATA ACA AC-3’ Reverse: 5’-GTA GTA CTT CCC GTC AAA GG-3’; Occludin: Forward: 5’-ATG GAC TGC GTC ACG CAG AG-3’ Reverse: 5’-CCA CAA ACA TGG CCA GGA AG-3’; ZO-1: Forward: 5’-AGC ACA GCA ATG GAG GAA AC-3’ Reverse: 5’-GGT CCT CCT TTC AGC ACA TC-3’; CHRNA3: Forward: 5’- TGC TGT CTC TGC TGC GAC TG-3’ Reverse: 5’-ACA TGG ACA CCT CGA AAT GG-3’; CHRNA4: Forward: 5’-GGA GCG GCT CCT GAA GAA AC-3’ Reverse: 5’-CAC TCC TGC TTC ACC CAT AC-3’; CHRNA5: Forward: 5’-CAC ACT CAG TGC TCC ATT CC-3’ Reverse: 5’-CGA ACC CAT CTT TCG TAG TC-3’; CHRNA7: Forward: 5’-GAT GGC CAG ATT TGG AAA CC-3’ Reverse: 5’-CGA TGT AGC AGG AAC TCT TG-3’; CHRM1: Forward: 5’-GGC AGT GCT ACA TCC AGT TC-3’ Reverse: 5’-CGT GCT CGG TTC TCT GTC TC-3’; CHRM3: Forward: 5’- TCC GAG CAG ATG GAC CAA GAC-3’ Reverse: 5’- CTG TGA CCC GGA AGC TTG AG-3’.Total RNA from cells was isolated using the RNAeasy Plus mini kit  and reverse transcribed using GoScript Reverse Transcription System . Quantitative Real-Time PCR (qRT-PCR) was done using the ViiA7 RT-PCR System  and FastStart Universal SYBR Green Master  according to manufacturer’s instruction. Relative gene expression was calculated using the 2Longitudinal colonic strips were washed in ice-cold PBS and embedded as a Swiss Roll in Tissue-Tek O.C.T. Compound . Cholinergic nerve fibers were visualized in 5µm cryosections using a purified mouse IgG2b anti-human acetylcholinesterase antibody  or a purified mouse IgG2a anti-human β3tubulin  together with an anti-mouse HRP-AEC staining kit  according to the instruction manual. Slides were scanned with an Olympus BX63 Light microscope. The density of cholinergic fibers in epithelial regions of the rectosigmoid was evaluated by four double-blinded individuals and has been previously described in detail .Between all steps, tissue was washed three times with PBS supplemented with 0.2% Tween-20 . Cryosections (5µm) were fixed with 4% paraformaldehyde  for 5min, followed by a blocking step with goat serum (40min). Primary human antibodies, purified mouse IgG1 anti-IL-8 , purified mouse IgG1 anti-alpha7nAchR  and FITC-labeled mouse IgG2b anti-EpCAM  were incubated for 1h at room temperature. Purified primary antibodies were detected by secondary antibody goat anti-mouse IgG1 A555  for 40min. Antibodies were diluted in Antibody-Diluent . Finally, nuclei were visualized by 4′,6-Diamidin-2-phenylindol (DAPI) staining , and slides were mounted using Prolong Diamond Antifade Mountant . Secondary antibody controls were included as negative controls. Images were taken with a fluorescence microscope  and analyzed using CellSense software (Olympus).+ cells. Frequencies of IL-8+ EpCam+ cells were shown as the percentage of total EpCam+ cells. Patients included into the analysis had the following ages (in month) and HSRC presentations : Fiber-low: 3/S-HSCR; 5/L-HSCR; 3/S-HSCR. Fiber-high: 3/S-HSCR; 4/S-HSCR; 3/S-HSCR. CD G (Non-HSCR): 5/anal atresia; 6/anal atresia; 7/anal atresia.For the quantification of IL-8, images were analyzed using Cellprofiler-3.1.9 software. To quantify IL-8 expression in epithelial cells in the two patient groups , 4 to 8 epithelial regions (in average 1500 IEC) per patient from the rectosigmoid were examined. Primary objects were defined by nuclear DAPI staining. Secondary objects were defined by extracellular EpCam expression using the watershed propagation module after applying a global threshold to identify epithelial cells. IL-8-positive cells were identified as a second primary object using Three Classes Otsu thresholding method, and subsequently related to EpCam4-x105 cells/ml at 37°C and 5% CO2). For cytokine induction cells were seeded into 96-well flat-bottom plates . Next day, the medium was exchanged, and cells were stimulated with 100ng/ml lipopolysaccharide  in the presence or absence of agonists and antagonists/inhibitors. Antagonists and inhibitors were added 20min prior to AChR agonists and thus 20min prior to LPS stimulation. The following agonists/antagonists were used: acetylcholine chloride , nicotine , muscarine chloride hydrate , GTS-21 , mecamylamine HCL , alpha-Bungarotoxin , tyrphostin AG490  2, Merck, Darmstadt, Germany), and wortmannin , Merck, Darmstadt, Germany). After 20h, cell-free supernatants were collected for IL-8 analysis and cell viability was analyzed using CellTiter 96 Aqueous One Solution Cell Proliferation Assay . For phospho (p) NFĸB -p65 and RNA extraction cells were stimulated for 4h prior to cell lysis. Supernatants and cell lysates were stored at -80.The human epithelial colorectal adenocarcinoma cell line SW480  was maintained in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% FCS and 1% Penicillin-Streptomycin  under humidifying conditions  containing protease inhibitor cocktail . Patients included into the analysis had the following ages (in month) and HSRC presentations: Fiber-low: 3/S-HSCR; 5/L-HSCR; 3/S-HSCR; 3/S-HSCR; 2/S-HSCR. Fiber-high: 3/S-HSCR; 4/S-HSCR; 3/S-HSCR; 3/S-HSCR; 4/L-HSCR. CD AG: 5/TCA; 11/L-HSCR; 5/TCA; 5/L-HSCR. CD G: 3/S-HSCR; 5/S-HSCR; 2/S-HSCR.IL-8 was determined in cell culture supernatants and IEC lysates using a human IL-8 ELISA Ready-SET-Go Kit  according to the instruction manual. Patient-derived IEC were lysed in Cell Extraction Buffer (5 x 10Phospho (p)-p65 was determined in SW480 cells using a human NFĸB p65 Human InstantOne™ ELISA Kit  according to the instruction manual. ELISA plates were measured using a Synergy H4 Hybrid Reader and GEN5 2.00 software (BioTeK).Data were analyzed using Prism GraphPad 6.0 software. Data are reported as the mean ±standard error of the mean (SEM). Statistical significance was determined using the Kruskal-Wallis test, one-way ANOVA multiple comparison analysis as well as paired t test and considered statistically significant with * p≤ 0.05; ** p≤ 0.01; *** p≤ 0.001 and **** p≤ 0.0001. Unless otherwise noted, figures showed pooled patient data from several independent experiments or a representative of independent repeated experiments tested in triplicates.Neuroimmune Interactions in the Gut) study. In rectosigmoid tissue of non-HSCR patients AChE activity is limited to the myenteric plexus located between the longitudinal and transverse muscle layers   and tubulin+ (arrow) but AChE- nerve fibers in the mucosa and submucosa , undergoing surgery for miscellaneous reasons, from the prospective, multicenter NIG  . Dependi+ fiber density and IEC cytokine production we performed Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) analysis of isolated IEC from surgically removed colonic tissue of HSCR and non-HSCR patients. IEC purity (EpCam+CD45-cells) was confirmed by FACS analysis  and anti-inflammatory cytokines (IL-10 and TGFβ1). In aganglionic rectosigmoid tissues, IEC isolated from fiber-low segments expressed significantly higher IL-8 levels compared to the fiber-high group  of 3 fiber-low and 3 fiber-high patient tissue samples using the Cellprofiler-3.1.9 software. We detected increased frequencies of IL-8-positive IEC in fiber-low compared to fiber-high and DC tissue  and M3 (CHRM3) receptors as well as nicotinic α3 (CHRNA3), α4 (CHRNA4), α5 (CHRNA5) and α7 (CHRNA7) subunits . We perfvia the activation and phosphorylation of the transcription factor subunit nuclear factor ‘kappa light chain enhancer’ of activated B cells (NF-ĸB) p65  p65 , 41. We -ĸB) p65 –48. To iHere, we classified the aganglionic rectosigmoid of HSCR patients dependent on a prominent (fiber-high) or sparse (fiber-low) mucosal cholinergic innervation and showed that the presence of cholinergic innervation is associated with decreased pro-inflammatory IL-8 levels in patient-derived IEC. Muscarinic 1 as well as nicotinic alpha-7 AChRs were upregulated in IEC isolated from aganglionic rectosigmoid compared to IEC isolated from ganglionic control tissue. Using the human IEC cell line SW480, we demonstrated that activation of a7nAChR suppressed LPS-induced IL-8 response involving the JAK2/3 signaling molecule.+ cells of 90% with less than 2% contamination with CD45+ cells. Using qRT-PCR and ELISA analysis we showed elevated expression as well as protein levels in IEC isolated from fiber-low compared to fiber-high aganglionic rectosigmoid. IL-8 immunofluorescence verified the expression in EpCAM+ IEC. We found elevated TNF-α levels in IEC derived from fiber-low compared to aganglionic and ganglionic DC. Comparable expression levels among all groups were detected for IL-10 and TGF-β. Borovikova’s study on the effect of CAIP in LPS-stimulated human macrophages showed that cholinergic signaling preferentially acts on the diminishment of pro-inflammatory cytokines without affecting the anti-inflammatory response  and the Medical Faculty of Heidelberg (S-388/2015). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.Concept and design: SK. Acquisition of data: IM, UK, VG, ST, US, TK, ES, PS, DC, PR, BR, SH-C, SK, and the members of the NIG study group. Analysis and interpretation of data: IM, UK, VG, and SK. Drafting of the manuscript: IM and SK. Revision of the manuscript: all authors. All authors contributed to the article and approved the submitted version.This work was supported by the Stay on Track funding (awarded to SK) and the Research Fund Junior Researcher  from the University of Basel, Switzerland.