Vancomycin-resistant enterococci (VRE) are a serious threat to human health, with few treatment options being available. New therapeutics are urgently needed to relieve the health and economic burdens presented by VRE. A potential target for new therapeutics is the VanRS two-component system, which regulates the expression of vancomycin resistance in VRE. VanS is a sensor histidine kinase that detects vancomycin and in turn activates VanR; VanR is a response regulator that, when activated, directs expression of vancomycin-resistance genes. This review of VanRS examines how the expression of vancomycin resistance is regulated, and provides an update on one of the field’s most pressing questions: How does VanS sense vancomycin? Amycolatopsis orientalis and soon emerged as a promising new treatment for infections caused by penicillin-resistant staphylococci and other Gram-positive bacteria [S. aureus prompted its use as an antibiotic of last resort [In the early 1950s, the glycopeptide vancomycin was isolated from bacteria . Early sbacteria ,4. Thus,bacteria . Impuritbacteria . At the t resort ,11,12. Vt resort ,14. ThisE. faecium is listed among the so-called ESKAPE pathogens  [VRE infection was first identified as an emerging clinical problem in the late 1980s, nearly 30 years after vancomycin made its debut ,16. Todaer spp.) , and theer spp.) . VRE lever spp.) ,23,24. Ad-alanyl-d-alanine (d-Ala-d-Ala) residues of the muramyl pentapeptide portion of lipid II, a precursor in peptidoglycan synthesis  or d-alanyl-d-serine (d-Ala-d-Ser), thereby reducing the affinity of vancomycin for its ligand [d-lactate or d-serine to d-alanine (the naming convention for these ligases is described in d-Ala-d-Ala dipeptidase (VanX or VanXY). VanH and VanT convert pyruvate to d-lactate and L-serine to d-serine, respectively [d-Ala by the appropriate ligase to form d-Ala-d-Lac or d-Ala-d-Ser [d-Lac/d-Ser)-UDP-MurNAc pentapeptide is incorporated into lipid II and displayed on the exterior of the cell, effectively eliminating the cell’s vulnerability to vancomycin. d-Ala-d-Ala dipeptides produced by the normal cell-wall biosynthetic machinery are cleaved by VanX/VanXY, preventing them from being included in the nascent peptidoglycan chain [Resistant enterococci have acquired a suite of resistance genes. Several of the associated gene products work together to alter the  binding . In diffs ligand ,35,36,37ectively ,39,40, wla-d-Ser ,51,52,53la-d-Ser . The resan chain ,56,57. Man chain ,63,64,65VRE isolates are assigned to one of nine types, which are genotypically and phenotypically distinct. These types are denoted by the letters A–E, G, L, M, and N, and their characteristics are summarized in E. faecalis or E. faecium, with the latter being the more prevalent species [E. faecium are responsible for the majority of nosocomial VRE infections and are particularly difficult to treat due to their resistance to all commonly-used glycopeptide antibiotics [Most VRE isolated from human infection sites are  species ,81. A-tyibiotics . B- and ibiotics ,105,106.d-Ala-d-Lac or d-Ala-d-Ser to achieve vancomycin resistance. VRE types A, B, D, and M fall into the former group, and types C, E, G, L, and N into the latter. The use of d-Ala-d-Lac versus d-Ala-d-Ser controls the level of resistance observed. d-Ala-d-Lac is bound 1000-times less tightly by vancomycin than d-Ala-d-Ala [d-Ala-d-Lac are resistant to high concentrations of vancomycin [d-Ala-d-Lac is also bound more weakly by teicoplanin, explaining why VRE belonging to types A, D, and M are also teicoplanin-resistant [The VRE types can be categorized based on whether they use la-d-Ala ,74,107, ncomycin ,109,110.esistant ,87,108. d-Ala-d-Ser is also bound less tightly by vancomycin and teicoplanin than d-Ala-d-Ala, but the difference in affinity is less dramatic than is seen for d-Ala-d-Lac, with the remodeled precursors being bound ~3- to 8-fold less tightly [d-Ala-d-Ser  exhibit only low-to-moderate levels of resistance to vancomycin and teicoplanin [ tightly . Consistcoplanin ,111,112.d-Ala-d-Lac or d-Ala-d-Ser are only incorporated into the cell wall when vancomycin is present; in the absence of the antibiotic, d-Ala-d-Ala is used [Expression of vancomycin resistance genes can be inducible or constitutive. VRE exhibiting inducible expression include types A, B, C, E, G, L, and M. For these organisms, precursors containing  is used ,109,113. is used ,112,114.d-Ala-d-Lac or d-Ala-d-Ser ligase gene has been used to classify different VRE types [vanA, that of B-type vanB, and so on. VRE isolates can be typed based on van ligase sequence using a variety of PCR techniques [van genes differ sufficiently to warrant subtyping. For example, C-type VRE are subdivided into C1-, C2/3-, and C4-types [Historically, the sequence of the RE types ,52,53,93chniques ,116,117.C4-types ,47,80,85C4-types ,122,123.d-Ala-d-Ala remodeling, as discussed in vanR and vanS [vanY, which encodes a d,d-carboxypeptidase that complements the action of VanX by removing the terminal d-Ala residue from UDP-MurNAc pentapeptides that have escaped remodeling [d,d-carboxypeptidase activity, because their VanXY proteins have dual d,d-dipeptidase and d,d-carboxypeptidase activities [vanZ in A-type VRE and vanW in B- and G-type VRE [vanZ seems to play a role in teicoplanin resistance [Several other genotypic characteristics define the VRE types, including the composition and organization of the resistance-gene cluster . All VREand vanS , which cmodeling ,127,128.tivities . Other atype VRE ,130. Thesistance ,132. Tn1546 [In some VRE, sequences of the regions flanking the resistance operon reveal that the operon was acquired en bloc by transposition. For example, the A-type resistance operon lies within the well-characterized transposon Tn1546 ,133. ResTn1546 ,138,139.Tn1546 ,94,96,99Many types of VRE express the vancomycin-resistance phenotype only after exposure to the antibiotic, making the regulation of resistance an intriguing potential target for treatment of VRE. Specifically, compounds that inhibit the expression of resistance could function as antibiotic adjuvants , enhancivanRS regulatory genes, which encode the VanRS two-component system. A two-component system (TCS) is a type of signaling system found in prokaryotes, archaea, and certain eukaryotes, including plants and fungi. Notably, they are not found in metazoans [Regulation of the resistance phonotype in VRE depends upon the etazoans ,144,145.etazoans . TCSs coetazoans ,148. Upoetazoans ,149. Wheetazoans ,151. In etazoans ,153. ThiVanS is a Class-I HK, belonging to the same family as EnvZ . MembersB fall into this group. VanSB is categorized as “periplasmic-sensing” HK, because its periplasmic domain contains 103 amino acids. Consistent with this classification, the VanSB periplasmic domain is predicted to adopt a PAS-like structure [In the EnvZ family of HKs, the periplasmic domain is thought to detect the activating signal, although in some cases signals may be sensed by other domains . The VanS periplasmic domain (together with the TM domain and HAMP domain/linker region) lies within the N-terminal half of the protein, which displays considerably more sequence variability than the C-terminal half. The size of the VanS periplasmic domain also differs greatly between the different VRE types, ranging from 12 to 103 residues in length. This heterogeneity in length and composition suggests that periplasmic domains from different VanS orthologs may adopt different structures and thus sense vancomycin differently. Based on the length of the periplasmic domain , VanS prtructure  . In addition, the TM domain transduces the signal to the catalytic domain, thereby bridging the sensing and catalytic events in the HK . BeginniMolecular structures are key contributors to our knowledge of sensing and signal transduction. However, obtaining structural information for membrane-bound domains like the TM domain is challenging. Nonetheless, a general idea of how signal transduction can function through the TM domain has been developed. This model, based on HK models derived from crystallographic, NMR, and disulfide cross-linking experiments, suggests that signals are transduced by some combination of rotations, translations, and scissoring motions of the TM helices ,168,169.B and VanSM this region is predicted to adopt a HAMP-domain fold [HKs, adenylyl cyclases, methyl-carrier proteins, and phosphatases [All VanS proteins contain a membrane-proximal region connecting TMH2 and the DHp domain, which is responsible for propagating the signal to the DHp and CA domains; deletion of this region abrogates HK activity ,171. Thiain fold ,172. HAMphatases . HAMP-dophatases ; in dimephatases ,175,176.Following the linker region/HAMP domain is the conserved kinase region of the HK, consisting of the DHp and CA domains. These domains are ~70 and ~110 amino acids in length, respectively. The DHp domain earns the first half of its name by contributing to the dimerization of HK protomers; for example, in the EnvZ family of HKs, the DHp domain forms a long helical hairpin, with the two α-helices of each protomer dimerizing to form a four-helix bundle ,178. ReaA and the cytosolic portion of VanSA display low micromolar affinity for VanRA, with KD values of 1.9 ± 0.7 µM and 6.8 ± 1.4 µM, respectively [For the A-type VanRS proteins, the aforementioned binding interaction has recently been quantified. Both full-length, detergent-solubilized VanSectively . Perhapsectively .In