Campylobacter encompasses 39 recognized species and 13 subspecies, with many belonging to a group known as ‘emerging Campylobacter pathogens’. Within Campylobacter, seven species have been identified to harbour a complete T6SS cluster but have yet to be comparatively assessed. In this study, using systematic bioinformatics approaches and the T6SS-positive Campylobacter jejuni 488 strain as a reference, we explored the genus-wide prevalence, similarity and make-up of the T6SS amongst 372 publicly available ‘complete’ Campylobacter genomes. Our analyses predict that approximately one-third of Campylobacter species possess a T6SS. We also putatively report the first identification of a T6SS in four species: Campylobacter cuniculorum, Campylobacter helveticus, Campylobacter armoricus and Campylobacter ornithocola. The Campylobacter T6SSs cluster into three distinct organizations (I–III), of which two break down into further variants. Thirty T6SS-containing genomes were found to harbour more than one vgrG gene, with Campylobacter lari strain NCTC 11845 possessing five. Analysis of the C. jejuni Pathogenicity Island-1 confirmed its conservation amongst T6SS-positive C. jejuni strains, as well as highlighting its diverse genetic composition, including additional putative effector–immunity pairs (e.g. PoNe and DUF1911 domains). Effector–immunity pairs were also observed neighbouring vgrGs in several other Campylobacter species, in addition to putative genes encoding nucleases, lysozymes, ATPases and a ferric ATP-binding cassette uptake system. These observations highlight the diverse genetic make-up of the T6SS within Campylobacter and provide further evidence of its role in pathogenesis.Bacterial type VI secretion systems (T6SSs) are contractile nanomachines that deliver proteinic substrates into target prokaryotic or eukaryotic cells and the surrounding milieu. The genus  Accessoffectors . Recogniffectors , yet dueffectors . The forffectors , whilst ffectors .Campylobacter encompasses a collection of 44 proposed species, of which 39 names have been published and recognized by the International Code of Nomenclature of Prokaryotes, and 13 subspecies observed to colonize a range of environments [Campylobacter is the leading cause of bacterial foodborne gastroenteritis worldwide, with C. jejuni as the causative agent of the majority of infections in humans and is therefore of significant clinical relevance [Campylobacter species belong to a group known as ‘emerging Campylobacter pathogens’, including Campylobacter concisus, Campylobacter fetus and Campylobacter lari, which opportunistically infect both animals and humans [Campylobacter species still possess only one available genomic sequence, such as Campylobacter ornithocola and Campylobacter rectus [C. jejuni has over 200 genomes of quality level ‘complete’ in the National Center for Biotechnology Information (NCBI) RefSeq database [Campylobacter, several species are described to harbour a T6SS, with the earliest reported and predominantly investigated in C. jejuni [C. jejuni T6SS to numerous physiological functions, including pathogenic interaction , cytotoxicity and resistance to oxidative stress [C. jejuni genomes, determining the presence of a Campylobacter jejuni Integrated Element 3 (CJIE3)-variant termed Campylobacter jejuni Pathogenicity Island-1 (CJPI-1) harbouring the T6SS amongst a significant proportion of strains [Campylobacter species [The genus ronments . This abronments . Campyelevance . Severald humans . A smallrectus . In compdatabase . Within jejuni . Studiese stress . Recentl strains , as well strains . Further species .Campylobacter species, its (i) evolutionary origins, (ii) genus-wide prevalence, (iii) interspecific similarity, (iv) intraspecific genetic transfer, (v) range of associated effectors and (vi) contribution to pathogenesis remain largely unknown. We have therefore performed a bioinformatic analysis of 372 publicly available Campylobacter genomes encompassing 33 species and 17 unassigned strains, investigating the prevalence, genetic composition and make-up of the T6SS. The major T6SS components TssC, Hcp and VgrG, and a T6SS-harbouring genetic element were investigated using the T6SS-positive C. jejuni 488 strain as a reference. A systematic prediction of genes neighbouring identified vgrGs was also conducted. We identified several potentially insightful inter- and intraspecific similarities and differences between T6SS-harbouring genomes, thus expanding our current understanding of the T6SS diversity and roles.While several studies have called attention to the existence and importance of the T6SS in a growing number of pathogenic Campylobacter genomes were collected from the NCBI RefSeq genome database release 205 (March 2021) at assembly level ‘complete genome’ or higher [C. jejuni 488 strain (a human isolate from Brazil) [Nucleotide and protein sequences of r higher . The T6SMarch 202 at assem Brazil) .blastp (blast+ v2.12.0) [Campylobacter genomes, using an expected value  of 1e-10. Amino acid sequences of the 14 T6SS loci (two VgrGs) from the T6SS-positive C. jejuni 488 strain [Campylobacter genomes. A similarity percentage was calculated by dividing the bit-score value for each amino acid alignment by twice the specific lengths of the individual query amino acid sequence to facilitate T6SS loci identification similarly to that reported previously [blast [v2.12.0)  was empleviously  and compeviously , with dey [blast , with deCampylobacter genomes in the local dataset, blastp [C. jejuni and Campylobacter coli isolates by PubMLST [Campylobacter [Campylobacter protein database. Then, extracted sequences were aligned using muscle [https://itol.embl.de) [To construct a phylogenetic tree of all , blastp  was empl PubMLST  and is tbacter . The prog muscle , with deg muscle  using thembl.de) .blastp [Campylobacter genomes, using an E-value=1e-10. Protein sequences CJ488_00968 (Hcp) and CJ488_00974 (TssC) from C. jejuni 488 were aligned against the local Campylobacter protein database. Extracted sequences were then aligned using muscle [n=500) parameters, conducted in the Molecular Evolutionary Genetics Analysis X  software package [blastp  was emplg muscle  with def package . These a package .blastp [Campylobacter genomes, using an E-value=1e-10. The N-terminal amino acid sequence of VgrG1 and VgrG2 possesses >99 % amino acid identity [C. jejuni 488 was aligned against the local Campylobacter protein database to identify VgrG homologues. Sequences were then filtered to remove any of the traits: (i) sequence matched to a genome that was not considered to harbour a T6SS (e.g. C. jejuni CLB104), (ii) possessed a fragmented VgrG ORF (e.g. AEI23_RS09810 and AEI23_RS05295 from C. jejuni CJ018CCUA), (iii) internal stop codons present in the sequence (e.g. EL232_RS07845 from C. jejuni NCTC 13265), (iv) missing methionine start to the sequence, (v) less than 100 aa in length, and/or (vi) the VgrG domain was not identified using NCBI-CDD blast (e.g. AEI20_RS09140 from C. jejuni CJ071CC464). A total of 19 sequences were removed from further analysis. Domain prediction was conducted using NCBI-CDD [blastp  was emplNCBI-CDD  with defC. jejuni genomes from the local Campylobacter dataset, the CJPI-1 genes CJ488_0930 (CJPI-1 integrase), CJ488_0941 and CJ488_1004 from C. jejuni 488 were used in an in silico identification method as proxies [CJ488_0941 and CJ488_1004 are homologues of C. jejuni RM1221 strain genes cje1105 and cje1153, respectively, harboured in the CJIE3. blastn [CJ488_0930, CJ488_0941 and CJ488_1004 against the local nucleotide dataset created for the Campylobacter genomes. A similarity percentage was calculated according to Fridman et al. [CJ488_0930, CJ488_0941 and/or CJ488_1004. To be regarded as possessing a T6SS-harbouring plasmid, (1) a minimum similarity of 50 % was required to just the gene CJ488_0930 and (2) the presence of at least 12 T6SS loci (T6SS-positive). Comparative genomic analysis of CJPI-1-harbouring C. jejuni genomes was conducted using the Artemis Comparison Tool (v18.1.0) [blastn (megablast) webtool [C. jejuni 488 acting as the query and the predicted CJPI-1 sequence in the other C. jejuni strain as the subject.To identify the presence of CJPI-1 amongst the . blastn  was emplv18.1.0) . Query c webtool , with thvgrGs are commonly identified to possess toxic activity and are characterized as cognate effectors [vgrG genes identified in the previous analysis stage (94 sequences) were systematically extracted and collated. Amino acid sequences were then submitted to the NCBI CDD-blast  [Genes neighbouring ffectors . Proteinresults) , SignalPresults)  and TMHMresults)  servers blastp [Campylobacter genomes, using an E-value=1e-10. The amino acid sequence of the PAAR-like domain (amino acids 25–150) from CJ488_00990 in C. jejuni 488 was aligned against the local Campylobacter protein database. Homologous sequences were then subject to domain prediction using NCBI CDD-blast and Pfam [blastp  was emplblastp  amongst and Pfam , with deIdentified PAAR-like domain-containing protein sequences were then aligned using ClustalOmega  and visuCampylobacter, we first constructed a local database of Campylobacter genomes of assembly level ‘complete genome’ or higher (n=371) from the NCBI RefSeq database [C. jejuni 488 strain as a reference, creating a total of 372 genomes (Table S1). Our local database consisted of 33 Campylobacter taxa and 17 strains unassigned to any known species. Genome size ranged from 1.465 Mbp (Campylobacter insulaenigrae NCTC 12 927) to 2.572 Mbp (C. rectus ATCC 33 238). C. jejuni was the most abundant species in our database, contributing 212 genomes to the dataset, followed by C. coli (n=33) and C. fetus (n=20) (n=281) followed by avian species (n=34)  . Metadats (n=34) .vgrG locus VgrG2 from T6SS-positive C. jejuni 488 against the locally constructed database, identifying 62 (16.66 %) genomes to possess at least 12 of the 13 major T6SS components, and therefore classifying them as T6SS-positive (Tables S1 and S2). No T6SS-positive genomes were found to present in more than one copy of the complete T6SS cluster. Campylobacter cuniculorum 2010D-8469 and C. cuniculorum LMG 2588 were the only genomes predicted to harbour two tagH genes. C. jejuni FDAARGOS_421, NCTC13265, RM1221, RM1221 (2) and CLB104, C. coli AR-0411, and C. concisus P3UCB1 and P3UCO1 strains were identified to possess up to two of the screened T6SS components but were not considered to harbour a T6SS. In total, 11 species (33.33 %) in our database were identified as possessing a T6SS operon, as well as a further five strains unassigned to a known species. Again, the most prevalent species was C. jejuni, with 23 strains predicted to harbour a T6SS, followed by C. concisus (n=14) and C. coli (n=7)  . Phyloget groups .n=281), 17.65 % within all avian species isolates (n=34), and 17.54 % within all other species or unknown isolates (n=57). Among the 11 species observed to encode a T6SS, to the best of our knowledge, we putatively report the first identification of a T6SS in four species: C. cuniculorum, Campylobacter helveticus, Campylobacter armoricus and C. ornithocola. C. armoricus CA639, C. helveticus 2013D-9613 and ATCC 51209, and C. ornithocola LMG 29815 were predicted to harbour all 13 T6SS components , whilst both C. cuniculorum strains, mentioned previously, harbour all 13 and an additional putative tagH gene. Both encoded tagH genes in the C. cuniculorum strains possessed 99.67 % nucleotide sequence identity when compared to each other.Of the T6SS-positive genomes, 46 (74.19 %) were isolated from humans as hosts, six (9.68 %) from avian species, and ten (16.13 %) from other or unknown species  and environmental isolation. Therefore, among the genomes under study, 16.37 % were identified as T6SS-positive within all human isolates (tssL to tssG) which cluster together in the same orientation. Observable differences between the variants are determined by the location