Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase involved in various cancers. In its basal state, the structure of ALK is in an autoinhibitory form stabilized by its A-loop, which runs from the N-lobe to the C-lobe of the kinase. Specifically, the A-loop adopts an inhibitory pose with its proximal A-loop helix (αAL-helix) to anchor the αC-helix orientation in an inactive form in the N-lobe; the distal portion of the A-loop is packed against the C-lobe to block the peptide substrate from binding. Upon phosphorylation of the first A-loop tyrosine (Y1278), the αAL-helix unfolds; the distal A-loop detaches from the C-lobe and reveals the P+1 pocket that accommodates the residues immediately after their phosphorylation, and ALK is activated accordingly. Recently, two neuroblastoma mutants, F1174L and R1275Q, have been determined to cause ALK activation without phosphorylation on Y1278. Notably, F1174 is located on the C-terminus of the αC-helix and away from the A-loop, whereas R1275 sits on the αAL-helix. In this molecular modeling study, we investigated the structural impacts of F1174L and R1275Q that lead to the gain-of-function event. Wild-type ALK and ALK with phosphorylated Y1278 were also modeled for comparison. Our modeling suggests that the replacement of F1174 with a smaller residue, namely leucine, moves the αC-helix and αAL-helix into closer contact and further distorts the distal portion of the A-loop. In wild-type ALK, R1275 assumes the dual role of maintaining the αAL-helix–αC-helix interaction in an inactive form and securing αAL-helix conformation through the D1276–R1275 interaction. Accordingly, mutating R1275 to a glutamine reorients the αC-helix to an active form and deforms the entire A-loop. In both F1174L and R1275Q mutants, the A-loop rearranges itself to expose the P+1 pocket, and kinase activity resumes. Anaplastic lymphoma kinase (ALK) is a member of the superfamily of the insulin receptor protein tyrosine kinase; ALK participates in embryonic nervous system development during embryogenesis with decreased expression after birth . AccumulMany fingerprint features distinguish the active and inactive structures of a kinase. For example, in an active state, the conserved DFG motif adopts a DFG-in with the phenylalanine pointing inward, which causes the ATP site to become available; by contrast, in an inactive state, the conserved DFG motif adopts a DFG-out position where the phenylalanine points outward and obstructs the ATP site ,15,16. Ikcat (in min−1) values of 9.32 ± 0.85 for the wild type (WT), 119 ± 13 for R1275Q, 365 ± 61 for F1174L and 425 ± 63 for pY1278 [Mutations within the ALK domain that promote constitutive, ligand-independent activation are frequently involved in many diseases. Three “hot spot” residues are reportedly involved in 85% of all mutations, namely R1275 (43%), F1174 30%) and F1245 (12%), where glutamine or leucine is typically substituted for R1275; leucine, isoleucine, valine, cysteine or serine replace F1174; and F1245 is replaced with leucine, isoleucine, valine or cysteine ,23. Exper pY1278 . Figure r pY1278 ,29,30,31% and F12+ ions were added to neutralize WT, the pY1278 variant, the F1174L variant and the R1275Q variant, respectively. Each solvated ALK system underwent three stages of energy minimization; each stage consisted of 5000 steps of the steepest descent algorithm and 5000 steps of the conjugate gradient algorithm with a non-bonded cutoff of 8.0 Å. In Stage 1, all atoms in the ALK domain were restrained, thereby enabling the added TIP3P water molecules to reorient. In Stage 2, atoms in the protein backbone were restrained, and thus, the atoms in the amino acid side chains were rendered to interact with the added water molecules. In Stage 3, the whole solvated system was minimized without restraint to minimize conformational conflict.Three available crystal structures of the ALK, namely apo WT (PDB code: 3L9P) , the F11The MD simulations in this study were performed in accordance with the standard protocol, which specifies gradual heating, density equilibration, equilibration and production procedures in the isothermal isobaric ensemble  MD. A minimized solvated system was used as the starting structure in subsequent MD simulations. In the 100-ps heating procedure, the system was gradually heated from 0–300 K within 40 ps; this was followed by density equilibration at 300 K for 100 ps and then constant equilibration at 300 K for 1000 ps. Following the equilibration procedure, each complex system underwent two independent 100-ns production runs at a 2-fs time step. We recorded a snapshot every 10 ps throughout the production runs. An 8 Å cutoff was applied to treat nonbonding interactions such as short-range electrostatics and van der Waals interactions; the particle-mesh-Ewald method  was applThe Cα root-mean-square deviation (RMSD) values for the four studied ALK systems, each system undergoing two independent 100 ns simulation runs, in the production duration given as a function of time are plotted in As shown in As discussed, R1275 on the αAL-helix plays a dual role, namely securing the electrostatic interactions between the αC- and αAL-helices and preserving the helical conformation of the αAL-helix through the R1275–D1276 interaction. In the R1275Q variant displayed in In this MD simulation study, we used the X-ray crystal structures of ALK in its WT, F1174L and R1275Q variants to elucidate the activation mechanism of the F1174L and R1275Q mutations. We also generated a structure of WT-carrying phosphorylated Y1278 to obtain structural features of the active ALK. The DFG motif in each of the four studied ALK systems adopted the DFG-in conformation, and thus, the DFG phenylalanine was able to participate in R-spine assembly regardless of the inactive state of WT ALK. Both DFG-in and the presence of R-spine were characteristic of the active state of kinases. The WT ALK structure was known to be an intermediate between the active and inactive states. Because of the DFG-in conformation, the DFG phenylalanine pointed inward and rendered the ATP binding site available, as in active kinase structures. However, the N-terminal portion of the A-loop folded as the αAL-helix remained in close contact with the αC-helix and gradually influenced the spatial arrangement of the C-terminal portion of the A-loop to seal the P+1 pocket generated by the P+1 loop. With respect to the pY1275, F1174L and R1275Q variants, the A-loop moved either upward or to the right and accordingly exposed the P+1 pocket for the substrate. In summary, we conducted protein cavity detection using fpocket  and plot


Vibrio vulnificus (VvRRSP). Structural and biochemical analyses revealed that VvRRSP is a metal-independent TIKI family endopeptidase composed of an N-terminal membrane-localization and substrate-recruitment domain (N lobe) connected via an inter-lobe linker to the C-terminal active site–coordinating core β-sheet–containing domain (C lobe). Structure-based mutagenesis identified the 2His/2Glu catalytic residues in the core catalytic domain that are shared with other TIKI family enzymes and that are essential for Ras processing. In vitro KRas cleavage assays disclosed that deleting the N lobe in VvRRSP causes complete loss of enzymatic activity. Endogenous Ras cleavage assays combined with confocal microscopy analysis of HEK293T cells indicated that the N lobe functions both in membrane localization via the first α-helix and in substrate assimilation by altering the functional conformation of the C lobe to facilitate recruitment of cellular substrates. Collectively, these results indicate that RRSP is a critical virulence factor that robustly inactivates Ras and Rap1 and augments the pathogenicity of invading bacteria via the combined effects of its N and C lobes.Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are secreted by Gram-negative bacteria and function as primary virulence-promoting macromolecules that deliver multiple cytopathic and cytotoxic effector domains into the host cytoplasm. Among these effectors, Ras/Rap1-specific endopeptidase (RRSP) catalyzes the sequence-specific cleavage of the Switch I region of the cellular substrates Ras and Rap1 that are crucial for host innate immune defenses during infection. To dissect the molecular basis underpinning RRSP-mediated substrate inactivation, we determined the crystal structure of an RRSP from the sepsis-causing bacterial pathogen  Vibrio vulnificus, secrete multifunctional autoprocessing repeats-in-toxin (MARTX)10–Numerous Gram-negative bacterial pathogens have the ability to deliver bacterial “effector” proteins into the host cytoplasm to modulate a multitude of host cell functions . Direct Within host cells, different effectors display distinct cytopathicity or cytotoxicity, and the overall toxicity depends on the combined virulence of individual MARTX toxin effectors , 14. BecVibrio species (Vibrio vulnificus CMCP6 is a Ras/Rap1-specific endopeptidase (RRSP)  . Notablye (RRSP) . RRSP cae (RRSP) , 19 thate (RRSP) , 21.V. vulnificus CMCP6 depends on the C-terminal subdomain (residues 3672–3855) harboring residues Asp-3721 and Arg-3841 that are critical for maintaining the structural stability of the RRSP , and we demonstrated that it is a metal-independent TIKI family endopeptidase. We further dissected the structural features, identified catalytic and substrate recognition residues, and unveiled the roles of the subdomains in the functions of the enzyme.In this study, we determined the crystal structure of an RRSP from the sepsis-causing pathogen V. vulnificus strain CMCP6 (A). However, the resulting crystals failed to diffract to high resolution. Deletion of N- and C-terminal flexible regions resulted in a core construct (residues 3596–4072) that produced crystals diffracting X-rays to 2.7 Å . The structure of each of the four protein chains is essentially identical, except for the N-terminal α1 helix spanning residues 3596–3609 . Size-exclusion chromatography revealed that VvRRSP exists as a monomer in solution ; hence, the structure of chain A was considered representative of the subunit structure and was used in further analysis. The enzyme activity of VvRRSP (residues 3596–4072) was confirmed by in vitro KRas cleavage assay, demonstrating that the construct was functional and processed Ras and Rap1 .A previous study reported that the MARTX toxin effector RRSP is an enzyme possessing site-specific processing activity against the small GTPases Ras and Rap1 that act as cellular switches during signal transduction, including innate immune responses . Computain CMCP6 A. Howeveto 2.7 Å , with foVvRRSP structure can be divided into an N-terminal lobe  and a C-terminal lobe , and the N lobe can be further subdivided into the membrane localization N1 domain (MLD), consisting of four helices (α1–α4), as characterized previously . This bundle  forms a hydrophobic core, in which Tyr-3613, Ile-3616, Leu-3620, Val-3623, Ile-3632, Leu-3638, Ile-3642, Tyr-3645, Leu-3658, Leu-3661, Val-3665, and Leu-3669 are arranged to maintain structural integrity . The structural arrangement of the MLD in VvRRSP is markedly different from that of the two structures of isolated MLDs determined previously by nuclear magnetic resonance  , suggesting that this domain may be highly flexible. The VvRRSP MLD is similar to the membrane localization C1 domain in the Pasteurella multocida toxin (PMT) . Residues Phe-3636 on the α3 helix and Phe-3670 on the α4 helix make critical contributions to the structural stabilization of the MLD by engaging in extensive hydrophobic contacts with the groove lined by Met-3674, Gln-3678, Asp-3682, Ala-3685, Pro-3689, Ala-3693, and Phe-3696 on the α5 and α6 helices of the N2 domain .The eviously , and theig. S1B)  and crysA), the C1 domain (α8–α12 and β1–β3), and the C2 domain (α13–α22 and β4–β11) that adopts a typical α + β TIKI fold . The C lobe is firmly integrated with the N lobe, mainly via hydrophobic interactions between Leu-3710, Met-3713, and Trp-3717 on α7 in the N2 domain; Ile-3845, Ile-3848, and Tyr-3849 on α12 in the C1 domain; His-3996, Met-3999, Tyr-4000, and Gly-4003 on α20; and Gly-4039 and Val-4041 on β9 in the C2 domain (E). A previous study reported that Asp-3721 and Arg-3841 are critical for structural stability of RRSP (E).The C lobe can also be separated into two subdomains A, the C12 domain E. A prev of RRSP . The str of RRSP E.F). In particular, residues Tyr-3723 and Leu-3724 play a critical role in maintaining structural integrity between the N2, C1, and C2 domains (F). The inter-lobe linker appears to function as a hinge to bestow flexibility between the two lobes. To evaluate the importance of the loop, residues Tyr-3723 and Leu-3724 were mutated to alanine, and in vitro KRas cleavage assays revealed that RRSP enzyme activity was completely abolished (G). Thus, these two residues might be essential for controlling flexibility.A long loop  connects the two lobes F. In par domains F. The inbolished G. Thus, VvRRSP and TIKI superfamily proteins , HopBA1 , Bcr135 , and ChaN , all of which contain a parallel β-sheet core surrounded by α-helices .Structural comparison using PDBeFold identified structural similarity between proteins , includiVvRRSP has a six-stranded β-sheet, flanked by helices on both sides . Five of the six β-strands of the C2 domain  form the characteristic parallel β-sheet core observed in all TIKI proteins .TIKI superfamily proteins share a common core domain consisting of a central β-sheet surrounded by α-helices . LikewisVvRRSP with the Ere family protein Bcr135 revealed that functional residues are highly conserved (A–E). The predicted functional residues His-3902, His-4030, Glu-3900, and Glu-3930 (forming the 2His/2Glu motif) in VvRRSP are located near β4, β5, and β7 strands within the parallel β-sheet core of the C2 domain . We subsequently investigated whether these 2His/2Glu residues are essential for hydrolysis of Ras and Rap1 by mutating them to leucine. In vitro KRas cleavage assays demonstrated that none of RRSPH3902L, RRSPH4030L, RRSPE3900L, or RRSPE3930L catalyzed the proteolytic cleavage of KRas, indicating that all four 2His/2Glu residues are essential for catalytic activity (F). Mutant proteins did not undergo any significant conformational changes compared with WT RRSP according to the results of circular dichroism (CD) spectrometry analysis .It has been proposed that enzymatically active TIKI family proteins share a catalytic mechanism with the erythromycin esterase (Ere) family, in which a conserved His–Glu pair is essential for substrate hydrolysis . Structuonserved , A–E. Thactivity F. MutantA) or Rap1  in HEK293T cells transfected with each VvRRSP mutant. The results clearly showed that mutation of Glu-3930 or His-4030 completely abolished enzymatic activity, consistent with the results of in vitro KRas cleavage assays. Interestingly, mutation of Glu-3900 or His-3902 inhibited enzymatic activity significantly, but not completely, in cells (A). Consequently, ectopic expression of RRSPE3930L or RRSPH4030L for 16 h did not cause the cell-rounding that was observed with wildtype (WT) RRSP (B), as typically observed in RRSP-expressing cells (E3900L or RRSPH3902L expression (B).We further evaluated the importance of the 2His/2Glu residues for the hydrolysis of endogenous Ras A or Rap1in cells A. ConseqWT) RRSP B, as typng cells . Mild cepression B.VvRRSP in this study. We therefore tested whether the VvRRSP mutations affected cellular localization, and no difference was found between WT and mutant proteins at 16 h post-transfection; all proteins were mainly localized to the HEK293T cell membrane, where their substrates reside . We further evaluated the ability of the mutants to cleave endogenous Ras at 16 h post-transfection by confocal microscopy analysis, and WT RRSP efficiently processed endogenous Ras, whereas mutants lacking essential catalytic residues were inactive (C). Thus, these results collectively demonstrate that VvRRSP is a Ras/Rap1-specific endopeptidase that requires the essential 2His/2Glu catalytic residues in the C2 domain.Green fluorescent protein (GFP)-fused MLD of RRSP localizes to the plasma membrane in HeLa cells , and we inactive C. Thus, 30DEYDPTIEDSY40 in K/H/N-Ras, and 30EKYDPTIEDSY40 in Rap1A/B) of the cellular substrates Ras and Rap1 following sequence-specific substrate recognition  that might be involved in forming contacts with the RRSP active site. In addition to the catalytic His/Glu residues, a highly conserved basic residue is found near the active site residues in all TIKI proteases (VvRRSP is Arg-4001 (B and C). We therefore presumed that Arg-4001 may be a substrate recognition residue. Proteolytic processing assays with RRSP mutated at Arg-4001 revealed complete loss of catalytic activity, indicating that this residue may indeed play a role in substrate recognition (D).RRSP cleaves between residues Tyr-32 and Asp-33 in the Switch I region , indicating flexibility and a potential involvement in substrate recognition. Given that the catalytic residues of VvRRSP were successfully assigned, we hypothesized that substitution of catalytic residues with nonfunctional residues might stabilize the active site and the adjacent flexible region. We subsequently determined the structure of the RRSPE3930L/H4030L mutant at a resolution of 2.3 Å (E). Like WT RRSP, the active site of the VvRRSPE3930L/H4030L mutant is arranged similarly to that of BcR135 . Luckily, the flexible region spanning residues Arg-3988–Gln-3992 in molecule A of the mutant is ordered and adopts a loop structure close to the active site (E). To evaluate the involvement of this region in the function of RRSP, we selected Arg-3988 based on its interactions with the Switch I region of Ras, dominated by negatively charged residues . This residue was mutated to glutamate, and the overall conformation was confirmed by CD spectroscopy . In vitro KRas cleavage assays showed that the mutation completely inhibited enzymatic activity (D), suggesting that the flexible loop may be involved in substrate recognition.During refinement of the RRSP structure, we noticed that the electron density map around residues Arg-3988–Gln-3992 located close to the active site in the C2 domain is disordered E, indicaof 2.3 Å . The strof 2.3 Å E. Like Wive site E. To evaactivity D, suggesin vitro pulldown assays revealed that the nonfunctional RRSP was similarly unable to maintain interaction with KRas . No differences in the active sites were observed between WT and mutant RRSP structures. Therefore, these results suggest that residues Glu-3900, Glu-3930, His-3902, and His-4030 in the active site may spontaneously adopt the catalytically active conformation in the presence of substrate.Because we could not observe an interaction between WT RRSP and Ras, we used the inactive RRSP mutant to examine interactions with KRas. However, VvRRSP and homologs revealed the highest similarity with the C1/C2 domains of PMT , even though VvRRSP shares only 26% amino acid sequence identity with PMT. PMT also belongs to the TIKI superfamily, but it lacks the catalytic 2His/2Glu motif in the common β-sheet core . Accordingly, endopeptidase activity