prokaryotic HKs, the CA domain adopts an α/β sandwich topology known as the Bergerat fold ,186. WitS. coelicolor [VanR belongs to the OmpR family of RRs ,188 and elicolor . These oA). In OmpR-related RRs, this aspartate is situated at the end of the third β-strand of an α/β sandwich [The receiver domain accepts the phosphoryl group from VanS, with phosphorylation occurring on a conserved aspartate residue , only a single promoter is used [A and VanRB have been shown to bind their DNA targets in both the phosphorylated and unphosphorylated states; however, transcription of resistance genes is achieved only when VanR is phosphorylated [The effector domain of VanR is a winged-helix DNA-binding domain ,192, witof vanRS ,193,194. is used ,94,193. orylated ,152,153.orylated ,195,196.orylated ,200,201.The activities of the VRE VanR proteins will be more clearly understood once structures are determined for these proteins. Although it is possible to formulate models of VanR architecture by examining structures of related RRs, the specific details revealed by true experimental structures may provide insights into treating VRE. For example, therapeutics that disrupt the VanR-VanS or VanR-DNA interactions might restore vancomycin sensitivity to VRE, and are thus worth investigating. The expression of vancomycin resistance is initiated when VanS detects vancomycin in the periplasmic space. The mechanism by which this occurs remains one of the principal open questions in the field. Addressing this question has proven challenging, at least in part because VanS is an integral membrane protein, and therefore a difficult subject for biochemical and biophysical analysis. Furthermore, different VanS orthologs may employ different vancomycin-detection mechanisms, meaning that insights gleaned from one system cannot necessarily be translated to another. Broadly speaking, VanS could detect vancomycin via two distinct mechanisms: It might detect the antibiotic directly, by binding to it A, or indStreptomyces coelicolor, which expresses the VanS ortholog VanSSc. A direct interaction between vancomycin-VanSSc was deduced using a vancomycin photoaffinity probe, which was shown to label native protein in S. coelicolor membranes, as well as recombinant VanSSc in E. coli membranes [Sc [Direct binding provides the most conceptually straightforward model. A direct-binding mechanism has been most convincingly demonstrated for the non-VRE species embranes . Unlabelanes [Sc . Bacillus subtilis or CorS in Pseudomonas syringae [In contrast to the direct-binding model, indirect-detection models include any mechanisms that do not involve a direct physical interaction between vancomycin and VanS. One such indirect model suggests that VanS senses increased lipid II levels resulting from vancomycin’s inhibition of transglycosylase and transpeptidase enzymes  could activate their reporter, as well as cell-wall hydrolytic enzymes such as lysozyme and mutanolysin. These treatments cause the build-up of a wide range of different peptidoglycan precursors and breakdown products. The structural heterogeneity of these molecules again makes it unlikely that a single binding site in VanSA directly recognizes them. However, membrane stress is a common consequence of all of these treatments, and may therefore be a more credible candidate for the activating signal. Consistent with this idea, the membrane-perturbing agent chlorhexidine gluconate also activates VanSA, as revealed by RNA-seq analysis in E. faecium [vanRS strain showed no increase in vanHAX transcript abundance, implicating VanS in sensing the chlorhexidine [Late-stage intermediates in cell-wall biosynthesis, such as lipid II, are not the only potential candidates for activating VanSacZ gene . They fo faecium . Controlhexidine . A activation described above, activation can also be probed in the purified enzyme, by measuring its autophosphorylation, phosphotransfer, and dephosphorylation activities. If vancomycin directly activates VanSA, it should increase autophosphorylation and phosphotransfer activity, decrease phosphatase activity, or both. However, detergent-solubilized VanSA displays no change in any of its activities in the presence of vancomycin [A activity can be ruled out, since when VanSA is reconstituted in either amphipols or nanodiscs, its autophosphorylation and dephosphorylation activities also do not change in the presence of vancomycin [A. In addition to the cellular assays for VanSncomycin . Adversencomycin ,201. TheA, evidence also exists supporting a direct-binding model [A revealed a shift in the sedimentation coefficient of VanSA in the presence of vancomycin, suggesting that vancomycin induces a conformational change in the protein, presumably via a direct interaction [A, which has been interpreted as evidence for direct binding of the antibiotic, with a dissociation constant KD of approximately 70 μM [KD value is roughly one to two orders of magnitude higher than the antibiotic concentrations required to inhibit growth of antibiotic-sensitive Enterococci [A’s autophosphorylation activity is highly sensitive to detergents [Despite this large body of evidence favoring an indirect-detection model for VanSng model ,220,221.eraction ,223. Addly 70 μM ,224. Interococci ,220,221,tergents ,225.A, we note that models can be conceived that combine elements of both mechanisms. For example, VanSA activation might entail recognizing a vancomycin-lipid II complex, rather than the antibiotic alone. Support for this idea comes from the S. coelicolor system, where VanS activation only occurs when vancomycin binds its d-Ala-Ala target [Finally, as we weigh indirect vs. direct sensing mechanisms for VanSa target , even tha target .A, tantalizing data also exist that support a direct-binding model. Ultimately, this question will not be resolved without further study.In summary, while the preponderance of evidence currently points toward an indirect-detection mechanism for VanSB is less well-studied than that of VanSA, but the cumulative weight of the evidence points to a direct-sensing mechanism. First, VanSB is activated only by vancomycin [A. In the preceding section, we noted that it is difficult to conceive of how VanSA’s small periplasmic domain would be able to recognize the structurally diverse set of molecules that activate resistance, providing suggestive support for an indirect-binding mechanism for VanSA. Conversely, VanSB’s narrow specificity for its activator makes it plausible that the protein does bind vancomycin directly.The vancomycin-sensing mechanism of VanSncomycin , in starB’s ligand preference maps to its periplasmic domain, with mutations in this region altering ligand specificity and rendering B-type E. faecalis resistant to teicoplanin [B periplasmic domain does not exhibit a high degree of sequence homology to any domains of known structure; however, threading experiments predict that it can adopt a PAS-domain fold . This would be consistent with the lack of homology with other proteins, since PAS domains typically exhibit low pairwise similarities with one another, and contain no highly conserved residues [B would not be unprecedented among HKs, as a number of other sensor kinases possess periplasmic PAS domains, including CitA, PhoQ, and DcuS [B is the only enterococcal VanS ortholog for which a periplasmic ligand-binding domain is predicted. Combined with the specificity of VanSB for vancomycin, this observation suggests that VanSB may sense vancomycin by direct binding, which could make it an outlier among the VRE VanS proteins.VanScoplanin ,228. Theresidues . A PAS dand DcuS ,164,229.In the canonical model for VanRS function, exposure to vancomycin leads to expression of the vancomycin-resistance genes, via the intermediate steps of activation and autophosphorylation of VanS and subsequent phosphotransfer to VanR. However, in practice, vancomycin induces expression of resistance in only a subset of VRE types: A, B, E, G, L, M, and some C. In contrast, in other VRE types  vancomycin-resistance genes are expressed constitutively, regardless of whether the antibiotic is present. By comparing and contrasting the inducible and constitutive systems, we can gain insights into mechanisms of VanRS signaling.As noted earlier, VanS possesses both kinase activity  and phosphatase activity (dephosphorylation of VanR). In inducible systems, vancomycin induces expression of resistance by tipping the kinase/phosphatase balance in favor of the former . HoweverMuch of our knowledge about the inducibility of vancomycin resistance is derived from analysis of mutant VanS proteins with constitutive kinase activities/loss of phosphatase activity ,193. A hd-Ala-d-Ala target of teicoplanin. To our knowledge, the enzymatic consequences of these mutations have not been experimentally tested, but clues about their effects can be found in other HKs, in which similar substitutions within and near the H box abrogate phosphatase activity [B H box. Supporting this notion, a recent mutational study of VanSA showed that substitution of residue T168 (corresponding to residue T237 of VanSB) decreased VanSA phosphatase activity without affecting autophosphorylation activity [Mutations affecting inducibility of vancomycin resistance were first identified in B-type VRE grown under teicoplanin selection ,228,230.activity ,232. Henactivity .B, constitutive resistance to both vancomycin and teicoplanin results [Loss of all VanS activity should also lead to a constitutively resistant