of the gene vgrG and tagH-tssM-hcp genetic cluster. In both I-a and I-b, the genes tagH-tssM-hcp are localized at the N-terminus of the main cluster adjacent to tssL, whilst the location of the vgrG varies. Alternatively, in organizations I-c and I-d, multiple genetic loci separate the main cluster from the genes tagH-tssM-hcp, and in the case of I-d, several vgrGs and effectors (see below) are harboured within this genetic region.After determining the prevalence of the T6SS, we next investigated the organization of the T6SS clusters amongst the T6SS-positive genomes. During a systematic assessment of the clusters, we putatively predict three T6SS organizations (I–III) , of whicT6SS II possesses a unique organization that differs significantly from the T6SS I:tssL-tssK-tssJ are encoded in the reverse orientation to their homologues in the T6SS I and III organizations. As a result, all genes of the cluster are encoded in the same transcriptional direction and suggest a coregulated expression.Genes tssM-tagH-hcp are found at the C-terminus of the main cluster adjacent to the gene tssG.Genes hcp and vgrG.Regions of genetic variation are found between genes tssM-tagH at the C-terminus of the main cluster, similar to T6SS II, but the orientation of tssL-tssK-tssJ was similar to T6SS I. Observable differences between variants III-a and III-b lie with the location of the genes hcp and vgrG. In T6SS organization III-a, the genes hcp-vgrG are found distant from the C-terminus of the main cluster separated by more than 10 genes, whereas in organization III-b both hcp and vgrG are encoded at the N-terminus with several genes separating hcp and vgrG.The final organizations T6SS III-a and III-b were unique to one strain in the database, respectively. The organizations of III-a and III-b both possessed genes C. jejuni strains were identified to possess the T6SS organization I-a, as well as Campylobacter sp. CFSAN093227, CFSAN093238 and CFSAN093246, C. cuniculorum 2010D-8469 and LMG 2588, and all C. coli strains, except for strain ZV1224 which exclusively possessed organization I-b (Table S1). Organization I-c was observed in the genomes of C. helveticus 2013D-9613 and ATCC 51209. Genomes of strains C. armoricus CA639, C. ornithocola LMG 29815, C. lari RM16712, Campylobacter sp. 2014 D-0216, Campylobacter peloridis 2016D-0074 and LMG 23910, Campylobacter subantarcticus LMG 24374 and LMG 24377, and C. lari NCTC 11845 were identified to harbour the T6SS organization I-d. The number of vgrG genes carried by the genomes with T6SS organization I-d varied from one to five within the genetically variable region. All C. concisus genomes were classified to present the T6SS II organization . Analysis of the core component TssC was also performed between the three T6SS organizations and their variants, confirming higher homology within the distinct T6SS organizations than to each other, suggesting the organization variants are related  secretion and thus is an important constituent of T6SS functionality [C. jejuni and is therefore likely to present species-specific traits to prevent immunity during T6SS-mediated conflict [n=62) by producing two maximum-likelihood phylogenetic trees based on the core-sheath component TssC and secreted component Hcp as they each appear to contribute distinct roles to the activity of the Campylobacter T6SS. Both trees produced similar topological arrangements of T6SS-positive species and strains  of VgrG1 (CJ488_00978) from C. jejuni 488. In total, 113 homologous sequences were identified initially, of which 19 were subsequently removed due to criteria described in the Methods. The remaining 94 sequences were subject to further analysis. The average number of vgrG sequences identified amongst the T6SS-positive genomes was greater than one (n=1.633), with 30 (48.39 %) genomes harbouring more than one VgrG . Notably, five vgrGs were identified in C. lari NCTC 11845, located in the genetic region between genes hcp and tssL of T6SS organization I-d. Lengths of VgrGs also varied greatly, with C. concisus H9O-S2 harbouring a VgrG of 1025 aa, whilst the shortest of 430 aa was identified in multiple strains. VgrG sequences were then analysed to identify conserved and additional domains. As expected, all sequences across all strains were found to harbour the conserved domains VgrG (COG3501) and VI_Rhs_Vgr superfamily (cl37255) in the N-terminus (Table S3). Several strains were predicted to harbour the 5_superfamily (cl33691) domain previously observed in the sequence of VgrG2 from C. jejuni 488 [Recently, we identified the presence of two distinct ni 488 . To asseC. jejuni strains under study. In silico identification and visualization in Artemis [C. jejuni strains (n=23) in our dataset harboured a T6SS either within the CJPI-1 (n=18) or on a plasmid (n=5) (Tables S1 and 4). Comparative analysis of the newly identified CJPI-1 amongst C. jejuni strains against CJPI-1 from C. jejuni strain 488 revealed considerable diversity in length and genetic composition between harbouring strains . Additionally, several non-C. jejuni species were putatively identified to possess synonymous T6SS-harbouring islands, as well as T6SS-encoding plasmids observed in Campylobacter sp. CFSAN093246 and C. coli strains 14 983A, FDAARGOS_1028, meC0281, YH502 and YH503.Twelve (5.63 %) out of 212 C. jejuni 488, four putative effectors and two immunity genes were predicted to be encoded within a genetically variable region between the genes vgrG1 and vgrG2, representing the first potential secreted substrates to be utilized by the T6SS in Campylobacter [vgrGs in the previously identified T6SS-harbouring Campylobacter species. To achieve this, we systematically extracted the protein sequences of the five upstream (+5) and downstream (−5) genes adjacent to identified vgrG homologues across all T6SS-positive species under study. Extracted sequences were then subject to functional characterization through domain, signal peptide and transmembrane helix analyses (Table S5).Recently identified within CJPI-1 of C. jejuni M129) and was commonly found downstream of the cognate vgrG. The second pair encoded domains belonging to the PoNe (cl41756) and DUF1911 (cl07503) superfamilies (e.g. A6J90_RS09495 and A6J90_RS06400 in C. jejuni FDAARGOS_262) and was identified upstream of the cognate vgrG . Other putative effectors were also frequently observed near the vgrGs encoding domains belonging to the Tox-AHH (PF14412), lysozyme (cl13324), endolysin (cl00222), peptidoglycan hydrolase (cd12797) and lipase (cl21494) families (Table S5). Tox-AHH domain-containing proteins encoded by +5 or −5 genes were observed in the genomes of 10 C. jejuni strains, five C. coli strains, C. sp. CFSAN093227 and C. sp. CFSAN093238, and C. lari NCTC11845 and RM16712. Lysozyme/endolysin domain-containing proteins were predicted in the genomes of four C. concisus strains and C. sp. CCUG 57310. Interestingly, C. sp. CCUG 57310 was also predicted to encode a lysozyme immunity (cl34764) domain-containing protein, CORI_RS01180, adjacently upstream of its lysozyme (cl00222) domain-containing protein, CORI_RS01185. Additionally, five putative ATPase- (cl34796) encoding proteins were also predicted near vgrGs in the genomes of C. peloridis 2016D-0074 and LMG 23910 and C. lari NCTC11845, as well as putative ATP-binding cassette (ABC) Fe3+-siderophore transport system  domain-containing proteins in C. concisus P27CDO-S2 (C. concisus strains (Table S5).We predicted two effector–immunity pairs amongst the T6SS-harbouring population. The first pair consisted of proteins harbouring domains belonging to the Ankyrin (PF12796/cl33707) and Tox-REase-7 (cl21441) superfamilies  either −1 or +1 genes neighbouring vgrGs were predicted as Ankyrin domain-containing , TssG  or Hcp  proteins. Similarly, predicted proteins encoded by either +2 or −2 genes with identifiable domains (n=85) were predicted as TssF , Tox-REase-7 , TssM  or Lysozyme/Endolysin  proteins (Table S5).Of note, the most frequently observed proteins that encoded identifiable domains . This was further confirmed by aligning the homologous sequences and visualizing the presence of the conserved cysteine and histidine residues . The PAAR-like domain-containing sequences were then analysed for additional domains using NCBI-CDD and Pfam [n=14) and PoNe  superfamily were consistently found in the C-terminus of select PAAR-like domain-containing proteins (n=19) superfamily in their N-terminus.A bioinformatic screening for the PAAR-like domain identified in the protein CJ488_0990 (amino acids 25–150) from and Pfam . Severalproteins . FurthervgrGs. Sixteen proteins encoded by genes neighbouring vgrGs were predicted to contain the PAAR-like domain, of which nine (56.25 %) also possessed C-terminal PoNe toxin domains. The PAAR-like domain-containing proteins encoded by genes +5 to −5 of the identified vgrGs were found in the genomes of C. jejuni 14 980A, FDAARGOS_262, NCTC11351 and NCTC13268, C. peloridis 2016D-0074 and LMG 23910, C. lari NCTC11845 and RM16712, and C. subantarcticus LMG 24377 (Table S5).PAAR-like domain-containing proteins identified in the previous stage of analysis were also cross-referenced with the genes extracted from +5 upstream to −5 downstream of the Campylobacter T6SS has been explored in C. jejuni, contributing to different physiological mechanisms to promote pathogenesis [Campylobacter genomes that 33 % of Campylobacter species under study putatively possess a T6SS. We note that Campylobacter ureolyticus DSMZ 20703 was previously reported to encode homologues of T6SS components [Campylobacter were also absent from the NCBI RefSeq release 205 database, including Campylobacter aviculae, Campylobacter estrildidarum, Campylobacter taeniopygiae [, Campylobacter portucalensis [, Campylobacter novaezeelandiae [, Campylobacter massiliensis [Campylobacter vulpis [Campylobacter species to date, and therefore we stress that the results of our study are exclusive to species only included in the local database.The T6SS is a multifunctional weapon with a broad range of targets due to its array of deployable toxic effectors . The funogenesis . In thismponents , but it calensis , Campyloelandiae , Campyloiliensis  and Cavulpis , of whicCampylobacter species is evident [Campylobacter species. Moreover, the reverse can also be stated for the absence of a T6SS in select species. Species and strains other than those included in our ‘complete’ genome dataset may possess a T6SS, such as the recently identified Campylobacter species mentioned above, warranting further investigation. Unsurprisingly, our study demonstrates the presence of the T6SS within Campylobacter is not solely restricted to one or two species, similarly to other T6SS-harbouring genera [It is also important to state that although many T6SS-harbouring species included in this analysis possess only one sequenced isolate, we cannot consider the genomes to be representative of the species. Variation in intraspecific T6SS prevalence within  evident ; therefog genera . Its widg genera . Ultimatg genera .Campylobacter species. have been identified to encode a complete T6SS [C. cuniculorum, C. helveticus, C. armoricus and C. ornithocola. We hope that reporting the presence of a T6SS in these species advances the knowledge about their putative virulence, in addition to developing strategies to control their distribution and pathogenicity. We also anticipate this novel finding to be the ‘tip of the iceberg’ for Campylobacter as more ‘emerging’ strains are sequenced and investigation into T6SS prevalence is conducted. Interestingly, both C. cuniculorum strains predicted to harbour a complete T6SS cluster were observed to encode an additional tagH gene. The function of this gene has not been independently characterized in Campylobacter species [Pseudomonas aeruginosa [Since 2012, seven ete T6SS . Here, w species , but itsginosa .C. jejuni isolates from humans has been observed to vary between low- and middle-income