against KRas was not observed for the PMT C1/C2 domain .Comparison of VvRRSP during refinement of its structure, we biochemically examined whether it belongs to the TIKI metalloprotease family. The enzymatic activity of VvRRSP toward KRas was comparable in the presence or absence of EDTA (A), indicating that metal ions are not involved in RRSP function. We also searched for other inhibitors that might be associated with blocking the enzymatic activity of RRSP, and a protease inhibitor mixture solution inhibited enzymatic activity of VvRRSP (B). Subsequent analysis of the separate components revealed that benzamidine was responsible for inhibition of KRas cleavage by VvRRSP (C and D). It is reported that KRas directly interacts with benzamidine (E) (VvRRSP and benzamidine (data not shown). Together, these results indicate that VvRRSP is a metal-independent TIKI family endopeptidase.Tiki, involved in the Wnt signaling pathway, is a representative TIKI protease with metal ion-dependent activity . However of EDTA A, indicaf VvRRSP B. Subseqy VvRRSP , C and Dzamidine E 29), s, sVvRRSPVvRRSP has an N-terminally extended N lobe as part of its MLD. Thus, to further assess the role of the N lobe, we constructed various deletion mutants (A) and evaluated them by in vitro KRas cleavage assay (B). Deletion of the N lobe and inter-lobe linker (Δ3580–3736), leaving only the C lobe, resulted in complete loss of enzymatic activity toward KRas (B). Similarly, the C2 catalytic domain alone (residues 3856–4072) was unable to hydrolyze KRas (B). Interestingly, deletion of the MLD N1 domain (Δ3580–3671) significantly reduced the efficiency of KRas cleavage activity compared with WT RRSP (B). Thus, these results suggest that the N lobe and inter-lobe linker are crucial for substrate recruitment or recognition via integration with the C lobe to induce the functionally competent conformation of VvRRSP. It is worth mentioning that the inter-lobe linker may act as a flexible hinge to properly arrange the two lobes into a functional conformation that is enzymatically active. In particular, Tyr-3723 and Leu-3724 on the linker may be key residues regulating flexibility (F and G). Only deletion of α1 (Δ3580–3609) within the MLD failed to affect the enzymatic activity (B).TIKI proteases have a wide variety of domains appended to the conserved TIKI fold structure that might act to promote enzymatic function . VvRRSP  mutants A and evage assay B. Deletiard KRas B. Similayze KRas B. Intere WT RRSP B. Thus, xibility , F and Gactivity B.in vitro assay results, the cleavage of endogenous Ras was entirely blocked in HEK293T cells transfected with plasmid expressing the N lobe deletion mutant (residues 3737–4089), and the activity of the MLD-deleted RRSP (residues 3672–4089) was significantly impaired (C). We further analyzed the cellular localization and enzymatic activity of VvRRSP by confocal microscopy. As reported previously (D). However, the MLD-deletion mutant (residues 3672–4089) or the C lobe mutant (residues 3737–4089) was dominantly expressed in the cytoplasm, again without enzymatic activity (D). Remarkably, the α1 helix appears to be critical for membrane localization of RRSP. Without the α1 helix (Δ3580–3609), membrane localization does not occur, resulting in reduced enzymatic activity, indicating that proper membrane localization of RRSP is required for enzymatic function (D). It is worth noting that in the structure the α1 helix points away from the MLD helix bundle (A). Taken together, these results demonstrate that the N lobe of RRSP performs dual roles: localization to the membrane via the α1 helix of the MLD and promoting enzymatic function.Consistent with the impaired C. We fureviously , the MLDeviously D. Howeveactivity D. Remarkfunction D. It is x bundle A. Taken Although mainly studied in the context of broad medical research in tumor initiation, progression, and metastasis, Ras proteins also play fundamental roles in immunity and inflammation . The Ras31–37–N-acetylglucosamination, and proteolytic cleavage . It remains uncertain whether VvRRSP shares the same catalytic mechanism with TIKI proteins because the His-4030–Glu-3930 pair was assigned as the essential catalytic residues in VvRRSP, rather than the His-3902–Glu-3930 pair (A and B). However, we cannot rule out the possibility that VvRRSP shares a catalytic mechanism with TIKI proteins because a water molecule was observed close to Glu-3900 and Glu-3930 in WT VvRRSP  and in the center of the active site of the catalytically nonfunctional mutant .The RRSP effector from the highly virulent La cells , and it La cells . Howeverproteins , includiproteins . It has proteins , 27. His . This structural feature may render the 14-amino acid residue α1 helix suitable for membrane anchoring (A). One side of α1 is composed of hydrophobic residues, and the other side has positively charged residues that may enhance membrane interaction . Superimposition of VvRRSP molecules in the asymmetric unit reveals different conformations for the MLD fingers (C), indicating flexibility. Indeed, part of the α1 helix is involved in crystal packing contacts, its flexibility is independent of the rest of the helices of the N1 domain in the RRSP structure, and it may be stabilized by these contacts . Thus, we cannot rule out the possibility that the α1 helix conformation in the structure of VvRRSP may be due to the crystal packing contacts. The membrane-interacting conformation of the N lobe likely stabilizes the functional conformation of RRSP in association with the active site-containing C lobe, thereby promoting recruitment of substrates from the membrane.The structure of 2 domain D. This snchoring A. One si fingers C, indicaVvRRSP might be broadly applicable for the development of strategies to combat powerful virulence mechanisms.Pathogenic bacteria have evolved mechanisms to effectively combat host defenses. Expanding our understanding of how bacteria manipulate immune systems in humans will provide promising strategies for controlling infection. Targeting the Ras and Rap1 signaling switches could offer an efficient way to subvert host signaling systems and augment the virulence of pathogenic bacteria. Because RRSPs are conserved among pathogens infecting mammals, fish, and plants, our detailed structural and functional analysis of Table S1. Escherichia coli strains were grown in Luria-Bertani (LB) medium at 37 °C with appropriate antibiotics. HEK293T cells were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C with 5% CO2 in Hyclone Dulbecco's modified Eagle's medium  supplemented with 1% antibiotic/antimycotic (Gibco-BRL) and 10% fetal bovine serum (Gibco-BRL).Bacterial strains and plasmids used in this study are listed in V. vulnificus strain CMCP6 was kindly provided by Dr. Joon Haeng Rhee from Chonnam National University Medical School and utilized as a template for amplifying the DNA fragment encoding the effector domain (residues 3580–4072) of the MARTX toxin, named VvRRSP. The amplified DNA fragment was subcloned into the NcoI and XhoI restriction enzyme sites of the pHis-Parallel1 expression vector, a protein expression vector that attaches an N-terminal His6-tag cleavable using recombinant tobacco etch virus (rTEV) protease. To investigate the critical residues responsible for endopeptidase activity, constructs for expression of RRSPE3900L, RRSPE3930L, RRSPH3902L, RRSPH4030L, RRSPY3723A, RRSPL3724A, RRSPR3988E, and RRSPR4001F were generated using site-directed mutagenesis. For protein structural studies, a DNA fragment corresponding to an N-terminal deletion mutant (residues 3596–4072) was incorporated into the same expression vector using the same method. In addition, to investigate the domains related to substrate recognition and binding, DNA fragments expressing truncated forms of VvRRSP, namely residues 3610–4072, 3672–4072, 3737–4072, 3580–3855, and 3856–4072, were subcloned into the pHis-Parallel1 expression vector. For analysis of localization and cytotoxicity, a DNA fragment encoding residues 3580–4089 was amplified and inserted into the NheI and ApaI sites of the pAcGFP vector for expression of C-terminal GFP fusion proteins. Several plasmids expressing mutated or truncated forms of VvRRSP fused with a C-terminal GFP, including RRSPE3900L, RRSPE3930L, RRSPH3902L, and RRSPH4030L, or residues 3580–3671, 3672–4089, 3610–4089, and 3737–4089, were generated by site-directed mutagenesis or subcloning as described above. For analysis of endopeptidase activity against endogenous Ras in cells, DNA fragments encoding VvRRSP residues 3580–4089 were amplified and inserted into the XbaI and XhoI sites of the pEXPR-IBA103 vector for expressing the RRSP protein with Strep-tag in the C terminus. Constructs for expressing RRSPE3900L, RRSPE3930L, RRSPH3902L, RRSPH4030L, 3580–3671, 3737–4089, and 3672–4089 were also generated as described above for pEXPR-IBA103-RRSP (residues 3580–4089). The k-ras gene encoding KRas was amplified by PCR using the pCMV-SPORT6-KRas construct obtained from the Korea Human Gene Bank (KHGB) as a template, and the resulting PCR products were subcloned into the NcoI and XhoI sites of the pHis-Parallel1 expression vector. The DNA fragment encoding Raf1 (residues 54–131) was cloned from pCMV-SPORT6-Raf1 obtained from KHGB into the BamHI and XhoI sites of the pHis-Parallel1 expression vector.Genomic DNA from Table S2, and all mutated and cloned plasmids were confirmed by DNA sequencing .A chemically synthesized DNA fragment encoding the C1/C2 region of PMT  was also subcloned into the pHis-Parallel1 expression vector to investigate enzymatic