phenotype, as VanR can still be activated by endogenous small-molecule phosphoryl donors such as acetyl phosphate. Consistent with this idea, when a stop codon is inserted after codon 30 in VanS results .For C-type VRE, some isolates  express resistance constitutively, while others (C2/3 and C4) exhibit inducible resistance. The constitutive phenotype appears to map to substitutions in the DHp and CA domains. Comparison of VanS sequences from constitutive and inducible strains revealed several notable substitutions associated with constitutive behavior: R200L, D312N, D312A, and G320S . ResidueC substitutions D312N, D312A, and G320S fall between the F and G2 boxes of the CA domain. In the related Class-I HK EnvZ, mutations to the F box primarily affect phosphotransfer, while mutations to the G2 box affect all three enzymatic activities [B [C phosphatase activity.The VanStivities . Thus, tities [B . TentatiB to be activated by teicoplanin. For example, an E. faecalis strain selected for growth in the presence of teicoplanin was found to contain a A30G mutation in its VanSB protein, which conferred teicoplanin resistance by making the resistance genes inducible by teicoplanin [B’s periplasmic domain, suggesting that the A30G mutation may alter glycopeptide recognition by the periplasmic domain. Alternatively, it is possible that wild-type VanSB can bind to teicoplanin, but is unable to transduce this detection event to the protein’s catalytic region. If this is true, the A30G mutation, lying as it does at the junction between the protein’s first transmembrane helix and its periplasmic domain, may enhance the efficiency of signal transduction. Consistent with this notion, other teicoplanin-resistant VanSB mutations have been found either in the HAMP domain (D168Y) or between the HAMP and DHp domains (E221G) [Certain point mutations within the sensor region cause VanScoplanin . Residue (E221G) ,233. BecA, including L50V, E54Q, and Q69H, all of which fall within the predicted periplasmic domain [A recognizes glycopeptides via its periplasmic domain, with the ability to sense teicoplanin being specifically lost in the mutant strains. However, it is difficult to reconcile this model with the observation that vancomycin does not alter the enzymatic activities of VanSA in vitro [A.The response of A-type VRE to teicoplanin can also be altered by substitutions in the sensor region of VanSc domain . In thesin vitro , suggesthttps://www.ncbi.nlm.nih.gov/ipg; accessed on 1 December 2020) for “vanS AND enterococcus” and “vanR AND enterococcus.” We also searched for VRE type-specific entries that might have been missed in the initial search. Results were filtered to exclude entries containing “partial” sequences. The VanS and VanR protein sequences from S. coelicolor were included in the final sequence analysis, as were those from the glycopeptide producer A. teichomyceticus, bringing the total sequence counts to 120 and 109 for VanS and VanR, respectively. S. coelicolor was included in the analysis because its VanS and VanR proteins have been extensively characterized, while A. teichomyceticus was included because it produces teicoplanin, and the operons conferring resistance to glycopeptide antibiotics are thought to have originated from such antibiotic-producing species [Amycolatopsis orientalis) does contain a TCS that has been suggested to be involved with vancomycin resistance [To obtain a comprehensive view of the relationships among the enterococcal orthologs of VanS and VanR, we constructed phylogenetic trees for both proteins . Nonredu species . The natsistance ; howeverd-Ala-d-Lac/d-Ala-d-Ser ligase, and we followed this convention to assign VRE types for our VanS and VanR protein sequences. Although several VRE types have been divided into subtypes, we chose to subtype only C-type VRE, as the C subtypes are phenotypically distinct. Proteins were assigned to type C1 if they belonged to a strain with a vanC gene of 98–100% sequence identity to C1-type E. gallinarum strain BM4174, and 69–71% identity to C2/3 and C4 E. casseliflavus strains ATCC25788 and F32, respectively. C2/3-type proteins were classified as such if the vanC gene had 71% sequence identity to BM4174, 99–100% to ATCC25788, and 94% to F32. Proteins were assigned to type C4 if the vanC gene had 68–71% identity to BM4174, 94–96% to ATCC25788, and 96–98% to F32. The first of each type to be characterized was chosen as the reference strain [Conventionally, VRE are typed based on the gene sequence encoding their e strain ,47,85.E. faecium, E. faecalis, E. mundtii, E. alcedinis, E. sacchoralyticus, E. asini, E. sp. CU9D, E. diestrammenae, E. malodoratus, and E. florum. While these sequences were included because of their similarity to known VanRS proteins, they belong to strains that have not been explicitly identified as VRE [d-Ala-d-Lac/d-Ala-d-Ser ligases; hence it is currently unknown whether the corresponding gene products function as true VanRS proteins.This analysis contained proteins belonging to several vancomycin-susceptible enterococci ,245,246,d as VRE ,246, andAlthough VRE are not typed based on their VanS and VanR sequences, it is unsurprising that in our phylogenetic analysis the VanS and VanR proteins cluster according to VRE type , suggestA. teichomyceticus and S. coelicolor. Type B is the VRE type for which the VanR and VanS proteins are most similar to their counterparts in A. teichomyceticus and S. coelicolor .What is the mechanism of VanS activation for clinically relevant VanS orthologs? To date, while much progress has been made toward elucidating mechanisms for VanSB? It now appears evident that VanSB binds vancomycin via its periplasmic domain, leading to direct activation; however, the precise location must be mapped.What are the vancomycin-binding determinants for directly-activated VanS proteins such as VanSSc is the first for any VanRS protein, but structural characterization lags for the VRE orthologs of VanR and VanS. Structures of these proteins will prove invaluable in any efforts to disrupt vancomycin sensing in VRE.What are the structural consequences of activation for VanRS proteins? A recent structure of VanRVanRS was established as the regulatory TCS of vancomycin resistance expression in 1992 . Since tDisrupting the expression of vancomycin resistance is a potentially powerful new approach to restoring vancomycin susceptibility to VRE. Antibiotic adjuvants can be imagined that would abrogate VanS’s vancomycin-sensing activity or VanR’s DNA-binding activity; either should restore vancomycin susceptibility to VRE. An additional possibility is the development of novel glycopeptide antibiotics that retain vancomycin’s mechanism of action, but evade detection by VanS. Ultimately, however, any effort to modulate the expression of vancomycin resistance leads directly to the VanRS TCS.


The present study evaluated the effectiveness of computer-aided detection (CAD) system in screening automated breast ultrasound (ABUS) and analyzed the characteristics of CAD marks and the causes of false-positive marks. A total of 846 women who underwent ABUS for screening from January 2017 to December 2017 were included. Commercial CAD was used in all ABUS examinations, and its diagnostic performance and efficacy in shortening the reading time (RT) were evaluated. In addition, we analyzed the characteristics of CAD marks and the causes of false-positive marks. A total of 1032 CAD marks were displayed based on the patient and 534 CAD marks on the lesion. Five cases of breast cancer were diagnosed. The sensitivity, specificity, PPV, and NPV of CAD were 60.0%, 59.0%, 0.9%, and 99.6% for 846 patients. In the case of a negative study, it was less time-consuming and easier to make a decision. Among 530 false-positive marks, 459 were identified clearly for pseudo-lesions; the most common cause was marginal shadowing, followed by Cooper’s ligament shadowing, peri-areolar shadowing, rib, and skin lesions. Even though CAD does not improve the performance of ABUS and a large number of false-positive marks were detected, the addition of CAD reduces RT, especially in the case of negative screening ultrasound. Mammographic screening has reduced the rate of breast cancer mortality . Recent Real-time B-mode ultrasonography has emerged as an alternative imaging technique for breast cancer screening . UltrasoAutomated breast ultrasonography (ABUS) was proposed as a supplementary screening modality recently, for increased cancer detection combined with digital mammography (DM), especially in dense breasts ,11,12. IHowever, due to the large number of images in a single scan, the reading time (RT) of a full ABUS examination can be prolonged and cancers may be easily overlooked . For thiIn this study, we evaluated the effectiveness of computer-aided detection (CAD) system in screening automated breast ultrasound (ABUS) through diagnostic performance and reading time (RT). We also investigated and analyzed the characteristics of CAD marks and the causes of false-positive marks, to distinguish between true and false marks.This retrospective study was approved by the institutional review board (IRB) of our institution. The need for informed consent was waived by the ethics committee due to the retrospective design. All procedures involving human participants were in accordance with the ethical standards of IRB issued by our institution, and assessments were carried out in accordance with the tenets of the Declaration of Helsinki of 1975, and