and high-income countries [Campylobacter T6SS and humans, as well as disease severity.Prevalence of the T6SS in ountries . In thisCampylobacter. Discrepancies between the T6SS variant organizations of I and III imply genetic reshuffling, potentially arbitrated by horizontal gene transfer, has allowed translocation of the vgrG gene. This may have occurred through its possession of a ‘rearrangement hotspot’ element, previously observed in C. jejuni [Campylobacter genomes under study and T6SS-positive isolates also highlighted the conservation of T6SS cluster organizations within distinct species. T6SS organization II and variants of organization III were exclusively found in one species, respectively, whereas T6SS organization I was identified in 10 species , genomic location , effector utilization and conservation of energy may contribute to their determination. T6SS organization II potentially demonstrates this prospective benefit of energy conservation by encoding its T6SS genes in the same transcriptional direction, thus relying on a singular promoter to co-regulate the expression of the entire T6SS cluster as one operon. Furthermore, as observed in this study and our previous work, T6SS cluster-containing mobile genetic elements could also lead to the dissemination of organizations among species with homologous recombination potentially facilitating chromosomal integration [Numerous T6SS-harbouring genera possess several T6SSs within their genomes clustered into distinct organizations . Here, wjejuni . Phyloge species . To deduvgrGs and hcps distributed between main and orphan clusters, exhibiting diverse functions [vgrG genes were putatively identified by our group in the CJPI-1 of the C. jejuni 488 strain, with homologues to both in C. coli genomes [vgrGs, as well as a conserved domain across all VgrG protein sequences. All additional vgrGs were found distant from the main cluster. We speculate that harbouring diverse VgrGs within the genus Campylobacter provides unique advantages to encoding species and strains, particularly related to the diverse environments they inhabit. Typically, interchange of the spike complex VgrG trimer leads to the specific interaction of effectors via the protein’s C-termini, widening the range of cellular targets [Encoding multiple T6SS clusters also leads to the presence of several structural component homologues across genomes. T6SS-harbouring species can encode multiple unctions . Two dis targets .C. jejuni population, in conjunction with exploring its prospective variation and make-up. Through comparative approaches, we found that 18 T6SS-harbouring C. jejuni strains encoded their T6SS within the CJPI-1, with the remainder harbouring theirs on discrete plasmids. We note considerable variation in the length and genetic composition of CJPI-1 between the strains under study, suggesting multiple events of genetic exchange have resulted in the acquisition or loss of specific genes. A select few were observed to possess additional toxin–antitoxin (TA) modules (data not shown) not detected in our previous study, whilst others harboured supplemental effector–immunity pairs in the downstream genetically variable region [Campylobacter species to harbour mobile genetic elements and plasmids that code for T6SSs, meriting further investigation.Pathogenicity islands are a unique subset of mobile genetic elements, conferring specific virulence phenotypes to harbouring bacteria governed by the genes carried . In thise region ; accordivgrGs are frequently identified as secreted substrates for the cognate spike proteins, presenting a strategic investigative route to uncover associated toxic effectors [Campylobacter vgrGs identified, we observed several putative effector and immunity proteins. Predicted effectors encoded nuclease domains Tox-REase-7, Tox-AHH and PoNe, as well as lysozyme/endolysin and lipase domains. These nuclease domains have been previously identified in several polymorphic protein delivery systems, including the T6SS [C. jejuni strains [Genes neighbouring ffectors . After sthe T6SS . Immunit strains .3+-siderophore import system immediately downstream of a cognate vgrG (CVT17_RS00555 – CVT17_RS00570) in C. concisus strain P27CDO-S2 /FepA (COG4771) ligand-gated outer membrane receptor protein [P. aeruginosa which facilities iron acquisition [3+) acquisition is a vital component of iron homeostasis in bacteria [3+ acquisition strategies commonly involve the secretion of ferric-chelating siderophores and internal translocation via receptor uptake systems [Campylobacter, such as the CfrA/CeuBCDE system in C. jejuni [Campylobacter. Periplasmic binding protein FepB was also found downstream of vgrGs in four other C. concisus strains under study, but the permease, ATPase and outer membrane receptor were not. It remains unknown how C. concisus utilizes this ferric uptake system, as well as which ferric-siderophore is responsible for environmental Fe3+ acquisition. One hypothesis is that the C. concisus FepABCD system is scavenging siderophores produced by other competing bacteria. On the other hand, it is also possible that a distant chromosomal gene to the vgrG is encoding the metal scavenging siderophore, potentially secreted by the cognate secretion system; however, it is important to note that the FepABCD system is encoded in the opposing transcriptional orientation to the vgrG.In addition to the predicted effectors, we also identified a putative ABC Fe27CDO-S2 . Investi protein . To dateuisition . Iron isbacteria . During  systems . Severaljejuni . To the C. jejuni 488 [Campylobacter database to investigate its distribution and genetic context. We identified the presence of the PAAR-like domain across several species and strains within the genus Campylobacter, with a subdivision encoding toxic domains in their C-termini and others harbouring lipase domains at their N-termini. The existence of this domain across T6SS-positive species indicates a shared function of PAAR-like-containing proteins, further supported by the presence of conserved cysteine and histidine residues. A PolC (cl35100) superfamily domain, associated with the gram-positive bacterial DNA polymerase III gene polC [Campylobacter PAAR-like domain-containing protein is fulfilling both the role of sharpening the spike complex and simultaneously facilitating the transport of toxin domains to the secretion apparatus [PAAR domains are encoded in several bacterial pathogens, frequently harboured in the N-terminus of proteins with C-terminal toxin domains . A PAAR-ni 488  was screene polC , was alspparatus .Campylobacter to date. We demonstrate genetic similarities and differences between the T6SS of Campylobacter species, as well as variation in the number of vgrGs, PAAR-like proteins and putative effectors harboured by T6SS-containing genomes. We also report the putative first identification of a T6SS in four Campylobacter species, with other T6SS-harbouring species belonging to the growing group of ‘emerging Campylobacter pathogens’. Understanding the arsenal of effectors this secretion system utilizes and the mechanisms by which the apparatus can be genetically transferred will provide a foundation for future experiments characterizing the Campylobacter T6SS.In summary, this study provides the most in-depth analysis of T6SS prevalence and diversity within the genus Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


VP1 region, and five Chinese EV-A76 isolates were selected for whole genome sequencing based on VP1 nucleotide divergence. Similarity plots and boot-scanning analyses revealed frequent intertypic recombination in the nonstructural region of the EV-A76 isolate, as found with the EV-A89 donor sequence . The breakpoint of recombination is around nucleotide 3960, and the recombinant fragments covered part 2C and all P3 regions. This study increases publicly available EV-A76 nucleotide sequence and further our understanding EV-A76 molecular epidemiology.Enterovirus 76 (EV-A76) is a serotype of enterovirus A and has been rarely reported. In this paper, we present the genetic characteristics of 15 EV-A76 isolates reported to circulate in the Xinjiang Uighur autonomous region of China in 2011. Sequence analysis revealed that all Chinese EV-A76 isolates had high similarity (> 98.3%) in the  Enterovirus, within family Picornaviridae, order Picornavirales, contains 15 species, among which four main species have been reported to cause human disease: Enterovirus A, B, C, and D (EV-A to EV-D). EV-A76 is a rare reported serotype within EV-A, and at present, only a few countries have reported cases or outbreaks of EV-A76, and only six complete genome sequences are available in the GenBank database (as of October 2022) [The genus An enterovirus surveillance programme was launched in southern Xinjiang in 2011 with the aim of evaluating enterovirus carriage among patients with acute flaccid paralysis (AFP) and their contacts . A totalThis study did not involve human experimentation. All sample collection and experimental procedures were approved by the Second Ethics Review Committee of the National Institute for Viral Disease Control and Prevention and the Chinese Center for Disease Control and Prevention.Stool samples were collected from AFP patients and their contacts during the enterovirus surveillance programme initiated in southern Xinjiang in 2011, and viruses were isolated from original stool specimens by propagation in human rhabdomyosarcoma (RD) and human laryngeal epidermoid carcinoma (HEp-2) cell lines according to the standard protocol .VP1. The PCR products obtained were purified using the QIAquick Gel extraction kit (Qiagen), and the amplicons were bidirectionally sequenced using an ABI PRISM 3130 Genetic Analyzer . The Enterovirus genotyping tool and BLAST server were used to identify Enterovirus type [VP1 region.Viral RNA was extracted from the viral isolates using a QIAamp Viral RNA Mini Kit . RT–PCR was performed with Primscript One-Step RT–PCR Kit Ver. 2  according to the manufacturer’s instructions. The primer pair EV76-VP1S/EV76-VP1A  were used in PCR amplification to yield fragments of approximately 1.0 kb spanning VP1 nucleotide divergence. The complete genome sequence was amplified using the “primer-walking” strategy and overlapping segments representing the entire genome were amplified by one-step RT–PCR using specific primers (Supplementary Table 1). The 10 VP1 sequences and five full-length genome sequences detected in this study have been deposited in GenBank under accession numbers ON646231-ON646245, ON646232, ON646239, ON646240, ON646243, and ON646245 are whole genome sequences.Five Xinjiang EV-A76 isolates were selected for whole genome sequencing based on The evolutionary characteristics of the EV-A76 strains described in this study were investigated using the Maximum Clade Credibility (MCC) tree and Bayesian skyline plot generated by BEAST software (version 2.5) [VP1, P1, P2, and P3 coding regions   using th2C and all P3 regions of the genome, which is consistent with the results of the aforementioned phylogenetic trees. Thus, the co-circulation of various EV-A (e.g. EV-A76 and EV-A89) may be a key factor in facilitating the development of recombinant EV-A76.To further investigate the source of EV-A76 recombination, The nucleotide alignment containing the genome sequence of Xinjiang EV-A76 strains, EV-A76 prototype strain, and EV-A89 strain KSYPH-TRMH22F/XJ/CHN/2011 [In conclusion, 15 EV-A76 strains isolated from an outbreak in Xinjiang, China in 2011 were reported in this study, and sequence analysis showed that they belonged to the same origin. Five of these viruses were subjected to whole genome sequencing and bioinformatics analysis, and all of them were found to undergo intertypic recombination. This study increases publicly available EV-A76 nucleotide sequence, expands whole-genome sequences in GenBank, and provides further insight into the EV-A76 molecular epidemiology.Click here for additional data file.


