Fabp4-cre driven adipose-specific Aff4 knockout mice and find that AFF4 deficiency impedes adipocyte development and white fat depot formation. Mechanistically, we discover AFF4 regulates autophagy during adipogenesis. AFF4 directly binds to autophagy-related protein ATG5 and ATG16L1, and promotes their transcription. Depleting ATG5 or ATG16L1 abrogates adipogenesis in AFF4-overepressing cells, while overexpression of ATG5 and ATG16L1 rescues the impaired adipogenesis in Aff4-knockout cells. Collectively, our results unveil the functional importance of AFF4 in regulating autophagy and adipogenic differentiation, which broaden our understanding of the transcriptional regulation of adipogenesis.Transcriptional elongation is a universal and critical step during gene expression. The super elongation complex (SEC) regulates the rapid transcriptional induction by mobilizing paused RNA polymerase II (Pol II). Dysregulation of SEC is closely associated with human diseases. However, the physiological role of SEC during development and homeostasis remains largely unexplored. Here we studied the function of SEC in adipogenesis by manipulating an essential scaffold protein AF4/FMR2 family member 4 (AFF4), which assembles and stabilizes SEC. Knockdown of AFF4 in human mesenchymal stem cells (hMSCs) and mouse 3T3-L1 preadipocytes inhibits cellular adipogenic differentiation. Overexpression of AFF4 enhances adipogenesis and ectopic adipose tissue formation. We further generate  Obesity is a major health problem jeopardizing millions of individuals worldwide. From a pathological perspective, obesity occurs in the process of white adipose tissue expanding its mass through the enlargement of adipocyte size or advanced differentiation of adipocyte precursors to mature adipocytes. Studies have documented the dysregulated adipocyte metabolism of adipose tissue and associated disorders. However, our understanding of adipocyte development in which mesenchymal stem cells (MSCs) commit their fate and preadipocytes undergo differentiation and maturation is scarce. Here, we identify the super elongation complex (SEC) scaffold protein AFF4 as an essential regulator of adipogenesis. We reveal that AFF4 promotes adipocyte formation by regulating the cellular autophagic process. AFF4 directly regulates the transcription of the autophagy-related protein ATG5 and ATG16L1, which are essential for autophagosome formation. This finding further elucidates the physiological role of SEC during tissue development, besides its recognized role in cancer occurrence. The prevalence of obesity is a major challenge to chronic disease prevention and intervention. The World Health Organization stated that there are more than one billion overweight adults around the world. As one of the major health problems, obesity has been identified as a key contributor to heart disease, type 2 diabetes, strokes, liver steatosis and other chronic diseases , 2. ApprIt is well recognized that obesity is closely related to genetic risk factors and unhealthy lifestyle such as calorie excess and lack of physical activity . Pathoge+) adipocyte precursors and its inactivation is a prerequisite for adipogenic differentiation of Pref1+ cells into platelet-derived growth factor receptor α-expressing (PDGFRα+) cells [The adipogenesis process is tightly controlled by systemic signals and transcriptional factors . Systemi+) cells . Furtherin vitro and increased ectopic bone formation in vivo [The super elongation complex (SEC) consists of eleven-nineteen Lys-rich leukemia (ELL) family proteins, positive transcription elongation factor b (P-TEFb) and the mixed lineage leukemia (MLL) translocation partners, which is required for robust and productive gene transcription through the release of paused Pol II . AF4/FMR in vivo . NeverthCCAAT/enhancer binding protein delta (CEBPD), CEBPA, Adiponectin (ADIPOQ) and lipoprotein lipase (LPL) was significantly down-regulated upon AFF4 depletion in hMSCs (delta-like 1 (DLK1) expression, an early negative adipogenic fate regulator, in AFF4-depleted cells during differentiation  and western blot . Knockdoin hMSCs . We did ntiation . In agreL1 cells .Cebpd, Cebpa, Adipoq and Lpl, were elevated in AFF4-overexpressing cells (-overexpressing 3T3-L1 cells into immunodeficient nude mice formed larger size (mm2) of fat pads  mice using CRISPR/Cas9 technique, and bred with Fabp4-Cre mice to conditionally delete Aff4  mice were viable and born at Mendelian’s ratio. CKO mice exhibited lower body weight at all examined time points from 2 weeks of age, compared with the control littermates (CON)  . The tises (CON) . The WATipocytes .Next, we cultured preadipocytes from WAT in CON and CKO mice. More lipids were detected in CON group after adipogenic differentiation . QuantitAFF4 depletion and uncovered a remarkable down-regulation in autophagy related pathway  or CQ and investigated the adipogenic capacity. Both 3MA and CQ reversed the augmented lipids formation and triglyceride accumulation in AFF4-overexpressing cells . The expATG5, ATG16L1 and ATG4A was significantly reduced after AFF4 depletion, while that of ATG7, ATG9A, ATG12, ATG4B and ATG2B was upregulated  protein family is one of the most important proteins to assist autophagosome formation . Therefoegulated . We then ATG16L1 .ATG5 and ATG16L1. Notably, the enrichment of AFF4 at the promoter of ATG5 and ATG16L1 was significantly decreased in response to the depletion of AFF4 , is a member of the FABPs family and plays an important role in adipose tissue differentiation and lipid metabolism –24. Usinturation , 26, 27.ipoq-Cre . ConsideAdipogenesis is regulated by a variety of signal pathways such as the insulin signaling, glucocorticoid signaling and BMP signaling , 23. AutAtg5-/- MEFs failed to form lipid droplets and gradually turned apoptotic after 3 days of adipogenic induction, suggesting that Atg5 was involved in the early stages of adipogenic differentiation. Indeed, treatment of CQ to MEFs alone inhibited adipogeneis [Fabp4-Cre;Atg7fl/fl mice were smaller and thinner than the control mice but with similar food intake. The total fat was decreased by about 30% compared with control mice [Several studies reported the regulation of autophagy in adipogenesis and proposed that autophagy aided in adipogenesis by degrading cellular organelles –33. Duripogeneis . Anotherrol mice . CollectFabp4-Cre;Atg7fl/fl mice as well [On account of the close relationship of adipogenesis and mitochondria, we further examined the mitochondria after AFF4 knockout. The number of mitochondria was increased in preadipocytes of CKO mice . A simil as well . We thusAs the scaffold protein of SEC, AFF4 contributes to the stability of SEC. Lin et al. found AFF4 was required for the association of CDK9 and cyclin T1 with SEC . AnotherAff4-knockout mice died in utero, with alveoli development severely impaired. The 13% mice survived exhibited an infertility with defective spermiogenesis [Aff4 in specific tissue.AFF4 functions in development besides its important role in cancer and virus infection , 35. Misogenesis . To our Aff4-CKO mice is largely unexplored. Further study illustrating the regulation of AFF4 on adipose lipid and energy metabolism, and related diseases like diabetes, is warranted.One limitation of this study is the lack of clarification of AFF4 function in SEC and the promoter-proximal paused Pol II release. Though we verified a direct regulation of AFF4 on ATG5 and ATG16L1, whether and how AFF4 regulates the recruitment of P-TEFb to their promoters need to be further explored. Besides, the metabolic prolife of In summary, our present study unveils the unknown function of AFF4 in adipogenesis and its intriguing mechanism by targeting autophagy through ATG5 and ATG16L1. This study broadens the knowledge of transcriptional regulation of adipogenesis and might provide insights into diseases such as obesity and lipodystrophy.All animal experiments were approved by and performed under the guidance of the Subcommittee on Research and Animal Care (SRAC) of Sichuan University.Primers for qPCR and genotyping were listed in Aff4fl/+ mice were generated by Biocytogen company . Fabp4-Cre transgenic mice were purchased from Jackson Laboratory (strain number 018965). Mice were maintained at 23 ± 2°C with a humidity of 35 ± 5% under a 12 h light and 12 h dark cycle, with ad libitum access to water and food. Body weight was measured every week after 2 weeks of age.Nude mice (6 weeks of age) were purchased from the Chengdu Dossy Experimental Animals CO. LTD. 2 incubator.The hMSCs were obtained from Cyagen  and 3T3-L1 were obtained from ATCC  and tested negative for mycoplasma contamination before use. hMSCs were cultured in Minimum Essential Medium α and 3T3-L1 cells were in high-glucose Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Adipogenic differentiation medium contains 0.5 μM IBMX,1 μM dexamethasone and 10 μg/mL insulin. For hMSCs and 3T3-L1 cells, adipogenic differentiation lasted for 14 days to form mature adipocytes . All medWhite adipose tissue was harvested from CON and CKO mice (8 weeks of age) and then treated with type I collagenase for 30 min at 37°C . The digsiAFF4, sc-91842; mouse siAff4, sc-140897). Each siRNA consists of mixes of three to five target-specific 19- to 25- nucleotide siRNAs. siRNAs targeting ATG5 and ATG16L1 were purchased from Sangon . For siRNA mediated knockdown, cells were transfected using Lipofectamine RNAimax as previously reported [Small interfering RNAs (siRNAs) targeting AFF4 were purchased from Santa Cruz . Cells transfected with an empty GV358 vectors (Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin) were used as control . Cells wCells were washed and fixed with 4% paraformaldehyde for 20 min. After washed with PBS, the cells were treated with 60% isopropanol and stained with Oil Red O for 20 min, then rinsed three times with distilled water and observed under a microscope . For quaFor triglyceride measurement, differentiated cells were washed twice with PBS and lysed by lysis buffer. The triglyceride levels of the cells were measured using the triglyceride assay kit according to the manufacturers’ protocol.Cells and tissues were lysed on ice using protein extraction kit with protease and phosphatase inhibitor cocktail  for 20 min. The protein concentration was determined using the BCA protein assay kit . Proteins were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membrane  membranes. The membranes were blocked with 5% non-fat milk at room temperature for 1 h and then incubated with corresponding primary antibodies overnight at 4°C. Next day, the membranes were incubated with goat anti-rabbit HRP-conjugated secondary antibodies at a dilution of 1:5,000 at room temperature for 1 h.