its revision in 2013.3 volume data sets of the breast in one sweep using a mechanically driven linear array transducer (14L5). Adequate depth and focus can be obtained using predefined settings for different breast cup sizes. All ABUS examinations were performed by a single trained radiographer. To ensure coverage of the entire breast, three overlapping acquisitions including antero-posterior, medial, and lateral views were performed. The scan thickness was displayed at 1 mm intervals without overlap. A dedicated ABUS workstation was used to reconstruct the transverse slices into a 3D volume that can be read in a multiplanar hanging, with sagittal and coronal reconstructions.The ABUS examinations were performed with the ACUSON S2000 Automated Breast Volume Scanner system . This ABUS system acquires 3D B-mode ultrasound volumes over an area of 15.4 × 16.8 × 6 cmA prototype workstation was designed and developed specifically for high-throughput ABUS screening in this observer study . In this prototype, each user action was logged with timestamps, which were subsequently used to estimate the time spent per case. The workstation was integrated with a commercially developed CAD software , which is designed to detect suspicious candidate regions in an ABUS volume highlighted with the so-called CAD marks .In addition, the QVCAD software provides an “intelligent” minimum intensity projection (MinIP) of the breast tissue in a 3D ABUS volume that can be used for rapid navigation through ABUS scans for enhancement of the possible suspicious regions. The CAD-based MinIP integrated with a multiplanar hanging protocol for ABUS displays the conventional ABUS planes. By clicking on the dark spot, the 3D multiplanar hanging automatically snaps to the corresponding 3D location. The crosshair is focused on a breast lesion that is marked by the CAD software with a green circular marker. The same lesion is also enhanced and visualized as a dark spot in the MinIP. A screenshot of the CAD-aided reading environment is presented in The number of CAD markers displayed per ABUS volume could be adjusted by changing the values of the false-positive rate (FPR) in the configuration setting of the CAD software. According to the manual from the manufacturer, FPR was defined as the total number of false-positive CAD markers in non-cancer volumes divided by the total number of non-cancer volumes. In this study, we set the FPR to 0.2 , which was its default setting as in previous studies ,17,18.The study included a total of 846 women aged 40–49 years who underwent ABUS screening from January 2017 to December 2017. The CAD (QVCADTM) system was used in all ABUS examinations and its diagnostic performance was evaluated retrospectively.We evaluated glandular tissue component (GTC), which was classified as minimal (<25% of the fibroglandular tissue (FGT)), mild (25–49% of the FGT), moderate (50–74% of the FGT), or marked (≥75% of the FGT) in each woman based on bilateral breast images .We analyzed whether CAD addition shortened the RT. The RT was determined by the expert breast radiologists based on their subjective perception in each of the following cases: (1) CAD with ABUS = ABUS only, (2) CAD with ABUS > ABUS only, (3) CAD with ABUS < ABUS only. We defined there is a difference when RT was shortened by more than 1 min.Furthermore, we analyzed the characteristics of CAD marks including the size of the marked lesion, lesion type (mass or non-mass), tissue composition under ultrasound, and the causes of false-positive marks. The false-positive mark was defined as the mark located on the typical benign lesion or pseudo-lesions that require no additional studies following ABUS. The number of marks per patient and per lesion and the frequency of false-positive marks were also evaluated.Two board-certified expert breast radiologists determined the characteristics of CAD marks based on consensus. In addition, the pseudo-lesions were also evaluated by two expert breast radiologists with consensus. The characteristics of pseudo-lesions were analyzed including the number, size, and location .All women with suspicious lesions were recalled and US-guided 14G core-needle biopsy was performed. Patients who were not disease-positive were followed up in 2 years with radiologic examination using mammography or ultrasonography.A total of 846 women participated in the study, and the median age at enrollment was 44 years (mean age ± standard deviation = 43.9 ± 3.0 years). Based on ABUS screening, five breast cancers were diagnosed pathologically over a two-year follow-up . The senBased on the lesion type detected, the large mass lesions were more than the non-mass lesions (60 vs. 11). Based on tissue composition under ultrasound, the number of minimal-to-mild cases in GTC was higher than moderate-to-marked cases (668 vs. 178). The rate of CAD positivity in moderate-to-marked lesions was higher than in minimal-to-mild. In the absence of the CAD mark, the readers determined that the reading time for CAD with ABUS was less than for ABUS only and easier to make a decision . Table 2Of 846 patients, 1032 CAD marks were marked in 534 lesions of 348 patients with a mean CAD mark per person of 0.8 (SD ± 1) (range 0–6) . No CAD The characteristic CAD marks were determined by two reviewers by consensus as suspicious malignant lesions , benign lesions (13.3%. n = 71), and clear pseudo-lesions .Among 530 false-positive marks, 459 marks were marked on the clear pseudolesions ; the mosThe false-positive marks on pseudo-lesions were frequently detected in the upper portion than in the mid-to-lower portion, and in the outer portion than in the mid-to-inner portion of breast . There wIn this study, we evaluated the effectiveness of computer-aided detection (CAD) system in screening automated breast ultrasound (ABUS) through diagnostic performance and reading time (RT). A total of 846 patients displayed 1032 CAD marks and 534 CAD marks based on lesions. The sensitivity, specificity, PPV, NPV, and accuracy of CAD were 60.0%, 59.0%, 0.9%, 99.6% and 59.0% for 846 patients, respectively, while those of 1032 CAD marks were 60.0%, 48.3%, 0.6%, 99.6%, and 48.4%, respectively. The relatively higher NPV compared with other parameters indicates that the exam can be concluded with a negative study if no CAD mark is detected on ABUS. The presence of marks in multiple views did not suggest malignancy in this study. In the absence of the CAD mark, the readers determined that the reading time for CAD with ABUS was less than for ABUS only and easier to make a decision.Several studies have reported that the performance of ABUS was comparable to that of hand-held ultrasound ,21,22. IHowever, while the ABUS can yield standardized and structured images regardless of the experience of the operator, it takes much more time and effort to interpret the exams . For thiIn addition to the diagnostic performance of CAD on ABUS, the previous studies evaluated the RT of CAD on ABUS ,28,29. YIn this study, we investigated and analyzed the characteristics of CAD marks and the causes of false-positive marks, to distinguish between true and false marks. Among 530 false-positive marks, 459 were identified clearly for pseudo-lesions; the most common cause was marginal shadowing, followed by Cooper’s ligament shadowing, peri-areolar shadowing, rib, and skin lesions, all of which were easily distinguishable radiologically. The false marks for pseudo-lesions were detected more frequently in the upper rather than in the mid-to-lower portion and in the outer rather than in the mid-to-inner portion, probably because of bulkiness and flexibility of the upper and outer portion of the breast.ABUS is a standardized examination with multiple advantages in both screening and diagnostic settings, including increased detection of breast cancer, improved workflow, and reduced examination time. However, ABUS has disadvantages and even some limitations. Disadvantages regarding image acquisition are the inability to assess the axilla, vascularization, and lesion elasticity. The limitations of interpretation include motion- or lesion-related artifacts due to poor positioning and the lack of contact . In the There are several limitations to this study. First, we used only image data obtained with equipment from a single vendor, with a small number of participants. In addition, this study was performed only in academic institutions by a limited number of users, board-certified expert breast radiologists, and does not represent varying clinical environments. Second, the absence of the numerical result of RT is the limitation of this study. The RT was determined by the expert breast radiologists based on their subjective perception. Finally, in our study, the expert radiologists’ decision was a gold standard for suspicious lesions or pseudo-lesions. However, a large number of marks await the radiologist’s rational judgment. Therefore, CAD users should be familiar with marks in various situations before using them, and the review summarizes the characteristics of CAD marks only without radiological evaluation. The knowledge of the characteristics of CAD marks and the causes of false-positive marks could improve the diagnostic performance of radiologists.In conclusion, even though CAD addition does not improve the performance of screening ABUS and is associated with a large number of false-positive marks, CAD addition improves the negative predictive value and reduces RT, especially for negative screening ultrasound.