USP11 ablation inhibits leukemia progression in vivo, sparing normal hematopoiesis. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell–specific protein-tyrosine kinase (LCK) and enhance its activity. Impairment of LCK activity leads to increased glucocorticoid receptor (GR) expression and glucocorticoids sensitivity. Genetic knockout of USP7 improved the antileukemic efficacy of glucocorticoids in vivo. The transcriptional activation of GR target genes is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Our data unveil how dysregulated deubiquitination controls leukemia survival and drug resistance, suggesting previously unidentified therapeutic combinations toward targeting leukemia.Dysregulation of kinase signaling pathways favors tumor cell survival and therapy resistance in cancer. Here, we reveal a posttranslational regulation of kinase signaling and nuclear receptor activity via deubiquitination in T cell acute lymphoblastic leukemia (T-ALL). We observed that the ubiquitin-specific protease 11 (USP11) is highly expressed and associates with poor prognosis in T-ALL.  The deubiquitinases USP7 and USP11 enhance LCK activity and hamper response to glucocorticoids in T-ALL. Overcoming resistance to systemic therapy is an unmet need in acute leukemia  connectivity. These findings provide a rationale for the use of deubiquitinase or LCK inhibitors and GCs as combination treatments in leukemia.We performed an unbiased analysis of deubiquitinase levels and association with disease prognosis, coupled to biochemical, molecular, and in vivo disease modeling studies. We demonstrate that USP11 plays an oncogenic role in lymphoid malignancy and controls LCK activity in association with USP7 via LCK deubiquitination. Impairment of LCK activity impedes T cell signaling, leading to up-regulation of GC receptor (GR) expression, which sensitizes the leukemia cells to GCs. In addition, USP7 inhibition enhances transcription activation of GCs target genes, such as USP11, USP6, USP5, and USP44 . Additional analysis demonstrated that USP11 protein levels were higher in T-ALL patients and cell lines compared to normal T cells  and serine/arginine-rich splicing factor 6 (SRSF6) by the deubiquitinase USP7 in T-ALL (nd USP44 . USP11 p T cells . Reverse T cells . Our datUSP11 using two short-hairpin RNAs (shRNAs), shUSP11.1 and shUSP11.2, leads to inhibition of cell growth  after disease establishment . USP11 ablation inhibited tumor growth, suggesting a critical role for USP11 in T-ALL maintenance . These data demonstrate that USP11 ablation suppresses T-ALL growth in vivo.To further characterize the essential role of USP11 in T-ALL progression in vivo, we used a xenograft model. Usp11 gene affects normal hematopoiesis and thymic development, we used a germline Usp11 knockout mouse  or in the size or cellularity of the thymus  identified 51 proteins as direct USP11 substrates . USP7, USP11, Src-Y416, and LCK protein levels showed a positive correlation in T-ALL patients .LCK has been shown to play a critical role in the T cell development and activation through regulation of TCR signaling , indicating that USP7 and USP11 might cooperate in regulating LCK.To test whether USP11 and USP7 regulate LCK deubiquitination, our KεGG-MS analysis revealed that the total numbers of ubiquitinated peptides of LCK were increased upon hibition . We idenC275S, reduced the overall ubiquitination of LCK in 293 T cells . Of note, inhibition of USP7 or combined attenuation of USP7 and USP11 levels also affected phosphorylated LCK levels . We observed 356 proteins with lower expression levels [down (DN)] and 99 proteins with higher expression levels (UP) upon USP11 silencing (fig. S7A). Like shUSP11, USP7i also presents a reduction in protein levels . Dasatinib treatment, in contrast, led to very limited changes in protein expression (three DN and five UP proteins) (fig. S7C). Of note, attenuation of USP7 or USP11 leads to similar global changes in protein levels (fig. S7D). In line with the previously suggested role of USP11 in DNA damage repair   presented changes; 51 of them showed a decrease and 101 had increased phosphorylation . USP7i lUSP11 silencing. Notably, these datasets exhibit significant overlap with each other on increased or decreased transcripts  was performed upon dasatinib treatment or nscripts . As LCK egulated  s levels . MoreoveR levels . SimilarNR3C1 up-regulation was confirmed in eight of the eight primary T-ALL patient samples in the dasatinib treatment group with or without DXM (NR3C1 expression difference was observed upon LCK inhibition in unstimulated human CD3+ cells (fig. S9L).LCK activity controls the response to GCs in lymphoid tumors . Notably, USP7 inhibitor and DXM or prednisone showed various synergistic effects in inhibiting T-ALL growth, ranging from mild to strong, in seven of the eight primary T-ALL patient samples tested  and patient samples ] levels in the presence of GCs . Hi-C-bench analysis . USP7i alone does not significantly affect gene expression, chromatin accessibility, or genomic connectivity in BCL2L11 or TSC22D3 loci . WeXM alone . Consistwith DXM . Analysi2D3 loci .Dysregulation of deubiquitination as a modulator of oncogenic state in hematopoietic malignancies is largely uncharacterized. In this study, we have demonstrated a previously unidentified role for the deubiquitinase complex of USP7 and USP11 in regulating LCK kinase signaling and therapy response in T cell leukemia. USP-mediated deubiquitination maintains LCK in the active pY394 conformation that inhibits activation of apoptotic pathways upon application of GCs. LCK activity is mainly involved in regulation of TCR signaling . Human “thymocytes” refer to magnetically isolated thymocytes from human thymi derived from pediatric heart surgery (age from 7 days to 6 months). This refers to the previously published study by our groups; see the “Sorting of human thymocyte populations from human thymus” section in  or from Astarte Biologics . Primary human T-ALL samples were collected under an informed consent after the study, and potential consequences were explained to the patients under the supervision of the Institutional Review Board of Padova University, the Associazione Italiana Ematologia Oncologia Pediatrica, the Berlin-Frankfurt-Münster (AIEOP-BFM) ALL 2000/2006 clinical trials, and the Institutional Review Board of Ghent University. The risk group is classified by detection of minimal residual disease (MRD) based on detection of leukemic blasts with patients. Patients were considered MRD standard risk if MRD was negative at days 33 and 78, medium risk if MRD was positive either at day 33 or 78 with up to 1 leukemic cell per 1000 healthy cells (< 10−3) on day 78, and high risk if more than 1 leukemic cell per 1000 healthy cells (>10−3) is detected on day 78. The study was registered at http://clinicaltrials.gov . Mouse thymocytes were isolated from young animals (4 to 6 weeks old) and represent total population of thymocytic T cells (mainly including double negative and double positive for CD4 and CD8 markers). The Usp11 knockout mice were purchased from European Mouse Mutant Archive-Infrafrontier and the Wellcome Trust Sanger Institute . The insertion of the L1L2_Bact_P cassette created a deletion of size 2743 starting at position 20578261 and ending at position 20581004 of chromosome X at Usp11 allele (Genome Build GRCm39). Mx1-Cre Usp7 conditional knockout mice (https://ocg.cancer.gov/programs/target/data-matrix). For the DXM-treated Usp7 knockout model in vivo study  and the animal welfare committee of Ghent University. The cells are periodically tested for the presence of mycoplasma using the Lonza Walkersville MycoAlert Mycoplasma Detection Kit. The cell lines have been authenticated using short-tandem repeats profiling (JURKAT and DND41) or using polymerase chain reaction (PCR) to detect the TCRb-NOTCH1 translocation  , rabbit anti–SHP-1 , rabbit anti–SHP-1 (Tyr564) , rabbit anti-BIM , rabbit anti-CYLD , rabbit anti–phospho-Lck (Tyr505) , rabbit anti-USP7 , rabbit anti-cleaved NOTCH1  , rabbit anti–phospho-Src family (Tyr416) , rabbit anti–glyceraldehyde-3-phosphate dehydrogenase , rabbit anti-histone H3 , rabbit anti-HSP90 , hemagglutinin antibody , and FLAG antibody . All antibodies for flow cytometry were from eBioscience. Secondary antibodies for immunofluorescence were goat anti-rabbit immunoglobulin G (IgG) H&L (Alexa Fluor 488)  and goat anti-mouse IgG H&L (Alexa Fluor 647) . Secondary antibodies for Western blots were horseradish peroxidase–conjugated anti-rabbit and anti-mouse IgG . DXM was purchased from Sigma-Aldrich. Chlorhexidine and doxycycline reagent were purchased from Thermo Fisher Scientific. Dynabeads human T-Activator CD3/CD28 and cell fractionation kit were purchased from Thermo Fisher Scientific. The latest generation of Progenra inhibitors (USP7i), P217564, was a gift from Progenra . USP7 inhibitor (P5091) generated by Progenra was also used yielding similar results to P217564. Dasatinib, bosutinib, and WH-4-023 were purchased from Selleckchem . USP11 peptide ligand was a gift from I. Dreveny’s laboratory. Hi-C kits were purchased from Arima Genomics.The following antibodies were used for Western blotting, immunofluorescence, or immunoprecipitation: mouse anti-actin , rabbit anti-USP11 , mouse anti-USP11 , rabbit anti-USP11 , rabbit anti-LCK , mouse anti-LCK , mouse anti-GR , rabbit anti-ZAP70 , rabbit anti-ZAP70  according to the manufacturer’s instructions. Eluted phosphopeptides were dried by SpeedVac, resuspended in 0.1% TFA, and desalted on 10 mg of SOLA HPR solid-phase extraction cartridges (Thermo Fisher Scientific). Peptides were dried by SpeedVac, labeled with TMT11 reagents, and multiplexed as described above. Phospho-tyrosine–containing peptides were enriched from the desalted sample using the P-Tyr-1000 rabbit monoclonal antibody (CST) according to the manufacturer’s instructions. Eluted peptides were cleaned by stage tip into an MS sample vial and dried by SpeedVac. The nonbound fraction was retained, desalted by SepPak, dried by SpeedVac, and fractionated by basic reversed-phase high-performance LC, as described above, into a 96-well plate. Each column was combined for a total of 12 final fractions, which were dried by SpeedVac, cleaned by stage tip, and resuspended in 5%FA/5% ACN for MS analysis.TMT11 multiplexed protein samples were analyzed with an LC-MS3 data collection strategy on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) equipped with a Proxeon Easy nLC 1000 for online sample handling and peptide separations as previously described (USP11-human: (forward) 5′-CGTTTCCGGGACCAGAATCC-3′ and (reverse) 5′-CATCGCCGTCCGTTCTCTTC-3′; NR3C1-human: (forward) 5′-GGAATAGGTGCCAAGGATCTGG-3′ and (reverse) 5′-GCTTACATCTGGTCTCATGCTGG-3′; ACTB-human: (forward) 5′-CTCGCCTTTGCCGATCC-3′ and (reverse) 5′-GGGGTACTTCAGGGTGAGGA-3′; BCL2L11-human: (forward) 5′-GGTCCTCCAGTGGGTATTTCTCTT-3′ and (reverse) 5′-ACTGAGATAGTGGTTGAAGGCCTGG-3′.The real-time primers are follows: shLCK.1: 5′-CCGGGAATGGGAGTCTAGTGGATTTCTCGAGAAATCCACTAGACTCCCATTCTTTTTTG-3′ ; shLCK.2: 5′-CCGGGCATGAACTGGTCCGCCATTACTCGAGTAATGGCGGACCAGTTCATGCTTTTTG-3′ ; shUSP11.1: 5′-CCGGCCGTGACTACAACAACTCCTACTCGAGTAGGAGTTGTTGTAGTCACGGTTTTT-3′ ; shUSP11.2: 5′-CCGGCCGTGATGATATCTTCGTCTACTCGAGTAGACGAAGATATCATCACGGTTTTTG-3′ ; shUSP7:5′-CCGGCCTGGATTTGTGGTTACGTTACTCGAGTAACGTAACCACAAATCCAGGTTTTT-3′; and nonmammalian shRNA control hairpin SHC002: 5′-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGT.Transfection and virus production were performed as previously described ] were used: shUSP11.a V3SH11252-228470650 (clone ID: V3IHSMCG_8408300). TRIPZ inducible lentiviral non-silencing shRNAs (RHS4743) were used as a shRNA control.Inducible shRNAs from Horizon Discovery [All mice were housed in a barrier facility, and procedures were performed as approved by the Northwestern University Institutional Animal Care and Use Committee (protocols Ntziachristos no. IS00002058 and Mazar no. IS00000556). For CUTLL1 or JURKAT T-ALL intravenous studies, 1 million cells in 100 μl of phosphate-buffered saline were injected into the tail vein of 8-week-old NOD.Cg-Prkdcscid male mice . Animals were monitored by in vivo imaging system (IVIS) every 3 days for luciferase signal detection. IVIS images were taken using the IVIS Spectrum in vivo imaging system (PerkinElmer). These experiments were performed by Center for Developmental Therapeutics of Northwestern University.For complete blood counts analysis, blood (50 μl) was collected from the tail vein in EDTA-coated tubes and analyzed by a Hemavet 850 complete blood counter (Drew Scientific). Thymocyte isolation from mice and flow cytometry analysis were performed as previously described  assay, immunoblots, and RPPA were performed as previously described , 1 μM dexamethasone (DXM), or their combination for 6 hours. A total of 30 M cells  were cross-linked with 1% formaldhyde 10 min followed by a 5-min treatment with 0.125 M glycine. Cells were lysed, and the cross-linked DNA was sheared into 200– to 400–base pair fragments using the truChIP chromatin hearing kit (Covaris) and Covaris M220. One percent of the sheared DNA was used as input, and the remaining material was incubated with 5 μg of rabbit GR (G-5) antibody. The eluted DNA was used as input for quantitative PCR (qPCR) for promoter and IGR (primer sets 1 and 2) and negative control (non–GR-bound) loci (https://meta-chart.com/venn). The raw data for the cohort of pediatric T-ALL samples showing an increased USP11 expression and the KGG study in regard to USP7 inhibition on T-ALL cells were analyzed from previous publication (https://synergyfinder.fimm.fi). Hi-C analysis was performed as before  , based on MS1 intensity or MS2 spectral count, is carried out by MaxQuant v1.6.0.16 . Intensity of reporter ions was calculated by Scaffold Q+. A 1% false discovery rate (FDR) cutoff was applied at the peptide level. Only proteins with a minimum of two peptides above the cutoff were considered for further study. Analysis of variance test with Benjamini and Hochberg FDR correction is applied to the comparison among the conditions using a 0.05 threshold for statistical significance.P value calculations were done using the Benjamini-Hochberg option. A P value of <0.05 was statistically significant. Gene essentiality data for cancer cell lines were obtained from the Project Achilles CRISPR-Cas9 screening dataset . Essentiality of individual genes is represented as the inverse of the CERES score for that gene  was done using the National Center for Biotechnology Information GEO2R online tool for microarray analysis. Quantile normalization was used to process microarray data. Adjusted


Apart from its transporter function, NTCP acts as the high-affinity liver-specific receptor for the hepatitis B virus (HBV), which attaches via its preS1-peptide domain of the large surface protein to NTCP, subsequently leading to endocytosis of the virus/NTCP-receptor complex. Although the process of NTCP-dependent HBV infection of hepatocytes has received much attention over the last decade, the precise molecular sites of the virus/NTCP interaction have not been fully identified. Inspection of the primary protein sequence of human NTCP revealed 139YIYSRGIY146 as a highly conserved tyrosine-rich motif. To study the role of Y139, Y141 and Y146 amino acids in NTCP biology, the aforementioned residues were substituted with alanine, phenylalanine or glutamate (mimicking phosphorylation) using site-directed mutagenesis. Similar to wt NTCP, the Y139A, Y141A, Y146A, Y141F, Y146F, and Y146E mutants were expressed at the plasma membrane of HEK293 cells and exhibited intact bile acid transport function. Y146A, Y146E, and Y146F demonstrated transport kinetics comparable to wild-type NTCP with Km values of 57.3–112.4 µM and Vmax values of 6683–7579 pmol/mg protein/min. Only Y141E was transport deficient, most likely due to an intracellular accumulation of the mutant protein. Most importantly, Y146A and Y146E mutation completely abrogated binding of the viral preS1-peptide to NTCP, while the Y146F mutant of NTCP showed some residual binding competence for preS1. Consequently, the NTCP mutants Y146A and Y146E, when expressed in HepG2 hepatoma cells, showed complete loss of susceptibility for in vitro HBV infection. In conclusion, tyrosine 146, and to some extent tyrosine 141, both belonging to the tyrosine-rich motif 139YIYSRGIY146 of human NTCP, are newly identified amino acid residues that play an essential role in the interaction of HBV with its receptor NTCP and, thus, in the process of virus entry into hepatocytes. Na Radio-labelled TC in HEK293 cells overexpressing wt and mutant NTCP as well as in Flp-In HEK293 cells (negative control), cells were incubated for 3 min with increasing concentrations  of [3H]TC and further transport experiment were conducted on three separate days in triplicates as described above.Qualitative transport experiments were performed in NTCP-transfected HEK293 cells with the fluorescent bile acid 4-nitrobenzo-2-oxa-1,3-diazole taurocholic acid (NBD-TC) in DMEM . After ireported . BrieflyTo study the HBV receptor function of NTCP, HEK293 cells expressing the wt or mutant NTCP constructs were incubated with the N-terminally myristoylated and C-terminally Alexa 568 fluorophore-coupled myr-preS1 viral peptide (further referred to as myr-preS1-AX568), consisting of amino acids 2–48 of the large HBV sub-genotype D3 surface protein  . BrieflyHBV was produced in vitro as reported before . HepG2 cThe cryo-EM structure of human NTCP (PDB: 7PQQ)  was visu50 calculations, statistical analysis was done by two-way ANOVA and Sidak’s multiple comparisons post-hoc test. A p-value of <0.001 was considered statistically significant.Data are shown as means ± SD. Prism software  was used for data presentation and statistical analysis. Statistical analysis was performed by one-way or two-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison post-hoc test as indicated in the figure legends. For ICInspection of the primary sequence of the human NTCP protein revealed a tyrosine-rich motif YIYSRGIY positioned between amino acids 139–146. Multiple sequence alignment analysis revealed that all three tyrosine residues of this motif, namely Y139, Y141, and Y146, are evolutionarily conserved among human NTCP, as well as chimpanzee, rhesus monkey, rat, and mouse Ntcps A. Based In order to address the importance of the 139YIYSRGIY146 motif for regulating the bile acid transport and HBV receptor function of NTCP, single mutants  and a triple mutant of all three tyrosine residues (Y139A/Y141A/Y146A) were generated by site-directed mutagenesis C. Of notTo analyze the effect of NTCP mutation on the preS1-binding ability to NTCP, HEK293 cells overexpressing NTCP wt and mutant proteins were incubated with the fluorescently labeled viral myr-preS1-AX568 peptide. Myr-preS1-AX568 binding to NTCP was visualized using fluorescence microscopy and revealed strong binding to wt NTCP-expressing HEK293 cells, while no binding was observed to empty vector (EV) transfected control cells A. Interehttp://www.hprd.org; accessed on 20 May 2020) [3H]TC  ,31 revea) [3H]TC E transpo) [3H]TC A and ful) [3H]TC C–E. Inte) [3H]TC A,B. Howe) [3H]TC A,B. Anot3H]TC in transiently transfected HEK293 cells (Km values of 44.8 ± 8.9, 82.4 ± 5.9, 112.4 ± 37.4, and 57.3 ± 5.6 µM and Vmax values of 5444 ± 336, 6683 ± 198, 7618 ± 1067, and 7579 ± 248 pmol/mg protein/min, respectively.Although the NTCP mutants Y146A and Y146E were binding negative for the myr-preS1-AX568 peptide, the bile acid transport function was completely preserved when TC uptake experiments were performed with NBD-TC and [93 cells C,D. Howe93 cells A. In add93 cells C. The stIn order to test whether mutations of the 139YIYSRGIY146 region would have any effect on HBV susceptibility, wt and mutant NTCP constructs were transiently transfected into HepG2 cells and further utilized for in vitro HBV infection studies. Western blot analysis confirmed successful expression of all NTCP mutant proteins in HepG2 cells A. The raTo date, little is known about the mechanistic insights into the process of initial NTCP-HBV complex assembly and NTCP-mediated virus entry into hepatocytes ,10. AlthIn mutation studies, effects of loss-of-function mutants are often difficult to interpret, as even single amino acid mutations can destabilize a protein in a way that ligand or substrate binding is no longer possible. However, there is, of course, also the possibility for a direct interaction of a particular amino acid residue with the above-mentioned molecules so that a loss of function phenotype can be explained by lack of a specific interaction site. In the case of the Y141E NTCP mutant, in the present study, this mutation seems to destabilize the NTCP protein in a way that means sufficient protein expression and sorting to the plasma membrane is not possible. Consequently, this mutant is no longer active in bile acid transport and myr-preS1-AX568 peptide binding A,C. In cProtein surface-exposed regions of NTCP play an essential role during initial docking of HBV to hepatocytes . Until nIn conclusion, we have provided evidence that the newly identified 139YIYSRGIY146 motif and its Y141 and Y146 aromatic amino acids are important virulence factors that play a fundamental role in the process of NTCP-mediated HBV tropism. Our comprehensive analyses of NTCP Y141 and Y146 mutants confirmed the importance of intramolecular interactions of these aromatic amino acids with previously proposed protein surface-localized NTCP regions involved in HBV tropism, such as 84RLKN87. The current study presents new insights into the HBV-NTCP interaction and further prerequisites for the development of novel HBV entry inhibitors aimed at blocking the initial virus-receptor complex assembly.