activity. All primers used for cloning and mutagenesis are listed in 6-tagged VvRRSP (residues 3596–4072) was overexpressed in E. coli BL21-CodonPlus (DE3)-RIPL cells by induction with 0.5 mm isopropyl β-d-thiogalactopyranoside (IPTG) at 21 °C for 18 h. Cultured cells were harvested by centrifugation at 5000 × g for 10 min at 4 °C, resuspended in ice-cold buffer A (50 mm Tris-HCl (pH 7.5), 300 mm NaCl), and lysed with a high-pressure homogenizer . Crude cell extracts were centrifuged at 25,000 × g at 4 °C for 1 h. The supernatant containing hexaHis-tagged RRSP (residues 3596–4072) was loaded onto a Ni-NTA–agarose column  pre-equilibrated with buffer A. Nonspecific resin-bound proteins were washed out with buffer A supplemented with 30 mm imidazole, and hexaHis-tagged RRSP was eluted with buffer A containing 250 mm imidazole. The hexaHis tag was cleaved by incubating the eluted protein with rTEV protease (Gibco) at 4 °C overnight. After protein concentration using a centrifugal concentrator with a 3-kDa cutoff , size-exclusion chromatography was performed using a Superdex 200 10/300 column . Fractions containing RRSP (residues 3596–4072) were pooled and loaded onto a second Ni-NTA– agarose column for further purification, and the flow-through containing the tag-free RRSP (residues 3596–4072) was concentrated to 15 mg/ml and stored at −80 °C after clarification by centrifugation at 16,000 × g for 30 min. Selenomethionine (SeMet)-substituted RRSP (residues 3596–4072) was expressed in E. coli B834 (DE3) cells (Merck Millipore Ltd.), a methionine auxotrophic strain, in minimal medium supplemented with 50 mg/ml SeMet and purified as described above for native RRSP (residues 3596–4072) in the presence of 5 mm methionine throughout all purification steps. RRSPE3930L/H4030L mutants were also expressed and purified as described for RRSP (residues 3596–4072). Purified proteins include a four-residue cloning artifact (Gly–Ala–Met–Ala) at the N terminus. For biochemical assays, expression of variants, including mutated and truncated forms, was carried out as described for RRSP (residues 3596–4072), but purification involved only one-step affinity column chromatography. Purified variants were stored at −80 °C after clarification by centrifugation at 16,000 × g for 30 min. Expression of KRas was induced in E. coli BL21-CodonPlus (DE3)-RIPL cells by treatment with 0.5 mm IPTG at 21 °C for 18 h. Harvested cells were resuspended in ice-cold buffer A and lysed using a high-pressure homogenizer . The recombinant N-terminally His6-tagged KRas in the supernatant was purified by Ni-NTA affinity chromatography, and the buffer was exchanged to 50 mm Tris-HCl (pH 7.5) and 300 mm NaCl for in vitro Ras cleavage assays. KRas fused with GSH S-transferase (GST) was also expressed and purified for in vitro pulldown analysis. The homogeneity of proteins was assessed by 12% SDS-PAGE and visualized using Coomassie Blue staining. Raf1 (residues 54–131) was made similar to that of KRas for pulldown analysis.For structural determination, Hism Tris-HCl (pH 7.5) and 300 mm NaCl at a flow rate of 0.35 ml min−1 at 4 °C. Purified RRSP (residues 3596–4072) was injected onto the column, and bovine serum albumin (BSA) was used as a molecular weight standard.A Superdex 200 10/300 GL gel-filtration column  installed on a fast protein LC (FPLC) system (Bio-Rad) was equilibrated with 50 mm ammonium sulfate, 0.1 m Tris-HCl (pH 8.0), and 12% glycerol proved optimal. Crystals of RRSP (residues 3596–4072) were frozen in cryoprotectant solution consisting of 1.6 m ammonium sulfate, 0.1 m Tris-HCl (pH 8.0), and 12% glycerol and exposed to X-rays in a nitrogen gas stream at −173 °C. Crystals diffracted X-rays to a resolution of 2.7 Å at beamline 5C of the Pohang Accelerator Laboratory (PAL) in Korea. Crystals of SeMet-substituted RRSP (residues 3596–4072) were grown and optimized under the same conditions, and single-wavelength anomalous diffraction data were collected at a resolution of 2.9 Å using the same beamline. Crystals of RRSPE3930L/H4030L were grown in 0.2 m LiSO4, 28% PEG 4000, and 0.1 m Tris-HCl (pH 8.0), and X-ray diffraction data were also collected at beamline 5C of PAL to a resolution of 2.3 Å. All X-ray diffraction data were processed and scaled with the HKL2000 software package , with drops consisting of 0.4 μl of protein (15 mg/ml) in buffer A and 0.4 μl of reservoir solution in 96-well MRC plates (Hampton Research). Initial crystals were produced in several different conditions. However, high-quality crystals appropriate for X-ray diffraction were only obtained after optimizing conditions, and 1.4  package . The str package . Initial package . The str package . COOT wa package , 51. Them) was incubated with RRSP or PMT at a ratio of 1:1 or 2:1 in 50 mm Tris-HCl (pH 7.5) and 300 mm NaCl. After incubating the KRas-RRSP or KRas-PMT mixture at 37 °C for 20 min, the reaction was stopped by addition of SDS-PAGE sample buffer. Proteins were separated on a 15% gel and visualized using Coomassie Blue staining. For analysis of inhibitors of RRSP activity, purified RRSP was preincubated with the indicated protease inhibitors at 37 °C for 30 min. After adding KRas, the mixture was further incubated at the same temperature for the indicated time. Samples were separated by SDS-PAGE and visualized by Coomassie Blue staining.Purified KRas (0.5 μm Tris-HCl (pH 7.5), 300 mm NaCl, 1 mm GTP, and 5 mm MgCl2 for 3 h at 4 °C. After intensive washing with incubation buffer, KRas fused with GST was eluted with the same buffer supplemented with 20 mm reduced GSH. RRSP variants eluted from the resin were analyzed by SDS-PAGE and visualized using Coomassie Blue staining.For pulldown assays, plasmids expressing KRas fused with GST were used as bait proteins, mixed with Raf1 (residues 54–131) or variants of RRSP, including mutated and truncated forms, and incubated with GSH-conjugated Sepharose beads  in 50 mHEK293T cells were grown to ∼70–80% confluency, and transfection was implemented using X-tremeGENE HP reagent  according to the manufacturer's instructions.m NaCl, 10% glycerol, and 20 mm HEPES (pH 7.2)) supplemented with complete protease inhibitor mixture (Roche Applied Science). After 30 min of incubation on ice, cell lysates were centrifuged at 16,000 × g for 30 min. Supernatants were mixed with LDS-PAGE loading buffer ; proteins were separated by SDS-PAGE and transferred onto a polyvinylidene fluoride membrane by the semi-dry blot method; and RRSP, Ras, and Rap1 proteins were detected by Western blotting using a chemiluminescence detection system . To detect Strep-tagged RRSP and its variants, a horseradish peroxidase (HRP)-conjugated mouse anti-Strep antibody  was used according to the manufacturer's protocol. To detect endogenous Ras, mouse monoclonal anti-Ras antibody  and HRP-conjugated anti-mouse IgG antibody  were used. To detect endogenous Rap1, mouse monoclonal anti-Rap1 antibody  and HRP-conjugated anti-mouse IgG antibody were used. Actin was detected using an HRP-conjugated rabbit monoclonal anti-β-actin antibody . All experiments were repeated at least three times, and data are expressed as the mean ± S.D. using Prism .HEK293T cells, transfected with plasmids expressing RRSP or its variants, were harvested at 16 h after transfection and lysed in lysis buffer .HEK293T cells were seeded into μ-slide 8-well plates  and transfected with plasmids expressing RRSP (residues 3580–4089) or its variants tagged with GFP at the C terminus. After 16 h, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 25 °C for 10 min. Fixed cells were washed with PBS and permeabilized with 100% methanol for 10 min at −20 °C. After further washing with PBS, cells were blocked with 2% BSA in PBS at 25 °C for 1 h and then incubated with Pan-Ras antibody  overnight at 4 °C. After washing twice with PBS containing 0.1% Triton X-100 (PBS-T) and once with PBS, cells were incubated with anti-mouse IgG secondary antibody Alexa Fluor 568  at 25 °C for 1 h. After washing three times, nuclei were stained with Hoechst 33342 (ThermoFisher Scientific) at 25 °C for 10 min and further washed with PBS-T. To visualize actin filaments, RRSP-transfected cells were stained with rhodamine-phalloidin  at 25 °C for 20 min. Mounting medium was applied to stained cells, and images were acquired using a Nikon laser-scanning confocal microscope (C2plus) and analyzed using NIS-Elements software.−1) in 50 mm Tris-HCl (pH 7.5) and 300 mm NaCl was subjected to far-UV CD measurements at 20 °C using a 1-mm path length quartz cuvette in a Jasco J-815 CD spectrometer . CD spectra were acquired over the wavelength range of 200–260 nm and converted into mean residue ellipticity (degree cm2 dmol−1). Blank spectra obtained using buffer without protein were subtracted.CD spectroscopy was used to compare the structural conformation of RRSP WT and its mutants. Pure protein (0.2 mg ml3930L/H4030L mutant have been deposited in the PDB (6A8J and 6A7H, respectively.The atomic coordinates and structure factor amplitudes of WT RRSP and the RRSPE the PDB  under acS. Y. J., J. H., and M. H. K. conceptualization; S. Y. J., J. H., B. S. K., and E.-Y. L. data curation; S. Y. J., J. H., B. S. K., E.-Y. L., and M. H. K. formal analysis; S. Y. J., J. H., B. S. K., E.-Y. L., B.-H. O., and M. H. K. validation; S. Y. J., J. H., B. S. K., and E.-Y. L. investigation; S. Y. J., J. H., and M. H. K. visualization; S. Y. J., J. H., and E.-Y. L. methodology; J. H. writing-original draft; B.-H. O. and M. H. K. writing-review and editing; M. H. K. supervision; M. H. K. funding acquisition; M. H. K. project administration.