-ΔΔCt method and calculated using GAPDH as the normalization control .10 μL CCK8 was added to each well of 96-well plate and incubated at 37°C for 4 h. The absorbance value of each well was detected by a microplate reader at 450 nm.5 cells overexpressing AFF4 or empty vector GFP were mixed with 50% matrigel suspensions. The mixture was injected subcutaneously flanking the sternum of the nude mice. Mice were then subject to high-fat diet. Six weeks after transplantation, the transplants were collected and fixed with 4% paraformaldehyde, and then dehydrated and embedded in optical cutting temperature (OCT) compound for tissue section.Approximately 2 ×10Frozen tissue was sectioned into slices with thickness of 20 μm  before staining. The slices were permeabilized with Triton X-100 for 10 min and stained with Nile Red solution (1:2000) for 15 minutes and then DAPI for 5 min at 37°C. Images were captured by confocal microscope .iWAT, rWAT, mWAT and eWAT obtained from CON and CKO mice were fixed in 4% paraformaldehyde for 24 h at 4°C. Then the WAT was dehydrated and embedded in paraffin. Tissue was sectioned into slices with thickness of 5 μm. The slices were stained and observed under light microscope.Cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature, and then permeabilized with Triton X-100 for 10 min. After blocking with 5% BSA for 1h, cells were incubated with primary antibodies at 4°C overnight. The next day, cells were treated with the goat anti-rabbit Alexa Fluor 550 secondary antibodies for 1 h. Nuclei were stained with DAPI for 5 min at room temperature. Images were taken by a confocal laser microscope .RNA-Seq2FC (Fold change) > 1 or < -1, with adjusted P value < 0.05 [RNA-Seq libraries were prepared using the Illumina TrueSeq mRNA sample preparation kit according to the manufacturer’s instruction and single-end sequenced on an Illumina HiSeq 3000 machine. We used FastQC (v0.11.5) to control the quality of RNA-Seq and aligned the reads to hg19 genome using HISAT2 (v.0.0.5). Gene expression level was considered significantly changed if loge < 0.05 . Gene Se4) for 2 h. The samples were dehydrated with increasing concentrations of ethanol  and transferred to acetone for 20 min. After placing in 1:1 mixture of absolute acetone and the final Spurr resin (SPI-CHEM) mixture for 1h at room temperature, the samples were transferred to 1:3 mixture of absolute acetone and the final resin mixture for 3 h and to final Spurr resin mixture overnight. The ultra-structures of cells were observed under a transmission electron microscope.Cells were washed with PBS and fixed in 2.5% glutaraldehyde for 4h, and then fixed with 1% osmium tetroxide (OsOChromatin immunoprecipitation (ChIP) assays were performed utilizing EZ-Zyme Chromatin Prep Kit and EZ-Magna ChIP HiSens Chromatin Immunoprecipitation Kit as previously described . The antP value of less than 0.05 was considered statistically significant.All values were expressed as mean ± SD. For normality test, Shapiro-Wilk test was applied using SPSS software. Differences were evaluated by two-tailed Student’s t-test for comparison between two groups or by one-way analysis of variance (ANOVA) followed by the Tukey’s post hoc test for multiple comparisons. A S1 FigAFF4 knockdown in hMSCs. (C) CCK8 assay of hMSCs proliferation. n = 6.  Western blot and RT-qPCR analysis of AFF4 expression 48 h after Aff4 knockdown in 3T3-L1 cells. (F) CCK8 assay of 3T3-L1 cells proliferation. n = 6. All quantified data are presented by as mean ± SD. The P values were calculated by one-way ANOVA followed by the Tukey’s post hoc test. Western blot and RT-qPCR analysis of AFF4 expression 48 h after (TIF)Click here for additional data file.S2 FigP values were calculated by one-way ANOVA followed by the Tukey’s post hoc test. Western blot and RT-qPCR analysis of AFF4 expression in Ctrl, oeScram and oeAFF4 in 3T3-L1 cells. (C) CCK8 assay of 3T3-L1 cells proliferation. n = 6. All quantified data are presented by as mean ± SD. The (TIF)Click here for additional data file.S3 FigAff4 conditional knockout strategy. The exon 2 and exon 3 are deleted after Cre-mediated recombination. (B) Representative images of mice genotyping.(A) Schematic representation of (TIF)Click here for additional data file.S4 FigsiCtrl and siAFF4 hMSCs. CQ, chloroquine.  Representative images and quantification of mitochondria in preadipocytes. n = 6. (D) TEM of preadipocytes from CON and CKO mice, yellow arrow indicating mitochondria. Scale bar, 5 μm.  Representative images and quantification of endoplasmic reticulum. n = 6. (G) TEM of preadipocytes from CON and CKO mice, yellow arrow indicating endoplasmic reticulum. Scale bar, 5 μm. All quantified data are presented by as mean ± SD. The P values were calculated by two-tailed Student’s t-test.(A) Western blot analysis of autophagy related markers in (TIF)Click here for additional data file.S5 FigsiCtrl and siAff4 3T3-L1 cells after adipogenic differentiation for 3, 5 and 7 days.(A) Western blot analysis of AFF4, ATG5 and ATG16L1 expression during adipogenic differentiation of 3T3-L1 cells for 0, 1, 3, 5 and 7 days. (B) Western blot analysis of autophagy related markers in (TIF)Click here for additional data file.S6 Fig(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.


Escherichia coli O80:H2 is still unknown. The aim of this study was to identify E. coli O80 in healthy cattle with an emphasis on melibiose non-fermenting E. coli O80:H2. Faecal materials collected from 149 bulls at 1 slaughterhouse and 194 cows on 9 farms were tested with O80 antigen-encoding gene PCR after overnight growth in enrichment broths. The 53 O80 PCR-positive broths were streaked on different (semi-)selective agar plates. Five E. coli colonies from 3 bulls and 11 from 2 cows tested positive with the O80 PCR, but no melibiose non-fermenting E. coli was isolated. However, these 16 E. coli O80 were negative with PCR targeting the H2fliC, eae, stx1, stx2 and hlyF genes and were identified by WGS to serotypes and sequence types O80:H6/ST8619 and O80:H45/ST4175. They were phylogenetically related to E. coli O80:H6 and O80:H45 isolated from different animal species in different countries, respectively, but neither to STEC and EPEC O80:H2/ST301, nor to other serotypes of the clonal complex 165. As a conclusion, healthy adult cattle were not identified as a source of contamination of humans and calves by STEC or EPEC O80:H2.The origin of human and calf infections by Shigatoxigenic (STEC) and enteropathogenic (EPEC)  Escherichia coli (EHEC) are a hybrid pathotype producing the Shiga toxins (Stx) of Shigatoxigenic E. coli (STEC) and the attaching–effacing (A/E) lesion of enteropathogenic E. coli (EPEC) [Enterohemorrhagic i (EPEC) . Since ti (EPEC) , they wii (EPEC)  in this E. coli [E. coli O80 and non-O80 serotypes, and different STs [The most frequent and pathogenic AE-STEC in humans belong to the following 6 serotypes: O26:H11, O103:H2, O111:H-, O121:H19, O145:H- and O157:H7 ,9,10. ToE. coli ,14,15,16rent STs ,18.AE-STEC and EPEC O80:H2 have also been frequently identified in young diarrheic and, more rarely, septicemic calves in Belgium since 2009 ,20. TheyE. coli. This is due to the deletion of the melibiose operon (mel) associated with the insertion of a 70 bp long DNA fragment (70mel) [Ruminants, especially cattle, are considered the most frequent source of human infection by the classical AE-STEC serotypes via foodstuffs contaminated by their faecal materials, since they can be asymptomatic carriers in their intestines . However (70mel) .E. coli O80 from healthy bulls at the slaughterhouse and healthy cows in farms, with emphasis on AE-STEC and EPEC O80:H2; (ii) identify the newly isolated E. coli O80 by PCR; and (iii) understand their phylogenomic relationships within the E. coli species after whole genome sequencing (WGS).The purpose of the present study was, therefore, to (i) isolate In November and December 2020, 149 slaughterhouse faecal samples were collected from young bulls during 3 visits to 1 slaughterhouse in the province of Liège, Belgium. The bulls originated from 43 different herds in the provinces of Liège, Limburg, Luxemburg and Namur, Belgium. Between October 2021 and February 2022, 194 faecal samples were collected from the rectum of healthy cows in late pregnancy, or maximum 1 month after calving, on 9 farms located in the provinces of Liège and Luxemburg, Belgium. One gram of each faecal sample was added to 9 mL of lauryl sulphate broth  and incubated overnight at 37 °C with shaking, for enterobacterial enrichment.wzy gene in the O80 antigen-encoding gene cluster  and total DNA was extracted by boiling for 10 min. In parallel, 2 mL of the enrichment broths was transferred into 2 CRYO tubes  with 2 mL of sterile 80% glycerol and stored at −20 °C and −80 °C, respectively. The DNA samples obtained were subjected to PCR targeting the E. coli 2/agar  for slaughterhouse samples or MacConkey agar  for farm samples. According to the manufacturers, Chromocult Coliform ES, Rapid E. coli 2/and MacConkey are selective for enterobacteria and coliforms in general, while TeK Chromocult Coliform ES and Chromagar STEC are selective for Te++-resistant coliforms, including a majority of STEC and EPEC.O80 PCR-positive enrichment broths were streaked on 5 (semi-)selective agar plates: Chromocult Coliform ES agar, Chromocult Coliform ES agar complemented with 2.5 mg/mL of potassium tellurite (TeK) and Chromagar STEC agar  for both slaughterhouse and farm samples, and either Rapid E. coli-like colonies were randomly picked up from the (semi-)selective agar plates. From the EHly blood agar plates, up to 10 colonies were picked up: 5 enterohemolyin-non-producing and 5 enterohemolysin-producing (if detected) colonies. In parallel, the O80 PCR-positive enrichment broths from farms were also streaked on melibiose-MacConkey agar plates. Melibiose-non-fermenting colonies (if detected) were picked up and subjected to species identification by API20E® , following the manufacturer’s instructions.All samples were also streaked on EnteroHemolysin (EHly) blood agar plates  to detect the production of enterohemolysin. After overnight incubation at 37 °C, up to 5 O80 PCR was performed on all picked-up colonies as described above, using the DNA samples obtained from 2 mL of an overnight culture in Luria–Bertani (LB) broth  at 37 °C with shaking.E. coli isolates were further tested by PCR using the FASTGENE2x Optima Hotstart kit  to detect the H2fliC gene encoding the H2 antigen and the following virulence-associated genes: eae encoding the intimin adhesin involved in the A/E lesion, stx1 and stx2 encoding Stx1 and Stx2a to Stx2d toxins, and pS88-located hlyF encoding the avian haemolysin . Libraries for Illumina sequencing were prepared using NEBNext UltraII FS DNA Library Prep Kit for Illumina  and sequenced on the Illumina Miseq platform (Illumina) to generate 300 bp paired-end reads. Assembly of the Illumina sequence reads was performed using the SPAdes (v3.13.0) assembler [The bovine O80 PCR-positive ssembler . Sequenccia, imm, cvaABC and cvi), iron acquisition  and virulence  [Gene annotation and in silico H antigen-genotyping were conducted by using DFAST  and Serond hlyF)   were searched for the genome sequences of d survey . Their Id survey . Using aE. coli O80 identified in healthy cattle, closed chromosome sequences of 104 E. coli strains representing each of 104 serotypes were selected and downloaded from the NCBI database and annotated by DFAST (Escherichia cryptic clade I strain TW10509 (No. AEKA00000000) was also downloaded and annotated to be used as an outgroup. The core genes (n = 2560) of all those E. coli were identified, and their concatenated sequence alignments were generated by Roary [To understand the phylogenetic positions of the by DFAST . The chrby Roary . Based oby Roary , a maximby Roary . Strainsby Roary  and the by Roary .E. coli O80. Three of the 5 isolates were isolated from 2 bulls on the Chromocult Coliform ES agar and the remaining 2 from another bull on EHly