The objective of this study is to report the complete-genome sequence of a field African swine fever (ASF) virus (ASFV), namely ASF/VN/CanTho-OM/2021, which caused a fatal outbreak in domestic pigs in the Mekong Delta. Complete-genome sequencing detected an 18 bp nucleotide deletion in the EP402R gene (encoding for serotype-specific proteins CD2v) of ASF/VN/CanTho-OM/2021, which was determined to belong to genotype 2 and serotype 8. This mutation pattern was confirmed as unique in GenBank; thus, ASF/VN/CanTho-OM/2021 can be considered a novel variant, with a potential change of sero-characteristics within genotype 2. An additional unique mutation of 78 bp nucleotide insertion was also observed in the B475L gene. Additionally, four copies of tandem repeat sequences were found in the intergenic region (IGR) located between I73R and I329L, previously assigned as the IGR III variant. This study is the first to report the complete genome of ASFV in the Mekong Delta, and it highlights the necessity of strengthening molecular surveillance to provide further knowledge on the evolution and incursion of ASFV in the Mekong Delta and Vietnam. Asfarviridae, a DNA arbovirus. The viral genome of ASFV has 170 to 195 kbp, encoding at least 160 open reading frames [African swine fever (ASF) is a highly infectious disease in pigs caused by the African swine fever virus (ASFV), which might result in a high mortality rate approaching 100% . ASFV isg frames ,4. CurreASF was first reported in Kenya in 1921 . Since tThe whole-genome sequence (WGS) of ASFV detected in this study, namely ASF/VN/CanTho-OM/2021, was deposited to GenBank and assigned the accession number ON402789. Based on the sequence alignment of the B646L gene (p72), ASF/VN/CanTho-OM/2021 showed 100% nucleotide identity with ASFVs previously detected in Vietnam, China and Georgia 2007/1, which all belong to genotype 2 (data not shown). Similarly, a phylogenetic analysis of the EP402R gene (CD2v) confirmed that ASF/VN/CanTho-OM/2021 belongs to serotype 8 . InteresSimilarly, ASF/VN/CanTho-OM/2021 has a repeated insertion of 78-nucleotide sequence in the B475L gene “GAAGAAACTATTCTTGCGATTAAACAGGATATATCTGAAGAAGATAATATTTTTGCGATTGATCAGGATAAACCTGAG” at positions 91,352 to 91,356 corresponding to 26-amino-acid insertion . The repIn addition, the nucleotide sequence of the IGR between the I73R and I329L genes of ASF/VN/CanTho-OM/2021 contains four copies of TRS “GGAATATATA”, which are identical to the nucleotide sequence of VNUA/Hanoi-ASF9/2021 (MZ812475.1) and China/Guangxi/2019 (MK670729.1), which all belong to IGR III variant classification . Therefohttps://www.fao.org/ag/againfo/programmes/en/empres/ASF/situation_update.html, accessed on 1 May 2022).In Vietnam, the first ASF outbreak was reported in February 2019. The disease quickly spread across all provinces, and ASF is currently endemic all over the country, with serial outbreak reports ,13. AccoOwing to the importance and widespread distribution of ASF in Vietnam, many studies have been conducted for the genetic characterization of circulating ASFVs and tracing incursion of viruses based on specific molecular markers ,11,12. MThe phylogenetic analysis based on B646L (p72) and EP402R (CD2v) sequences indicated that ASF/VN/CanTho-OM/2021 belongs to genotype 2 and serotype 8 . This reIntriguingly, an 18 bp nucleotide deletion of “CTACTACCCAATATCCCG” was detected in the EP402R gene, which resulted in a six-amino-acid deletion “LLPNIP” in the CD2v of ASF/VN/CanTho-OM/2021 . The shoBased on complete-genome analysis, this study detected another unique mutation in the B475L that had a repeated insertion of the 78-nucleotide sequence in the B475L gene “GAAGAAACTATTCTTGCGATTAAACAGGATATATCTGAAGAAGATAATATTTTTGCGATTGATCAGGATAAACCTGAG” . In factThe sequence of TRS insertions in IGR between the I73R and I329L genes is widely used as a genome marker to discriminate closely circulating ASFVs worldwide, including in Vietnam ,17,18. TThis study is the first to report the complete genome of ASFV in the Mekong Delta and detect a distinct variant of genotype 2. The disease remains highly endemic in the region, but little information is known about circulating viruses. Therefore, this study provides great significance for understanding the epidemiology, evolution and incursion of ASFV in the Mekong Delta and Vietnam.This study was conducted following a fatal virus outbreak in domestic pigs on a backyard farm in Can Tho city, a central administrative division of the Mekong Delta with a distance of about 1500 km from Ha Noi capital, in October 2021. All 11 infected pigs displayed obvious clinical symptoms of ASF infection. The samples were collected from organs (spleen and lymph nodes) of 2 dead pigs out of the 11 clinically infected pigs at the farm and used for conventional PCR targeting of the B646L gene to confirm the presence of ASFV, as described previously [® Genomic DNA Purification Kit . The WGS was performed at the KTest Science Company  using the Illumina MiniSeq platform (2 × 150 bp paired ends). Adapters, primers and low-quality sequences (average score < 20 and read length < 35 bp) were removed using fastp version 0.20.1 [NGS was used to obtain the WGS of the detected ASFV. Briefly, total viral DNAs were directly extracted from the collected organs using the Wizardn 0.20.1 . The trin 0.20.1  with defape [ips [msa [The complete genome in a single contig was submitted for downstream analyses such as DNA multiple alignments, trimming and phylogenetic tree construction. The full-length genome sequence of ASFV detected in this study and other ASFV reference strains obtained from GenBank were aligned using MAFFT version 7 with default settings . The quaape , ips [24ips [msa .Sequences in the genome contig of ASF/VN/CanTho-OM/2021 containing a mutation comparable to the Georgia 2007/1 genome were amplified by conventional PCR and sequenced using the Sanger method for verification. DNA alignments and chromatogram traces from Sanger sequencing were manually inspected using BioEdit version 7.0.5 .


Brassica oleracea var. acephala). An alanine and a leucine residue in the conserved domain of the smooth-margined S0835 were replaced by an aspartate and a phenylalanine, respectively, in the corresponding positions of the feathered-margined F0819. However, the expression pattern and function of this gene remain unclear. Here, we examined the expression patterns of BoALG10 using quantitative real-time PCR, and found that statistically significant differences in expression existed between F0819 and S0835 in nine developmental stages. The BoALG10 protein localized to the endoplasmic reticulum. The function of BoALG10 was then examined using complementary mutant assays. The overexpression strains phenocopied the smooth leaf margin after introduction of BoALG10S0835 into the feathered-margined inbred line F0819. Simultaneously, irregular dissections appeared in the leaf margins of knockout mutants KO-1 and KO-2, which were generated by CRISPR/Cas9 technology from the smooth-margined inbred line S0835. Microscopic observation showed that the leaf margin cells of the smooth-margined plants S0835 and OE-3 were arranged regularly, while the cells of the feathered-margined plants F0819 and KO-1 were of inconsistent size and distributed in an irregular manner, particularly around the indentations of the leaf. This elucidation of BoALG10 function provides a novel insight into the morphological regulation of leaf margin shape.The morphological diversity of leaf margin shapes is an identifying characteristic of many plant species. In our previous work, BoALG10  was predicted to be a key regulator of leaf margin shape in ornamental kale ( Published by Oxford University Press on behalf of Nanjing Agricultural University. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: 25 February 2022; Revised: 11 May 2022; Accepted 8 June 2022
<sec id="sec3"><title>Significance statement</title>
<italic>BoALG10</italic>, encoding α-1,2 glycosyltransferase in the N-glycan biosynthesis pathway, maintains the smooth leaf margin of ornamental kale. The mutation of <italic>BoALG10</italic> caused an N-glycan biosynthesis defect, giving rise to irregular lobes at the leaf margin.