L/SctVL, with a groove on the adjacent protomer’s cytoplasmic domain, which stabilises the nonameric ring assembly.Type three secretion is the mechanism of protein secretion found in bacterial flagella and injectisomes. At its centre is the export apparatus (EA), a complex of five membrane proteins through which secretion substrates pass the inner membrane. While the complex formed by four of the EA proteins has been well characterised structurally, little is known about the structure of the membrane domain of the largest subunit, FlhA in flagella, SctV in injectisomes. Furthermore, the biologically relevant nonameric assembly of FlhA/SctV has been infrequently observed and differences in conformation of the cytoplasmic portion of FlhA/SctV between open and closed states have been suggested to reflect secretion system specific differences. FlhA has been shown to bind to chaperone-substrate complexes in an open state, but in previous assembled ring structures, SctV is in a closed state. Here, we identify FlhA and SctV homologues that can be recombinantly produced in the oligomeric state and study them using cryo-electron microscopy. The structures of the cytoplasmic domains from both FlhA and SctV are in the open state and we observe a conserved interaction between a short stretch of residues at the N-terminus of the cytoplasmic domain, known as FlhA Type III secretion systems T3SS) are bacterial macromolecular machines that facilitate transport of protein substrates across the bacterial cell envelope , 2. The SS are baThe EA is made up of a core complex formed by the membrane proteins FliPQR (flagellar T3SS) or SctRST (virulence associated T3SS) which form a channel through which the unfolded protein substrates pass –8. The rin vivo [Shigella flexneri, Sf-SctV, as a nonameric ring matching the dimensions of a toroidal density observed by tomography, established the stoichiometry of FlhA/SctV as nonameric [L/SctVL. Variability has been observed in the conformation of the cytoplasmic domains, with structural transitions proposed to be linked to the secretion process. Open and closed states have been described, with the open state being competent for chaperone-substrate complex binding [FlhA/SctV has long been known to be the major component of the EA, but its stoichiometry was controversial for a long time , 17 and in vivo , 18. Fluin vivo , 18. Theonameric . This waonameric  whose st binding , while t binding .The transmembrane domain of FlhA/SctV is less well understood than the cytoplasmic domain. It is thought to contain approximately 8 transmembrane helices and conduct protons in order to use the pmf to power type III secretion . ConsistL/SctVL and the neighbouring subunit and confirm the conserved stoichiometry of the complex in the context of the full-length protein.We produced assembled rings of full-length FlhA/SctV from both flagellar and virulence associated systems and studied them using cryo-EM. Here, we present the structures of the nonameric cytoplasmic domain of both FlhA and SctV. The structures demonstrate the conserved interaction between FlhAE. coli membrane under overexpression conditions by employing superfolder GFP fusions [E. coli BL21 cells, and the cells were observed to be highly elongated, consistent with disruption of the membrane machinery (Vp-FlhA) and SctV from Yersinia enterocolitica (Ye-SctV) for further studies, as both could be produced in large quantities at high purity . Grids were blotted and plunged into liquid ethane using a Vitrobot Mark IV (FEI). The samples were imaged using a Titan Krios (FEI) equipped with a K2 detector (Gatan). Relion’s implementation of MotionCor2  was usedE. coli BL21 expressing FlhA-GFP or SctV-GFP were applied to a glass slide and imaged using a Zeiss 880 inverted microscope equipped with a plan-apochromat 63x/1.4 NA objective and an Airyscan detector. GFP fluorescence was excited using a laser (488 nm).8 μl of an overnight culture of S1 Raw imagesSamples as described in (PDF)Click here for additional data file. 3 May 2021Attachmentr2r_ed.docxSubmitted filename: Click here for additional data file. 24 May 2021Nonameric structures of the cytoplasmic domain of FlhA and SctV in the context of the full-length protein.PONE-D-21-14634Dear Susan,Thank you for submitting your revised manuscript and for addressing the reviewer's comments. I am pleased to accept your manuscript for publication. Please note that your manuscript will be formally accepted once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,EricEric CascalesAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 9 Jun 2021PONE-D-21-14634 Nonameric structures of the cytoplasmic domain of FlhA and SctV in the context of the full-length protein. Dear Dr. Lea:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. 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Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales are prevalent worldwide and pose an alarming threat to public health. The incidence and transmission of blaKPC-2 gene via horizontal gene transfer (e.g. transposition) have been well documented. However, the dynamics of transposon structure bearing blaKPC-2 and their exact effects on the evolution and dissemination of blaKPC-2 gene are not well characterized. Here, we collected all 161 carbapenem-resistant Enterobacterales (CRE) isolates during the early stage of CRE pandemic. We observed that the prevalence of KPC-2-producing Enterobacterales was mediated by multiple species and sequence types (STs), and that blaKPC-2 gene was located on three diverse variants of Tn1721 in multi-drug resistance (MDR) region of plasmid. Notably, the outbreak of KPC-2-producing plasmid is correlated with the dynamics of transposon structure. Furthermore, we experimentally demonstrated that replicative transposition of Tn1721 and IS26 promotes horizontal transfer of blaKPC-2 and the evolution of KPC-2-producing plasmid. The Tn1721 variants appearing concurrently with the peak of an epidemic (A2- and B-type) showed higher transposition frequencies and a certain superior ability to propagation. Overall, our work suggests replicative transposition contributes to the evolution and transmission of KPC-2-producing plasmid and highlights its important role in the inter- and intra-species dissemination of blaKPC-2 gene in Enterobacterales. Klebsiella pneumoniae carbapenemase (KPC) is a class A serine β-lactamase that efficiently hydrolyzes most β-lactam antimicrobial agents, including carbapenems, limiting treatment options in infected patients seriously  hsdR17 recA1 endA1 gyrA96 thi-1 relA1). HB101 (recA13 F- STRr) and E. coli J53 (AZr) were used as recipient strains in transposition experiments and conjugation experiments, respectively. The following antibiotics were added at the indicated concentrations: imipenem (IPM), 1 mg/L; streptomycin (STR), 25 mg/L; trimethoprim (TMP), 25 mg/L and sodium azide (AZ), 100 mg/L.Bacteria were routinely cultured at 37°C in Luria–Bertani (LB) medium or on LB agar. Plasmids were constructed in Enterobacterales (CRE) isolates were collected from August 2006 to December 2010 during routine identification and antimicrobial susceptibility testing by the Microbiology Laboratory, Huashan Hospital, Fudan University . This collection comprised all clinical isolates from the first occurrence of KPC-2-producing isolates (K. pneumoniae) to the prevalence of this cabapenemase (Dataset S1). For comparison, a susceptible collection of 112 carbapenem-sensitive K. pneumoniae (CS-KP) were also collected contemporaneously from similar departments (Dataset S2).A total of 161 non-duplicated, carbapenem-resistant https://bigsdb.pasteur.fr/klebsiella/klebsiella.html for K. pneumoniae and https://bigsdb.pasteur.fr/ecoli/ecoli.html for E.coli) and PubMLST website (https://pubmlst.org/organisms/citrobacter-spp for Citrobacter freundii and https://pubmlst.org/organisms/klebsiella-aerogenes for Klebsiella aerogenes). Genetic relatedness among the ST11 K. pneumoniae isolates was analyzed by XbaI-PFGE type as described previously [MLST was performed according to the protocol described on the Pasteur Institute MLST website  product [blaKPC-2-bearing genetic structures were determined by a series of PCR assays as reported previously [The  product . IncFII  product . The blaeviously .blaKPC-2-bearing plasmids were obtained by transformation or conjugation (Dataset S3). Plasmids of clinical isolates were extracted with a Qiagen Plasmid Midi kit  and examined by agarose gel electrophoresis, and then transformed into E. coli DH5α Electrocompetent cells by electroporation . Conjugation experiment was performed with E. coli J53 (AZr) as the recipient. Transformants and conjugants were selected on MacConkey agar containing IPM  and identified by VITEK 2 system  and were further subjected to PCR amplification of IncFII replicons, blaKPC-2 and genetic environment of blaKPC-2 according to our previous operations. For plasmids originated from clinical isolates of E. coli, each transformant was also identified by detecting the deletion of lacY gene as E. coli DH5α lacks the lac operon. Primers are listed in Table S1.Thirty-five Enterobacterales publicly available  and all of the plasmid sequences harbored in these strains  were downloaded from NCBI database in August 2021 (Chromosomes in Dataset S4 and plasmids in Dataset S5). The blaKPC-2 gene was identified by using nucleotide BLAST. Plasmid incompatibility type was determined by comparing with information in the Plasmid MLST locus/sequence definitions database (https://pubmlst.org/bigsdb?db=pubmlst_plasmid_seqdef& page = sequenceQuery).All complete genome sequences of 1721-like transposons, has been described previously [The primers and plasmids used in this study are listed in Table S1 and Table S2, respectively. pHS10842 (GenBank accession no. KP125892), a vector favorable for use in the exploration of the transposition mechanism of Tneviously .