agar.After overnight enrichment growth in lauryl sulphate broths, 35 out of the 149 faecal samples (23%) from young bulls at 1 slaughterhouse were positive with the O80 PCR. Using the non-specific methodology, 450 colonies were picked up, with the majority from Chromocult Coliform ES (40%) and Chromagar STEC (30%) agar plates a. After E. coli O80. Ten of the 11 isolates from farm samples were isolated from 1 cow on Chromocult Coliform ES (5 isolates) and MacConkey (5 isolates) agar, and the remaining 1 from a second cow in another farm on EHly agar.After overnight enrichment growth in Lauryl Sulfate broths, 18 out of the 194 faecal samples (9%) from cows in 5 out of the 9 farms (55%) were positive with the O80-antigen PCR. Using the non-specific methodology, 385 colonies were collected, with the majority from EHly (36%) and Chromocult Coliform ES (24%) agar plates b. After E. coli.The 18 positive enrichment broths of faecal samples from cows in farms were also streaked on melibiose-MacConkey agar plates. Although melibiose non-fermenting colonies could be isolated after overnight growth, none of them was identified to H2fliC, eae, stx1, stx2 and hlyF genes. However, none of these 5 genes was detected in any of the 16 O80 PCR-positive isolates.To further identify the O80 PCR-positive isolates to AE-STEC or EPEC O80:H2, PCR and WGS analysis were performed to detect the presence of the n = 10) or H45 (n = 6)  . All 10 ectively .E. coli O80:H6 obtained from the same cow in 1 farm were identical, as were the 4 E. coli O80:H45 obtained from 2 bulls at the slaughterhouse. Therefore, only 1 E. coli O80:H6 from this cow and 3 E. coli O80:H45 from 2 bulls and 1 cow were included in the phylogenomic and other genome sequence-based analyses.The core gene sequence analysis also revealed that the 10 E. coli O80:H6 and O80:H45 were compared with the genome sequences of 8 O80:H6 and 8 O80:H45 strains obtained from the NCBI and EnteroBase databases, and of 1 Belgian E. coli O80:H45 previously isolated from duck faecal material  or US turkey (n = 8) isolates, the E. coli O80:H45 (n = 12) were isolated from cattle, duck, pig and the environment, in different countries . Like the Belgian bovine isolates, all but 1 US turkey E. coli O80:H6 belonged to ST8619, and the remaining 1 belonged to ST12217, a single locus variant (SLV) of ST8619  in the Belgian bovine isolate, whereas the US isolates contained 3 to 5 replicons.Phylogenetically, the Belgian bovine isolate (SES6039) formed a distinct branch from the 8 US turkey isolates b. MoreovE. coli O80:H6. In addition, the cib gene encoding the Colicin Ib and the genes for the biosynthesis of pyelonephritis-associated pili (PAP) were detected in 1 and 2 US isolates, respectively. Conversely, no horizontally acquired AMR gene was detected in the Belgian bovine isolate by ResFinder , IncFIC(FII) and/or IncFII(29) replicons were detected and, in 1 of these 8 isolates (FDA1149481-S003-085S with the largest genome), an additional contig containing 3 replicons was detected.The 12 O80:H45 isolates  formed 3 distinct sub-clusters c that arE. coli O80:H45, some isolates contained additional virulence-related genes: afaAB  in a French isolate, cdtB  in a Canadian isolate, and different pS88-located genes in addition to sitA and iss  in the Belgian duck isolate , although many more colonies from farm (36%) and slaughterhouse (16%) samples produce an enterohemolysin on the EHly agar  the human stool samples tested by Bizot and collaborators were clinical samples, probably with high numbers of AE-STEC O80:H2, while the bovine faeces tested in this survey were sampled from healthy animals, most probably with much lower numbers of E. coli O80:H2, if any, and (ii) the melibiose-MacConkey agar tested with the human stool samples also contains piperacillin, which was not used during our survey. Identifying the antibiotic/heavy metal, including to Te++, resistance profiles of the bovine AE-STEC and EPEC O80:H2 sequenced to date [E. coli O80 could also be increased by designing an O80 antigen-specific capture method, like for other highly pathogenic AE-STEC serotypes [Therefore, melibiose-MacConkey agar plates  were str to date ,19,39 wierotypes ,41.E. coli O80:non-H2 from humans and animals have already been reported and can belong to one of the numerous E. coli pathotypes [E. coli O80:H6 and O80:H45 isolated during this survey, nor the 16 E. coli O80:H6 and O80:H45 whose genome sequences were downloaded from NCBI and EnteroBase databases, nor the additional Belgian duck E. coli O80:H45, are closely related to CC165 and members of phylogroup A STEC, or EPEC, or belong to any other classical E. coli pathotype in humans or animals, with the possible exception of the duck E. coli O80:H45. Indeed, with the exceptions of the afa and cdtB genes in 2 O80:H45 isolates, the majority of the potentially virulence-related genes detected in the other E. coli O80:H6 and O80:H45 are widely distributed in E. coli, including in the laboratory strain K-12. Therefore, their importance in their pathogenicity, if any of these E. coli O80:H6 and O80:H45 is unclear at this stage, although some of them (sitA and iss) are involved in the survival of ExPEC in blood stream and internal organs and are plasmid-located [Search of virulence-associated genes by VirulenceFinder  revealed-located ,43.E. coli O80:H45 harbours several pS88-located genes, including the hlyF gene (E. coli (APEC), AE-STEC and EPEC O80:H2 [E. coli O80:H6 and O80:H45 of this study (sitA gene detected in most E. coli O80:H45 (11/12) has 100% identity to the sitA gene of pS88 in the duck isolate vs. 97.9–98.0% identity in the other isolates. Similarly, the iss gene detected in all E. coli O80:H6 (9/9) and most E. coli O80:H45 (11/12) also possesses 100% identity to the pS88-located iss gene in the duck isolate vs. 90.4–95.9% identity in the other isolates. In addition, nearly half (13/27) of the pS88-located genes are present in the duck isolate, with >99% sequence identity and 100% coverage to the pS88-located genes, and not in the other isolates. Finally, this duck isolate contains 1 of the 2 replicons of pS88 (IncFIB(AP001918)). These different data strongly suggest that the duck isolate, in contrast to the other E.coli O80:H6 and O80:H45, including the Belgian bovine ones, harbour a pS88-like plasmid. pS88 plasmids have already been detected in APEC O80:H26 [E. coli O80 outside of the CC165. Nevertheless, this duck E. coli O80:H45 was isolated from the faecal material and its actual virulence potential is not known at this stage.Conversely, the duck lyF gene  which isC O80:H2 ,19,39,44is study  was ther O80:H26 , but to E. coli O80:H6 and O80:H45 genomics and to assess the virulence potential, if any, of the Belgian bovine E. coli O80:H6 and O80:H45, and of the Belgian duck E. coli O80:H45.As general and specific conclusions, neither AE-STEC nor EPEC O80:H2 were isolated during this survey, and healthy adult cattle were not identified as the source of contamination of calves and humans. Moreover, the bovine


UNC45A gene were recently identified in osteo-oto-hepato-enteric (O2HE) syndrome patients presenting with congenital diarrhea and intrahepatic cholestasis. Congenital diarrhea and intrahepatic cholestasis are also the prime symptoms in patients with microvillus inclusion disease (MVID) and mutations in MYO5B, encoding the recycling endosome–associated myosin Vb. The aim of this study was to determine whether UNC45A and myosin Vb are functionally linked.UNC45A is a myosin (co-)chaperone, and mutations in the CRISPR-Cas9 gene editing and site-directed mutagenesis were performed with intestinal epithelial and hepatocellular cell lines, followed by Western blotting, quantitative polymerase chain reaction, and scanning electron and/or confocal fluorescence microscopy to determine the relationship between (mutants of) UNC45A and myosin Vb.UNC45A-c.1268T>A variant, restored recycling endosome positioning and microvilli development. The O2HE patient-associated p.V423D substitution, encoded by the UNC45A-c.1268T>A variant, impaired UNC45A protein stability but as such not the ability of UNC45A to promote myosin Vb expression and microvilli development.UNC45A depletion in intestinal and hepatic cells reduced myosin Vb protein expression, and in intestinal epithelial cells, it affected 2 myosin Vb-dependent processes that underlie MVID pathogenesis: rat sarcoma-associated binding protein (RAB)11A-positve recycling endosome positioning and microvilli development. Reintroduction of UNC45A in UNC45A-depleted cells restored myosin Vb expression, and reintroduction of UNC45A or myosin Vb, but not the O2HE patient A functional relationship exists between UNC45A and myosin Vb, thereby connecting 2 rare congenital diseases with overlapping enteropathy at the molecular level. Protein instability rather than functional impairment underlies the pathogenicity of the O2HE syndrome–associated UNC45A-p.V423D mutation.  Two genes in which mutations are associated with different human diseases with shared intestinal symptoms are shown to be functionally linked, thereby connecting these diseases at the molecular level. The pathogenic mechanism of one of the patient’s mutations was demonstrated.UNC45 gene, vertebrates have two UNC45 gene homologues, UNC45A and UNC45B. The human UNC45B gene (located on chromosome 17) is predominantly expressed in muscle cells, whereas the human UNC45A gene (located on chromosome 15) is ubiquitously expressed (https://www.proteinatlas.org/ENSG00000140553-UNC45A/tissue).Uncoordinated (UNC)-45 belongs to the conserved UNC-45/Cro1/She4p (UCS) family of myosin (co-)chaperones.UNC45A gene were reported in 4 patients of 3 families who clinically presented with congenital (secretory) diarrhea, cholestasis, sensorineural hearing loss, and/ or bone fragility, referred to as osteo-oto-hepato-enteric (O2HE) syndrome (Online Mendelian Inheritance in Men #619377).UNC45B mutations,UNC45A variants were identified that affected amino acid residues across the UNC45A protein.Bi-allelic compound heterozygous mutations in the MYO1A, MYO6, MYO7A, MYO15, or MYH9 mutations.MYO5B mutations.MYO5B mutations have been shown to cause the mislocalization of bile acid transporters in the hepatocytes of some patients,,Notably, symptoms displayed by O2HE patients, except for bone fragility, have also been reported as direct consequences of mutations in myosin-encoding genes. Indeed, inherited non-syndromic sensorineural hearing loss is a prime symptom of patients with MYH9 gene do not develop diarrhea, and reciprocally, O2HE patients do not develop macrothrombocytopenia . Also, patients with inherited sensorineural deafness due to mutations in the various other myosin-encoding genes do not typically develop diarrhea. Defects in these deafness-related myosins are therefore not likely to underlie the intestinal symptoms in patients with O2HE. Bone fragility and diarrhea also do not appear to be strongly correlated in O2HE because one O2HE patient with diarrhea did not present with bone fragility, and one O2HE patient with bone fragility did not present with diarrhea.In this study we focused on the relationship between UNC45A and the intestinal symptoms. Of the unconventional human myosins, thus far only the myosin IIa protein has been demonstrated to be a client for UNC45A.Caenorhabditis elegans orthologue of myosin V).Saccharomyces