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Here, we identified point mutations located in the motor domain or the inhibitory CC1 domain, which resulted in gain-of-function alleles of unc-104 that exhibit hyperactive axonal transport and abnormal accumulation of synaptic vesicles. In contrast to the cell body localization of wild type motor, the mutant motors accumulate on neuronal processes. Once on the neuronal process, the mutant motors display dynamic movement similarly to wild type motors. The gain-of-function mutation on the motor domain leads to an active dimeric conformation, releasing the inhibitory CC1 region from the motor domain. Genetically engineered mutations in the motor domain or CC1 of UNC-104, which disrupt the autoinhibitory interface, also led to the gain of function and hyperactivation of axonal transport. Thus, the CC1/motor domain-mediated autoinhibition is crucial for UNC-104/KIF1A-mediated axonal transport in vivo.The UNC-104/KIF1A motor is crucial for axonal transport of synaptic vesicles, but how the UNC-104/KIF1A motor is activated  in vivo is not fully understood. Here, we identified gain-of-function mutations in the motor domain or CC1 domain that significantly enhance the synaptic vesicle transport. Further biochemical and structural analyses revealed that these mutations could disrupt the CC1/motor mediated autoinhibition. Thus, our work provides a mechanistic explanation for the role of some disease-related mutations in motor hyperactivation.UNC-104/KIF1A is the founding member of the kinesin-3 family. When not transporting cargos, most kinesin-3 motors adopt an autoinhibited conformation, and how the UNC-104/KIF1A motor is activated  C. elegans and is the primary kinesin motor for anterograde axonal transport of synaptic vesicle precursors [Neurons are highly specialized for the processing and transmission of cellular signals. Most proteins necessary for synaptic terminals are transported down axon after synthesis in the cell body. UNC-104/KIF1A was originally identified through genetic screens in ecursors . KIF1A-mecursors . In addiecursors . Given tecursors –9.unc-104 mutants have been identified from genetic screens in C. elegans [UNC-104/KIF1A belongs to the kinesin-3 subfamily  and can  elegans ,23. Thes elegans . Intrigu elegans ,24. HoweC. elegans neurons, while the wild type UNC-104 motor is distributed mainly in cell body, the gain-of-function motors tend to accumulate on particular places of neuronal processes. Once on neuronal processes, the mutant motor possesses similar motility as the wild type, suggesting the disruption of motor-domain-mediated autoinhibition results into more activated motors but not hyper-activated motors in vivo.In addition to the CC2-FHA and NC-CC1 interactions, we now revealed that the intramolecular interaction between the motor domain and the CC1 domain is also important for the autoinhibition of UNC-104/KIF1A motor. Here, the extensive genetic analysis based upon CRISPR/Cas9-mediated genome editing demonstrated that mutations disrupting the CC1/motor interactions result in hyper-activated axonal transport. Structure analysis further showed that the gain-of-function mutation on the motor region could cause an active dimeric conformation. Introducing interface-disrupting mutations into the motor domain or CC1 domain also lead to the gain of UNC-104 function in synaptic vesicle transport. In C. elegans, the cell bodies of DD motor neurons are situated on the ventral side, and they send out ventral-to-dorsal commissures to the dorsal nerve cord. After reaching the dorsal cord, each commissure splits into two branches, one extending anteriorly and the other posteriorly. These two branches form evenly distributed synaptic specializations on the dorsal muscles, which can be visualized with the synaptic vesicle marker GFP::RAB-3 driven by the unc-25 promoter [xd53 mutant  phenotype and display synaptic vesicle accumulation in the cell body region of neuronal cells [xd53 animals are healthy with relatively normal locomotion. Furthermore, the synaptic vesicle clusters in xd53 animals are found on the dorsal cord  worms, we further performed a suppressor screen to isolate secondary site mutations which restored the synaptic vesicle distribution. From this screen, the xd359, xd366, and xd475 mutants were isolated (unc-104(xd53 xd359), unc-104(xd53 xd366) and unc-104(xd53 xd475) worms, the even distribution of GFP::RAB-3 puncta on the dorsal cord was largely reestablished, similar to the wild type (unc-104(xd53 xd359)/unc-104(xd53), unc-104(xd53 xd366)/unc-104(xd53) and unc-104(xd53 xd475)/unc-104(xd53) heterozygote animals, the GFP::RAB-3 distribution pattern resembles that in wild-type animals  mutation site, each suppressor contains a secondary mutation in the coding region of the UNC-104 protein: C184Y in xd359, G421E in xd366 and L410F in xd475 ? One possibility is that the point mutations of xd359(C184Y), xd366(G421E) and xd475(L410F) can correct the protein folding deficiency specifically caused by xd53(lf L640F) and therefore suppress the synaptic vesicle distribution defect only in xd53(lf L640F). Alternatively, the suppressor mutations alone could lead to enhanced synaptic vesicle transport. In this case, the synaptic vesicle trafficking deficiency in other unc-104 loss-of-function mutants may also be suppressed by these dominant suppressors. Thus, we introduced the C184Y, G421E or L410F mutation into another loss-of-function allele, unc-104(xd130). The unc-104(xd130) allele was isolated from the same genetic screen in which unc-104(xd53) was identified. The mutated unc-104 gene in unc-104(xd130) encodes an UNC-104 motor containing a G1092E missense mutation in the C-terminal linker region  animals display an uncoordinated (Unc) locomotion phenotype (xd130(lf G1092E), which restored the even distribution of GFP::RAB-3 puncta on the dorsal cord (xd130(lf G1092E) resulted in a redistribution of the synaptic vesicle clusters to the dorsal cord region. We noticed, however, that the GFP::RAB-3 puncta in unc-104 were not evenly spread along the whole dorsal cord but instead tended to localize around the dorsal branching point of DD neurons (xd130(lf G1092E). We further examined the Unc phenotype and found that the unc-104(xd130) locomotion defect was considerably suppressed by the C184Y, L410F or G421E suppressor mutation  encodes an UNC-104 motor containing a D1497N mutation in the C-terminal PH domain, which may reduce the interaction with cargo [e1265 animals, the synaptic vesicles are retained at the cell bodies [unc-104, unc-104 , and unc-104 worms. Intriguingly, the abnormal synaptic vesicle accumulation defect of e1265(lf D1497N) could not be suppressed by xd359(C184Y), xd366(G421E) or xd475(L410F) (xd359(C184Y), xd366(G421E), and xd475(L410F) suppressors may specifically affect the non-cargo-binding aspect of UNC-104 motors. Alternatively, fewer activated motors in vivo might lead to fewer motors binding to cargo and motors not binding to cargo may undergo degradation [xd359(C184Y), xd366(G421E) or xd475(L410F) on xd53 or xd130 might be due to more active motors which lead to more motors being recruited on the cargo.How do the suppressors repress the mutant phenotype of r region . Comparer region , the GFPd region . Meanwhid region . In addihenotype . Using tsal cord . Introdu neurons . Hence, mutation . e1265 ith cargo . In e126l bodies  S3 Fig)unc-104(xradation . In thisxd53(lf L640F) and xd130(lf G1092E)? To assess the transport function of the UNC-104C184Y, UNC-104L410F and UNC-104G421E motors in vivo, we created xd359(C184Y), xd475(L410F) and xd366(G421E) mutant animals utilizing the CRISPR/Cas9 technique. The xd359(C184Y), xd475(L410F) and xd366(G421E) animals are healthy with normal locomotion. In DD motor neurons, we found that the synaptic vesicle clusters were evenly distributed along the dorsal cord in xd359(C184Y), xd366(G421E) and xd475(L410F) mutant animals  devoid of pre-synaptic structures [unc-104(C184Y) and unc-104(L410F) mutant animals, we found that in addition to the synaptic region, ectopic GFP::RAB-3 puncta appeared in the dorsal distal tip region (in vivo [unc-104(G421E) mutant animals did not display apparent ectopic synaptic vesicles at the distal region of either the dorsal or ventral process. We suspected that the G421E mutation has a relatively weak gain-of-function effect on UNC-104. Indeed, while the synaptic vesicle distribution in xd53(lf L640F) or xd130(lf G1092E) was restored or partially restored by G421E, the G421E mutation alone did not significantly augment the axonal transport in DA9 neurons. The length of the synaptic and asynaptic region is not altered by unc-104(C184Y), unc-104(L410F) or unc-104(G421E) unc-25::Gp region  and the p region , which i , which sxd53(lf L640F) mutants, synaptic vesicles abnormally accumulate in the commissure area of DA9 (xd130(lf G1092E) mutants also display ectopic synaptic vesicles on the commissure area and this phenotype could be suppressed by C184Y, L410F or G421E mutation (xd130(lf G1092E). Based on the average number of GFP::RAB-3 puncta, the xd130(lf G1092E) display a much stronger dendritic accumulation defect than unc-104(C184Y) or unc-104(L410F). Introducing the C184Y, L410F or G421E mutation could significantly reduce the synaptic vesicle clusters in the dendrite region in xd130(lf G1092E)  . Further G1092E) .C184Y and UNC-104L410F compared to wild-type UNC-104 . Compared to the wild type, the anterior movement and the cargo segment run length significantly decreased for UNC-104 lf L640F  function could be restored or even strengthened by C184Y mutation.We further created the UNC-104g region , suggest40F::GFP . In addiy region . In geneC184Y mutant motor is accumulated, we introduced the UNC-104C184Y::GFP into the neuronal marker, specifically labelling PLM neuron [en passant chemical synapses with neurons in the ventral nerve cord. We found that the UNC-104C184Y::GFP generally lacked in the PLM cell body, compared to wild type UNC-104::GFP  on the anterior process of PLN neuron , xd475(L410F) or xd366(G421E)  , further2+) and NC, but not of CC1, possibly due to the intrinsic flexibility of CC1 upon dissociation from the motor domain  mutant using the CRISPR/Cas9 technique. The L428 is located in the CC1 region and corresponding to the L415 site on KIF13B (unc-104(L428A) mutant animals, ectopic synaptic vesicles appear in the distal tip and dendrite region of DA9  mutant gene encodes an UNC-104 motor containing the L170Q mutation and the unc-104(xd407) allele encodes an UNC-104 motor containing the K427A mutation. To test whether xd408(L170Q) and xd407(K427A) behave as gain-of-function alleles of unc-104, we performed the following experiments. Firstly, we examined the synaptic vesicle distribution in DA9 neurons. We found that both the L170Q and K427A mutations lead to ectopic GFP::RAB-3 puncta appearing in the tip and dendritic region of DA9 neurons  and unc-104 mutant animals using CRISPR/Cas9 genome editing. We found that the uneven distribution of DD synaptic vesicle clusters in unc-104(lf 640F) could be rescued by L170Q or K427A mutation (arl-8 and xd408(L170Q) or xd407(K427A). We found that the synaptic vesicle transport defect of arl-8 in DA9 neurons could be efficiently suppressed by xd408(L170Q) or xd407(K427A) mutant (unc-104(lf 640F) mutant DA9 could be suppressed by L170Q or K427A mutation  may also be involved in the direct autoinhibitory contact between the motor domain and CC1 of UNC-104/KIF1A Figs  and 7A.  neurons . We furt animals . Secondlmutation . Thirdly) mutant . Furthermutation . Taken tunc-104. Combined with crystal structure and in vitro ATPase activity measurements, we further revealed that the CC1/motor intramolecular interaction could mediate the autoinhibition of UNC-104/KIF1A. Unlike the transgenic overexpression of UNC-104, we also showed that the endogenous wild type UNC-104 motors are diffusely distributed in cell bodies and neuronal processes. In contrast, gain-of-function motors are generally lacking from cell bodies. Instead, they accumulate on neuronal processes, particularly at