ΔtnpA1721Tn, IRR and tnpR fragments were amplified with primers JP934/JP935 and JP936/JP937, respectively. The IRR fragment share 20-bp sequences with AhdI restriction sites of pHS10842 and the tnpR fragments, respectively. The tnpR fragments also share 20-bp sequences with the IRR fragment and AflII restriction sites of pHS10842, respectively. The two fragments were subcloned into AhdI and AflII restriction sites of pHS10842 by use of the NEBuilder HiFi DNA assembly master mix , generating the derivative with the tnpA deletion of Tn1721, pHS10842-ΔtnpA1721Tn.For pHS10842-IS26 by digesting plasmid pHS10842 with SwaI and XmnI to removal a 560-bp fragment  and generate blunt ends. The DNA was ligated and transformed into competent cells of E. coli DH5α. pHS10842-ΔtnpA1721TnΔtnp26 was constructed in a similar strategy with pHS10842-ΔtnpA1721Tn.The transposase deletion was introduced into r TMPr STRr transconjugants per TMPr STRr transconjugant. For each transposition event, the movement patterns were determined by agarose gel electrophoresis and Southern hybridization [1721 and IS26 were determined by using primes specific for regions internal to Tn1721 and IS26 and primers specific for R388 as described previously [Transposition assays were performed as described previously ,17. Trandization . The exaeviously . Primers26 (8 bp) were calculated and plotted on a line graph. The pictures of the relative frequencies of the bases at each position were generated with the Pictogram program (http://genes.mit.edu/pictogram.html).The relative frequencies of the AT and GC contents of the region extending from 50 bp upstream to 50 bp downstream of the duplicated target site for IShttps://www.graphpad.com/). P < 0.05 was considered statistically significant.Statistical significance was assessed by Fisher’s exact test or Chi-square test with Yates’ correction using GraphPad Prism8 software , E. coli , Citro. freundii , K. aerogenes  and other species  . E. coli, Citro. freundii and K. aerogenes isolates were comprised of several STs. Together, these results suggested that blaKPC-2 is the chief culprit leading to carbapenem resistance and that the prevalence of blaKPC-2 in Enterobacterales was mediated by multiple species and STs, rather than clonal spread.All of the 161 CRE were identified as , 4.35%) A. Among identity B. For coblaKPC-2 genes were usually reported to be located on MDR region of plasmid, and IncFII plasmids contributed significantly to the global prevalence of blaKPC among K. pneumoniae [blaKPC-2 gene, we conducted a bioinformatic analysis of 5152 complete genome sequences of Enterobacterales publicly available . Among these, 311 strains were identified to be blaKPC-2-positive, and the overwhelming majority of blaKPC-2 genes were found on plasmids . Remarkably, most of the blaKPC-2-bearing plasmids belonged to IncFII group, but this group was less common in blaKPC-2-negative plasmids [174/300 (58%) versus 2195/10206 (21.51%), P < 0.0001; blaKPC-2-positive K. pneumoniae compared with that in blaKPC-2-negative group [151/204 (74.02%) versus 657/2701 (24.32%), P < 0.0001; blaKPC-2 genes are mostly located on plasmids in Enterobacterales, and that IncFII is the most common Incompatibility group, especially in K. pneumoniae.The eumoniae ,9. To deK. pneumoniae and the rate was slightly lower for E. coli (73.33%) and K. aerogenes (66.67%) (Table S3). None of Citro. freundii isolates was detected to IncFII-positive. However, the rate was much lower in carbapenem-susceptible collection in comparison with either CR-KP or CRE collection . Interestingly, 80% of plasmids carrying blaKPC-2 gene (28/35) belong to IncFII group. Finally, five plasmids in various sizes  were selected for complete sequence analysis to furtherly confirm the correlation between IncFII plasmids and blaKPC-2 gene.To evaluate this correlation in clinical isolates, we first performed IncFII replicon screening on all clinical isolates. Significantly, the detection rate of IncFII replicon was 84.82% in blaKPC-2 gene among 161 CRE identified three diverse variants of Tn1721 bearing blaKPC-2  (1721) that consists of two 38-bp terminal inverted repeats , transposase (TnpA), resolvase (TnpR) and resolution site (res). The B-type is composed of Tn1721, blaKPC-2, an additional inverted repeat (IRL2), as well as several unrelated elements . The sequence of A1-type (Tn1721-blaKPC-2) shares 100% identity with that of B-type, but it does not contain the additional IRL, IRL2. In the A2-type, Tn3 was disrupted by an IS26, forming the chimera, Tn1721-blaKPC-2-IS26. The A2-type Tn1721 accounted for more than half the samples , and A1- and B-type accounted for 19.25% (31/161) and 18.63% (30/161), respectively  B. All ofectively B.Table blaKPC-2 outbreak, we comprehensively analyzed all data obtained in molecular epidemiological investigation. blaKPC-2 bearing structure in Enterobacterales. Samples are plotted by month of isolation, wards, species, STs of most common resistant specie (K. pneumoniae) and transposable elements carrying blaKPC-2 gene. As shown in blaKPC-2 genes that were located in three distinct Tn1721-like transposons on plasmid, originated from K. pneumoniae, then became increasingly prevalent in this specie and spread in Enterobacterales further. The epidemic of blaKPC-2-carrying plasmid reported here had three stages, designated as (i) distributed period , (ii) epidemic period , and (iii) mixed epidemic period . In the distributed period, blaKPC-2 appeared sporadically in K. pneumoniae without any predominate STs, and all Tn1721-like transposons belonged to A1-type. At the second epidemic stage, KPC-2-producing plasmid were prevalent in K. pneumoniae ST11 that consisted of several different subtypes, and spread into other Enterobacterales. A2-type Tn1721 was the dominant structure carrying blaKPC-2. In the mixed epidemic period, KPC-2-producing isolates increased furtherly. This stage involved three distinct Tn1721 variants, and the number of emerging B-type was almost equal to that for A2-type during the same period.In order to clarify the progression of blaKPC-2-bearing transposon structure, and A2- and B-type Tn1721 appeared concurrently with the peak of blaKPC-2 epidemic.Altogether, outbreak of KPC-2-producing plasmid was correlated with the dynamics of blaKPC-2 is the HGT of mobile genetic elements (MGEs) surrounding this determinant. According to our comprehensive analysis of epidemiological data, it was presumed that replicative transposons, Tn1721 and IS26, can mobilize blaKPC-2 through transposition, promoting the dissemination of blaKPC-2 in Enterobacterales. In our previous study, A1- and B-type Tn1721 have been shown to transfer blaKPC-2 both internal and external to this element, and target transposition into 5-bp region that gradually exhibits a degenerated degree of AT-rich regions from both sides to the middle and that is immediately flanked by GC-rich regions [1721 (Tn1721-blaKPC-2-IS26).A critical step in the dissemination process of  regions . Here, w1721 and IS26 existed in this chimera. Tn1721 pattern was the same as the one detected in A1- and B-type Tn1721 previously, including cointegrate forming and resolving steps [1721 pattern, a different IS26 pattern via replicative transposition was also detected in several cases of A2-type Tn1721  A series of PCRs confirmed that P3 had the same blaKPC-2-bearing genetic structure (Tn1721-blaKPC-2-IS26) as that of pHS10842, and the junctions between donor and target in each case were amplified and sequenced with primers specific for regions internal to IS26 and primers specific for R388.Two distinct patterns of movement mediated by Tnng steps . In addie Tn1721 B. Only o of data A, as fol26 preferentially targets AT-rich regions with AA and TT nucleotide tandems.Consistent with previous reports , 8-bp ta1721 was measured to be 3.8 × 10−6 . The tnpA deletion of either Tn1721 or IS26 decreased half of the frequency, and both tnpA deletions led to a functional inability to mobilize blaKPC-2. Hence, blaKPC-2 embedded between Tn1721 and IS26 was transferred at an apparently higher frequency owing to the existence of both elements.The transposition frequency of A2-type Tn8 × 10−6 . This reblaKPC-2 through replicative transposition, and that A2- and B-type Tn1721 showed higher transposition frequencies and had a certain superior ability to propagation.Taken together, our findings indicate that all three transposons were capable of transferring 1721 variants have been shown various capacities of transferring blaKPC-2 via replicative transposition, an attractive hypothesis is that the A2- and B-type Tn1721 occasionally appearing in the late-stage stood out from the rest through selection for their ability to facilitate dissemination of blaKPC-2. As discussed above, the outbreak of KPC-2-producing plasmid was correlated with the dynamics of blaKPC-2-bearing Tn1721 structure, and A2- and B-type Tn1721 arising concurrently with the peak of blaKPC-2 epidemic showed apparently higher transposition frequencies. Such an association and different movement capabilities imply that replicative transposition of Tn1721 variants contributes to the evolution and dissemination of KPC-2-producing plasmid in Enterobacterales. This mechanism could enable blaKPC-2 gene to search for suitable host spontaneously, dealing with the antibiotic pressure from the environment.Given that TnEnterobacterales that have spread extensively throughout the world, are an important cause of nosocomial infections, especially urinary tract infections, respiratory tract infections, and bloodstream-associated infections [blaKPC-2 gene [blaKPC-2 gene. Our investigation of clinical CRE provided a detailed description of the whole process from blaKPC-2 gene first occurrence, progress to final prevalence. We observed that the prevalence of KPC-2-producing Enterobacterales was mediated by multiple species and STs, and that blaKPC-2 gene was located on three diverse variants of Tn1721 in multi-drug resistance (MDR) region of plasmid. IncFII was the most common Incompatibility group. Further study indicated that replicative transposition contributed to the evolution and dissemination of KPC-2-producing plasmid.KPC-producing fections . During C-2 gene ,10,12. HblaKPC-2 gene is mostly located on transposable elements, such as Tn4401, Tn1721, and IS26 [4401 (Tn4401a to Tn4401h) have been identified, with Tn4401a and Tn4401b being the most widespread [tnpA or/and tnpR, or have deletions in the noncoding region upstream of blaKPC leading to enhanced or reduced expression of this carbapenemase [1721 variants are the dominant structures in Asia, particularly in China [1721 variants in current blaKPC-2 epidemic is too complicated and diverse to track the dissemination process of this gene, we focus on the early stage of blaKPC-2 epidemic. In addition to the dynamics of blaKPC-2-bearing transposon structure raised by this study, host and plasmid factors as well as antibiotic pressure from environment were also worth further study.Transposable elements play an important role in the genetic variation and evolution of bacteria. The and IS26 ,13. To ddespread ,19,20. Nin China ,15,22. G1721 variants were capable of mobilizing blaKPC-2 via replicative transposition, and A2- and B-type Tn1721 exhibited higher transposition frequencies than A1-type. Such various capacities are presumably associated with cointegrate resolution of Tn1721 in transposition assay. For the transconjugants with the Tn1721 pattern, only the cointegrate could be conjugated into the recipient  and screened on LB agar containing IPM, TMP, and STR in the transposition assay. Once the cointegrate resolved in donor strain, neither plasmid generated here could survive. Because P1 lacked the TMP resistance (TMPr) and the ability to conjugate into recipient (STRr), even though it possessed IPM resistance (IPMr), while P2 was an opposite case. In contrast, both the cointegrate and plasmid resolved from the cointegrate with Tn1721-blaKPC-2-IRL2 pattern possessed IPMr and TMPr, as well as had the ability to conjugate to recipient (STRr). For the transposition via IS26, there is no resolution process. It means that the cointegrate (P3) with IS26 pattern possessed IPMr and TMPr, as well as had the ability of conjugal transfer. Consequently, it survived on LB agar containing IPM, TMP, and STR. In other words, the plasmid with Tn1721-blaKPC-2-IRL2 pattern (in B-type) or IS26 pattern (in A2-type) had a certain superior ability of conjugal transfer and became more widespread. These results strongly supported our molecular epidemiological findings that the two peaks of blaKPC-2 epidemic followed the appearance of A2- and B-type Tn1721, reflecting an important role for replicative transposition of Tn1721 and IS26 in the evolution and transmission of blaKPC-2-carrying plasmid in Enterobacterales.Our findings indicated that all three TnEnterobacterales, and highlights its important role in the dissemination of antibiotic resistance genes between pathogenic bacterial species.In conclusion, our work demonstrates replicative transposition facilitates the evolution and transmission of KPC-2-producing plasmid in Click here for additional data file.Click here for additional data file.