Escherichia coli sequence type 131 (ST131), with its multidrug-resistance-associated H30R1 and H30Rx clonal subsets, causes most antimicrobial-resistant E. coli infections in the U.S., especially among veterans. The activity of ceftolozane-tazobactam (C/T), a new beta-lactamase inhibitor agent, against ST131 strains, and E. coli isolates from veterans, is undefined.E. coli isolates, collected in 2011 from 24 Veterans Affairs Medical Centers across the U.S. Categorical resistance and MICs were compared statistically with resistance category  and with PCR-defined ST131, H30R1, and H30Rx status.We determined broth microdilution MICs for C/T and five comparators–piperacillin-tazobactam (TZP) levofloxacin (LVX), gentamicin (GEN), ceftazidime (CAZ), and meropenem (MEM)–for 595 clinical H30 through H30R1 to H30Rx. For each comparator agent except MEM, although a significantly greater fraction of resistant than susceptible isolates were C/T-resistant, only a minority of comparator-resistant isolates were C/T-resistant .Resistance prevalence was ≤ 6% for C/T (6%) and MEM (0%), vs. from 8.0% (TZP) to 59% (LVX) for the other comparators. MICs generally increased by resistance category, from fluoroquinolone-susceptible through fluoroquinolone-resistant to ESBL, and by clonal subgroup, from non-ST131-E. coli clinical isolates from veterans, notwithstanding significant variation by resistance category and ST131-H30R1/H30Rx status, outperforming all non-carbapenem comparators. C/T should prove useful as a carbapenem-sparing therapy for multidrug-resistant E. coli ST131 infections, including in veterans.C/T was broadly active against  Escherichia coli sequence type 131 (ST131) is the single main cause of antimicrobial-resistant E. coli infections in the U.S. today, including those in veterans , weE. coli isolates from veterans. This is because the study population was deliberately enriched for FQ-R and ESBL isolates, which our results show are more likely to be C/T-resistant than are other E. coli.Regarding the observed 6% overall resistance prevalence for C/T, even this favorable statistic–which would qualify C/T for use in empirical treatment of pyelonephritis according to Infectious Diseases Society of America guidelines –doubtlesRegarding the progressive increase in categorical resistance to C/T and C/T MICs across the three resistance categories , this is consistent with the known presence within the FQ-R group of an ESBL-producing subset, with some ESBLs, possibly in conjunction with other resistance mechanisms, likely conferring resistance to C/T. Despite these trends, C/T still almost always exhibited better susceptibility statistics than did the other non-carbapenem study agents, including within the ESBL isolate group . The highest observed resistance prevalence for C/T, which was among TZP-resistant isolates, was only 38%, indicating that C/T could serve as an alternate agent for nearly two-thirds of TZP-resistant isolates, let alone for nearly all isolates resistant to the other non-carbapenem comparators.H30Rx isolates than among the non-H30 and the H30R1 isolates, which did not differ greatly from one another. By contrast, for GEN and LEV, resistance prevalence and MICs were higher among the H30R1 and the H30Rx isolates (which did not differ greatly from one another) than among the non-H30 isolates.Regarding the clonal distribution of resistance to C/T and the comparators, the observed patterns were consistent with class-specific resistance mechanisms that are known to be clonally associated , 21, 22.H30 isolates than the H30R1 isolates, as reflected in the upper limit of the range  higher C/T MICs among the non-he range . This maCTX-M-27 , 23.in vitro nature, since MICs may not correspond with clinical efficacy, its focus on veterans , the lack of attention to genetic resistance mechanisms, the uncertain current relevance of isolates from 2011, and the geographic restriction to the U.S. Study strengths include the broadly distributed (within the U.S.) strain collection, the comparisons with the clinically relevant ST131 clonal subgroups H30R1 and H30Rx, and the inclusion  of a range of relevant comparator agents.The study is limited by its E. coli isolates from U.S. veterans from 2011 we found that C/T is broadly active, albeit with significant variation in relation to co-resistance and to H30R1 and H30Rx subclone status, and outperformed all non-carabapenem comparators. Thus, C/T should prove useful as a carbapenem-sparing agent against multidrug-resistant E. coli infections in veterans, including those caused by ST131-H30R1 and H30Rx strains.In summary, within broadly representative set of clinical S1 Study Dataset(XLSX)Click here for additional data file.


Escherichia coli sequence type (ST) 131 is a worldwide pandemic pathogen and a major cause of urinary tract and bloodstream infections. The role of this pandemic lineage in multidrug resistance plasmid dissemination is still scarce. We herein performed a meta-analysis on E. coli ST131 whole-genome sequence (WGS) databases to unravel ST131 plasmidome and specifically to decipher CTX-M encoding plasmids-clade associations. We mined 880 ST131 WGS data and proved that CTX-M-27-encoding IncF[F1:A2:B20] (Group1) plasmids are strictly found in clade C1, whereas CTX-M-15-encoding IncF[F2:A1:B-] (Group2) plasmids exist only in clade C2 suggesting strong plasmid-clade adaptations. Specific Col-like replicons , and Col8282) were also found to be clade C1-associated. BLAST-based search revealed that Group1 and Group2 plasmids are narrow-host-range and restricted to E.coli. Among a collection of 20 newly sequenced Israeli ST131 CTX-M-encoding plasmids (2003–2016), Group1 and Group2 plasmids were dominant and associated with the expected clades. We found, for the first time in ST131, a CTX-M-15-encoding phage-like plasmid group (Group3) and followed its spread in the WGS data. This study offers a comprehensive way to decipher plasmid-bacterium associations and demonstrates that the CTX-M-encoding ST131 Group1 and Group2 plasmids are clade-restricted and presumably less transmissible, potentially contributing to ST131 clonal superiority.Extraintestinal multidrug resistant  Escherichia coli ST131 is a high-risk extraintestinal pathogenic E. coli (ExPEC) lineage that has spread explosively throughout the world4, mostly by patient-to-patient spread and contaminated food5. This lineage may colonize healthy humans and animals6 and is the causative pathogen of urinary tract and bloodstream infections9. E. coli ST131 nowadays is recognized as the major E. coli lineage responsible for the spread of multidrug resistance (MDR)3 and in specific CTX-M ESBL genes10.Multidrug resistant ESBL-producing fimH30 allele - H30R/C1 and H30Rx/C2, where the later harbors blaCTX-M-15 ESBL gene13. Plasmid-bacterium associations in ST131 clades connected CTX-M-15-encoding IncF[F2:A1:B-] (FAB formula) plasmids to clade C215, and non-CTX-M-15-encoding IncF[F1:A2:B20] plasmids to clade C111. The major CTX-M genes described in clade С1 were CTX-M-14 and −27 alleles of the CTX-M-9 group16, with this later allele- C1-M2716, recognized recently as a new subclade with global occurrence18.The global expansion of this lineage is driven by spread of two major fluoroquinolone-resistance clades which possess 20. However, if there are highly adaptive plasmid-bacterium associations within ST131 lineage, the main driving force for MDR spread related to this pandemic clone is due to clonal expansion rather than to horizontal plasmid spread15. The existence of successful plasmid-ST131 lineage associations raises a question on the ability of this clone to serve as a reservoir for inter- and intra-species resistance spread. In spite several comprehensive studies on ST131 CTX-M-encoding plasmids, some questions are still understudied, including how frequently clade-associated plasmids are found in the respected clades? Are there plasmid horizontal transmission events between clades? What is the distribution of CTX-M genes across ST131 clades, and if there are correlations between specific clades, plasmid replicon types, and CTX-M gene alleles? Does ST131 serve as a MDR reservoir by transferring its most widespread plasmids to other bacterial species?MDR often disseminates in bacteria through horizontal gene transfer of mobile genetic elements, especially plasmid transmissionTo address these questions, we explored ST131 plasmidome by performing a meta-analysis encompassing all ST131 whole genome sequencing data  from the NCBI Sequence Read Archive (SRA). We assessed correlations between ST131 clades, plasmids types and CTX-M gene alleles. We focused on the high prevalent Group1 and Group2 plasmids, Group1 are clade C1-associated-CTX-M-27 IncF[F1:A2:B20] plasmids, and Group2 are clade C2-associated-CTX-M-15-encoding IncF[F2:A1:B-] plasmids.E. coli ST131 plasmidome; it improves our understanding of plasmid-associated traits within this lineage and sheds light on the ability of this pandemic clone to serve as a source for MDR plasmids.In addition, we sequenced a collection of Israeli ST131 plasmids (n = 20) encoding CTX-M-15, CTX-M-14 and CTX-M-27 alleles, studied their diversity and verified plasmid-clade associations observed in the global dataset. We found a new CTX-M-15 phage-like plasmid group and screened the SRA collection for it. This study reveals insights on E. coli ST131 lineage, we retrieved ST131 WGS data from the NCBI Sequence Read Archive . We analyzed in silico each ST131 isolate for various bacterial host and plasmid features including the clade type, fimH and CTX-M alleles, plasmids’ replicon types and specifically IncF replicons of Group1  and Group2 plasmids . The plasmidome of ST131 was highly diverse and revealed multiple combinations of plasmid types, which we present on a plasmid family-basis, i.e. combining IncFIA, IncFIB, IncFIC, and IncFII specific replicons and all their type into the IncF family , whereas Group2 plasmid carriers were from clade C2 (n = 103), almost all possessing fimH30 . For simplicity, possible Group1 and Group2 plasmid carriers will be further referred as ‘SRA Group1’ and ‘SRA Group2’ isolates, respectively.All SRA ST131 isolates that we positively filtered as possibly harboring Group1 plasmids, strictly belonged to clade C1 Searching for Group1 and Group2 replicons only independent with the CTX-M alleles revealed their presence in 245 and in 164 SRA isolates, respectively , Group1 replicon could be rarely found in clade A and Group2 replicon in Clade B as