the neuronal terminals.Till now, two kinds of intramolecular interactions could mediate UNC104/KIF1A autoinhibition. One is the intramolecular interaction between CC2 and FHA domains, another is NC-CC1 interaction . Here, wC. elegans three decades ago [unc-104. The xd53(L640F) mutation resides in CC2, while the xd130(G1092E) mutation is located in the flexible linker region between CC3 and the C-terminal PH domain. Further modifier screens identified that disrupting the CC1/motor mediated autoinhibition could suppress the reduction of UNC-104 activity caused by different mutations (L640F and G1092E) implying that the uninhibited state of the MD-NC-CC1 region is sufficient to drive UNC-104/KIF1A motor activation. Consistent with this assumption, the structural analysis demonstrated that the T192Y-MD-NC-CC1 mutant adopts an active dimeric conformation  could not be suppressed by gain-of-function mutation C184Y, G421E or L410F. This observation supports the notion that cargo binding is a prerequisite for the cargo transport activity of a UNC-104 motor. Noticeably, two intragenic suppressors of the mutation in the PH domain have been identified. It appears that while loss of cargo binding reduces motor levels, these two suppressors could restore the UNC-104 level and cargo binding [Genetic screens have been widely used to explore the molecular, cellular and developmental mechanisms underlying various biological processes. In fact, UNC-104/KIF1A, the axonal transport kinesin for synaptic vesicles and the founding member of the kinesin-3 family, was originally identified through a locomotion behavior screen in ormation . The los binding . It willC184Y tends to accumulate on neuron processes. In PLM neurons, the UNC-104C184Y motor is enriched at the terminal region of the anterior branch but not the posterior end of PLM. Our previous studies demonstrated that actin is concentrated at the terminal region of the anterior PLM process, in which the PLM forms gap junctions with interneuron BDU [C. elegans neurons [C184Y::GFP dots does not resemble the distribution pattern of gap junction connections. For PLN neuron, it is completely unclear whether any special cytoskeleton arrangement exists in the two places on which the UNC-104C184Y::GFP is accumulated. Hence, why UNC-104C184Y motor specifically accumulates on above positions and whether the abnormal accumulation leads to any functional consequence of C. elegans neurons remain to be determined. Considering the synaptic vesicle transport is enhanced in unc-104(C184Y) mutants and the cell-body-distribution of UNC-104C184Y motors is greatly reduced, it is a reasonable proposal that the C184Y mutation could lead to more motors in an activated state.The gain-of-function motor UNC-104uron BDU . Gap jun neurons . Howeverunc-104 that caused hyperactive axonal transport of synaptic vesicles  mutant animals display typical gain-of-function features of unc-104 and subcellular localization of UNC-104(L428A)::GFP resembles C184Y::GFP. Furthermore, when two new mutations in the UNC-104 motor domain or CC1 predicted to disrupt the inter-domain autoinhibitory interface were introduced into UNC-104 motor, the point mutations resulted in gain of UNC-104 function and hyperactive axonal transport in vivo , xd359(C184Y), xd475(L410F), xd366(G421E), xd407(K427A), xd496(L428A), xd53(lfL640F), xd130(lfG1092E), e1265(lf D1497N), xd53 xd408, xd53 xd359, xd53 xd475 , xd53 xd366 , xd53 xd407,xd130 xd359 , xd130 xd475, xd130 xd366 , xd500 xd359, xd501 xd475, xd502 xd366, xdKi3(unc-104::gfp knock-in), xdKi6(C184Y::gfp knock-in), xdKi60(L428A::gfp knock-in),xdKi4(lf L640F::gfp knock-in), and xdKi61. Other strains are: xdIs164(Punc-25::GFP::RAB-3),xdEx968 (Pmec-7::mCherry) and xdEx2678 (Plad-2::mCherry). wyIs85(Pitr-1::GFP::RAB-3) was provided by Kang Shen (Stanford University). The unc-104 (xd53) and unc-104 (xd130) worms were isolated from xdIs164 worms treated with EMS. Thexd53 xd359, xd53 xd475 , xd53 xd366  worms were obtained from an EMS-based genetic screen usingunc-104(xd53);xdIs64 strain. Briefly, the distribution of synaptic vesicles in F2 progeny was examined under a fluorescence microscope, and suppressor mutant animals were recovered to produce progeny. A total of 7,200 mutagenized haploid genomes were screened and total eight suppressors were isolated from this screen.escribed . Mutantsunc-104cDNAs were amplified by PCR and cloned into dpSM vectors . psc325 vector contains the unc-25 promoter. Transgenic animals were made by standard microinjection procedures. In general, plasmid DNAs of interest were used at 1–50 ng/μl, and the co-injection marker was Pmyo-2::RFP or Podr-1::RFP or Pord-1::GFP. All transgenic strains were crossed to N2 wild type 4 times before use. The CRISPR/Cas9-mediated genome editing strains were generated by SunyBiotech and were verified with DNA sequencing. All strains were outcrossed with N2 for 4 times before use. The detailed information is included in Animals were mounted on 2.5% agar pads in M9 buffer containing 1% 1-phenoxy-2-propanoland. Fluorescence photographs were taken using a Leica SP8 confocal microscope unless specifically indicated. The GFP::RAB-3 fluorescence images of DD/VD neurons were taken at the young adult stage. The GFP::RAB-3 fluorescence images of the DA9 neuron were taken at the first day of adulthood. Straightened dorsal cord images were obtained using ImageJ software. The lengths of the synaptic region and the asynaptic region were measured by ImageJ. For each genotype, more than 20 worms were imaged.Time-lapse images of GFP::RAB-3 were taken at the L4 stage, using an inverted Zeiss Axion Observer Z1 microscope equipped with a Plan-Apochromat 63x/1.4 objective, an Andor EM-CCD digital camera, and a Yokogawa CSU-X1 spinning disk system. 201 frames were taken for each movie with a speed of 6 frames per second and an exposure time of 160 ms. For movie acquisition, worms were mounted on 2.5% agar pads and anesthetized with 1-phenoxy-2-propanol. Movies were analyzed using ImageJ. The “register” plugin was used for movie correction. Subsequently, “segmented line” was used to select the region for kymograph analysis. Finally, kymographs were constructed with the “kymograph” plugin. For each genotype, 30 worms were imaged.Each contiguous line with a constant slope was scored as a single transportevent and its run length  were calculated. Run length is defined as a singlemotility occurrence typically right after a pause or a reversal and,vice versa, ends when the motor is again pausing or reversing.The FRAP experiments were performed on the young adult animals using a laser scanning confocal microscope  equipped with a Plan-Apochromat 63x/1.4 NA oil immersion. For bleaching, worms were mounted on 2.5% agar pads and anesthetized with 1-phenoxy-2-propanol. Photobleaching, image acquisition, and image analysis were performed on ZEN system. Image acquisition was conducted for regions with 792 x 792 pixels (0.09μm/pixel) using 488-nm semiconductor lasers at 10% power with the pinhole diameter 56μm. Prior to the bleaching, 3 frames of images were acquired. Photobleaching was performed on a rectangle region with size 55 x 22 pixels using 488-nm lasers at 20% power for 250ms. After photobleaching, images were collected till no further recovery was detected. The fluorescence recovery of the bleached region was calculated as follows. Firstly, average fluorescence intensities within the bleached region and non-bleached regions  were measured. Second, the lowest intensity value (immediately after bleaching) within the photo bleaching region was subtracted from all measurements. The average intensities of the 3 measurements that precede the bleaching were then determined, establishing a pre-bleach value; all intensities were normalized by dividing by that value. The halftime of recovery is the time point where the fluorescence recovered to half of the maximum recovery.post hoc test (the test used is stated in each figure legend). * P<0.05; ** P<0.01; *** P<0.001.To compare multiple groups, one-way ANOVA was used with an appropriate multiple comparisons Worms were assessed for Unc phenotypes and locomotion defects by examining their motility on NGM plates. Young adult worms were transferred to new plates and movement was recorded under a dissection microscope (Nikon) equipped with a HQ CCD camera (Aoka).For velocity assay, movements were recorded at 1–2 frames/s. The obtained movements were tracked using a custom programmed ImageJ’s “multitracker” plugin.Young adult worms were cryofixed on a Leica Microsystems HPM 100 and frozen in liquid nitrogen. After high-pressure freezing, the samples were washed 4 times in acetone and stained with 1% uranyl acetate for 1 hr. The infiltration was performed with increasing concentrations of Embed 812. Finally, samples were embedded in fresh resin at 60°C for 3 days. Thin sections (60 nm) were produced with a diamond knife (Diatome) on an ultramicrotome . Serial sections were pictured with a JEM-1400 electron microscope at 80 kV. Pictures were recorded using a Gatan832 4k × 2.7k CCD camera.Worms were collected and washed in M9 buffer. Lysis buffer  was added and worms were disrupted with a Dounce homogenizer  on ice for 15 min. Debris was removed by centrifuging at 12000 rpm for 15 minutes at 4°C. For western blotting, protein samples were boiled and separated in an 8% SDS-PAGE gel. After transfer, the nitrocellulose membranes  were incubated with 5% dry milk and probed with an anti-GFP antibody . The signals were visualized with a Supersignal West Pico Substrate kit (Pierce). The stained bands were analyzed by ImageJ Software. Graphical data are presented as mean ± SEM. n = 3 independent experiments.6-tag. Point mutations were introduced using the standard PCR-based mutagenesis method and confirmed by DNA sequencing. Recombinant proteins were expressed in Escherichia coli BL21 (DE3) host cells at 16°C. The His6-tagged fusion proteins were purified by Ni2+-Sepharose 6 Fast Flow  affinity chromatography followed by size-exclusion chromatography  in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, 1 mM DTT. For analytical gel-filtration analysis, protein samples were concentrated to ~2.5 mg/ml and loaded onto Superdex-200 10/300 GL columns .DNA sequences encoding rat KIF13B MD-NC-CC1 and various mutants were cloned individually into the pET32a vector with a carboxyl HisMeasurements of the microtubule-stimulated ATPase activities of KIF13B MD-NC-CC1 and various mutants were performed using the HTS Kinesin ATPase Endpoint Assay Biochem Kit . Briefly, all the measurements were based on the malachite green phosphate assay to probe inorganic phosphate generated during the reaction. A standard curve of phosphate was made to estimate the amount of phosphate generated. The basal ATPase activities (without microtubules) of these KIF13B fragments were also measured using the same method. Each protein sample had three replicates and each measurement was repeated at least three times independently. The kinesin-1 heavy chain (KHC) supplied in the Kit was used as the control.2Fo-Fc and Fo-Fc electron density maps using COOT [Crystals of the KIF13B T192Y-MD-NC-CC1 mutant (~25 mg/ml) were obtained in 0.1 M Bis-Tris propane, pH 8.2, 30% (w/v) PEG 20000 using the sitting drop vapor diffusion method at 16°C. Before being flash-frozen in liquid nitrogen, crystals were soaked in the mother liquor supplemented with 15% (v/v) ethylene glycol for cryo-protection. The diffraction datasets were collected at the beamline BL17U1 of the Shanghai Synchrotron Radiation Facility with a wavelength of 0.989 Å at 100K . Dataseting COOT . The string COOT . The staS1 Figwt) and unc-104(xd53). White arrows indicate DD neurons. White brackets indicate regions lacking GFP::RAB-3 puncta. The schematic drawings on the right show the synaptic vesicle distribution in DD neurons. (C and D) EM images of dorsal synapses in wt and unc-104(xd53). M, muscle. White arrows indicate active zones. Scale bar represents200 nm. (E-H) The distribution of Punc-25::GFP::RAB-3 (green) puncta in wt, unc-104, unc-104, and unc-104 worms. Scale bar represents 50 μm.