cerevisiae.Danio rerio), depletion of either intestinal unc45aMYO5B orthologue goosepimplesYeast-two-hybrid experiments indicated that UNC45 may interact with the motor domain of Hum2  in the UNC45A genes in Caco-2 cells (A). These frameshift mutations were predicted to result in premature termination codons and subsequent loss of expression of the UNC45A protein. Western blot analyses confirmed the absence of the UNC45A protein when compared with control cells (B).To investigate a functional relationship between UNC45A and myosin Vb, we used the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas)9–based technology to knockout the -2 cells A. These ol cells B.Figure UNC45A knockout (KO) Caco-2 cells (4 independent clones) showed a reduced expression of the myosin Vb protein when compared with control cells, as evidenced by Western blot (C and D). The specificity of the myosin Vb antibody was validated in MYO5B KO cells (C). Quantification of Western blots of 3 independent experiments revealed 60%–80% reduction in full-length myosin Vb protein expression in Caco-2 UNC45A KO cells (D). Occasionally a reduction in UNC45A expression in MYO5B KO cells was observed (C), but this was not statistically significant over multiple independent experiments, suggesting that the relationship between UNC45A and myosin Vb expression is unidirectional. The effects of UNC45A depletion were not limited to myosin Vb protein expression because also a reduced expression of myosin 1A and 1C was observed (E and F). The expression levels of 2 other gene products that have been associated with MVID, ie, the STX3- and STXBP2-encoded syntaxin-3Figure 1G and H).ern blot C and D. KO cells C. QuantiKO cells D. Occasiobserved C, but thobserved E and F. UNC45A KO cells when compared with control cells (A). Treatment of UNC45A KO cells with the peptide aldehyde Cbz-leu-leu-leucinal (MG132), which is a potent proteasome inhibitor, caused an increase in the amount of ubiquitinylated proteins as evidenced by the increased reactivity toward the anti-ubiquitinylated proteins antibody FK2 (demonstrating its activity) but did not restore expression of full-length myosin Vb protein (B and C), suggesting that myosin Vb was not synthesized in UNC45A depleted cells. This was confirmed by immunofluorescence microscopy with antibodies against myosin Vb (D).Quantitative polymerase chain reaction (using 3 different primer sets) revealed no reduction in myosin Vb mRNA in ol cells A. Treatm protein B and C, yosin Vb D.Figure UNC45A KO Caco-2 cells (3 independent clones) restored myosin Vb expression when compared with untreated UNC45A KO cells and wild-type (WT) cells (E and F). Together, the data unequivocally demonstrate that loss of UNC45A caused a reduction in myosin Vb protein expression.The reintroduction of human UNC45A carrying a FLAG (DYKDDDDK)-tag at its carboxyl-terminus (UNC45A-FLAG) in T) cells E and F. ,,,UNC45A KO Caco-2 cells. As shown in Figure 3A, RAB11A labeled vesicular structures throughout the cytoplasm and in the periphery of most control Caco-2 cells. By contrast, RAB11A showed a condensed juxta/supra-nuclear labeling pattern in UNC45A KO cells . This redistribution of RAB11A-positive RE in UNC45A KO Caco-2 cells was similar to that observed in MYO5B KO Caco-2 cells . The subcellular distribution of RAB11A-positive RE has previously been reported to depend on microtubules.UNC45A KO cells .We next examined the impact of UNC45A loss on myosin Vb-dependent processes. We and others have previously shown that the loss of myosin Vb expression or function affects the spatial distribution of rat sarcoma-associated binding protein (RAB)11A-positive recycling endosomes (RE) in enterocytes in vivoUNC45A KO Caco-2 cells, the UNC45A-FLAG protein was reintroduced. Immunolabeling of RAB11A showed that the reintroduction of UNC45A-FLAG in UNC45A KO cells reduced the condensed RAB11A labeling phenotype (E), which was still present in cells that did not or minimally express the UNC45A-FLAG protein .To determine the causality between the loss of UNC45A and the observed redistribution of RAB11A-positive RE in henotype E, which  protein E, arrowsUNC45A KO Caco-2 cells. The functionality of the encoded myosin Vb was previously demonstrated.Figure 3F shows the expression of myosin Vb as evidenced by Western blot analysis. In contrast to the expression of endogenous myosin Vb, the expression of myc-myosin Vb-FLAG appeared unaffected by the loss of UNC45A because expression was achieved in both control cells and UNC45A KO cells. Immunolabeling for RAB11A showed that introduction of myc-myosin Vb-FLAG in UNC45A KO cells reduced the condensed RAB11A labeling phenotype , which was still present in cells that did not express the myc-myosin Vb-FLAG protein .To determine the causative role of loss of myosin Vb in the redistribution of RAB11A-positive RE upon UNC45A depletion, a myc-FLAG–tagged human myosin Vb protein (myc-myosin Vb-FLAG) was overexpressed in Collectively, these results demonstrated that loss of UNC45A in Caco-2 cells reduced the expression of myosin Vb and, consequently, caused the microtubule-dependent redistribution of RAB11A-positive RE.,,,MYO5B KO and UNC45A KO Caco-2 cells were less abundant and shorter when compared with WT Caco-2 cells (A). Notably, in agreement with the electron microscopy results, F-actin- and ezrin-stained microvilli at the apical surface appeared, in a patchy manner, less abundant and shorter in both UNC45A KO and MYO5B KO Caco-2 cells when compared with WT Caco-2 cells (A–C). To determine the causality between the loss of UNC45A and the observed alterations in microvilli organization in UNC45A KO Caco-2 cells, UNC45A-FLAG was reintroduced. Immunolabeling of F-actin showed that cells that expressed UNC45A-FLAG rescued the microvilli phenotype (D). The microvilli phenotype was also rescued in UNC45A KO Caco-2 cells in which myc-myosin Vb-FLAG was expressed, but not in the cells that did not express the myosin Vb protein (E).The loss of myosin Vb expression and redistribution of RAB11A-positive RE have been associated with atrophic or altered organization of brush border microvilli,-2 cells . Immunof-2 cells A. Notabl-2 cells A–C. To dhenotype D. The mi protein E.Figure Taken together, loss of UNC45A and the subsequent reduction in myosin Vb expression resulted in less and/or less organized apical microvilli. The effect of UNC45A depletion on apical microvilli in Caco-2 cells is in agreement with the patchy brush border atrophy observed in the O2HE patient’s intestine.UNC45A mutations have also been associated with intrahepatic cholestasis in some O2HE patients, we examined the effect of UNC45A depletion on myosin Vb expression in hepatocellular HepG2 cells, widely used model cell lines for the study of hepatocellular biology. HepG2 cells form bile canalicular lumens with bile canalicular surface domains that are enriched in microvilli-associated actin-filaments and bile canalicular proteins such as ABCC2 (MRP2), and which are surrounded by RAB11A-positive RE (A). Gene sequencing of CRISPR-Cas9–treated HepG2 cells revealed homozygous frameshift mutations in the UNC45A gene in HepG2 cells (B). This frameshift mutation was predicted to result in a premature termination codon and subsequent loss of expression of the UNC45A protein. Western blot analyses confirmed the absence of the UNC45A protein when compared with control cells (C). UNC45A KO HepG2 cells (2 independent clones) showed a reduced expression of the myosin Vb protein when compared with control cells, as evidenced by Western blot (D). Quantification of Western blots of 3 independent experiments revealed an 80%–100% reduction in myosin Vb protein expression in HepG2 UNC45A KO cells (E). UNC45A mRNA expression levels were unaffected (F). The reintroduction of UNC45A in UNC45A KO HepG2 cells restored myosin Vb expression levels (G). Although loss of myosin Vb expression has been reported to affect the spatial distribution of RAB11A-positive RE in intestinal cellsUNC45A KO HepG2 cells, the typical subapical localization of RAB11A, the apical localization of ABCC2, and appearance of F-actin–stained apical microvilli were likewise indistinguishable from control HepG2 cells (H and I). Thus, although these results underscore the importance of UNC45A expression in maintaining myosin Vb expression levels, only in Caco-2 cells did the reduction in myosin Vb expression correlate with a redistribution of RAB11A-positive RE, consistent with the reported differential involvement of myosin Vb in RAB11A-positive RE positioning in these cell types.,Because itive RE A. Gene sG2 cells B. This fol cells C. UNC45Aern blot D. QuantiKO cells E. UNC45Aaffected F. The ren levels G. AlthouG2 cells H and I. UNC45A, presented with isolated congenital intractable diarrhea with no signs of cholestasis, bone fragility, or deafness.Figure 7A). Val423 is conserved in mouse UNC45A as well as in human and mouse UNC45B . In the UNC45A orthologue of the invertebrate fruit fly Drosophila melanogaster and nematode Caenorhabditis elegans, the amino acid at this position is a leucine or isoleucine, which share the hydrophobic characteristics of valine (conservative replacement). UNC45A and UNC45B of the vertebrate zebrafish Danio rerio show a leucine and valine at this position, respectively, supporting that a hydrophobic branched-chain amino acid is favored at this position in the protein and that the 3 branched-chain amino acids  are interchangeable .One of the 4 O2HE patients, carrying compound heterozygous c.1268T>A and c.784C>T mutations in UNC45A (C) and show by means of Western blot analyses that the reintroduction of the UNC45A-c.1268T>A variant in UNC45A KO Caco-2 cells (D) resulted in very little if any rescue of UNC45A expression and, accordingly, did not rescue full-length myosin Vb protein expression (D). MG132 treatment of UNC45A KO Caco-2 cells in which the UNC45A-c.1268T>A variant was reintroduced significantly increased the expression of a full-length (∼103 kDa) UNC45A-p.V423D (D and E). This indicated that this mutant protein was fully formed but was unstable and degraded by the proteasome. Moreover, MG132 treatment of UNC45A KO Caco-2 cells in which FLAG-tagged UNC45A-p.V423D was re-expressed (D and E) but not of UNC45A KO as such  and increased the expression level of myosin Vb. Interestingly, when examining UNC45A-p.V423D expression by immunofluorescence microscopy, we noted that although UNC45A-p.V423D was not visibly expressed in most cells that were not treated with MG132, a small number of cells did express the FLAG-tagged UNC45A-p.V423D . Notably, these FLAG-positive cells also showed increased expression of myosin Vb  and a rescue of the microvilli phenotype as evidenced by F-actin staining . FLAG-positive cells  also showed a rescue of RAB11A-positive RE distribution . We conclude that (1) the c.1268A>T-encoded p.V423D mutation in the UNC45A protein caused its instability and subsequent proteasomal degradation, and (2) the p.V423D mutation, as such, did not impair the function of the UNC45A protein with regard to its ability to promote myosin Vb expression, RAB11A-positive RE positioning, and microvilli development.Although predicted to be pathogenic, the mechanism that underlies the pathogenicity of the p.V423D mutation is not known. Here we used site-directed mutagenesis to introduce the c.1268T>A variant in human UNC45A