already been observed in the SRA collection (Supplementary Table E. coli only (Supplementary Table Mining the Nucleotide collection (nt/nr) using the target replicons and CTX-M genes returned 20 plasmids with Group1 replicon . Co-existence of these two replicons on the same plasmid partially explains the high correlation rate of these two traits that we have observed in the SRA analysis  from clade C1, and Group2 - plasmid uk_P46212  from clade C2. BLAST-based comparison of plasmids from the NCBI Nucleotide database showed high similarity of sequences within groups and proved that the reference plasmids may serve as representative sequences for these groups: the average coverage of pEC-81009 by Group1 plasmids was 93.4%; and 95.7% for uk_P46212 covered by Group2 plasmids  covered Group1 reference plasmid to a higher breadth compared to Group2 reference , and reads of Group2 plasmids (n = 103) to the Group2 reference  that colonized a dog and harbored Group1 plasmid  and uk_P46212 with Group2 plasmids . This is the first report on Group 3 plasmids in ST131 background, found in clades C2 and A. To determine the distribution of Group3 plasmids in ST131 population, ST131 CTX-M-15-encoding SRA isolates (n = 252) were mapped to Group3 reference, pECOH89. We found 18 isolates covering more than a half of the reference sequence, the coverage range was 83.2–94.3%. These isolates belonged to C1  and C2  clades.Out of the 20 sequenced plasmids, two nearly identical phage-like plasmids formed a new group, Group3. They shared 100% coverage and 99% identity with pECOH89  and one CTX-M-14-encoding plasmid (p56), were novel and each belonged to a distinct plasmid group Fig. .ccdAB, pemIK and sok/hok combined with doc/phd in Group1 plasmids and vagCD in Group2 plasmids. The Israeli plasmids varied significantly in the number of conjugation transfer genes  consisted of a plasmid backbone and an accessory region Fig. . The maj11, the colicin ColIa immunity-encoding genes cjrABC, the enterotoxin-encoding senB gene, the copper-sensitivity suppressor scsCD, and merR, the mercury resistance regulator. Group2 plasmids encoded ARGs (five to 10 genes) and the iron transport system mentioned above.In the accessory region, Group1 plasmids encoded antibiotic resistance genes (ARGs) cassettes (1–10 genes), and possessed diverse virulence-associated traits including an iron transport system “IncF island 1” described previouslyGroup 3 plasmids consisted of phage-related and hypothetical proteins. These plasmids lacked conjugation region and stability systems. However, the appearance of this group in ST131 isolates of clades A and C suggests their possible horizontal transfer.Hypothetical and uncharacterized proteins (i.e. not-present or uncharacterized genes in UniProtKB/Swiss-Prot database) encoded by our sequenced plasmids, comprise around 15–18% of length of IncF plasmids, and 40% of length of Group3 phage-like plasmids. These DNA regions are interesting to explore, and require the development of computational tools and molecular studies in order to predict their potential role, which may reveal their contribution to ST131 lineage.The phylogenetic tree of grouped plasmids Fig.  was baseThis study presents the first meta-analysis performed on SRA ST131 WGS data in order to explore ST131 plasmidome. We mined 880 SRA isolates to reveal correlations between plasmid and ST131 chromosomal features. We limited our analysis to several widespread CTX-M-carrying plasmid groups; however, the same approach could be applied to identify the distribution of any bacterial genetic markers.16. IncF plasmids are characteristic for ST131, and the evolution of this lineage has been described in parallel to IncF plasmids arrivals and loss4.ST131 plasmidome consisted mostly of IncF and Col-like plasmids, each presented in more than 90% of the studied SRA isolates. Less common plasmid families were found in 39% of the isolates, usually accompanied by the high-prevalent IncF and Col-like plasmids. One of our consistent findings was a clear segregation between specific IncF plasmid groups and ST131 clades. We found that: (i) Group1 plasmid features  were restricted to ST131 clade C1 (with one exception in clade A); (ii) Group2 plasmid specific traits  were restricted to clade C2 isolates exclusively. This study provides an extensive support to previously described combinations of ST131 clades and IncF plasmidsE. coli ST131 superiority have yet to be studied. In E. coli K12, a non-ST131 E. coli background, the acquisition of IncF Group2 plasmid resulted in minor chromosomal modifications and fitness cost diminished after several generations, while acquisition of IncC-IncR plasmid lead to major genomic rearrangements with consistent high fitness cost22. In K. pneumoniae, adaption to IncF pKpQIL-like plasmids was associated mostly with changes in gene expression levels, together with variations in other physiological features such as conjugation rates, competitiveness, or biofilm formation levels23. In another study that looked on the effect of CTX-M-15- and CTX-M-14-encoding plasmids on non-ESBL-producing ST131 strains, bacterial growth rates were not affected, whereas biofilm formation and serum resistance decreased after plasmid acquisition24. Unfortunately, the clades used in that study were not defined. To extend our understanding on plasmid-clade adaptation processes, comprehensive comparative studies on ST131 clades C1 and C2 transformed with Group1 and Group2 plasmids are required.The contribution of Groups 1 and 2 IncF plasmids on 26, and a wide distribution of Col-like plasmids was shown in the recently emerging clone E. coli ST119327. However, further study is required to identify E. coli ST131 isolates carrying small Col156 plasmids, as well as to accurately distinguish them from isolates that co-encode Col-like replicons with other replicon groups. Less frequent plasmid families were identified in different ST131 clades. A major caveat of performing a meta-analysis that is based on Illumina WGS data is the problem of sorting chromosomal and plasmid reads. Thus, we were not able to divide SRA ST131 isolates into isolates that harbor multiple plasmids or one plasmid co-encoding fused replicons. This problem highlights the necessity of increasing the collection of available complete sequenced plasmids.In contrast to the well-described IncF plasmids, the role of small Col-like plasmids in ST131 is still unknown in spite of their wide occurrence in this lineage. Examination on the most widespread Col replicons revealed correlations between Col156, Col(MG828), and Col8282 plasmids and clade C1, however all these mentioned replicons were found in both clades C1 and C2 co-existing with the strictly divided IncF Group1 and Group2 plasmids. Col156 plasmids were recognized previously as important ARGs transmittersE. coli ST34928, and in this meta-study, we found Group3 plasmids only in 18 ST131 SRA isolates carried in clades C1 and C2. These plasmids appear to be rare in SRA isolates compared to Group1 (n = 80) and Group2 (n = 103) SRA isolates, which highlights the clade-restricted nature of specific IncF plasmids.Data from this study expanded the global collection of CTX-M-encoding ST131 plasmids by 20 new complete sequences from diverse sources in Israel. Our clinical ST131 isolates belonged to clades C1 and C2 and harbored mostly Group1 and Group2 plasmids. However, we identified several plasmids with unique replicon types. Some of these plasmids showed high sequence homology to the grouped plasmids, like pB14 (IncF[F1:A2:B-]) and pU60 (IncF[F-:A1:B-]), possibly derivatives of Groups 1 and 2, respectively. A comparison of the genetic modifications and the host features of plasmids diverged from the globally widespread plasmid groups will improve our understanding on plasmid evolution, host range, and plasmid-host adaptation. Our CTX-M-encoding ST131 plasmid collection revealed a complicated epidemiology of ST131 in Israel. We found Group2 plasmids in clade C2 isolates; the phylogenetic analysis we performed divided Group2 plasmids into two subgroups in agreement with ST131 virotypes. In addition, we identified a new phage-like plasmid group (Group3), described herein for the first time in ST131. In our collection, Group3 plasmids were found in ST131 clades A and C2. This Group3 plasmid was originally described in 29. Based on the involvement of active conjugation machinery on bacterial fitness cost30, we speculate that plasmids that are associated with a specific ST131 clade that spread vertically may result in the reduction of tra genes. Nevertheless, the translational consequences of this reduction on fitness cost, plasmid transferability, and conjugation rates should be further elucidated experimentally.In our newly sequenced plasmids, we noticed conjugation gene loss in both Group1 and Group2 plasmids, where the number of known conjugation genes dropped significantly (from 33 to seven genes with some intermediate numbers in Group1 plasmids and from 36 to five genes for Group2 plasmids). Conjugation genes loss was previously described in Group2 plasmid pEK95832 however, further research should be performed to extend our knowledge.Interestingly, we demonstrated that Group1 and Group2 plasmids may possibly be found in other ST131 clades as in the case of Group1 plasmid p13.1 isolated from ST131 clade A colonizing a dog. Our collection of ST131 clade A isolates was limited to only two samples, thus the plasmidome richness of rare ST131 clades is still unknown. Systematical studies have started to describe the epidemiology of ST131 from animal and environmental reservoirsE. coli ST131 or in other lineages and species. Although the entire ST131 plasmid network is not fully resolved, the combination of clinically important CTX-M-encoding MDR IncF plasmids within specific ST131 clades became conclusive. IncF plasmids could be ‘locked’ within their clades, however they still may exchange ARGs transmitted by IS elements33, which keeps ST131 as a threatening MDR reservoir.In conclusion, we showed that ST131 plasmidome is shaped by clade-restricted plasmids. Available WGS databases serve as a challenging source to explore plasmid networks and spread patterns within https://blast.ncbi.nlm.nih.gov/Blast.cgi). Then, sequences with full-length and 100% identical target regions were added to this study.Complete sequence plasmids of Group1 and Group2 were retrieved from the NCBI Nucleotide collection (nr/nt). The concatenated target replicons and CTX-M genes , separated by gaps of 50 Ns were submitted for the web BLASTn search  were loaded excluding three samples that appeared also in the Nucleotide collection with identical BioSample numbers. The accurate identification of E. coli sequence types, ST131 clades, fimH-alleles, together with IncF replicon types, and the CTX-M alleles was performed using two complementing approaches: (I) reads were trimmed (Trimmomatic-0.36), assembled (SPAdes-v3.10.1) and then a complete target sequence was found by local BLASTn-based analysis, and/or (II) trimmed reads were mapped (bwa 0.7.17-r1188) to the reference genomes and analyzed using samtools-1.7 and bcftools-1.8, checking for 100% coverage and a total absence of SNPs in the area of interest. Samples were identified as ‘ST131’ if all seven Achtman MLST genes and pabB from Pasteur scheme were identical to ST131 alleles. Clade-specific regions were cropped from ST131 isolates - clade C0 CD306, clade C1 EC81009, clade C2 JJ1886 and EC958  from the NCBI Sequence Read Archive (SRA) were searched for “E. coli ST131”. 