(A and B) The distribution of GFP::RAB-3 puncta (green) in wild type ((TIF)Click here for additional data file.S2 Figunc-104(lfG1092E), unc-104, unc-104, and unc-104 worms. Dashed circles indicate the spots on which 15 worms of each genotype were placed. Scale barrepresents1 mm. (E) Quantification of the velocity in various genotypes. ***P<0.001, one-way ANOVA with Tamhane’s T2 test. Mean ± SEM, N> = 15 worms for each genotype.(A-D) Snapshots were taken at 10 s, 5 min and 20 min of (TIF)Click here for additional data file.S3 Figxd359(C184Y), xd475(L410F), xd366(G421E) and e1265(lf D1497N). xd359, xd475 and xd366 are indicated. (B) The even distribution of GFP::RAB-3 puncta on the dorsal cord is not restored in unc-104, unc-104 and unc-104 animals.(A) Schematic drawing of the domain organization of UNC-104 motor protein. The mutation sites of (TIF)Click here for additional data file.S4 Figunc-25 in xd359 (C184Y) (A), xd475 (L410F) (B) and xd366 (G421E) (C). Scale bar represents 50 μm. (D) The schematic drawing shows the synaptic vesicle distribution of DD neurons. (E-F) The abnormal synaptic accumulation defect in unc-104(lf L640F) could be suppressed by C184Y (G) or G421E (H) mutation on UNC-104. (I)Quantification of the misaccumulated GFP::RAB-3 puncta in the asynaptic region and commissure region. (J) Quantification of the length of the asynaptic region. **P<0.01; NS, not significant. One-way ANOVA with Tamhane’s T2 test. Mean ± SEM, N> = 20 worms for each genotype.(A-C) The distribution of GFP::RAB-3 puncta (green) driven by P(TIF)Click here for additional data file.S5 FigC184Y motors. (B) Quantification of the expression level of the motor proteins in (A). Mean ± SEM, two-tailed paired Student’s t test. NS, not significant. (C) Quantification of the percentage of the worms with GFP accumulation on the anterior tip region of PLM neuron. Mean ± SEM, two-tailed unpaired Student’s t test. Total 300 worms were examined for each genotype. N = 6. (D) Quantification of the percentage of the worms with normal PLM morphology. Mean ± SEM, two-tailed unpaired Student’s t test. About 130 worms were examined. N = 3. (E) Quantification of the percentage of the worms with normal synapse formation. Mean ± SEM, two-tailed unpaired Student’s t test. 250 worms were examined. N = 5. ***P<0.001; NS, not significant.(A) Western blot of worm lysates probed with a GFP antibody to detect the expression level of wild-type and UNC-104(TIF)Click here for additional data file.S6 Fig2+ (contoured at 1.5σ level).(A) Analytical gel-filtration analysis of wild type and various mutants of the MD-NC-CC1 region of KIF13B . (B) Microtubule-stimulated ATPase activity of the UNC-104 MD-NC-CC1 fragment containing the C184Y, L410F, G421E, or L428A mutation. Bars represent Mean ± SD. n = 4 independent experiments, * P<0.05, one-way ANOVA with LSD test. (C) Structure of the T192Y-MD-CC1 mutant fragment of KIF13B. The inset shows the omit electron-density maps of ADP and Mg(TIF)Click here for additional data file.S7 Figarl-8, arl-8;unc-104(C184Y),arl-8;unc-104(L410F)and arl-8;unc-104(G421E) worms. The synaptic region is shown on the top. Scale bar represents 25 μm. (F) Quantification of the misaccumulated GFP::RAB-3 puncta in the asynaptic region and commissure region. (G) Quantification of the length of the asynaptic region. ***P<0.001, one-way ANOVA with Tukey test. Mean ± SEM, N> = 20 worms for each genotype.(A-E) The GFP::RAB-3 puncta distribution in wild type, (TIF)Click here for additional data file.S8 Figwt) unc-104(L428A). Time and length are on the y axis and x axis, respectively. (B) The number of stable GFP::RAB-3 particles in a 1-μm section within 1 min. (C) The number of anterograde transport events. (D) The ratio of anterograde transport events to retrograde transport events. **P<0.01, ***P<0.001. Mean ± SEM, two-tailed paired Student’s t test. N = 30 worms for each genotype. (E-H) The even distribution of GFP::RAB-3 (driven by Punc-25 promotor) puncta on the dorsal cord is restored in unc-104 and unc-104 animals. (I-L) The synaptic vesicle transport defect of arl-8 mutants could be suppressed L170Q or K427A mutation (M) Quantification of the misaccumulated GFP::RAB-3 puncta in the asynaptic region and commissure region. The synaptic region is shown on the top. Scale bar represents 25 μm. (N) Quantification of the length of the asynaptic region. ***P<0.001, one-way ANOVA with Tamhane’s T2 test. Mean ± SEM, N> = 20 worms for each genotype.(A) Representative kymograph images showing transport events in wild type ((TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


PIK3CA mutations. METHODS/STUDY POPULATION: Non-canonical PIK3CA mutant constructs generated via site-directed mutagenesis are subcloned into doxycycline-inducible vector pLVX-Puro. Serum-dependent HNSCC cell line (PCI-52-SD1) is then stably transfected with vectors and undergo doxycycline-induction. Cell survival is determined by depriving cells of fetal bovine serum for 72 hours and quantifying remaining cells with 3--2,5-diphenyltetrazolium bromide (MTT) assays. Cell proliferation and migration is evaluated with colony formation assays and transwell assays respectively. RESULTS/ANTICIPATED RESULTS: To date, the survival behavior of eight non-canonical mutants was assessed. Three mutants – Q75E, V71I, and E970K – exhibited 18.7-26.7% greater survival rate relative to cells transfected with wild-type. Five mutants – R519G, Y606C, W328S, C905S, and M1040I – demonstrated survival rates that differed only by −4.3% to +6.6% relative to wild-type. We hypothesize the three activating mutants that exhibited increased survival will also demonstrate increased cell proliferation and migratory behavior whereas the three neutral mutants will not differ from control. DISCUSSION/SIGNIFICANCE OF IMPACT: Ongoing HNSCC PI3K inhibitor trials could be more effective if all PIK3CA hyperactivation mutations are known. Identifying non-canonical mutation effects could result in greater efficacy if drugs are restricted only to those with activating mutations. CONFLICT OF INTEREST DESCRIPTION: JRG and DEJ are co-inventors of cyclic STAT3 decoy and have financial interests in STAT3 Therapeutics, Inc. STAT3 Therapeutics, Inc. holds an interest in a cyclic STAT3 decoy oligonucleotide. The remaining authors declare no conflicts.OBJECTIVES/GOALS: To characterize the oncogenic potential of HNSCC cell lines harboring 17 non-canonical


Immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptors (IRs) enable discrimination between self- and non-self molecules on the surface of host target cells. In this regard, they have a vital role in self-tolerance through binding and activating intracellular tyrosine phosphatases which can inhibit cellular activation. Yet, self-MHC class I (MHC I)-specific IRs are versatile in that they can also positively impact lymphocyte functionality, as exemplified by their role in natural killer (NK) cell education, often referred to as ’licensing‘. Recent discoveries using defined mouse models of cytomegalovirus (CMV) infection have revealed that select self-MHC I IRs can increase NK cell antiviral defenses as well, whereas other licensing IRs cannot, or instead impede virus-specific NK responses for reasons that remain poorly understood. This review highlights a role for self-MHC I ‘licensing’ IRs in antiviral immunity, especially in the context of CMV infection, their impact on virus-specific NK cells during acute infection, and their potential to affect viral pathogenesis and disease.  NK cells are essential mediators of host defense against viruses and tumors ,2. PatieNK cell effector activities are regulated by a diverse array of germline-encoded activation and immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptors (IRs) . Many NKIn addition to their negative regulatory role, IRs can dynamically increase NK cell functionality when they encounter and bind self-MHC I ligands ,11. ThisMany viruses employ a variety of strategic mechanisms to manipulate MHC I and MHC I-like molecules to evade CD8+ T cell and NK cell immunity . EndThough well known for their role in self-tolerance through dominant inhibition of cellular activation, self-MHC I-specific IRs help to educate NK cells, thereby maintaining or enhancing effector functions as well ,20,21. CAlthough a mechanistic basis for licensing remains to be uncovered, several studies have advanced our understanding of the requirements for IR-dependent tuning of NK cell reactivity. A functional IR ITIM domain is essential to bind and activate SH2 domain-containing phosphatases , and to SHP-1 activity is required in licensing since SHP-1-deficient NK cells fail to reject MHC I-deficient tumors and poorly respond to activation receptor crosslinking ,37. InteITIM signals drive phosphatase activity and also enhance the functionality of licensed NK cells. One explanation that could account for these seemingly paradoxical observations is that ITIM-regulated signaling molecules are spatially and temporally poised to promote cellular activation during release of inhibition. In this regard, an IR SHP-1 signaling axis leading to Crk phosphorylation may be an important mechanism by which self-MHC I-specific NK cell IRs modulate the movement and