</sec>Solanum lycopersicum) were recently shown to significantly improve the flavor and quality of the fruits , and CUC3 together with miR164A  and BnA10.RCO [43] both positively regulate the formation of serrated leaf margins in B. napus, with cis-regulatory variations in these genes leading to the appearance of lobed or serrated leaf margins. In this study, we analyzed the spatiotemporal expression patterns of BoALG10 in the ornamental kale genotypes F0819 (feathered margin) and S0835 (smooth margin), revealing that it was expressed throughout development in both lines with no differences in tissue specificity. We speculated that the discrepancy of BoALG10 expression pattern was not sufficient to explain their differences in leaf margin phenotypes. In previous research, we found that two amino acid differences occurred in the conserved domain between F0819 and S0835 . We previously isolated a gene, BoALG10, by fine mapping a genomic region with a putative regulatory effect on leaf margin shape in ornamental kale . The leaf margin was almost smooth and the vein was simple with few secondary vascular veins in Arabidopsis. Nevertheless, the leaf vein pattern was very complex with many secondary veins in ornamental kale. Once the gene mutated, leaves became smaller in Arabidopsis; in contrast, some lobes appeared in ornamental kale. In Arabidopsis, ALG10 is necessary for leaf development, and also in ornamental kale. In the inbred line with a smooth leaf margin, S0835, BoALG10 functioned normally to transfer the last glucosyl molecule to the adipose-linked donor, facilitating N-glycan biosynthesis. The mutation of BoALG10 in F0819 resulted in its defective function and the disruption of adipose-linked oligosaccharide biosynthesis. The defective N-glycan biosynthetic pathway cannot maintain the smooth leaf margin morphology, and thus irregular lobes appeared in the leaf margin of the mutant ornamental kale . First-strand cDNA was synthesized with the AMV First-Strand cDNA Synthesis Kit .et alQuantitative real-time PCR (qRT–PCR) was performed as described by Feng BoALG10 were PCR-amplified using the primers GFP-BoALG10F 5′-GAACACGGGGGACTCTTGACATGGGGAGACTAGCGCTTG-3′ and GFP-BoALG10R 5′-CCTTTACTAGTCAGATCTACCATCCAAATAAACCTTTGGACACCATC-3′, and then fused with green fluorescent protein (GFP) under the control of the cauliflower mosaic virus 35S promoter using a NEBuilder HiFi DNA Assembly , for which the pCAMBIA1302 vector frame had been digested using the restriction endonuclease NcoI. The recombinant expression vector 35Spro:BoALG10-GFP and the ER marker vector ER-rk (CD3–959) combined with mCherry [33] were introduced into Agrobacterium tumefaciens strain GV3101 using the method described by Feng et al. [35Spro:BoALG10-GFP and ER-rk constructs were equally mixed and then infiltrated into 4-week-old N. benthamiana leaves, with the pCAMBIA1302 vector used as the blank control. The fluorescent signals were observed under a laser scanning confocal microscope  after a 48-hour incubation in the dark.The full-length coding sequences of g et al. . The 35SArabidopsis arenosa (CAE6085553.1), Arabidopsis lyrata ssp. lyrata (XP_002873050.2), Arabidopsis suecica (KAG7553847.1), A. thaliana (NP_195861.2), Brassica cretica (KAF3568203.1), B. napus (XP_013729813.1), B. napus (XP_013746118.1), Brassica oleracea var. oleracea (XP_013623580.1), Brassica rapa (XP_009130699.1), Camelina sativa (XP_010452078.1), Capsella rubella (XP_006287552.1), Eutrema salsugineum (XP_006398701.1), Microthlaspi erraticum (CAA7021219.1), Raphanus sativus (XP_018473028.1), and Sinapis alba (KAF8105067.1). ClustalW was used for amino acid sequence alignment, and the phylogenetic tree was constructed using MEGA 7.0 software with the neighbor-joining tree method [Protein sequences homologous to BoALG10 were downloaded from the National Center for Biotechnology Information (NCBI) and used to construct a phylogenetic tree: etica KAF68203.1, cea XP_0123580.1, BoALG10 coding region from the S0835 inbred line was PCR-amplified from cDNA using the primers F 5′-GGACTCTTGACCATGGGGAGACTAGCGCTTGCAGC-3′ and R 5′-ACCTGTAATTCACACGTGCTACCAAATAAACCTTTGGACACC-3′. The PCR product was inserted between the 35S promoter and the Agrobacterium NOS terminator sequence using the NEBuilder HiFi DNA Assembly (New England Biolabs) to generate the overexpression plasmid 35Spro:BoALG10. The A. tumefaciens GV3101 line containing 35Spro:BoALG10 was introduced into F0819 using the methods described by Feng et al. [The plant binary vector pCAMBIA3301 was digested at 37°C for 3 hours in a 50-μL reaction volume comprising 1 μL of the restriction enzymes NcoI and PmlI (New England Biolabs), 10 μL of the pCAMBIA3301 plasmid (50 ng/μL), and 38 μL of RNase-free water. The g et al. .BoALG10, the CRISPR/Cas9 system was used to generate boalg10 knockout mutants. The single target sites were selected using the CRISPOR online tool (http://crispor.tefor.net/crispor.py). The BoALG10-CRISPR cassette was generated using a PCR amplification from pCBC-DT1T2 using the primers DT1-BsF: ATATATGGTCTCGATTGATAGTTCCCGATCCCTACAGTT, DT1-F0: TGATAGTTCCCGATCCCTACAGTTTTAGAGCTAGAAATAGC, DT2-R0: AACGCGGTCTACTAATGCTGTTCAATCTCTTAGTCGACTCTAC, and DT2-BsR: ATTATTGGTCTCGAAACGCGGTCTACTAATGCTGTTCAA, which were designed as reported previously [BoALG10. The PCR fragments were purified and then cloned into the pEASY-T vector , before being subjected to Sanger sequencing . The A. tumefaciens GV3101 line containing pHSE401-BoALG10 was introduced into S0835 using the methods described by Feng et al. [To further verify the function of eviously . The PCRg et al. .The phenotypes of the transgenic and knockout plants were compared with the WT F0819 and S0835, respectively, in the growing periods. Then, to confirm the integration of the overexpression cassette, specific fragments were PCR-amplified using the primers OEF: 5′-ATGGGGAGACTAGCGCTTGC-3′ and OER: 5′-CACAGCACTGAGGATATATC-3′. The relative transcript levels of BoALG10 were measured in all transgenic plants using the same qRT–PCR method as that described above. To determine the sequences surrounding the target sites, we PCR-amplified using the primers CasF: 5′-TTTGGATTTGACTCCTCTCTG-3′ and CasR: 5′-GATATATCCTCAGTGCTGTG-3′. The PCR fragments were purified and then cloned for sequencing three times as described above.A layer of clear nail polish was applied evenly to the adaxial surface of fresh leaf blades from F0819, S0835, OE-3 , and KO-1 (a feathered-margined knockout line) plants at the 10-leaf stage. After the nail polish had dried completely, temporary blotting film was used to transfer the nail polish impression of the leaf margin to a microscope slide. The impressions of the leaf margin cells were imaged using an optical microscope  under 100× magnification.We are grateful to professor Qijun Chen from China Agricultural University for the CRISPR/Cas9 vectors. This research was supported by the National Natural Science Foundation of China (32171850 and 31770739).BoALG10. P.Z. revised the manuscript. All the authors reviewed and approved this submission.X.F. and P.Z. designed the experiments. X.F. wrote the manuscript and performed substantial experiments containing nucleic acid extraction, overexpression and CRISPR/Cas9 vector construction, and genetic transformation. X.Y. conducted the subcellular localization of BoALG10 and identified the transgenic plants. M.Z. executed microscopic observation of epidermal cells. X.L. executed spatiotemporal expression analysis of The data that support the results are included in this article and its supplementary materials. Other relevant materials are available from the corresponding author upon reasonable request.The authors declare that they have no conflicts of interest.Horticulture Research online.supp_data_uhac137Click here for additional data file.