37 found ≥80% identity with a plasmid replicon from the CGE PlasmidFinder 2.0 Enterobacteriaceae collection35. Virotypes were identified based on the presence or absence of four genes included in the ST131 virotyping scheme38 and virulence genes were searched using BLASTn-based analysis and KMA-1.1.7. Reference plasmid coverage was identified by mapping SRA reads (bwa 0.7.17-r1188) followed by samtools depth analysis.Plasmid families were identified based on methods described above or by read mapping using KMA-1.1.7P-values of coefficients were corrected in a P-value multiple test based on the step-down method using Bonferroni adjustments . Principal-component analysis transformed characteristic binary data into contributing loading vectors placing SRA isolates on the scatter plot.In an attempt to find plasmid-bacterium associations, we constructed a binary table of ST131 isolates characteristics that describes the presence or absence of each studied allele. The association coefficient between each pair of ST131 characteristics was determined using phi coefficient . P-values of < 0.001 were considered significant. All statistical analyses were performed using Python modules.The samples of coverage results of grouped SRA isolates and the two reference plasmids were proved not to be normally distributed by the Shapiro-Wilk and Anderson-Darling tests; thus sample means were compared using the two-tailed Mann-Whitney test. E. coli ST131 isolated in Israel (2003–2016) were included in this study . E. coli ST131 clades were determined by Matsumura’s scheme34. For clades C1 and C2, presence of O25b serotype42 and fimH30 allele43 were additionally verified. For non-fimH30 ST131 isolates, fimH gene was amplified and sequenced . A unique presence of fimH30:Rx SNP was also proved for E. coli ST131 clade C244. Virotypes were identified by multiplex PCR38.Selection of the 20 CTX-M-encoding E. coli DH10B, and resulted transformants were selected on LB plates supplemented with 100 μg/mL ampicillin. CTX-M-encoding plasmids were isolated from transformed E. coli DH10B and checked for the presence of accompanying plasmids from the clinical strains by gel-electrophoresis and plasmid incompatibility group typing45. To ascertain the purity of blaCTX-M-encoding plasmid for sequencing, additional transformations were carried out until purity was confirmed. The resulted CTX-M-encoding plasmids were cleaned from residual chromosomal DNA using Plasmid-Safe ATP-dependent DNase . Antibiotic susceptibility testing of all 20 E. coli DH10B transformants carrying a single CTX-M-encoding plasmid was performed using Vitek-2 .CTX-M-encoding plasmids were purified from the 20 ST131 isolates using Plasmid Midi Kit . Plasmids were individually transformed by electroporation  into freshly prepared electro-competent 46. Contamination of E. coli DH10B chromosomal DNA was filtered out using bowtie2–2.3.2 and samtools-1.7, if necessary. Where it improved assembly statistics, read coverage was normalized to 50x using BBMap/BBTools-36.20. Scaffolds were generated by SPAdes-3.10.1 assembling Illumina long paired reads47.Plasmids were sequenced by Illumina HiSeq 2500  at the Technion Genome Center  using a 2 × 250 bp paired-end libraries with an estimated fragment size of 800 bp. Adapter sequences and low-quality reads were removed using Trimmomatic-0.3648. Plasmids were sequenced using standard protocol SQK-RAD004 v. RSE_9046_v1_revB_17Nov2017 according to the manufacturer’s instructions. Libraries were prepared using the SQK-RAD004 ONT Rapid sequencing kit and loaded onto the MinION flow cell FLO-MIN106 (R9.4 SpotON). Adapter sequences were trimmed using Porechop (v0.2.3) and low quality reads were removed using NanoFilt (v.2.20). The hybrid read sets (both Illumina and Nanopore reads) were assembled using Unicycler (v0.4.0)49. SPAdes scaffolds of other plasmids were ordered using a home-made Python script according to the MinION sequenced reference or the GenBank highly related plasmid. Plasmid sequences were annotated with the RAST tool50. Predicted proteins were verified and updated using UniProtKB/Swiss-Prot database. ARGs and their transport cassettes were retrieved using ResFinder-2.136, supported by CARD51 and ISfinder52. Virulence genes were identified using VirulenceFinder-1.253 and VFDB54. Replicon types were identified using pMLST-1.4 and PlasmidFinder-1.235. Plasmid maps were generated using Easyfig-2.2.355.Four ST131 plasmids  were additionally sequenced using Nanopore technology  to complete plasmid assemblyThe complete sequences of 20 plasmids have been deposited at the GenBank under project number PRJNA494636  v458.Phylogenetic analyses of complete sequence plasmids (n = 40) were performed based on SNPs alignments generated by kSNP3.0Supplementary file.


The growth hormone/insulin-like growth factor 1 (GH/IGF-1) axis has recently been recognized as an important factor related to the longevity of many organisms. MicroRNAs (miRNAs or miRs) could also participate in diverse biological processes. However, the role of miRNAs in the decline of pituitary GH during the growth process remains unclear. To better characterize the effects of miRNAs on the pituitary, we used a miRNA microarray to investigate the miRNA profile in the rat pituitary from postnatal development throughout the growth process. Then, in vitro experiments were conducted to analyze the miRNAs’ potential roles related to GH regulation. Taken together, the microarray results indicated that there were 22 miRNAs differentially expressed during pituitary development. The bioinformatics analysis suggested that the most differentially expressed miRNAs may participate in multiple pathways associated with the pituitary function. Furthermore, the in vitro findings demonstrated that miR-141-3p was involved in GH regulation. The pituitary is a small but complex gland situated beneath the hypothalamus in the sella turcica. It is the “master” endocrine organ that functions as a relay between the hypothalamus and peripheral target organs via five major cell types, including corticotropes, thyrotropes, gonadotropes, somatotropes, and lactotropes ,2. SomatGH is one of the major pituitary hormones and the primary regulator of organism growth and metabolism ,4,5. MorAging is a natural and inexorable biological process. The changes accompanying aging manifest on both molecular and organismic levels . For exaGH expression [MicroRNAs are small noncoding RNAs that play important roles in post-transcriptional gene regulation . In animpression . miR-375pression . Furtherpression , liver [pression , muscle pression , and brapression  during tInvestigators have identified the differential expression of several miRNAs in various developmental stages in the porcine pituitary, have predicted potential target genes, and defined their biological roles using a gene ontology (GO) analysis . HoweverGH1, growth hormone-releasing hormone receptor (GHRHR), and somatostatin receptor 2 (SSTR2) mRNA expression were all markedly age-dependent. Body weight was the lightest on Day 7, increased rapidly until Day 90, and was followed by a gradual reduction in the increasing ratio  with the results of the microarray. Although the expression of miR-384-3p seemed to be inconsistent on Day 250, it had the same uptrend with the microarray.To verify and evaluate the reliability of the results obtained from the microarray analysis, we selected 15 miRNAs to be tested using a real time quantitative polymerase chain reaction (qRT-PCR) assay . These mIGF1R, miR-141-3p/suppressor of cytokine signaling 7(SOCS7), miR-141-3p/ G protein subunit alpha 13 (GNA13), miR-141-3p/MAPK1, miR-141-3p/sex determining region Y (SPY)-box 5 (SOX5), miR-141-3p/ aspartate-beta-hydroxylase (ASPH), miR-183-5p/MAPK9, and miR-29a-3p/GNA13. Then, TargetScan was used to analyze the conservation of the target relationship among species  and pmirGLO-SOCS7-3′UTR-WT. The luciferase activity in the construct of mutations and the deletion in the 3′UTR of SOCS7 did not show any change.The information gathered from IPA allowed us to identify 19 candidate genes b which c species b–e, and er assay f. The reGH1 mRNA  simultaneously via a miRNA microarray. In summary, we validated and confirmed 22 differentially expressed miRNAs in the rat pituitary. In addition, most of the altered miRNAs were identified to participate in signaling pathways associated with pituitary functionality. There were 15 miRNAs that were predicted to target 19 candidate genes by IPA. To determine the actual positive and functional miRNA/target pairs, the mRNA expression of the miRNAs and genes was detected by qRT-PCR and the correlation coefficient was calculated. The miR-141-3p/SOCS7, miR-141-3p/MAPK1, miR-141-3p/SOX5, and miR-29a-3p/GNA13 pairs were all negatively correlated. Finally, we chose miR-141-3p for further study and preliminarily found that miR-141-3p inhibits pituitary GH expression. Although the miRNA profile had been analyzed in the pituitary of several species [In the present study, we conducted an analysis of rat growth from birth through adolescence to adulthood by measuring the body weight and serum IGF-1 levels as well as detecting the expression of genes related to growth, including  species ,31,32, nSprague-Dawley (SD) male rat growth chart on the Taconic Biosciences website, excepting that the plateau that occurs by Day 90 is a little early. The other three ages reflected different typical stages of rat growth. Day 7 is