spatial distribution of activation receptors in the NK cell plasma membrane ,26,27,42KIR (iKIR)/HLA gene pairs have been associated with disease outcomes in several human chronic viral infections, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), and CMV [The effects of IR-dependent tuning of NK cell functionality described above likely regulate the quality and magnitude of NK cell-mediated antiviral immunity in both humans and mice. Moreover, human genetic variation within these diverse NK cell recognition receptors and their polymorphic MHC I ligands may influence the immune response to particular pathogens. Indeed, discrete inhibitory  and CMV ,45.iKIR/HLA gene pairs in antiviral immunity. Notably, the HIV-encoded immunoevasin Nef selectively downregulates HLA-A and HLA-B cell surface expression to evade T cell recognition [Genetic association studies of individuals with HIV infection were among the first to identify a role for ognition . HIV-infognition . Howeverognition . These fKIR3DL1 allotypes. High KIR3DL1 cell surface expression in combination with HLA-B*57 bearing a Bw4 motif with Ile at position 80 (Bw4-80I) corresponds to delayed progression to acquired immune deficiency syndrome (AIDS) in comparison to patients lacking HLA-B*57 [KIR3DL1 and HLA-B allotypes exhibit higher reactivity against autologous HIV-infected CD4+ T cells in vitro relative to licensed NK cells encoding low receptor/ligand surface expression KIR3DL1/HLA-B allotypes [Indeed, HIV-infected patient outcomes can be related to HLA-B-binding HLA-B*57 . Relatedllotypes . Thus, allotypes , greaterKIR3DL1  allele variant was shown to be significantly associated with elite virus control in HIV-infected HLA-B*57+ patients [Beyond surface expression differences, KIR3DL1 structural polymorphism may also regulate NK functionality through binding or clustering interactions with HLA-B. KIR3DL1 alleles differing in affinity or specificity for HLA-B were shown to differently affect NK cytotoxicity towards HLA-Bw4+ target cells . Moreovepatients . HIV serpatients . Hence, Whereas Nef targets HLA-A and HLA-B to subvert T cell recognition, the HIV-encoded Vpu immunoevasin specifically mediates HLA-C downregulation and limits in vitro HLA-C restricted T cell responses to HIV-infected CD4+ T cells . In suppiKIR/HLA gene pairs and disease outcome have also been observed in hepatitis C virus (HCV) infection. Homozygosity for certain KIR2DL3 and HLA-C1 allotypes is associated with resolution of HCV infection in a cohort of individuals exposed to low infectious doses [KIR3DL1 and an HLA-Bw4 allele with threonine at position 80 (HLA-Bw4-80T) is significantly enriched in seropositive-HCV RNA-negative individuals in comparison to seropositive-HCV RNA-positive individuals [Associations between us doses . A separividuals . Intriguividuals ,58. Moreividuals . While aSimilar to HIV and HCV, herpesviruses such as CMV have evolved an arsenal of immunoevasins to evade detection by cytolytic effector cells of the immune system, namely CD8+ T cells and NK cells. Several of the proteins employed by herpesviruses regulate cell surface expression of MHC I or MHC I-like molecules in infected cells, which underscores the importance of the IR/MHC I recognition axis for control of viral spread ,60. DespA direct role for NK cells in antiviral immunity to CMV is supported by a study showing that a T-B+NK+ severe combined immunodeficiency (SCID) patient could control CMV infection in the absence of T cell immunity . NotablyKIR2DL3/2DS2+/HLA-C1+ CMV seronegative solid organ transplant (SOT) recipients receiving organs from CMV seropositive HLA-C1+ donors experience significantly decreased hazard of CMV viremia [HLA-C1 expression in KIR2DL3/2DS2+ SOT recipients or donors is associated with increased risk of CMV viremia [The significance of the expansion of licensed NK cells in response to CMV remains unclear. Certain licensed iKIR+ NK cell subsets may be more responsive to CMV reactivation, enabling efficient control of CMV spread. In support of this possibility,  viremia . Notably viremia , suggestTaken together, the above findings suggest self-specific iKIR/HLA interactions may endow NK cells with increased responsiveness during HIV, HCV, or CMV infection, perhaps by enabling detection of virus-modified self-MHC I. Additional studies are needed to define whether particular iKIR/HLA gene pairs also affect disease risk associations in other viral infections, especially for viruses known to modify MHC I expression or conformation. In this regard, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-encoded immunoevasin ORF8 was recently shown to re-direct MHC I to lysosomes for degradation .IRs that can efficiently distinguish self-MHC I from virus-induced self-MHC I alterations or virus-encoded MHC I mimics may enable more effective NK cell responses and viral clearance. Herein, we highlight murine CMV (MCMV) infection studies in mouse models demonstrating the importance of self-MHC I-specific IRs in antiviral immunity. These studies reveal a combined effect of polymorphism in both MHC I and Ly49 receptors on NK cell antiviral immunity which hints that similar mechanisms may underlie iKIR/HLA associations with human disease.k protein found that NK cells expressing the Dk-specific-Ly49G2 inhibitory receptor undergo selective expansion during acute MCMV infection [k) and Ly49G2 IR alleles. Both Dk-binding MA/My (G2M) and C57L (G2L) Ly49G2 IRs can license NK cells and enable selective NK expansion in Dk-bearing host mice during MCMV infection [B6) IR which does not bind Dk cannot [k mice before infection thus abolishes MCMV control [Ly49 haplotypes lack a Ly49h gene, MHC I-dependent MCMV resistance in these strains is fundamentally distinct from NK-mediated MCMV control in B6 mice.Prior work using MA/My or C57L-derived mouse strains expressing the MHC I Dnfection ,68,69. Tnfection ,70,71, wk cannot . Depleti control ,68,69,73L’s role in MCMV immunity, we crossed the C57L NKC (NKCL) and Dk onto the B6 background. We further used CRISPR/Cas9 editing to ablate Ly49G2 from B6.NKCL-Dk mice, generating Ly49G2null mice. We discovered G2null mice exhibit significantly increased mortality during MCMV infection, despite an otherwise intact Ly49 repertoire and normal NK cell development [null NK activation, differentiation, or proliferation in comparison to wild-type NK cells thus suggests G2L recognition of MCMV targets is vital to deliver highly efficient antiviral immunity.To further elucidate G2elopment . MCMV coB6 or Ly49AB6 IRs can likewise enhance NK-mediated antiviral immunity in Dd-transgenic B6 mice during MCMV infection [d, license NK cells, and promote rejection of missing-self targets [Parikh et al. have since shown the G2nfection . Both IR targets ,33,75,76 targets , suggest targets ,68. PresL, G2B6, or Ly49AB6 self-specific IRs in NK-mediated MCMV control suggests each may have a key role in recognition of viral targets, possibly via detection of missing-self MHC I MCMV targets. However, this model does not readily explain why NK cells bearing other Dk-specific .A requirement for G2 Ly49O/V ) or lice Ly49O/V ) IRs in b-specific Ly49C IR was shown to impede virus-specific NK cell immunity driven by the Ly49H activation receptor [b-licensed Ly49C+ NK cells exhibit high basal reactivity at steady-state, efficiently reject missing-self MHC I targets [b is efficiently downregulated by viral immunoevasins gp40 and gp48 in MCMV-infected cells [b [b  is unknown. Nonetheless, polymorphic IRs with differing affinities or specificities for MHC I thus may differently recognize virus-induced changes in cognate self-MHC I expression or conformation on infected targets.Unlike the self-MHC I licensing IRs described above, the H-2Kreceptor , which breceptor . This re targets ,78, and ed cells ,82. Whetcells [b , its brocells [b , or bindd) mice can control an attenuated MCMV strain lacking the m04 gene which encodes gp34 [A clue to understanding their role in MCMV immunity was revealed in a study from Babić et al. showing that licensed IR+ NK cells in BALB/c . Transplant recipients frequently undergo pharmacological immunosuppression to prevent complications associated with transplant rejection such as graft versus host disease . Howeverk expression in both hematopoietic and non-hematopoietic cells is required for optimal NK cell stimulatory activity, rejection of missing-self targets, as well as efficient control of MCMV infection [d-transgenic NKCB6 mice [Evaluation of reconstituted Ly49G2+ NK cells in radiation bone marrow chimeras demonstrated that Dnfection ,73. In snfection . Both heCB6 mice ,88. SeleCB6 mice ,88. Furtd tetramers [b molecules modify the strength of Ly49C inhibitory signals [Whether self-specific IRs recognize missing- or altered-self MHC I, the signal-driving mediators of MHC I-dependent NK cell antiviral immunity are less well defined. Many types of viruses target MHC I antigen processing and presentation pathways to reduce presentation of viral antigens . Yet, ceetramers . Similar signals . In huma signals . Likewis signals . Self-MHSelf-specific KIR activation receptors have similarly been shown to exhibit selectivity for pathogen-derived peptides. For example, KIR2DS2 discriminates HLA-C-presented flaviviral peptides . Moreovetrans) and on the same cell (in cis), when NK cells encounter healthy cells an equilibrium  describing IR—self-MHC I interactions thus exists  that dominantly turn off activation receptor signals and prevent NK effector functions. A shift in this equilibrium so that cis binding prevails upon encountering missing-self MHC I targets, however, results in reduced IR access to the immune synapse in trans [Because Ly49 IRs bind self-MHC I on targets (in iewed in ; and seein trans , less inin trans , and incin trans  that alsk itself [k or another MCMV-modified Dk ligand. Still, the Ly49R-specific mAb 12A8 given to mice before infection abolishes Dk-licensed Ly49G2+ NK antiviral immunity during MCMV infection [Prior studies suggest Ly49P and Ly49R activation receptors contribute to MCMV control in MA/My mice, whereas the Ly49L activation receptor is linked with MCMV control in BALB.K mice ,100,101.k itself ,77, it infection . Activatnfection . Nonethenfection ,87,88,89d-transgenic B6 mice [L NK cells in MCMV-infected Dk mice by blocking an interaction between the activation receptor and its ligand on infected targets, this treatment might instead alter the cis trans equilibrium for G2 binding with Dk described in k proteins in cis and trans [Whereas blockade of the NKG2D activation receptor abrogates licensed NK cell immunity in BALB/c or BALB.K mice infected with attenuated Δm04/gp34 MCMV , license B6 mice . Becausend trans . Usage oThere is evidence to suggest that similar mechanisms of IR-licensed or Ly49H-like NK cell activation occur in response to viral infection in humans. IR-licensed human NK cells coexpressing the CD94/NKG2C heterodimeric activation receptor clonally expand in CMV-seropositive individuals . NotablyCMV UL40 peptides were shown to elicit potent degranulation and cytokine production by NKC2C+ ‘adaptive’ NK cells derived from CMV-seropositive patients . In vitrIntriguingly, enhanced proliferation of NKG2C+ NK cells in response to disparate UL40 peptides coincides with increased accumulation of NKG2C+ NK cells co-expressing self-specific iKIR . HoweveriKIR/HLA class I associations in human patients with chronic virus infections may likewise be related to highly efficient NK sensing via licensing IRs. Given their role in self-tolerance, we envision licensed IR-dependent antiviral immunity might also promote adaptive immunity through better overall protection of host cells, lymphoid structures, antigen presenting cells and the development of highly specific T cells and B cells. Resolving these questions may facilitate tailor-made strategies to enhance NK cell cytotoxicity against specific viruses and tumors which downregulate MHC I molecules to evade T cell immunity.With the seminal discovery that select IRs confer essential MCMV control, now there is evidence that licensed NK cells play a key role in antiviral immunity, though not all IRs are created equal. This distinction is almost certainly linked to IR polymorphism which affects MHC I protein binding and IR-dependent tuning of licensed NK cell effector functions, including functions conserved in mice and humans. Nonetheless, it remains unknown why only certain licensing IRs adequately respond to missing-self MHC I cues during wild-type or attenuated MCMV infection and contribute to viral control. Whether this ability is related to a particular IR’s affinity for MHC I, its ability to license or tune NK effector functions, or its capacity to efficiently detect altered-self MHC I proteins remains an open question. Notwithstanding, multiple virus-responsive activation receptors are rendered impotent in the absence of these vital licensing IRs during MCMV infection. These findings are suggestive that


Here, we report the characterization of Pgam1 knockout mice. We observed that homozygous knockout mice of Pgam1 were embryonic lethal. Although we previously reported that both PGAM-1 and -2 affect global glycolytic profile of cancers in vitro, in vivo glucose parameters were less affected both in the heterozygous knockout of Pgam1 and in Pgam2 transgenic mice. Thus, the impact of PGAM on in vivo glucose metabolism is rather complex than expected before.Glycolytic metabolism is closely involved in physiological homeostasis and pathophysiological states. Among glycolytic enzymes, phosphoglycerate mutase (PGAM) has been reported to exert certain physiological role  Glucose is utilized as energy source, from bacterias to higher eukaryotes. Glycolysis provides energy supply in the form of ATP through oxidation of carbon atoms in glucose. As it also supports the synthesis of macromolecules , glycolyDysregulation in glycolysis, either the pathological enhancement or impairment, is closely related to human disease states. For example, most of cancer cells display enhanced glycolytic feature, known as the Warburg effect. On the other hand, impaired glycolysis is much involved in dysfunction in various tissues and degenerative disorders ,3. In huin vivo. However, both isoforms display the significant similarity in their sequences (79% identity) and enzymatic activities in vitro [Glycolytic enzyme phosphoglycerate mutase (PGAM) converts 3-phosphoglycerate into 2-phosphoglycerate as an isomerase . Two isoin vitro –13.Besides its role in glycolysis, PGAM would play a key role connecting glycolysis to physiological homeostasis. PGAM is post-translationally regulated by several modification, including the phosphorylation, acetylation and ubiquitination by environmental stimuli or under stress conditions –15. Morein vivo is still unclear. Here we characterized the profiles of glucose metabolisms in mice with ablation of Pgam1 or global overexpression of Pgam2 in vivo. Homozygous knockout of Pgam1 is embryonic lethal, while its global heterozygous knockout mice displayed comparable glucose parameters in vivo, compared to those in wild type. Moreover, global overexpression of Pgam2 unlikely affects the in vivo profile for glucose metabolisms. Thus, the impact of PGAM on in vivo glucose metabolism is rather complex than expected before.However, the global impacts of PGAM All procedures for animal experiments were approved and conducted in accordance with the principles and guidelines of the Animal Care and Use Committees of Kyoto University Graduate School of Medicine (Med Kyo 15556 and Med Kyo 15558).Pgam2-Tg mice were generated previously [Pgam2-Tg is a strain of transgenic C57BL/6 mice that overexpresses the Pgam2 with a 3xFLAG tag under the cytomegalovirus immediate-to-early enhancer element and chicken β-actin promoter (CAG) [eviously . Pgam2-Ter (CAG) .Pgam1 is previously reported [Pgam1 knockout mice, Pgam1+/- mice were generated by crossing between Pgam1flox/+ mice and CAG-Cre Tg mice, which ubiquitously express Cre recombinase under the CAG promoter. Subsequently, Pgam1+/-; CAG-Cre micewere crossed with wild-type mice and the genotypes of progenitor mice were analyzed by using PCR. Resulting Pgam1+/- mice without CAG-Cre transgene were applied for further experiment.Generation of the conditional knockout mouse of reported . For theIntraperitoneal glucose tolerance test (IPGTT) was performed as previously described . After 12, 0.2 mM NADH, 3 mM ADP, and 10 μM 2,3-diphosphoglycerate) with enzyme mixture . The levels of NADH were monitored at 37°C, from adding to 1 mM 3-phosphoglyceric acid. Enzymatic activity was measured as NAD+ release.PGAM enzymatic activity was measured as previously described . BrieflyThermal Cycler Dice Real-Time system  and. Gene expression levels were normalized to Rpl13a mRNA and presented as values relative to wild-type mice.Total RNA was extracted with TRIzol (Invitrogen). cDNA pools were prepared by the ReverTra Ace qPCR RT kit . Real-time quantitative PCR was carried out using Thunderbird SYBR qPCR mix (Toyobo). The level of expression of target gene was monitored by the Rpl13a:Fw 5’- TGC TGC TCT CAA GGT TGT TCG -3’, Re 5’-GCC TTT TCC TTC CGT TTC TCC-3’, Hk1:Fw 5’-AAG AAT GGC CTC TCC CGG-3’, Re 5’-CGC CGA GAT CCA GTG CAA TG-3’, Hk2:Fw 5’-ATA TGG TTG CCT CAT CTT GG-3’, Re 5’-CTC CCT CCC TCC CAA TG-3’, Hk3:Fw 5’-ATT CCT GGA TGC ATA CCC CGT -3’, Re 5’-GCC GCT GCA CCT AAA ACC TTT -3’, Gpi:Fw 5’-CCA ATG CAG AGA CAG CAA AGG-3’, Re 5’-CAC TTT GGC CGT GTT CGT AGA-3’, Pfkl:Fw 5’-GCT GCA ATG GAG TTG TG-3, Re 5’-GTA GCC AGG TAGC CAC AG-3’, Pfkm:Fw 5’-TGG AGC GAC TTG CTG AAT GAT -3’, Re 5’-TCA TTG TCG ATT GAG CCA ACC -3’, Aldo a:Fw 5’-CTG GCC ATC ATG GAA AAT GC-3’, Re 5’-TCA AGT CAT GGT CCC CAT CAG-3’, Aldo b:Fw 5’-ATC GGC GGA GTG ATC CTT TT-3’, Re 5’-TCC AAC TTG ATG CCC ACC A -3’, Tpi:Fw 5’-TGC CAA ACA ATG AGC ACT GC-3’, Re 5’-ATC AGA AGC ATG TGA CCG GTG-3’, Gapdh:Fw 5’-AGC CTC GTC CCG TAG ACA AAA-3’, Re 5’-TGG CAA CAA TCT CCA CTT TGC-3’, Pgk1:Fw 5’-TTT GGA CAA GCT GGA CGT GAA-3’, Re 5’-GCT TGG AAC AGC AGC CTT GAT-3’, Pgam1:Fw 5’-GTT GCG AGA TGC TGG CTA TGA-3’, Re 5’-CAC ATC TGG TCA ATG GCA TCC-3’, Pgam2:Fw 5’-TGG AAT GAG GAG ATC GCA CCT -3’, Re 5’-TCG GAC ATC CCT TCC AGA TGT -3’,Eno1:Fw 5’-TAT TGC GCC TGC TCT GGT TAG-3’, Re 5’-GGA TGG CAT TTG CAC CAA AT-3’, Eno3:Fw 5’-GGA GAA GAA GGC CTG CAA TTG -3’, Re 5’-CCC AGC CAT TAG ATT GTG CAA -3’,Pkm1:Fw 5’-CTG TTT GAA GAG CTT GTG GCG -3’, Re 5’-CTG CTA AAC ACT TAT AAG AGG CC -3’.The primer sequences are shown as below. All data are expressed as the mean ± standard error of the mean (SEM) Comparisons between two independent groups were analyzed using an unpaired Student’s two-tailed t-test.in vivo, conditional KO mice for Pgam1 were established [Pgam1 heterozygous KO (Pgam1+/−) mice were generated through crosses between CAG-Cre Tg mice and Pgam1flox/+ mice. Among 155 progenies obtained after mating between Pgam1+/− mice, the ratio of wild-type; heterozygous KO; and homozygous KO was 52:103:0 (Pgam1 homozygous KO (Pgam1−/−) mice were embryonic lethal, consistent with the lack of observed Pgam1−/− embryos at embryonic day 13.5 (E13.5)  . The ear enzymes –25 , but not in the heart, brown adipose tissue (BAT), and muscle, compared to those in control  and serum glucose level), in addition to body weight, visceral-fat weight, muscle mass, total-cholesterol, and other biochemical blood markers (in vivo parameters for glucose metabolism were less affected by heterozygous Pgam1 knockout.It was reported that brain-specific knockout of Glut4 sitivity . As glyc[in vivo . To examhenotype . In comp markers . Collectin vivo. We previously reported that heart-specific Pgam2-transgenic (Tg) mice displayed almost normal glycolytic features in the heart [in vivo. As it has been demonstrated that the overexpression of either PGAM isoform confers similar physiological impact [in vivo parameters of glucose metabolism in Pgam2-Tg mice, in which a Pgam2-FLAG transgene is driven by the CAG promoter. Pgam2-Tg mice exhibited a significant increase of PGAM protein in the whole body [Pgam2-Tg mice also displayed their similar features with respect to body weight and IPGTT both under physiological  [Pgam1−/− mice (in this study). In this setting, PGAM would be rate-limiting in glycolysis. Moreover, we recently reported that the global enhancement of glycolytic profiles in cancerous cells, the Warburg effect, is provoked in vitro by the cooperation of PGAM with Chk1 kinase under oncogenic Ras expression [Pgam1 deficient T cells are impaired in maintenance of such glycolytic enhancement, followed by immunological dysfunction [Pgam2-Tg mice under chemical carcinogenic protocol [Pgam2-Tg mice under standard conditions were comparable to control [in vivo for its physiological impact, as several post-translational regulations of PGAM were reported in vitro [Different from our observations in genetic model mice for PGAM, several reports suggest that impaired glycolysis results in diabetes or impaired glucose metabolism both in human patients ,5 and ins study) . As PGAMs study) , the ress study) , rather pression . Besidespression . Pgam1 dfunction . Thus, tprotocol , while t control . It is pin vitro .in vivo.Further investigation might provide valuable clue to understand the impact of PGAM on glucose metabolisms S1 Data(XLSX)Click here for additional data file.