C and sho-2 cells D resultepression D. MG132 -p.V423D D and E. xpressed D and E bsuch cf, B and inc-p.V423D F, green.yosin Vb F, red anstaining F, white.,UNC45A gene with an UNC45A gene carrying the c.1268T>A mutation in intestinal and liver epithelial cell lines resulted in a significant reduction in the expression of MVID-associated myosin Vb. We demonstrated that the patient’s p.V423D substitution in UNC45AIn this study we tested the hypothesis that UNC45A and myosin Vb, the losses of which are associated with 2 rare diseases with shared enteropathy,MYO5B gene transcription was not affected. Inhibition of proteasome activity did not restore full-length myosin Vb expression in UNC45A-depleted cells, as evidenced by Western blot analysis. Because the anti-myosin Vb antibody recognizes an epitope that is located in the coiled-coil domain  that is distal to the motor domain, we could not determine whether an incompletely synthesized myosin Vb protein was formed. The expression of an exogenous, amino-terminal myc-tagged myosin Vb could not be used to detect myc-positive myosin Vb (motor domain) fragments in UNC45A–depleted cells because its expression appeared insensitive to UNC45A depletion. Thus, our results support a role for UNC45A in the regulation of myosin Vb at the protein level. Additional studies, which are beyond the scope of this study, are needed to unravel the precise mechanism via that UNC45A controls endogenous myosin Vb expression.In vitro transcription/translation-coupled assays suggested that UNC45A regulated the folding of the smooth muscle myosin motor domain post-translationally.UNC45A gene expression in intestinal cells not only reduced myosin Vb protein expression but also affected 2 myosin Vb-dependent processes, microvilli development and RAB11A-positive RE positioning. Both processes have been implicated in the pathogenesis of MVID.,,UNC45A-depleted Caco-2 cells is in line with such a model.We demonstrated that prohibiting endogenous ,Notably, UNC45A has been identified as a microtubule-destabilizing protein,MYO5B,,,,,STX3,,STXBP2,,,,,,,myo5b KO mice.UNC45A-depleted Caco-2 and HepG2 cells showed reduced myosin Vb expression while preserving these downstream in vivo phenotypic differences between intestinal and liver cells (this study), which underscores that these phenotypes are not cell line anomalies and predictive of the in vivo situation. Finally, the microvillus atrophy as observed in UNC45A-depleted Caco-2 cells is in agreement with the reported microvillus abnormalities in the enterocytes of the O2HE patient intestine.The observed reduced myosin Vb expression and RE positioning could not yet be validated in O2HE patient intestinal tissue because of lack of access to patient material. Nonetheless, Caco-2 and HepG2 cells have been proven to be highly predictive models for the cellular phenotypes in MVID(-like) disease. Indeed, the depletion of myosin Vb, syntaxin-3, or syntaxin-binding protein-2 in Caco-2 cells faithfully reproduced the key in vivo phenotypes of intestinal epithelial cell in MVID patients resulting from mutations in The unidirectional link between UNC45A and myosin Vb expression as demonstrated in this study suggests that reduced expression of myosin Vb and perturbation of myosin Vb-dependent processes may underlie (part of) the intestinal symptoms in O2HE patients. Furthermore, as a regulator of myosin Vb expression, changes in the expression or functionality of UNC45A may contribute to the heterogeneity and complexity of clinical presentations associated with microvillus inclusion disease.5) the diarrhea resolved at unspecified later age, and her sister (patient B.II.35) reportedly did not present with diarrhea. The reason for this heterogeneity in diarrhea presentation is not clear but is also observed in MYO5B-associated MVID.UNC45A mutations determine the diarrheal phenotype in O2HE. For example, the siblings B.II.4 and B.II.35 carried mutations located in the very C-terminal end of the armadillo-like domain of UNC45A, which has different functions when compared with the N-terminal tetratricopeptide repeat domain and/or central domain of UNC45A in which the mutations of the other 2 patients were identified. The reported difference in diarrhea presentation between siblings B.II.4 and B.II.35 further suggests that also non-genetic factors may play a role.O2HE patients also display heterogeneity in the clinical presentation. Three of the 4 O2HE patients described by Esteve et alMYO5B mutations, bi-allelic mutations in the MYO5B gene that are predicted to result in the loss of myosin Vb expression have not been identified in patients with isolated intrahepatic cholestasis.,,UNC45A mutations reported in O2HE patients who did present with cholestasis will lead to a similar loss of myosin Vb expression and whether any myosin Vb remaining will function properly in the absence of UNC45A or may behave as a dysfunctional/mutant-like myosin Vb. It is also possible that UNC45A mutations affect other myosin proteins that have been proposed to play a role in bile acid secretion  did not ablate the function of the mutant UNC45A protein with regard to its ability to promote myosin Vb expression and microvilli development. In support of this, V423 is not located in the functional tetratricopeptide repeat or UCS domains of the UNC45A protein, and valine (similar to leucine and isoleucine) is assumed to be non-reactive and as a hydrophobic branched-chain amino acid more likely to be involved in protein folding and structure.We demonstrated that the Taken together, this study revealed a functional relationship between UNC45A and myosin Vb protein expression, thereby connecting 2 rare congenital diseases with overlapping intestinal symptoms at the molecular level. Protein instability rather than functional impairment underlies the pathogenicity of the O2HE syndrome–associated UNC45A-p.V423D mutation.2 for Caco-2 and Hek293 and 7.5% CO2 for HepG2. For experiments, cells were grown on poly-L-lysine–coated coverslips and fixed after 3 days or 14 days in culture.Human Caco-2 cells (American Type Culture Collection HTB-37), HepG2 cells (American Type Culture Collection HB8065), and human embryonic kidney (HEK)293 cells (American Type Culture Collection CRL-1573)  were cultured in high-glucose Dulbecco modified Eagle medium  supplemented with 10% heat-inactivated fetal calf serum , 2 mmol/L L-glutamine and antibiotics  (Thermo Scientific Fisher), and incubated at 37°C in a humidified atmosphere with 5% COAntibodies used in this study were ABCC2/ MRP2 , RAB11A (mouse) , RAB11A (rabbit) , myosin Vb , FLAG , UNC45A (rabbit) , UNC45A (mouse) , beta-tubulin , c-myc , ezrin , beta-actin , syntaxin-3 , and munc18-2 . Fluorescently labeled phalloidin  was from Invitrogen.MYC deletion forward 1: GCAGCAAATGATATCCTG, MYC deletion reverse 1: CTCTCCATCTTGGTTGGG, deletion forward 2: GATTACAAGGATGACGACGATAAGGTTTAA, MYC deletion reverse 2: GAGCGGCCGCGTACGCGT, UNC45A p. Val423Asp forward: CTGAGCGGTGaCATGGAGAGTGTG, UNC45A p.Val423Asp reverse: CTCCAAGGCCCGGTTGCC, UNC45A CRISPR 1 forward: CACCGAGACTACGGGGGCGCCCTGG, UNC45A CRISPR 1 reverse: AAACCCAGGGCGCCCCCGTAGTCTC, UNC45A CRISPR 2 forward: CACCGGTTCAAATGTGGAGACTACG, UNC45A CRISPR 2 reverse: AAACCGTAGTCTCCACATTTGAACC. Caco-2 UNC45A KO2 and HepG2 UNC45A KO2 were both generated with oligos UNC45A CRISPR 2 forward and UNC45A CRISPR 2 reverse.UNC45A sequence was amplified from plasmid pCMV6-Entry-UNC45A (Origene RC206953) through polymerase chain reaction. Amplified UNC45A-myc-FLAG was inserted into pENTR1a vectors, and then the myc tag was deleted. Patient-derived point mutations were produced on the myc tag deleted pENTR1a plasmid. The Q5 Site-Directed Mutagenesis Kit  with primers designed in the NEBaseChanger tool was used for myc tag deletion and point mutation creation. UNC45A-FLAG and mutant UNC45A-FLAG were cloned into lentiviral vectors.UNC45A.Full-length tagged human 6 HEK293T were plated in a well of a 6-well plate. Vectors of 1200 ng pLenti-plasmid, 1000 ng pCMVdR8.1, and 400 ng pVSV-G were mixed into 100 μL OptiMEM with 7.8 μL Fugene . After 5 minutes of incubation, the mixture was added into the medium of HEK293T. The next morning the medium was removed, and virus production medium: DMEM with 10% fetal calf serum and Dulbecco modified Eagle medium: F12 (no serum) in the ratio of 1:3 was added. After a 2-day incubation the virus-containing supernatant was filtered through a polyvinylidene difluoride membrane–based 0.45 μm filter  and harvested as viral particles. After seeding in 6-well plate at least for 4 hours, the target cells were cultured with 500 μL viral particles and 1 mL medium supplemented with 8 μg/mL polybrene. After overnight incubation, the virus-containing medium was replaced with antibiotics-containing medium .With a second-generation system based on pCMVdR8.1 and pVSV-G, lentiviral particles were generated to transduce cells. For virus production, 1 × 10Cell pellets were lysed with lysis buffer  supplemented with protein inhibitors . Bicinchoninic acid (Sigma B-9643) and 4% (w/v) CuSO4.5H2O were used to determine protein concentration. Equal amounts of protein lysates were mixed with sample buffer 2% sodium dodecyl sulfate, 5% β-mercaptoethanol, 0.125 mol/L Tris–HCl, pH 6.8, 40% glycerol, 0.01% bromophenol blue) and heated at 95°C for 5 minutes and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with Odyssey-blocking buffer  and incubated with primary antibody for 2 hours at room temperature or overnight at 4°C and secondary antibody for 2 hours at room temperature. Blots were then detected by Odyssey Imaging System (LI-COR) and quantified with Odyssey software.Cells were fixed with 4% paraformaldehyde for 20 minutes and permeabilized in 0.2% (v/v) Triton X-100 for 10 minutes at room temperature and then blocked in phosphate-buffered saline solution containing 1% (w/v) fetal calf albumin in 37°C incubator for more than 1 hour. Cells were incubated with primary antibodies overnight at 4°C, and secondary antibodies, 4′,6-diamidino-2-phenylindole (DAPI), Alexa-633–labeled phalloidin for 2 hours at room temperature. Coverslip were mounted in Dako mounting medium and dried overnight. All images shown in figures were taken by a SP8X DLS confocal , 63×, oil-immersion objective and analyzed using a combination of ImageJ and Adobe Photoshop. x-y mages are shown as maximum intensity projections. Yellow lines indicate position of x-z, y-z sections.Cells grown on glass coverslips were fixed by adding 1 volume 2% glutaraldehyde in 0.1 mol/L sodium cacodylate to the cells still in culture medium. After 10 minutes this mixture was replaced by pure fixative for another 20 minutes. Cells were washed with 0.1 mol/L sodium cacodylate and post-fixed in 1% osmium tetroxide in 0.1 mol/L sodium cacodylate for 1 hour, followed by 3 times washing with water. Cells were dehydrated with ethanol  and dried using critical point drying from carbon dioxide. Samples were glued on stubs using double-sided carbon adhesives and sputter coated with 10 nm chrome before imaging in a Zeiss Supra55 SEM  at 3 KV.MYO5B expression level was determined by real-time quantitative polymerase chain reaction using ABsolute aPCR SYBR Green Master Mix  in a Step-One Plus Real-Time PCR machine . Primers to target MYO5B sequences were TCGGGGTCCTGGACATCTAT (qRT1 forward), CCAGTTTGAAAACATGCGAGTTG (qRT1 reverse), TTGGAAGTGTGGCGATTCAG (qRT2 forward), GCAGTCGGCAGAAGTTGCTT (qRT2 reverse), TCCCCTGAATGAATTTGAAG (qRT3 forward), GGTCATTCCGCTCTTGTAGT (qRT3 reverse).As described,t tests were performed to determine statistical significance with Graph Pad Prism software . P values were ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001.All experiments were performed at least 3 times independently. Two-tailed unpaired Student Qinghong Li Zhe Zhou Yue Sun Chang Sun Karin Klappe Sven van Ijzendoorn