In addition,in vivo study confirms that the circVPS13A-miR-182-GDNF network regulation can attenuate hyperglycemia-induced EGC damage of duodenum in streptozotocine (STZ)-induced DM mice. The findings of this study may provide novel insights into the protective role of circVPS13A in DM-associated EGC damage and clues for the development of new therapeutic approaches for the prevention of GI complications of DM.Gastrointestinal (GI) complications of diabetes mellitus (DM) significantly impact on patients’ quality of life. Enteric glial cells (EGC) are the key cell type of enteric nervous system (ENS), which contributes to the destruction of gut homeostasis in DM. Circular RNAs (circRNAs) are a novel type of RNAs abundant in the eukaryotic transcriptome, which form covalently closed continuous loops. In this study, the contribution of circRNAs to EGC damage in DM is investigated. Transcriptome sequencing analysis and functional study show that circVPS13A is significantly down-regulated in hyperglycemia-treated EGC, and circVPS13A overexpression attenuates EGC damage in both Diabetes mellitus (DM) widely influences organs in the body, and gastrointestinal (GI) complications are more common in diabetic patients than in healthy subjects -3 . ThuCircular RNAs (circRNAs) are a new class of noncoding RNAs, which are likely related to the occurrence and development of various human diseases ,11 . ThIn this study, we found that circVPS13A is significantly down-regulated in hyperglycemia-treated EGC based on transcriptome sequencing data, and further investigated the role of circVPS13A in the diabetes-induced enteric glia damage. This study may lead to a new therapeutic approach for the treatment of diabetes-associated GI complications.CRL-2690 enteric glial cells were purchased from ATCC  and cultured in DMEM medium  supplemented with 10% fetal bovine serum (Gibco) and 25 mM D-glucose . Hyperglycemia concentration was obtained by adding D-glucose to a final concentration of 300 mM. For experiments, cells were transfected with GDNF siRNA1 , GDNF siRNA2 , pcDNA3.1-circVPS13A  using Lipofectamine 2000  according to the manufacturer’s protocols.Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay Kit  was used to detect apoptotic cells. Cells or tissues were fixed with 4% paraformaldehyde for 30 min and treated with 0.5% Triton X-100-PBS for 5 min according to the manufacturer′s instructions. Following this, the sections were then incubated with a terminal deoxynucleotidyl transferase reaction mix for 60 min at 37°C, followed by DAPI staining for 5 min. TUNEL‐positive cells (red staining cells) were observed via fluorescence microscopy .C57BL/6J mice were purchased from Changzhou Cavens Experimental Animal Center . DM was induced by intraperitoneal injection of STZ (Sigma). Mice received STZ  injection for 5 consecutive days. Control mice were administered with the same volume of sodium citrate buffer. All mice were kept in an air-conditioned room with a 12-hour light-dark cycle and fed with standard laboratory chow, with free access to water. Seven days after the final STZ injection, blood samples were collected from the tail vein for the measurement of blood glucose level. The mice with a blood glucose level of over 300 mg/dL were considered as DM. All animal experiments were performed in accordance with the institutional guidelines of Jiangsu Institute of Nuclear Medicine  for the Care and Use of Laboratory Animals, and this study was approved by the Ethics Committee of Jiangsu Institute of Nuclear Medicine.CRL-2690 cells were incubated with 25 mM glucose or 300 mM glucose for 24 h. After incubation, RNA samples were prepared from cells for transcriptome sequencing by Novogene  . The clustering of samples was processed through the cBot Cluster Generation System  according to the manufacturer’s instructions. After cluster generation, the library was sequenced on the Illumina Hiseq platform and 125 bp/ 150 bp paired-end reads were generated. HTSeq v0.6.0 was used to count the number of reads mapped to each gene. FPKM of each gene was calculated based on the gene length with reads count mapped individually.circVPS13A F: 5′-TGAAATTCTTGCAGAAATGTTG-3′, R: 5′-GTGCTCCTGGTCTTTGCACAAT-3′;GDNF F: 5′-TTCAAGCCACCATCAAAAGAC-3′, R: 5′-GTAGCCCAAACCCAAGTCAGT-3′; ratGAPDH F: 5′-TGCCACTCAGAAGACTGTGG-3′, R: 5′-TTCAGCTCTGGGATGACCTT-3′; mouseGAPDH F: 5′-TTGTCTCCTGCGACTTCAACAG-3′, R: 5′-GGTCTGGGATGGAAATTGTGAG-3′;miR-182 F: 5′-ATCACTTTTGGCAATGGTAGAACT-3′, R: 5′-TATGGTTTTGACGACTGTGTGAT-3′; andU6 F: 5′-GCTTCGGCAGCACATATACTAA-3′, R: 5′-AACGCTTCACGAATTTGCGT-3′. All reactions were run in triplicates. The level of miRNA was normalized to that ofU6, and the relative levels of circular RNA were normalized to that ofGAPDH. Total RNA from CRL-2690 cells was digested with RNase R  followed by qRT-PCR detection of circVPS13A and VPS13A mRNA expressions.Total RNA was isolated from cells using TRIzol reagent  and subjected to reverse transcription using a PrimeScript RT Master Mix Kit (TaKaRa). Quantitative PCR was performed using a Realtime PCR kit (TaKaRa) with the following primers:Cells were treated with RIPA lysis buffer for 15 min prior to total protein extraction. Protein concentration was determined by BCA assay. Samples (20 μg) were subject to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane . The membrane was blocked with 5% bovine serum albumin (BSA) for 1 h and then incubated with the anti-GDNF antibody  or anti-GAPDH antibody , followed by incubation with the corresponding HRP-conjugated secondary antibody . The protein bands were visualized using an ECL assay kit (Beyotime) and quantified using ImageJ . GAPDH was used as the loading control.After treatment, the culture medium was collected and processed for ELISA. Culture medium (100 μL) was reacted with the GDNF ELISA kit  according to the manufacturer′s instructions. The absorbance was measured at 450 nm using a microplate reader .http://www.targetscan.org/) was used to predict the target of miR-182. Luciferase report assay was conducted for the measurement of miR-182 function. To evaluate the effect of miR-182 on circVPS13A, HEK293 cells were plated in 6-well plates and co-transfected with 100 pM of either miR-182 mimic (5′-UUUGGCAAUGGUAGAACUCACACCG-3′ or negative control (5′-UUCUCCGAACGUGUCACGUTT-3′) , 40 ng of either pGL3-circVPS13A or pGL3-circVPS13A-MUT ( Genepharma), and 4 ng of pRL-TK  using Lipofectamine 2000 (Life Technologies). To assess the effect of miR-182 on GDNF, HEK293 cells were co-transfected with 100 pM of either miR-182 mimic or control, 40 ng of either pGL3-GDNF-3′UTR or pGL3-GDNF-3′UTR MUT (Genepharma), and 4 ng of pRL-TK (Promega) using Lipofectamine 2000 (Life Technologies). Forty-eight hours after transfection, cells were collected and analyzed using the Dual-luciferase Reporter Assay System (Promega).TargetScan (CRL-2690 cells were transfected with 3′-end biotinylated miR-182 (Genepharma) at a final concentration of 50 nM for 24 h. Then, the cells were lysed and centrifuged as described previouslyRNA FISH experiments were carried out using the kit purchased from Genepharma according to the manufacturer′s instructions. The probes of circVPS13A and miR-182 (labeled with Cy3 dye) were as follows: circVPS13A, CAAGAATACCATGTATAGCATAAGCCCACTTTAGGGCTTGTGCTCCTGGTCTTTGCAC; miR-182, CGGTGTGAGTTCTACCATTGCCAAA. All hybridizations were done overnight in the dark at 37°C in a humidified chamber. For protein FISH experiments in cells, CRL-2690 cells were cultured on coverslips, fixed with 4% paraformaldehyde for 20 min and incubated in PBS overnight at 4°C, followed by the detection of circVPS13A and miR-182 as previously reportedcircVPS13A F, 5′-TGAAATTCTTGCAGAAATGTTG-3′, R, 5′-GTGCTCCTGGTCTTTGCACAAT-3′;miR-182 F, 5′-ATCACTTTTGGCAATGGTAGAACT-3′, R, 5′-TATGGTTTTGACGACTGTGTGAT-3′.The chip-based digital PCR (dPCR) was conducted on a QuantStudio 3D Digital PCR System (Life Technologies) for the absolute quantification of the copy number of circVPS13A and miR-182. cDNA was synthesized from RNA isolated from EGC under indicated conditions, and digital PCR was conducted using the following reaction conditions: hot start at 96°C for 10 min, denaturation at 98°C for 30 s, annealing/extension at 62°C for 2 min for a total of 39 cycles, followed by a final extension step at 65°C for 2 min. The data analysis was performed with QuantStudio 3D AnalysisSuite Cloud Software version 3.1.2. The primers used are:The section of mouse duodenum was cut into 35-μm slices with a cryostat. Then, the sections were incubated with anti-GFAP antibody (Sigma) overnight at 4°C. On the following day, the sections were washed and incubated with Alexa Fluor 488-conjugated anti-mouse IgG for 1 h. After wash with PBS, the sections were incubated with ProLong gold Antifade reagent containing DAPI (Invitrogen) for visualization of the nuclei. Immunofluorescence images were captured with an Olympus DP73 fluorescence microscope (Olympus).11 GC/ml) or AAV9-miR-182 agomir (1×1011 GC/ml) was conducted using a 10-μL Hamilton syringe into the wall of the duodenum at two sites near the myenteric plexus as previously reportedVirus injections were performed at 2 weeks after the establishment of DM mice model. The mice were divided into three groups: DM group, DM+circVPS13A OV (Overexpression), DM+circVPS13A OV+miR-182 TF (Transfection). Mice were anesthetized using isoflurane (2%–4%) and kept at a constant body temperature. For the experiments, circVPS13A was subcloned into pHBAAV-CMV- circRNA-EF1-ZsGreen vector, and the vector or micrON™ miR-182 agomir was packaged by AAV9, then the injection of 2 μL AAV9-circVPS13A  or 300 mM glucose  for 24 h. First, cell viability was assessed by TUNEL assay and the results showed that HG significantly reduced cell viability in CRL-2690 cells A. The RNeatment E. Meanwheatment F,G. Thesin vitro andin vivo DM models (in vivo (in vitro andin vivo DM models.To understand the function of circVPS13A under hyperglycemia, circVPS13A was up-regulated by circVPS13A overexpression in both models A. First, models B,C. In a models B,C. Thenin vivo D,E. ThesTo understand the mechanism of the regulatory role of circVPS13A under hyperglycemia, the target genes of circVPS13A were analyzed using bioinformatics tools and dual-luciferase reporter assay. First, bioinformatics data predicted that circVPS13A exerts its function at the post-transcriptional level by acting as a miR-182 sponge A. Then, GDNF is one of the candidate genes to be selected for further evaluation due to its role in glia activation. Luciferase reporter assay verified the direct regulation of miR-182 in GDNF expression; while miR-182 mimic significantly reduced GDNF expression at the protein level (GDNF silencing attenuated the protective effect of miR-182 inhibitor or circVPS13A overexpression on hyperglycemia-induced EGC cytotoxicity and apoptosis in CRL-2690 cells (in vitro andin vivo DM models.Targetscan database was used to predict the target genes of miR-182.n level A–C. In an level D,E. Furtn level D. Meanwh0 cells , indicat0 cells . These din vitro andin vivo DM models. Furthermore, circVPS13A acted as a miR-182 sponge to sequester and inhibit miR-182 activity, which led to increased expression of GDNF. This study might provide novel insights into the understanding of the pathogenesis of DM-related GI complications.Impaired function of the GI tract is the common complication of DM, and EGC dysfunction contributes to the destruction of gut homeostasis under pathophysiological conditions ,22 . Thet al.in vitroandin vivo, which is comparable to that of circVPS13A overexpression.circRNAs exert their functions by acting as the sponges for miRNAs or proteins, competing with the linear mRNA, or regulating the transcription of their parental genes ,24 . ciet al.in vivoet al.GDNF silencing attenuated the protective effect of circVPS13A overexpression or miR-182 inhibition on hyperglycemia-induced EGC cytotoxicity and apoptosis in CRL-2690 cells. In addition, ourin vivostudy confirmed the protective effect of circVPS13A overexpression on EGC damage. However, future studies are required on theGNDF knockdown mouse model to further investigate the molecular mechanism of circVPS13A/miR-182. In addition, besides modulating circVPS13A-miR-182 signaling to enhance GDNF in EGC, normalizing GFAP+ enteric glial cells could also recover GDNF expression indirectly in duodenum, since enteric glial cells are a source of GDNF.To further investigate the effects of miR-182, targetscan database and luciferase reporter assay were performed to confirm whether GDNF, an important factor in glia activation, is the direct target of miR-182. GDNF is known to participate in the regulation of EGC function, and altered GDNF expression leads to a higher susceptibility towards apoptosis, resulting in the disruption of the mucosal integrity and severity of inflammationIn conclusion, in this study we revealed that circVPS13A is an important regulator of EGC damage in DM. circVPS13A overexpression increases cell viability and inhibits apoptosis of EGC by sponging miR-182 and inducing GDNF release . Therefo