The performance exhibits a short‐circuit current density of 14.8 mA cm−2, an open‐circuit voltage of 473.0 mV, and a fill factor of 58.7%, yielding a power conversion efficiency (PCE) of 4.1% under standard air mass 1.5 global . The cells retain ≈90.0% of the initial PCE even after illuminating under AM 1.5G (100 mW cm−2) for 2321 min. Here, a new approach is provided for combining selenide and iodide as anions, to fabricate highly efficient, highly stable, green, and low‐cost solar cells.Although antimony selenoiodide (SbSeI) exhibits a suitable bandgap as well as interesting physicochemical properties, it has not been applied to solar cells. Here the fabrication of SbSeI solar cells is reported for the first time using multiple spin‐coating cycles of SbI 3 solutions on Sb2Se3 thin layer (deposited by using a single‐source precursor solution) is reported. The champion cell exhibits a power conversion efficiency of 4.10% under standard air mass 1.5 global (100 mW cm−2). These cells show good humidity, photo‐, and thermal stabilities.The fabrication of SbSeI solar cells using multiple spin‐coating cycles of SbI Most of the aforementioned properties exist in materials containing metal cations with ns2 valence electron configuration, owing to their high bandwidth conduction band and high Born effective charge derived from large spin–orbit effects as well as their soft Polaris ability. Popular light harvesters including halides or chalcogenides of Pb2+, Sn2+, Ge2+, Sb3+, and Bi3+ contain metal cations with the ns2 valence electron configuration. However, most of them are affected by one or several issues, such as low efficiency, low stability, toxicity, and high cost. Hence, efficient, stable, green, and low‐cost light‐harvesters must be developed.Small effective mass, large dielectric constant, high band dispersion level, and valence band maximum with antibonding states are desirable properties for highly efficient and defect‐tolerant light harvesters.1 Most2 electronic configuration, metal chalcohalides have received extensive attention owing to their interesting physical and chemical properties. Because of the distinct bonding preferences of chalcogenide and halide atoms, to form stable sites in compounds, the competition among atoms might yield unique structures and properties. Additionally, a wide bandgap range can be obtained in these materials because halide and chalcogenide coexist as anions; hence, they can be used in various applications, such as solar cells, radiation detection and transparent electronic devices. As an important member of metal chalcohalides, SbSI has been applied to solar cells as a light harvester. SbSI solar cells with TiO2 and poly dithiophene)‐alt‐4,7‐] (PCPDTBT) as electron transporting layer (ETL) and hole transporting layer (HTL), respectively, exhibit a relatively poor power conversion efficiency (PCE) of 3.05%. The unsatisfactory PCE might be due to the wide bandgap (≈2.1 eV) of SbSI. Compared with SbSI, SbSeI exhibits a much narrower bandgap (1.67 eV), which is more suitable for light harvesting. Furthermore, compared with the difference in ionic radii between S2− (184 pm) and I− (220 pm), the better match in ionic radii between Se2− (198 pm) and I− (220 pm) might produce superior and more stable crystal phases.As important material exhibiting the nsoperties.8 Beca SbSeI has attracted significant attention owing to its important optical and semiconducting properties, which have been applied in X‐ray and γ‐ray detections as well as optoelectronics. Based on relativistic quasi‐particle self‐consistent GW theory, Butler et al. reported that SbSeI exhibited a multivalley electronic structure, where several electron and hole basins occurred near the band extrema, which might cause nonconventional photophysical behaviors. The ionization potential of SbSeI was predicted to be 5.3 eV based on first‐principles calculations. Appropriate contact materials should be selected to optimize performance. Brandt et al. reported that the lifetime of SbSeI exceeded 1 ns, which is the threshold to yield promising early‐stage photovoltaic device performance. Although extensive studies have been performed regarding SbSeI, experimental studies applying SbSeI in solar cells are nonexistent.Furthermore, SbSeI is a representative A(V)B(VI)C(VII) type compound. Similar to SbSI, SbSeI is ferroelectric and a good photoconductor.14 SbS2 through multiple spin‐coating cycles of SbI3 solutions on Sb2Se3, which was formed by thermal decomposition after depositing a single‐source precursor (Se‐SSP). Here, [(SbL2Cl2)Cl]2·(CH3)2CO], where L is N,N‐dimethyl selenourea, as Se‐SSP was synthesized according to our previous work. SbSeI was characterized by X‐ray powder diffraction (XRD), ultraviolet–visible (UV–vis) absorption spectroscopy, X‐ray photoelectron spectroscopy (XPS), high‐resolution transmission electron microscopy (HR‐TEM), field emission scanning electron microscopy (FESEM) and ultraviolet photoelectron spectroscopy (UPS). The effects of deposition cycles including the spin‐coating and thermal decomposition of SbSeI on the device performance were investigated by measuring the electrochemical impedance spectroscopy (EIS) and temperature‐dependent current–voltage curves. The cells with TiO2 and PCPDTBT as the ETL and the HTL, respectively, exhibited a PCE of 4.10%. Furthermore, the cells showed good stabilities under ambient conditions (≈80% relative humidity (RH)), at 85 °C in air (<30% average RH), and under standard air mass 1.5 global (AM 1.5 G) conditions (100 mW cm−2) without a UV light filter.In this study, we deposited SbSeI on the mesoporous (mp)‐TiOous work.18 SbSFigure 2/SbSeI in the range of 10°–60°; it is well‐indexed to the SbSeI orthorhombic phase with a Pnma (62) group (JCPDS No. 76‐1354). To observe the main peaks, the XRD patterns in the range of 18°–22° and 28°–32.5° are shown in Figure 2Se3  except for lattice parameters. The UV–vis absorption spectrum of the glass/mp‐TiO2/SbSeI is shown in Figure  As shown in Figure S4 (Supporting Information), the band gap of SbSeI remains unchanged even when excessive SbI3 is applied. Figure 2, revealing the crystal structure of SbSeI. The d‐spacing of the crystal lattice of 0.303 nm was assigned to the (112) plane of the orthorhombic phase of the SbSeI crystal. The uniform distribution of SbSeI on mp‐TiO2 was confirmed by scanning transmission electron microscopy (STEM) and energy‐dispersive X‐ray (EDX) elemental mapping /mp‐TiO2/SbSeI. Additionally, peaks corresponding to Sb, Se, and I were observed, which matched well with the reference spectra. The Ti 2p peaks were acquired from mp‐TiO2, whereas the C 1s peaks were owing to adventitious carbon contamination. Figure S8 in the Supporting Information shows the process for depositing SbSeI.g Figure . The EDX2/SbSeI is shown in Figure 2. Some SbSeI needles were observed, as SbSeI tends to grow in the form of long needles along the c‐axis of the material. Figure  Furthermore, an FTO layer, a TiO2 blocking layer, an mp‐TiO2/SbSeI/HTM(L) layer, and an Au layer were observed. The uniform morphology of the mp‐TiO2/SbSeI/HTM(L) indicated the uniform distribution of SbSeI in mp‐TiO2 and the efficient infiltration of HTM(L) into the pores of mp‐TiO2. To further confirm the uniform distribution of SbSeI in mp‐TiO2, the line EDX data  layer and the EDX mapping analysis . Because UPS is more surface sensitive than XPS, XPS and UPS were used to measure the energy levels. In particular, XPS was used to detect the highest occupied molecular orbital (HOMO) regions to avoid the effect of surface contamination. Figure EF) of SbSeI was 4.65 eV, with the valence band maximum (VBM) located at 1.04 eV below the EF ). The EF of SbSeI was measured twice; it was discovered that their values were similar, indicating that the EF value was reliable . Combining these results and the bandgap value  curves of the SbSeI solar cells, where SbSeI was prepared using 8, 