Our results showed that HAug could induce a cross-strain protective immune response in mice against both H3N2 and H7N9 subtypes with better antibody-dependent cellular cytotoxicity (ADCC) than the HAmg- and HAfg-immunized groups, which suggested that highly conserved epitopes that were masked by surface glycosylation may be exposed and thus promote the induction of broad antibodies that recognize the hidden epitopes. This strategy may also supplement the direction of deglycosylated modified HA for universal influenza vaccines.Recent efforts have been directed toward the development of universal influenza vaccines inducing broadly neutralizing antibodies to conserved antigenic supersites of Hemagglutinin (HA). Although several studies raise the importance of glycosylation in HA antigen design, whether this theory can be widely confirmed remains unclear; which influenza HA with an altered glycosylation profile could impact the amplitude and focus of the host immune response. Here, we evaluated the characteristics and efficacy of deglycosylated modified HA proteins, including monoglycosylated HA (HA The influenza virus is a rapidly evolving pathogen that remains a serious threat to public health worldwide despite the availability of licensed vaccines. Influenza vaccination is one of the most effective means of preventing influenza infection and disease. Currently, influenza vaccines are composed of H1N1 and H3N2 of influenza A virus and either lineage  or both lineages  of influenza B virus, which were recommended by the World Health Organization (WHO). The low protective effect of the current influenza vaccines was mainly due to the significant mismatch between circulating and recommended strains, especially against H3N2 viruses with rapid antigenic drifts [Hemagglutinin (HA) protein mediates the membrane fusion of viral and host cells and thus is regarded as the primary vaccine target ,5,6,7. HThe immunodominant response elicited against influenza HA often targets variable epitopes on the head domain of HA, which is ready for escape mutation . Thus, tmg) trimmed by glycosidase could elicit a cross-strain immune response [ug) rather than HAmg. Similar results could be observed in ferret models.Glycosylation is a common modification that plays an important role in protein biology . Since tresponse . This st50  is 1 × 105 TCID50; A/Beijing/32/1992(H3N2) virus was serial passaging up to 14 passages, and the MLD50 is 5 × 104 TCID50; A/Shanghai/02/2013(H7N9)  × A/Puerto Rico/8/1934 (H3N2) reassortant was serial passaging up to 8 passages, and the MLD50 is 5 × 105 TCID50. All viruses were grown in MDCK cells using standard viral culturing techniques.Madin-Darby Canine Kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC). MDCK cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)-high glucose  supplemented with 10% low endotoxin fetal bovine serum (FBS) . The influenza virus strains A/Aichi/1968 (H3N2), A/Beijing/32/1992 (H3N2), and Wisconsin/67/2005 were kindly provided by BEI Resources. The influenza virus A/Shanghai/02/2013(H7N9) × A/PR/8/34 reassortant was obtained from the University of Hong Kong. The mouse-adapted strains A/Aichi/1968(H3N2), A/Beijing/32/1992(H3N2), and A/Shanghai/02/2013(H7N9)  × A/PR/8/34 reassortant were generated in our laboratory through successive passaging in the lungs of mice. A/Aichi/1968(H3N2) virus was serial passaging (up to 7 passages) through the lungs of BALB/c mice, and the MLDKpnI and XbaI restriction sites of the pCMV vector for human cell expression. HA protein was expressed and purified from 293F cells. All HA proteins were cloned, expressed, and purified as above.The H3 HA sequences were downloaded from the GISAID database. The transmembrane domain was replaced with the residues that are at the bacteriophage T4 fibritin trimerization segment, the thrombin cleavage site, and the His-tag at the C-terminus of HA. The nucleotide sequence of HA from A/Wisconsin/67/2005 (H3N2) was synthesized with codons optimized by Sangon . The gene was inserted into the mg was obtained by treatment with Endo H from New England Biolabs of the full-length HA, which was incubated with 200 Units of Endo H/mg protein at 37 °C for 16–18 h. HAug was obtained by treatment with PNGase F , which added 10 × G7 reaction buffer, 10% (v/v) Nonidet P-40, and 500 units of PNGase F at 37 °C for 16–18 h.Potential N-linked glycans were predicted using the NetNGlyc server. HAfg, HAmg and HAug proteins in 50 μL Tris buffer  mixed with 50 μL aluminum adjuvant at days 0 and 14. Serum samples were collected at day 28 for serological analysis. At day 42, immunized mice were challenged with 25 MLD50 of the homologous viruses A/Beijing/32/1992(H3N2) and A/Aichi/1968(H3N2) obtained from BEI Resources and heterologous virus A/Shanghai/02/2013(H7N9) × A/PR/8/34 reassortant obtained from the University of Hong Kong intranasally. PBS-immunized mice were used as a mock control. Mice were sacrificed at 3 dpi and 6 dpi to determine lung virus titers, which were determined by TCID50 assay. Survival rates and body weights were monitored daily for 14 days. Twenty-five percent weight loss was used as a humane endpoint for challenge. Any mice reaching that endpoint were euthanized.Female 6- to 8-week-old BALB/c mice (5 per group) were immunized twice with 15 μg HAfg, HAmg and HAug proteins in 300 μL Tris buffer mixed with 300 μL aluminum adjuvant on days 0 and 28. Serum samples were collected at day 42 for serological analysis. On day 56, immunized ferrets were challenged with 50 LD50 of the homologous viruses A/Beijing/32/1992(H3N2) intranasally. Veterinary clinical symptoms were observed every day. Nasal washes were collected every other day for 7 days. Virus titrations were obtained from nasal washes titrated by TCID50.Groups of four female 16- to 18-week-old ferrets were immunized twice in intramuscular route with 30 μg HASerum samples were collected 28 days after the booster immunization and treated with receptor destroying enzyme (RDE)  at 37 °C for 18 h followed by inactivation of the RDE via incubation at 56 °C for 30 min for serological analysis.ELISA was performed using standard ELISA procedures. Dilutions of immunized sera were incubated for 45 min at 37 °C on 96-well plates coated with 10 ng inactivated virus by ultracentrifugation. Binding was visualized by incubation with HRP-conjugated anti-IgG, -IgG1, or -IgG2a isotype antibodies  for 45 min at 37 °C. BM Chemiluminescence ELISA Substrate  was added to the plates. Plates were then read on a Lmax plate reader . Between each step, plates were washed with PBST.For the hemagglutination inhibition assay, RDE-treated sera were serially diluted 2-fold in 96-well plates and incubated with 8 HA units of virus for 30 min at room temperature. HAI titers were measured by the addition of 50 µL of 0.5% turkey red blood cells or 0.75% guinea pig erythrocytes by incubation for 45 min at room temperature.For the microneutralization assay, RDE-treated sera were mixed with 200 TCID50 of virus and were then incubated for 1 h at 37 °C. The mixture was added to confluent MDCK cells in MEM supplemented with 2 μg/mL 6-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK)-treated trypsin in a 96-well plate and incubated at 37 °C for 48 h. HA titers were used to determine the neutralization titers by HA assay.4 cells per well in 96-well plates (in duplicate) and incubated for 12 h at 37 °C with 5% CO2. The cells were then washed twice with PBS and replenished with 100 μL per well of assay media (DMEM).MDCK cells infected with virus at a multiplicity of infection (MOI) of 10 were used as target cells. The target cells were plated at a concentration of 3 × 102. The absorbance was read on a microplate reader to determine cell viability based on dye-reducing activity levels. In order to calculate the percent of CDC, the following calculation was used: cytotoxicity (%) = / × 100. Target cells treated with complement alone were used to determine the baseline reaction value. Target cells treated with complement and sera were used to determine the experimental reaction value, and target cells lysed in 1% Triton X-100 were used to determine the maximal reaction value2.6. Flow cytometryFor the ADCC assay, the target cells labeled with PKH67 (Sigma-Aldrich) were washed twice with PBS and plated in triplicate at 5 × 104 cells per well in 96-well plates, followed by the addition of serially diluted RDE-treated sera. After incubation for 30 min at 37 °C, human PBMCs were added to the target cell-antibody mixture at 1 × 106 cells per well. Following 3 h of incubation at 37 °C, 1 μL of 7-AAD (eBioscience) was added to the wells to stain dead cells. Cell death was determined on a FACS Aria III flow cytometer using BD FACS Diva software (BD Biosciences). For the CDC assay, serially diluted RDE-treated sera were added to each well, followed by 50 μL of human serum complement diluted in PBS (Quidel), and the cells were incubated for 2 h. Finally, 10 μL of cell-counting kit reagent  was added to each well, and the plates were incubated for 48 h at 37 °C and 5% CO6 cells/well and stimulated with 5 µg/mL HA, and anti-mouse CD28 (10 µL/mL) was added for co-stimulation. After 5 h of incubation with antigens (37 °C and 5% CO2), the cells were washed with PBS, and the remaining erythrocytes were lysed with a commercially available hemolysis buffer .The ratios of influenza virus-specific IFN-γ-secreting cells in CD8+/CD4+, and IL-4 secreting cells in CD4+ T-cell levels were determined by flow cytometry. Mouse spleens collected on day 28 after prime-boost immunization were dissected and passed through a 40 mm cell strainer . The splenocytes were seeded at 1 × 10Isolated splenocytes were washed with FACS staining buffer, stained for 30 min with fluorescein isothiocyanate (FITC)-labeled rat anti-mouse monoclonal antibodies (BD Biosciences) against the CD4 and CD8 cell surface markers, fixed and permeabilized with Cytoperm/Cytofx solution. Later, the cells were washed and treated with phycoerythrin-conjugated rat anti-mouse IFN-γ antibody for 30 min at RT in the dark. Analysis was performed on a FACS Calibur flow cytometer (BD Biosciences).p values were <0.05 using GraphPad Prism 8.0 . The significance of differences in neutralization/antibody titers and mean fractional body weights of surviving mice between different groups were analyzed by analysis of variance and Student’s t test. Statistical significance in all figures is expressed as *, p < 0.05; **, p < 0.01; and ***, p < 0.001, **** p < 0.0001.Statistical significance was assigned when To analyze the potential sites for glycan modification, we found that the number of glycosylation modifications of H3N2 subtype influenza A viruses increased from 7 to 11 during their evolution in humans from 1968 to the present . While afg) from A/Wisconsin/67/2005 (H3N2) expressed by human embryonic kidney cells (HEK293). Monoglycosylated HA (HAmg) is obtained