Respiratory syncytial virus (RSV) may cause serious lower respiratory tract disease (LRTD) in older adults, and there is currently no licensed vaccine. CYPRESS (NCT03982199) is a randomized, double-blind, placebo-controlled Phase 2b proof-of-concept trial of an Ad26.RSV.preF-based vaccine for the prevention of RSV-mediated LRTD in older adults. Adults aged ≥65 years were randomized 1:1 before the RSV season to receive Ad26.RSV.preF-based vaccine or placebo. Acute respiratory infection symptoms were collected through a patient eDiary and/or clinician assessment until the end of the RSV season. The primary endpoint was the first occurrence of RTPCR-confirmed RSV-mediated LRTD according to any of 3 case definitions: (1) ≥3 symptoms of lower respiratory tract infection (LRTI), (2) ≥2 symptoms of LRTI, or (3) ≥2 symptoms of LRTI or ≥1 symptom of LRTI with ≥1 systemic symptom. Immunogenicity was assessed in a subset of approximately 200 participants. A total of 2891 participants in each study arm received study treatment. Vaccine efficacy was 80% , 75% (50.1-88.5%), and 69.8% (43.7-84.7%) for case definition 1, 2, and 3, respectively . In the vaccine arm, geometric mean fold increase in antibody titers 14 days after vaccination was 13.5 for RSV neutralizing antibodies and 8.6 for RSV prefusion F-specific binding antibodies, and median frequency of RSV-F-specific INFγ T-cells increased from 34 to 444 SFC/10^6 PBMC; no relevant changes were observed in the placebo arm. The Ad26.RSV.preF-based vaccine was highly effective against RSV-mediated LRTD through the first RSV season and elicited robust immune responses in older adults.


Circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs) are differentially expressed in gastrointestinal cancers. These noncoding RNAs (ncRNAs) regulate a variety of cellular activities by physically interacting with microRNAs and proteins and altering their activity. It has also been suggested that exosomes encapsulate circRNAs and lncRNAs in cancer cells. Exosomes are then discharged into the extracellular environment, where they are taken up by other cells. As a result, exosomal ncRNA cargo is critical for cell–cell communication within the cancer microenvironment. Exosomal ncRNAs can regulate a range of events, such as angiogenesis, metastasis, immune evasion, drug resistance, and epithelial-to-mesenchymal transition. To set the groundwork for developing novel therapeutic strategies against gastrointestinal malignancies, a thorough understanding of circRNAs and lncRNAs is required. In this review, we discuss the function and intrinsic features of oncogenic circRNAs and lncRNAs that are enriched within exosomes. Solid cancers of the gastrointestinal system, such as colorectal cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, and pancreatic cancer, are among the most frequent types of malignancies. According to the GLOBOCAN database, gastrointestinal cancers are responsible for 26% of cancer incidence and 35% of cancer-related mortality worldwide . Due to ANXA1) proteins are cargo molecules in cancer cell-derived exosomes and activate epithelial–mesenchymal transition (EMT) in exosome-receiving cancer cells [MIF), to reshape the phenotype of drug-sensitive cancer cells into temozolomide-resistant cells by positively modulating phosphoinositide 3-kinase (PI3K)/AKT signaling. The pharmacological inhibition of MIF prominently enhances temozolomide efficacy in vivo [SNAI1) messenger RNA (mRNA). Anti-pancreatic cancer effects of gemcitabine can be improved by pharmacological interruption of exosome secretion using GW4869 [In solid cancers, the microenvironment is heterogeneous and composed of cellular and noncellular components, including cancer-associated fibroblasts (CAFs), immune cells, and the extracellular matrix ,5. Hypoxer cells . Another in vivo . In addig GW4869 .Circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs) are aberrantly expressed in cancer and impinge on the activity of target molecules, including microRNA (miRNA) and proteins, therefore controlling cancer progression, therapeutic resistance, etc. ,14,15,16PTEN) mRNA [These noncoding RNAs (ncRNAs) can be incorporated and differentially loaded into exosomes. For instance, circ_EPB41L2 quantity is low in exosomes secreted by colorectal cancer cell lines. Treatment of cancer cells with exosomes acquired from circ_EPB41L2-overexpressing cells retards cell proliferation, survival, migration, and invasion by negatively modulating the effect of miR-21-5p and miR-942-5p on phosphatase and tensin homolog (EN) mRNA . This reMAPK1) and maternal embryonic leucine zipper kinase (MELK), respectively [It was demonstrated that drug-sensitive parental cells cocultured with FOLFOX-resistant cells become desensitized to 5-fluorouracil (5-FU), implying that exosome-transferred molecules contribute to chemoresistance. Profiling of circRNAs revealed that circ_0000338 is one of the upregulated circRNAs in exosomes secreted from FOLFOX-resistant cells . Anotherectively ,26. Sincectively ,28, circPKM2), a glycolytic enzyme, facilitates oxaliplatin resistance via upregulating ATP-binding cassette subfamily B member 1  [Glycolysis has been found to be associated with therapeutic resistance toward several classes of anti-cancer drugs. Lactate, a glycolytic metabolite, can induce drug resistance by creating an acidic tumor microenvironment . In addiotein 1) . Recentlotein 1) . In thisotein 1)   serves as an activator of Ras homolog family member A (RhoA), promoting cell motility, invasion, and metastasis [Guanine nucleotide exchange factor H1 . Colony formation of cancer cells is attenuated by culturing with conditioned media derived from circ_EIF3K-silenced CAFs. The growth of cancer is also impaired in xenografts treated with exosomes from circ_EIF3K-silenced CAFs. These results denote the role of exosomal circ_EIF3K in cancer progression [Secretion of exosomes from CAFs is stimulated by hypoxic conditions . Furthergression   and musashi-1 (MSI1), respectively, by sponging miR-296-5p [Circ_0067835 is abundant in blood-derived exosomes from patients who received radiotherapy, suggesting an involvement of this circRNA in radiosensitivity modulation. Indeed, it was noticed that radiotherapy-induced apoptosis is increased in cancer cells treated with exosomes derived from circ_0067835-silenced cells . FurtherR-296-5p ,42 . In addition, exosomal circ_PACRGL stimulates neutrophil polarization towards a pro-tumorigenic N2 phenotype [Microarray analysis showed that circ_PACRGL is copiously detected in exosomes released from cancer cells . Functiohenotype , suggestCirc_0000337BACH1) levels [MAT2A) expression, thus accelerating migration and invasion in glioblastoma cells [JAK2) [GSK3B) expression and activate nuclear factor kappa B (NF-κB) signaling [Circ_0000337 has been discovered to be an oncogenic factor. In osteosarcoma, circ_0000337 augments proliferation and migration by elevating BTB domain and CNC homolog 1  levels . Besidesma cells . Circ_00ma cells . Moreovema cells . Non-ress [JAK2)   expression [It has been evaluated that miR-515-5p attenuates chemoresistance. The efficacy of vincristine and carboplatin is improved by miR-515-5p in retinoblastoma . Furtherpression . Owing tpression   functions as an oncogenic factor by advancing the proliferation and migration of gastric cancer cells via triggering Hippo signaling [Protein tyrosine phosphatase non-receptor type 14  [YAP1). The silencing of circ_PVT1 re-sensitizes resistant cancer cells to cisplatin by augmenting apoptotic cell death and suppressing autophagy [Circ_PVT1 is involved in the regulation of a variety of cellular events. Circ_PVT1 overexpression increases the invasive capacity of esophageal and gallbladder cancer cells ,67. In aMRP1) . In gastMRP1) . In theiutophagy . These fExosomal circRNAs play a part in angiogenesis regulation. Treatment with cancer cell-derived exosomal circ_0044366 significantly enhances the proliferation, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Besides this, exosomal circ_0044366 promotes cancer growth and angiogenesis in vivo. Mechanistically, exosomal circ_0044366 leads to an increment of vascular endothelial growth factor (VEGF) levels via inactivating miR-29a in endothelial cells  Figure .HUR) levels via inactivating miR-582-3p, resulting in an increment of VEGF expression in cancer cells in vitro. Cancer growth and angiogenesis are activated by circ_SHKBP1 in vivo [Circ_SHKBP1 levels are higher in cancer tissues and exosomes derived from patient serum and cancer cell lines . Exosoma in vivo   in cancer cells and enhances migration and invasion [Once activated, JAK/signal transducer and activator of transcription (STAT) signaling promotes the expression of target genes involved in diverse cellular events, such as cell survival, migration, invasion, and therapeutic resistance . Becauseinvasion . These oCDC42), miR-149-5p promotes apoptotic cell death, represses cell proliferation, and alleviates doxorubicin resistance in neuroblastomas [FOXM1) expression [PHF10) in gastric cancer [Both miR-149-5p and miR-409-3p perform a tumor-suppressive function. By targeting BCL2 and cell division cycle 42  and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), respectively, in cancer cells [Further functional studies showed that circ_NEK9 and circ_NHSL1 inactivate miR-409-3p and miR-149-5p, respectively. By suppressing such miRNAs, circ_NEK9 and circ_NHSL1 elevate the expression of microtubule-associated protein 7  [Circ_0061395  is notably overexpressed in hepatocellular carcinomas. It was also observed that circ_0061395 can be incorporated into exosomes . FunctioPIK3R3)   ,77. SincPIK3R3) ,79, bothPIK3R3) . Thus, iLRP6), which acts as a coreceptor of WNT ligands and activates WNT/β-catenin signaling [It was denoted that circ_FBLIM1 is overexpressed in hepatocellular carcinoma tissues and cell lines . Additioignaling . Hence, Another study showed that knockdown of circ_FBLIM1 hampers cell proliferation and invasion while promoting apoptotic cell death by augmenting miR-346 levels . Even thIGF2BP3), thus expediting cell proliferation and EMT by stabilizing FOXM1 transcripts [MTA1) [MMP2) axis [In lung cancer, circ_MMP2  can bind to insulin-like growth factor 2 mRNA binding protein 3 (nscripts . Besidess [MTA1) . Such ans [MTA1) . Circ_MMP2) axis   axis [Functional investigations indicated that this circRNA blocks apoptotic cell death and promotes in vivo cancer growth via regulating the miR-665/insulin