the neonatal period, Day 40 is the start time of a rapid growth period, and Day 250 is in a platform of growth associated with a stable weight. Therefore, investigations of miRNA expression patterns in the three time points may provide useful information about the miRNA temporal expressions in the pituitary. It has been reported that GH levels are extremely elevated in immature mammals and decline with age [GH1 mRNA levels with age was primarily consistent with previous reports except the data on Day 90. Iruthayanathan et al. [SSTR2 mRNA increased markedly and progressively with advancing age after birth. The study also detected a similar changing profile related to the mRNA of SSTR2. Korytko et al. [GHRHR gene expression and found that the GHRHR mRNA levels increased during the first 30 days of life and then declined with age. A consistent result that the expression of GHRHR mRNA increased from Day 7 to Day 40 was revealed. However, again, the level of GHRHR mRNA on Day 90 was low too early [The growth curve of the rats in this study reflected a general growth rate that parallels the results of the with age ,34. In on et al.  found thn et al. ,37,38. Gn et al.  analyzedoo early ,40. Due Recent reports have suggested that miRNAs might play a role in the aging process. For example, the expression of the miRNA lin-4 decreased with advancing age, whereas the overexpression of lin-4 extended the lifespan of Caenorhabditis elegans . Maes etSOCS7, MAPK1, and SOX5 were predicted targets of miR-141-3p. We found miR-141-3p negatively regulates GH. Since GH is not the direct target of miR-141-3p, it is most likely that miR-141-3p regulates GH1 expression via other targets. The relations between miR-141-3p and its target genes as well as their roles in GH regulation need to be verified in subsequent experiments. Furthermore, we identified no differentially expressed miRNAs directly targeting GH1. At present, there still no validation for miRNAs targeting GH1. Actually, we also used the whole set of miRNA expression profile data to predict miRNAs targeting GH1 by a miRanda analysis, and miR-543-5p, miR-199a-5p, and a novel miRNA Q62 were predicted to target GH1.Identifying the specific target genes of miRNAs is a critical step to investigate miRNA functionality ,48. IPA In conclusion, this study surveyed the miRNA expression in the pituitaries of rats throughout the entire lifespan using a microarray and provided novel insight into the alteration of miRNA expression during both development and aging. We found that 22 miRNAs were differentially expressed in the pituitary with age, some of which were predicted to be involved in the signaling pathways related to pituitary function. Moreover, we analyzed pairs of miRNAs/target genes. Finally, in vitro tests revealed that miR-141-3p inhibited GH expression in the pituitary. Taken together, our results shed new light on miRNA expression and GH regulation in the pituitary and indicate that altered miRNAs are potential meaningful biomarkers for studying the pituitary during postnatal development.Sprague-Dawley (SD) rats were purchased from the Guangdong Medical Laboratory Animal Center . The experimental animals were categorized into four different groups according their age: 7-day-old newborn rats , 40-day-old adolescent rats , 90-day-old young adult rats , and 250-day-old adult rats . All animal procedures were conducted under the protocol  approved by Institutional Animal Care and Use Committee (IACUC) of South China Agricultural University. All rats were weighed and then were anesthetized with diethyl ether and killed via a rapid decapitation. The blood was collected from the jugular vein in a 2 mL polyethylene tube and centrifuged at 3000 rpm for 20 min at 4 °C. After centrifugation, the serum was stored at −80 °C for preservation. The pituitaries were removed and stored at −80 °C until processing. The growth curve of the rats was determined by measuring the body weight.Specific pathogen-free male IGF-1 was extracted from serum to eliminate IGF-1 binding proteins (IGFBPs) following the procedure reported by Niu et al. . Briefly® miRNA cDNA Synthesis Kit  following the manufacturer’s protocol, respectively. The cDNA was diluted 5-fold with ddH2O. Quantitative real-time PCR was performed using STRATAGENE Mx3005P sequence detection system  as follows: denaturation (95 °C/10 min), PCR amplification and quantification  with a single fluorescence measurement at the end of the elongation step, repeated for 40 cycles. Each reaction contained 2 μL of cDNA, 0.3 μmol/L of each primer, 10 µL of 2× FastStart Universal SYBR Green Master (Rox) , and water to make up the final volume of 20 μL. The primers used for the miRNAs, genes, U6, and β-actin are listed in The total RNA from the pituitary was extracted using TRIzol reagent  following the manufacturer’s instructions. The quality of the RNA was monitored by spectrophotometry , and the 28 S and 18 S were examined by electrophoresis. DNase I  was used to remove DNA contamination. Reverse transcription of mRNAs and miRNAs was performed with poly-T primers using the M-MLV reverse transcriptase system  and One Step PrimeScripthttp://rana.lbl.gov/EisenSoftware.htm) [According to the results of the growth curve, serum IGF-1 level, and pituitary mRNAs expression, three  of the four groups were selected for the chip test. A total of 827 miRNAs, including 727 rat miRNAs from miRBase 20.0 and 100 randomly designed novel candidates, which refer to sequences homologous to human or mouse miRNAs, were used . For eacare.htm) .http://www.ingenuity.com/). The IPA tool has a miRNA target filter function which relies on experimentally validated interactions from the TarBase database [http://mirecords.biolead.org/), TargetScan (available online http://www.targetscan.org/), and manually curated miRNA-related findings within the IPA knowledgebase. IPA also generates the relevant diseases, location, molecule type, pathway, species, and tissue/cell line where the predicted miRNA targets are found. As mRNAs cannot be affected by miRNAs that are not co-expressed with them [Identification of the putative target genes for all of the differentially expressed miRNAs was performed using the miRNA Target Filter in IPA  and Rattus norvegicus miR-141-3p inhibitor (5′-CCAUCUUUACCAGACAGUGUUA-3′) were commercially synthesized from Shanghai GenePharma . The stable negative control (NC)  and inhibitor NC (iNC) (5′-CAGUACUUUUGUGUAGUACAA-3′) were used as negative controls. One day before transfection, the cells were seeded onto Poly-l-Lysine (PLL)-coated six-well plates at 2 × 106 cells/well in the absence of antibiotics. For transfection, the cells were grown to 50–80% confluence in the culture plates. The cells were then transfected with 100 pmol/well oligonucleotides by Lipofectamine 2000  according to the manufacturer’s protocol. At 6 h post-transfection, the cells were washed once with 1× PBS, cultured in complete culture medium, and grown for 48 h before collection. The miR-141-3p mimic, inhibitor, and their respective negative controls, NC and iNC, were obtained from Shanghai GenePharma .The rat pituitary cell line GH3, obtained from the American Type Culture Collection (ATCC), was maintained in F-12K Medium containing 15% horse serum and 2.5% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% COg centrifugation at 4 °C for 15 min. Supernatant protein concentrations were determined using a BCA Protein Assay Reagent Kit . After boiling for 5 min, the samples (30 μg each) were subjected to 5–10% SDS-polyacrylamide gel electrophoresis (PAGE) at 80 V for 20 min and 100 V for 70 min using Tris-glycine running buffer. The separated proteins were transferred onto PVDF membranes  by electroblotting in a transfer buffer . The membranes were blocked in 5% BSA in TBST buffer  for 2 h at room temperature and then incubated overnight at 4 °C with primary antibodies. The next day, the membranes were washed six times for 5 min each with TBST buffer followed by incubation with horseradish-peroxidase-labeled secondary antibodies for 1 h at room temperature. Anti-GH (MAB1566) were purchased from R&D Systems . Anti-β-actin(bsm-33139M), anti-rabbit IgG(bs-0295G), and anti-mouse IgG(bs-0296G) were obtained from Bioss . Immunoreactivity was detected using the ECL Plus chemiluminescence detection kit  in a FluorChem M system . The density of the bands was analyzed using the Image JSoftware .GH3 cells were lysed in RIPA buffer  containing 1 mmol/L protease inhibitor PMSF  followed by 12,000× SOCS7. The 3′UTR of SOCS7 contains the highly conserved binding sites of miR-141-3p, and the sequence containing the binding sites (113 bp) is as follows, the bold and underline sequence represents the binding sites.The Dual-Luciferase reporter genes were constructed using the pmirGLO Vector  and the 3′UTR sequence of rat 5′-TCGAGCATCAACACAGCTGTCCTTACACTAAATCTGTGCTATTGCATACACAGTGTTTATCAGTAGTTCAAAATGATTTATTTGCTT-3′.TGTAGCCATCTTTCTTTTCACTGCAG-SOCS7-3′UTR-WT. Meanwhile, a plasmid containing the mutant SOCS7 3′UTR, pmirGLO-SOCS7-3′UTR-MUT, was generated by mutating the core sequence of the miR-141-3p binding sites through DNA synthesis , and the sequence is as follows, the bold and underline sequence represents the mutational sites.Furthermore, the 3′UTR sequence was inserted into the pmirGLO Vector with XhoI and XbaI double digestion to construct the recombinant dual-luciferase reporter vector pmirGLO5′-TCGAGCATCAACACAGCTGTCCTTACACTAAATCTGTGCTATTGCATACAACAGAGGTATCAGTAGTTCAAAATGATTTATTTGCTT-3′.TGTAGCCATCTTTCTTTTCACTGCAGSOCS7 3′UTR, pmirGLO-SOCS7-3′UTR-DEL, was generated by deleting the core sequence of miR-141-3p binding sites through DNA synthesis, and the sequence is as follows:Similarly, a plasmid containing the deleted 5′-TCGAGCATCAACACAGCTGTCCTTACACTAAATCTGTGCTATTGCATACATGTAGCCATCTTTCTTTTCACTGCAGTATCAGTAGTTCAAAATGATTTATTTGCTT-3′.4 per well) were plated in a 96-well plate. At 60–70% confluency, cells were transfected with 3 pmol miR-141-3p mimics/NC and 100 ng pmirGLO-SOCS7-3′UTR-WT/pmirGLO-SOCS7-3′UTR-MUT/pmirGLO-SOCS7-3′UTR-DEL. Cells were collected 48 h after transfection, and luciferase activity was measured with the Dual-GLO luciferase reporter assay system  according to the manufacturer’s guideline.Hela cells , known as the product-moment coefficient of correlation, was determined for each pair of miRNAs and the corresponding predicted target gene using Microsoft Excel [The statistical analysis was performed using SPSS Statistics Software. A Student’s ariables . A Pearsft Excel ,59. The