after treatment with the glycosidase Endoglycosidase H (Endo H) with GlcNAc only, and unglycosylated HA (HAug) is obtained after treatment with the glycosidase PNGase F, which is denoted deglycosylated forms with amino acid change from asparagine (Asn) to aspartic acid (Asp) in each glycosylation site , indicating higher Th1-biased antibody responses to the heterotypic virus, while a higher IgG1/IgG2a ratio was induced by HAug group of antiserum against homotypic virus BJ/92(H3N2) strain , A/Beijing/32/1992 (BJ/92), A/Aichi/1968 (AI/68), and A/Shanghai/02/2013 (SH/13) A. Sera onfection ,24. Upre) strain . Sera fr) strain B,C. Inteug-vaccinated group  mouse-adapted viruses of A/Beijing/32/1992(H3N2), A/Aichi/1968(H3N2) and A/Shanghai/02/2013(H7N9). Mice vaccinated with PBS were used as controls. The efficacy of vaccine protection was evaluated for 14 days by recording the survival rate and body weight change  against heterosubtypic group two H7N9 SH/13 virus infection, in contrast to the 0% survival of HAfg, HAmg, and control groups.In order to verify the antiviral effect of deglycosylated HA proteins, six-week-old female BALB/c mice were intramuscularly immunized twice as indicated with a two-week interval with 15 μg HAand HAug A and chat change B,D,F. Fomg- and HAug-immunized mice had significantly reduced lung virus titers at 6 dpi in the case of the H3N2 virus challenge . Nasal washes were collected from the infected ferrets. Virus titrations of nasal washes were determined by TCID50 assay. Moreover, viral load detection showed that viruses replicated at a lower level in the nasal washes of HAmg- and HAug-immunized ferrets at 3 dpi to 7 dpi. At 7 dpi, viruses were not detected in the nasal washes of the HAug groups, whereas a significantly higher viral titer was maintained in the control ferrets. Thus, these results indicated that HAug-immunized ferrets could provide more effective protection against influenza virus infection.In addition, 28 days after the second vaccination, ferrets were challenged intranasally with 50 LDmg) was reported to elicit a cross-strain immune response, we speculated that deglycosylated modified HA might be beneficial in unlocking occluded and conserved epitopes, especially at the stem region, by removal of surface glycosylation for both globular and stem regions. The major influencing factors, especially for the stem region, contribute to the change in immunodominance patterns. On the other hand, although the prokaryotic expression system does not support glycosylation modification, unstable protein folding might not provide the natural conformation of occluded and conserved epitopes in HA. Thus, rational design for glycosylation modification to unlock occluded and conserved epitopes in eukaryotic expression would be a valuable approach in the future, which would also be applied to live attenuated vaccines to evoke a more robust humoral response to iron out flaws [Despite efforts and the availability of licensed commercial vaccines, seasonal influenza remains a major public health burden worldwide. Antigenic drift, mostly caused by the accumulation of mutations in HA, is the major cause of the mismatch and failure of seasonal influenza vaccines ,29. Univut flaws ,33.mg and HAug forms was constructed and evaluated. As a result of increased ADCC and CDC activity and more CD8+ and CD4+ memory T cells, the HAug vaccine conferred cross-protection against H3N2 and H7N9 subtype viruses. This is consistent with studies showing that ADCC plays an important role in influenza protection [ug provides a broader cross-protection ability against the H7N9 virus compared to HAmg, which indicated that an Fc-dependent mechanism induced by HAug might be responsible for protection when there is a lack of in vitro broadly neutralizing activity. The HAmg vaccines were found to be similar or, in most cases, worse than HAug in cross-strain protection. The reason behind this result is not clear and could be because the amino acid changes from Asn to Asp at each glycosylation site introduced by PNGase F would promote the exposure of protective but occluded epitopes and induce cross-strain protection, while the single N-linker GlcNAc left by Endo-H might cause steric hindrance for those epitopes that are hidden.In the present study, deglycosylated modified HA from H3N2 with HAotection . Interesmg vaccines of HA from the A/Brisbane/59/2007 H1N1 strain showed better cross-strain protection against various H1N1 viruses than the HAug vaccines, in which highly conserved sequences that were originally covered by glycans in fully glycosylated HA are exposed and thus induced more CD8+ and CD4+ memory T cells and IgG-secreting plasma cells. However, the HAug vaccines from the A/Brisbane/59/2007 H1N1 strain were speculated to contain partial glycans and further influence immunogenicity, which are not completely removed by enzymes. However, the HAug vaccines from the A/Wisconsin/67/2005 H3N2 strain used in our study contain 11 glycosylated sites and maintain few differences in the secondary structure, while the HAug from Bri/07 only has eight glycosylated sites. The difference in glycosylated sites and glycosidase treatment may result in the different immunogenicity between HAug and HAmg vaccines. Although the details to explain this phenomenon are not clear and need to be investigated, the above results could already indicate that deglycosylated modified HA would promote the exposure of protective but occluded epitopes and induce cross-strain protection.As mentioned above, the numbers of glycosylation sites of HA varied following viral evolution and depended on different subtypes and strains. In a previous study, the HAug vaccines would probably not contribute to the cross-protection, especially against A/Shanghai/02/2013 (SH/13). As the predominant antibody present in mice, IgG is the major subtype of HA-specific antibodies with a high affinity for the FcγRIII receptor on immune cells [ug vaccine increased ADCC and CDC activity along with better protection effectiveness. Indeed, the Fc-dependent functional antibodies would play an important role in unglycosylated HA vaccination, while the modified HA would induce multi-mechanism of protective immune, including more CD4 and CD8 T cell response, even some broadly-protective antibodies with unknown functional mechanisms, which need more exploration and evidence. In summary, our study shows the possibility of deglycosylated modified HA in inducing cross-protection by exposing occluded epitopes and offers insights into design options for more universal influenza vaccines.In our results, the low levels of cross-neutralizing activities of the HAne cells . It was ug play an important role in the protective immune response, but how to elicit these antibodies is not exactly understood and requires further study.This study focused on deglycosylated modified HA-based vaccine research. The characteristics of the protective epitope changed by PNGase F are still unknown and require further study. In addition, the antibodies with ADCC activities induced by HA


In recent years, an increasing number of studies have indicated that circular RNA plays crucial roles in regulating tumor development and chemoresistance. Using two high-throughput RNA sequence datasets, we previously found that circEXOC6B was downregulated in colon cancer. However, its role and mechanism in colorectal cancer (CRC) remained unknown.Real-time quantitative PCR was used to examine the expression of circEXOC6B in CRC tissues. In vivo and in vitro functional experiments were performed to determine the suppressor role of circEXOC6B in CRC progression. RNA pull-down, mass spectrometry, RNA-binding protein immunoprecipitation, co-immunoprecipitation, fluorescence in situ hybridization, and immunofluorescence were applied to investigate the possible mechanisms connecting circEXOC6B to CRC growth and 5-fluorouracil-induced apoptosis. Chromatin immunoprecipitation, dual-luciferase assay, western blot, and immunohistochemistry were used to explore the mechanisms underlying the HIF1A regulation of RRAGB transcription.circEXOC6B was downregulated in CRC tissues, and its lower expression was associated with poor prognosis of patients. Functional experiments showed that circEXOC6B inhibited growth and increased the 5-fluorouracil-induced apoptosis of CRC cells in vitro and in vivo. Mechanistically, circEXOC6B inhibited the heterodimer formation of RRAGB by binding to it, thereby suppressing the mTORC1 pathway and HIF1A level. In addition, HIF1A upregulated the transcription of RRAGB by binding to its promoter region. Altogether, the results demonstrated that a HIF1A-RRAGB-mTORC1 positive feedback loop drives tumor progression in CRC, which could be interrupted by circEXOC6B.circEXOC6B inhibits the progression of CRC and enhances the chemosensitivity of CRC cells to 5-fluorouracil by antagonizing the HIF1A-RRAGB-mTORC1 positive feedback loop. circEXOC6B is a possible therapeutic target for CRC treatment.The online version contains supplementary material available at 10.1186/s12943-022-01600-1. Colorectal cancer (CRC) is the second most common malignancy and the third leading cause of tumor-related deaths worldwide . The maiMammalian target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth and survival. It is activated by multiple intracellular and extracellular stimuli, such as growth factors and nutrients . ActivatWe previously found that the circular RNA (circRNA) circEXOC6B (hsa_circ_0009043) was significantly downregulated in colon cancer compared with normal tissue in two high-throughput sequencing datasets , 13. How2.Human CRC cell lines SW480, SW620, RKO, Caco-2, HCT116, LoVo and HT29 were purchased from the American Type Culture Collection . Cells were cultured in RPMI 1640 medium  containing 10% fetal bovine serum at 37 °C in a humidified atmosphere with 5% COThe 78 paired of CRC tissues and adjacent noncancerous tissues were collected from Nanfang Hospital, Southern Medical University . The consents were obtained from all patients and the use of clinical materials for research purposes were approved by the Ethics Committee of Southern Medical University . CRC tissues were taken from the general surgically resected CRC specimens, and the matched adjacent non-cancerous tissues were taken from the site around 5–10 cm away from the edge of the tumors. These specimens were immediately frozen in liquid nitrogen until use. All samples were diagnosed as adenocarcinoma by pathologists. All patients were not received preoperative chemoradiotherapy.The cDNA microarray of colon cancer tissues (HColA095Su02) was purchased from Shanghai Outdo Biotech Company. It contains 80 colon cancer tissues and 15 adjacent noncancerous tissues along with each patient’s gender, age, clinical stage, lymphatic metastasis status and survival information, that were recorded and archived in the National Engineering Center for Biochip. The use of cDNA microarray for research purposes was approved by the Ethics Committee of Shanghai Outdo Biotech Company.These assays were performed as previous described , 15. TheAll siRNAs were designed and synthesized by GenePharma  or RiboBio  Table S. sh-circSW620 and HCT116 Cells were treated with 5 μg/ml and 1 μg/ml 5-FU  for 24 h, then followed by CCK8, flow cytometry, TUNEL and western blot detection. For in vivo experiment, we established subcutaneous