growth factor 2 (F2) axis . Circ_TMF2) axis . TherefoInconsistent with the IGF2-targeting property of miR-665, other studies showed that miR-665 is overexpressed in hepatocellular carcinomas and prompts cell proliferation as well as metastasis ,88. ThusMYC) expression in oral squamous cell carcinomas [HAVCR2, also named TIM3), ultimately diminishing the anti-cancer effects of nivolumab, anti-programmed cell death 1 (PD1) antibodies [A recent study showed that circ_UHRF1 accelerates cell proliferation and EMT by elevating MYC proto-oncogene  in colorectal cancer, restraining cancer progression [SP1), thus impeding migration and invasion of gastric cancer cells [It has been proven that miR-203 and miR-502-5p are tumor-suppressive miRNAs in gastrointestinal cancers. For instance, miR-203 negatively modulates the level of eukaryotic translation initiation factor 5A2 (gression . In the gression . In addier cells .GUCD1) axis [In hepatocellular carcinoma, miR-203 and miR-502-5p commonly target SNAI1, an EMT-promoting factor, and they are sponged by circ_ZNF652; therefore, lung metastasis is provoked by circ_ZNF652 . FurtherD1) axis   axis , indicatZO-1), a master regulator of tight junctions [Endothelial tight junctions act as permeability barriers and restrict cancer metastasis. Transendothelial permeability of cancer cells is induced by the downregulation of Zona occludens-1  [MACC1) [MET) [In various cancer types, miR-338-3p functions as a tumor-suppressive miRNA. By inactivating MAPK signaling, miR-338-3p promotes apoptosis in osteosarcoma cells . In ovarSMO) . It was  [MACC1) , a trans1) [MET) . Additio1) [MET) . Exosoma1) [MET)   is stabilized by sirtuin 1 (SIRT1)-mediated deacetylation. SIRT1 knockdown reduces the invasion ability of cancer cells, together with a decrease in HIF-1α target genes, such as VEGF [Hypoxia-inducible factor-1α  expression [Accumulating evidence has shown that the expression of CCAL is substantially higher in cancer than in non-cancerous tissues and that CCAL promotes angiogenesis and metastasis through escalating, for example, FOXM1 and angiopoietin-like 4  [LncRNA profiling identified that 201 lncRNAs have aberrant levels, including 165 upregulated and 36 downregulated lncRNAs in colorectal cancer. LINC00659 is one of the highly expressed lncRNAs, and the knockdown of this lncRNA suppresses cell cycle progression and cell survival through inactivating PI3K/AKT signaling . MoreoveANXA2) , which iANXA2)   and CREB-binding protein (CBP) levels [USP22)-mediated stabilization of PD-L1 by deactivating miR-30a-5p and thereby inhibiting the anti-cancer immunity of CD8+ T cells [Numerous studies have suggested that oncogenic KCNQ1OT1 is implicated in migration, invasion, apoptosis, and therapeutic resistance. For example, KCNQ1OT1 significantly increases migration and invasion in hepatocellular carcinomas and osteosarcomas ,144. In ) levels . Of late T cells . Since K T cells   is induced by SNAI1 and facilitates cell migration and invasion [FAK), SRC proto-oncogene (SRC), and STAT3 is downregulated by TUBB3 silencing, thus restraining metastasis [Tubulin beta 3 class III  expression [In colorectal cancer, HOTTIP is overexpressed in mitomycin-resistant cancer cells and their exosomes. Exosomes from resistant cells weaken mitomycin-induced DNA damage in non-resistant cells via conveying HOTTIP, which impairs miR-214-mediated inhibition of karyopherin subunit alpha 3  levels, respectively [UCA1 is highly expressed in colorectal cancer tissues, cell lines, and patients’ serum exosomes . This suectively   [AFAP1-AS1, a highly expressed lncRNA in multiple cancers, was discovered to promote cancer progression, stemness, as well as therapeutic resistance. AFAP1-AS1 possesses stimulatory effects on cell proliferation, migration, and invasion in thyroid cancer as a consequence of sequestering miR-204-3p . In laryRBPJ) .CREB2, also known as activating transcription factor 2 (ATF2)), thereby boosting migration/invasion in vitro and lung metastasis in vivo [Besides this, a profuse amount of AFAP1-AS1 was detected in exosomes unleashed by M2 macrophages. Exosomal AFAP1-AS1 can enter esophageal cancer cells and interact with miR-26a to promote the level of cyclic AMP-responsive element-binding protein 2  activates the PI3K/AKT signaling-mediated NF-κB/SNAI1 axis, triggering the EMT process [FOXP1) supports cell survival via transcriptionally repressing the expression of proapoptotic genes, such as BCL2 interacting killer (BIK) [Neuropilin and tolloid-like 2 ( process . The sil process . In addier (BIK) . Furtherer (BIK) . Both NEer (BIK) . FAM225Aer (BIK)  . Thus, FMR1-AS1 exerts anti-apoptotic and pro-invasive effects and strengthens stemness in recipient cancer cells. In vivo observation also indicated that exosomal FMR1-AS1 fuels cancer growth [FMR1-AS1 is a highly expressed lncRNA and is sorted into exosomes in esophageal cancer . This exr growth  , in recipient cancer cells, hence expediting proliferation, migration, and invasion [LINC01711 is upregulated in esophageal cancer tissues and several cell lines. In addition, this lncRNA can be contained in exosomes produced by cancer cells, suggesting that exosomal LINC01711 can play an oncogenic role . It has invasion   expression, consequently increasing the occurrence of lung metastasis in hepatocellular carcinomas [In esophageal cancer, exosomal ZFAS1 is taken up by other surrounding cancer cells and exerts a positive influence on proliferation, migration, and invasion via the miR-124/STAT3 axis  Figure . ZFAS1 hrcinomas . ZFAS1 arcinomas . ExosomaEZH2) and, thus, epigenetically silences PTEN expression, leading to cisplatin resistance in gastric cancer [HMGB1) and incapacitates the invasive and migratory potential [PCAT1 and POU3F3 have been regarded as oncogenic lncRNAs. PCAT1 physically binds to enhancer of Zeste 2 polycomb repressive complex 2 subunit  [POU3F3 is also encompassed by exosomes generated from cancer cells and delivered to fibroblasts. POU3F3 ultimately accelerates the conversion of normal fibroblasts (NF) into CAFs, although the mechanisms of POU3F3-mediated fibroblast activation remain unclear. CAFs are then responsible for cisplatin resistance by secreting IL6  Figure . POU3F3 ROCK) . ROCK isROCK) , which sROCK) . For thiCEBPA-AS1 deactivates Notch signaling via sponging of oncogenic miR-10b-5p, and it increases apoptotic cell death in osteosarcomas . By contExosomal FRLnc1 from gastric cancer cells can effectively augment cell proliferation, viability, and migration in receiver cancer cells  Figure . AlthougP4HA2) [PCGEM1 stabilizes SNAI1 proteins via physically interacting with them, serving as a pro-migratory and -invasive lncRNA in gastric cancer . HypoxicP4HA2) . AccordiP4HA2) .HMGA1) via nullifying miR-218 activity [As mentioned in activity   and epigenetically inhibits PTEN expression. By regulating PGK1 and PTEN levels, exosomal LINC01559 can activate PI3K/AKT signaling and promote cell proliferation, migration, and stemness in gastric cancer cells [Moreover, it was noticed that exosomes from mesenchymal stem cells convey LINC01559 to gastric cancer cells . Exosomaer cells   mRNA to upregulate HIF-1α levels [SPRY4-IT1 has been proven to serve as either a tumor-suppressive or an oncogenic lncRNA. SPRY4-IT1 is downregulated in cisplatin-resistant lung cancer cells and takes part in the repression of EMT. Overexpression of SPRY4-IT1 reinforces cisplatin-induced apoptosis in resistant cells . Conversα levels . Thus, iAMPK) axis [Furthermore, SPRY4-IT1 is enriched in gastric cancer tissues, cancer cell lines, and serum exosomes . Hence, PK) axis   mRNA, retarding the growth of lung cancer along with ferroptosis [ADAM9) expression [ASMTL-AS1 has a dual role in cancer. Overexpression of ASMTL-AS1 stabilizes spermidine/spermine N1-acetyltransferase 1 (roptosis . ASMTL-Aroptosis . By contpression .NLK) expression via rendering miR-342-3p ineffective, fundamentally reinforcing malignant phenotypes, such as EMT and metastasis [In hepatocellular carcinoma, ASMTL-AS1 is upregulated in cancer tissues and further increased in residual tissues following insufficient radiofrequency ablation (RFA) . Such antastasis . Comparetastasis . These oCXCL17), which is targeted by miR-15a-5p [Exosomal lncRNAs from hepatocellular carcinoma cells are accountable for M2 polarization, following an uptake by macrophages. Coculturing cancer cells with M2 macrophages stimulated by exosomal DLX6-AS1 promotes migration, invasion, and EMT in vitro. Furthermore, by activating M2 polarization, exosomal DLX6-AS1 triggers lung metastasis in vivo. Exosomal DLX6-AS1 exerts its function by upregulating C-X-C motif chemokine ligand 17 , a cell death-promoting factor, augmenting cisplatin-induced apoptosis in osteosarcoma cells [LINC00161 upregulates interferon-induced protein with tetratricopeptide repeats 2  expression, LINC01133 is capable of inhibiting metastasis in breast cancer [TPD52) levels through sharing the same binding sites with miR-495-3p, acting as a pro-metastatic factor [CEBPB) and positively modulates cell proliferation via activating cyclin G1 expression in pancreatic cancer [Disparate functions of LINC01133 depending on cancer types have been reported. LINC01133 hampers gastric and bladder cancer progression via somatostatin upregulation and WNT signaling inactivation, respectively ,204. Cont cancer . On the t cancer . In ovarc factor . It was c cancer .AXIN2), activating β-catenin and promoting cell survival, proliferation, and EMT [POSTN) was discovered to increase LINC01133 via the epidermal growth factor receptor (EGFR)/MYC axis and promote exosome secretion in pancreatic cancer cells [Moreover, LINC01133 is highly expressed in pancreatic cancer cells as well as in their secreted exosomes. LINC01133 interacts with EZH2 to silence the expression of axis inhibition protein 2 ( and EMT . In thiser cells . These fAMOTL2) activates the SRC/ERK signaling pathway, playing a critical role in migration and proliferation of endothelial cells during angiogenesis [Angiomotin-like 2  [In addition to the inhibition of exosome cargo, restraint of exosome secretion has therapeutic benefits. However, pharmacological agents, such as GW4869, are non-specific inhibitors of exosome-mediated intercellular dialogues and have undesirable effects ,211. BesAGER) . TherefoOverall, an advanced understanding of the nature of ncRNAs and the mechanism underpinning the exosome itinerary will pave the way for overcoming gastrointestinal cancer.