tumor models using HCT116 with stable knockdown of circEXOC6B or control cells. When the subcutaneous tumors grew to 0.5 cm, nude mice were intraperitoneally injected with 20 μg 5-FU per gram of body weight, while the control group was injected with saline, 3 times a week, for 2 weeks.EdU Cell Proliferation Kit with Alexa Fluor 555  was used to detect the proliferation of cells according to the instruction. In short, 1 × EdU working solution was added into cells and incubated for 2 h. Then, cells were fixed, washed and incubated with the reaction solution at room temperature for 30 min. DAPI stained nucleus. The number of positive cells was counted and calculated.Fluorescent in Situ Hybridization Kit  was used to perform FISH on CRC cells according to the instruction and our previous study . In brieThese experiments were performed as previous described . BrieflyIn short, the primary anti-flag antibody  or mouse control IgG  were added into cell lysate and the mixture was incubated at 4 °C for 1 h. Thereafter, 20 μl A/G plus-Agarose beads  were added and incubated at 4 °C overnight. The proteins were eluded and detected using western blot with the primary anti-flag , anti-RRAGC , anti-RRAGD  and the secondary antibody .The total protein was separated by SDS-PAGE electrophoresis and transferred onto PVDF membrane . Then, the PVDF membrane was incubated with the following primary antibodies at 4 °C overnight: anti-RRAGB , anti-mTOR , anti-p-mTOR (Ser2448) , anti-S6K , anti-p-S6K (Thr389) , anti-HIF1A , anti-pro-caspase3 , anti-cleaved-caspase3 , anti-PARP , anti-cleaved-PARP , and anti-β-tubulin . Subsequently, the PVDF membrane was incubated with the corresponding HRP labeled goat anti-mouse  or goat anti-rabbit IgG  at room temperature for 1 h. FDBio-Dura ECL Kit  was used to visualize the protein bands.IHC staining and H-Score calculating were performed as our previous research . The priThe SimpleChIP® Enzymatic Chromatin IP Kit  was utilized to perform ChIP assay according to the specification. In brief, CRC cells were fixed, collected and lysed. Then, pelleted nuclei by centrifugation followed by DNA digestion. This digested chromatin was incubated with HIF1A antibody  and magnetic beads to perform immunoprecipitation. The chromatin was eluted from the magnetic beads and detected using PCR assay. The Dual Luciferase Reporter Assay System  was used to perform dual-luciferase reporter assay according to the instruction and previous research . HIF1A sCell Cycle Detection Kit  was used to detect the cell cycle progression of CRC cells according to the instruction. Annexin V-APC/PI Apoptosis Detection Kit  and One-step TUNEL cell apoptosis assay kit (red TRITC labeled fluorescence)  were used to detect the apoptosis according to the instructions.P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS: not significant.Statistical analysis was performed with GraphPad Prism6.0  and SPSS23.0 software . The measurement data are presented as mean ± standard deviation. A two-tailed Student’s t-test was used to detect the difference between two groups. The comparison of circEXOC6B expression in CRC tissues and matched noncancerous tissues was analyzed using the paired t-test. The relationships between circEXOC6B expression and clinicopathological characteristics were determined by χ2 test. Kaplan-Meier method and Log rank test were used to evaluate the difference of survival time between low and high circEXOC6B expression groups. Univariate and multivariate Cox proportional hazards model were performed to evaluate the prognostic value of circEXOC6B. Pearson’s correlation coefficient was used for correlation analysis of RRAGB and HIF1A expression. Differences were considered to be significant at *http://circbase.org/) showed that circEXOC6B is generated from exons 3–6 of its parental gene EXOC6B and that its mature length is 390 bp. We performed a series of experiments to verify that circEXOC6B is indeed a circRNA originating from EXOC6B. First, we designed two pairs of divergent primers (1 and 2), which could amplify 100 bp and nearly the full-length of circEXOC6B, respectively   and circ primers  and 2, w primers  and 2, w primers  and 2, w primers  and 2, wn = 39) and a low expression group (n = 39) according to the median expression of circEXOC6B. Statistical analysis revealed that the expression of circEXOC6B was negatively correlated with tumor size, lymphatic metastasis, and TNM stage   than patients with lower circEXOC6B expression   Fig. D. Next, ion Fig. E. Finall40) Fig. F. These To investigate the role of circEXOC6B in CRC progression, we knocked down circEXOC6B expression in CRC cells using siRNAs specifically targeting the splicing junction of circEXOC6B and upregulated circEXOC6B expression using a circEXOC6B overexpression vector Fig. A and S2AFurthermore, using a lentiviral approach, we stably silenced circEXOC6B expression in SW620 and HCT116 cells for further functional research Fig. SA. SubseqTo explore the molecular mechanisms underlying the circEXOC6B regulation of CRC progression, we first determined the subcellular localization of circEXOC6B in CRC cell lines and CRC clinical samples using FISH, PCR, and RT-qPCR. The results indicated that circEXOC6B mainly resided in the cytoplasm of CRC cells Figs. A and S4.Moreover, using the catRAPID database , we predGiven that formation of the RRAGB-RRAGC/D heterodimer is required for activation of the mTORC1 pathway, we deduced that circEXOC6B might suppress the mTORC1 pathway by interfering with heterodimer formation. Western blot results showed that overexpression of circEXOC6B reduced the expression of p-mTOR, p-S6K, and HIF1A while its downregulation increased their expression Fig. A and B. In addition, we performed rescue assays to further verify the involvement of RRAGB in the regulation of circEXOC6B in the mTORC1 pathway. Western blotting showed that the p-mTOR, p-S6K, and HIF1A elevation caused by RRAGB could be reversed by co-transfection with circEXOC6B Fig. D. SimilaWhen we determined the expression of HIF1A and RRAGB in six CRC cell lines using RT-qPCR, we surprisingly found that there was a positive relationship between HIF1A and RRAGB expression Fig. A. MoreovThe mTORC1 pathway enhances the translation of HIF1A. Therefore, a HIF1A-RRAGB-mTORC1 positive feedback loop exists in CRC to drive tumor progression, and circEXOC6B might antagonize the loop by binding to RRAGB. We used the HIF1A activator ML228 to upregulate HIF1A, and western blotting showed that the RRAGB level was increased. However, circEXOC6B was able to abrogate the effect and to decrease the expression of RRAGB and HIF1A Fig. F. In addhttps://www.cancerrxgene.org/)  Fig. S. Thus, c/) Fig. SA. Moreov/) Fig. SB. Consis/) Fig. SC.Fig. 6cFurthermore, we constructed subcutaneous xenograft tumors to test the role of circEXOC6B in 5-FU treatment in vivo. After intraperitoneal injection of 5-FU, the tumor volume in the sh-circEXOC6B group showed no difference from the untreated group. In contrast, the tumor size was significantly decreased in the sh-NC group after 5-FU treatment  in the promoter region of its target genes to upregulate their transcription . These tIn addition, circEXOC6B has been reported to be able to enhance the sensitivity of tumor cells to paclitaxel in ovarian cancer . Herein,In summary, our findings revealed the existence of a HIF1A-RRAGB-mTORC1 positive feedback loop that drives CRC development. circEXOC6B can bind to the heterodimer formation site of RRAGB to disrupt its physical interaction with RRAGC/D, which suppresses HIF1A-RRAGB-mTORC1 positive feedback Fig. . Thus, cAdditional file 1: Supplementary Fig. S1. Identification of circEXOC6B in CRC. (A) Heatmap showed the top five downregulated and five upregulated circRNAs in colon cancer screened by high-throughput sequencing (PMID25624062). (B) The consistent expression of circEXOC6B in GSE77661 and PMID25624062. (C) The schema showed that circEXOC6B is generated from the exon 3–6 of EXOC6B, and two pairs of divergent primers (1 and 2) were designed. PCR amplification with the Primer 2 followed by sanger sequencing confirmed circEXOC6B originated from the exon 3–6 of EXOC6B. The arrows showed the junctions between the exons of circEXOC6B. (D) PCR showed that circEXOC6B could only be amplified from cDNA in CRC cells using the divergent primer 2, and not from genomic DNA (gDNA). GAPDH as a control. (E) PCR indicated that circEXOC6B could resist RNase R digestion. GAPDH as a control. (F) CRC cells were treated with actinomycin D to inhibit new RNA generation, and RT-qPCR result revealed that circEXOC6B was more stable than GAPDH mRNA.Additional file 2: Supplementary Fig. S2. Downregulation of circEXOC6B promoted the growth of RKO and Caco-2 cells. (A) The expression of circEXOC6B was knocked down by transfection with siRNA. (B) CCK8 was performed to detect the proliferation of RKO and Caco-2 cells after knockdown of circEXOC6B expression. (C) and (D) Colony formation assay showed that the downregulation of circEXOC6B promoted the clonogenicity of CRC cells. (E) Flow cytometry indicated that the decreased circEXOC6B accelerated cell cycle progression of CRC cells.Additional file 3: Supplementary Fig. S3. Stable knockdown of circEXOC6B promoted the growth of CRC cells in vitro and in vivo. (A) The expression of circEXOC6B in SW620 and HCT116 cells with stable knockdown of circEXOC6B expression. (B) CCK8 and (C) Colony formation assays showed that the stable depletion of boosted the proliferation and clonogenicity of CRC cells compared with the control cells. (D) and (E) Subcutaneous tumor models demonstrated that the stable downregulation of circEXOC6B accelerated the growth of CRC cells in vivo. Scale: 20 μm.Additional file 4: Supplementary Fig. S4. The subcellular distribution of circEXOC6B in CRC cell lines and CRC clinical sample. (A) PCR and (B) RT-qPCR were performed to detect the subcellular distribution of circEXOC6B in CRC cell lines. GAPDH and U6 were used as control. (C) FISH was performed to show the subcellular distribution of circEXOC6B in CRC clinical sample. Scale: 20 μm.Additional file 5: Supplementary Fig. S5. RNA pull-down was performed in SW620 cells to identify the proteins interacting with circEXOC6B. The red frame marked RRAGB.Additional file 6: Supplementary Fig. S6. The possible binding sites of RRAGB with circEXOC6B was predicted using catRAPID database.Additional file 7: Supplementary Fig. S7. The positive correlation between HIF1A and RRAGB expression in CRC was validated using (A) GSE17538  and (B) The Cancer Genome Atlas (TCGA)  datasets.Additional file 8: Supplementary Fig. S8. IHC showed that HIF1A, RRAGB and p-mTOR levels in CRC clinical samples were lower in the high circEXOC6B group (n = 11) than in the low circEXOC6B group (n = 11). Scale: 50 μm.Additional file 9: Supplementary Fig. S9. The relative expression of circEXOC6B in LoVo, RKO, HCT116 and HT29 was higher than in SW620.Additional file 10: Supplementary Table S1. Primer sequences are used in PCR or RT- qPCR.Additional file 11: Supplementary Table S2. siRNAs are used for knockdown of circEXOC6B or HIF1A.Additional file 12: Supplementary Table S3. The relationship between circEXOC6B expression and the clinicopathologic characteristics of CRC patients.Additional file 13: Supplementary Table S4. Proteins might bind to circEXOC6B were identified by mass spectrometry (MS).