10, and 12 multicycles of spin‐coating and thermal decomposition. The device performances are summarized in Table JSC), open‐circuit voltage (VOC), and fill factor (FF) were 11.7 mA cm−2, 443.8 mV and 54.1%, respectively, resulting in a PCE of 2.81%. As the number of cycles increased to 10, the PCE increased to 3.77%, accompanied by simultaneous increments in the JSC, VOC, and FF. When the cycle further increased to 12, the PCE decreased to 1.23%, with significant decrements in the JSC, VOC, and FF. To understand the reason contributing to the changed device performances, we first measured the incident photon‐to‐electron conversion efficiency (IPCE) of these cells, which is closely related to the JSC. As shown in Figure JSC trend. Light‐harvesting efficiency (LHE), electron injection yield (EIY), and charge collection efficiency (CCE) are three key factors that determine the IPCE spectra. Furthermore, EIY and CCE can be combined as the charge‐transfer yield (CTY). Figure 2/SbSeI prepared through 8, 10, and 12 spin‐coating cycles and thermal decomposition. As the number of cycles increased from 8 to 10, the improved JSC could be attributed to the enhanced LHE, which was confirmed by the increased absorption. However, further increasing the number of cycles to 12 did not increase the absorption. Hence, the reduction in the PCE of the cells fabricated through 12 cycles was attributable to the reduced CTY. To investigate the CTY, we obtained the electrochemical impedance spectra of those cells. Figure Rs), whereas the low‐frequency section represents the recombination resistance (RREC). The fitting parameters of the Nyquist curves are shown in Table S1 in the Supporting Information. The cell prepared through 10 cycles yielded a greater RREC than that through 12 cycles, indicating a more efficient interface charge transfer in the cell prepared through 10 cycles. To understand the reason contributing to the different CTYs between the cells prepared through 10 and 12 cycles, we analyzed their trap states using temperature‐dependent current–voltage curves. Figure S13 in the Supporting information shows the current–voltage curves at various temperatures in the presence and absence of light irradiation. The average activation energy of the trapped electrons can be calculated using the Richardson–Dushman equation as followsE, k, and T are the electron activation energy, Boltzmann constant, and absolute temperature, respectively. Because the current at 0 V can be easily affected by noise, we used the current at −0.5 V bias to calculate ΔE. Figure The effect of SbSeI loading amount on the device performance was investigated. The performance of the Sb2, and the concentration of the Se‐SSP solution were optimized . Figure J–V curve of the best‐performing SbSeI solar cell, which exhibited a PCE of 4.10%, with a JSC of 14.77 mA cm−2, a VOC of 473.0 mV, and an FF of 58.7%. This PCE is the highest value among the solar cells that were first demonstrated by applying new light absorbers. . The corresponding IPCE spectrum is shown in Figure  Integrating the overlap of the standard AM 1.5 G solar photon flux with the IPCE spectrum yielded a current density of 14.66 mA cm−2, which was similar to the value measured from the J–V curve. Figure −2 and a stabilized PCE of 3.95% achieved by maintaining the bias voltage at the maximum power point (0.376 V). Here, a cooling fan was used to minimize the temperature rise due to continuous light irradiation. The PCE obtained from the steady‐state measurement was consistent with the J–V measurements, indicating that the PCEs of the SbSeI solar cells are reliable. Figure The thickness of mp‐TiOFigure VOC. Figure JSC, and almost unchanged VOC and FF. Additionally, the unencapsulated devices were illuminated under standard AM 1.5 G conditions (100 mW cm−2) without a UV light filter to test their photostability. After illuminating for 2321 min, the cells retained 90.0% of the initial PCE. These results show that the SbSeI solar cells exhibited good stabilities, regardless of humidity, temperature, and light.To check the humidity stability, the cells without encapsulation were stored under ambient conditions (≈80% relative humidity) at room temperature in the dark. As shown in 3 solution on Sb2Se3 and thermal annealing. Subsequently, Sb2Se3 was deposited using a Se‐SSP solution. SbSeI is of the orthorhombic phase with a Pnma (62) group and has a bandgap of 1.67 eV. The SbSeI cells fabricated by 8 and 12 spin‐coating cycles and thermal decomposition exhibited poor PCEs due to insufficient absorption and inefficient charge transfer, respectively. The inefficient charge transfer in the cells fabricated through 12 cycles was due to deeper level trap states. Sufficient absorption and efficient charge transfer were achieved in the cells fabricated through 10 cycles, and the champion cell exhibited a PCE of 4.10%. Furthermore, these cells demonstrated excellent humidity, thermal, and photo stabilities. This study provides an insight into using metal chalcohalides for solar cells and intermediate products to synthesize lead‐free perovskite materials.In summary, SbSeI solar cells were fabricated for the first time using multiple spin‐coating cycles of a SbI2 blocking layer (TiO2‐BL), pray pyrolysis of 20 × 10−3m titanium diisopropoxide bis(acetylacetonate) (Aldrich) solution was performed on an FTO glass substrate at 450 °C. After cooling, an mp‐TiO2 layer with a thickness in the range of 150–1800 nm was prepared by a screen‐printed TiO2 paste comprising TiO2 nanoparticles . Subsequently, the substrates were annealed at 500 °C for 1 h in air to crystallize TiO2 and remove organic materials.To deposit a 100 nm thick TiO The Se‐SSP solution in DMF (0.05–0.2 mol L−1) was spin‐coated on FTO/BL/mp‐TiO2 substrates at 1000–2000 rpm for 60 s. Subsequently, the substrates were thermally decomposed in Ar gas at 150 °C for 2 min. The spin‐coating cycle and thermal decomposition were repeated to obtain the appropriate amount of Sb2Se3. Subsequently, the samples were annealed in Ar at 300 °C for 10 min to crystallize Sb2Se3. To deposit SbSeI, the SbI3 solution (0.05–0.2 mol L−1) was spin‐coated on FTO/BL/mp‐TiO2/Sb2Se3 at 1000–2000 rpm for 60 s; subsequently, the samples were annealed in Ar gas at 150 °C for 2 min. The spin‐coating cycle and thermal decomposition for the SbI3 solution were identical to those for Se‐SSP. The concentration of the SbI3 solution was identical to that of the Se‐SSP solution. Finally, the samples were washed in DMF to remove the remaining SbI3.An Se‐SSP was prepared according to the method detailed in the previous study.18 The71BM  in 1 mL of 1,2‐dichlorobenzene was spin‐coated onto FTO/TiO2‐BL/mp‐TiO2/SbSeI at 2000 rpm for 60 s. Subsequently, the HTL was prepared by spin‐coating a poly doped with a poly(4‐styrenesulfonate)  solution at 2000 rpm for 60 s and then diluted threefold in MeOH. Finally, the anode was prepared by thermal depositing a 100 nm thick Au layer onto the samples as the anode. The active area of the device measured 16 mm2.To deposit the hole‐transporting material, a solution containing 10 mg of PCPDTBT  and 10 mg of PCα source (1486.6 eV) or an unfiltered He I (21.22 eV) gas discharge lamp. High angle annular dark field (HAADF) STEM images were obtained using an FEI Titan3 G2 60‐300 instrument equipped with a probe‐side spherical aberration corrector operating at an accelerating voltage of 200 kV. EDX elemental mapping was conducted using FEI's Super‐X EDS detection system. Elemental analysis was conducted using a Thermo Scientific Flash 2000 analyzer. The cross‐sectional image and corresponding EDX line and mapping data were acquired using a focused ion beam instrument  and energy dispersive spectroscopy. The J–V curves and IPCE spectra were measured using a solar simulator  with a source meter (Keithley 2400) at 100 mA cm−2 illumination AM 1.5 G and an internal quantum efficiency system , respectively. The electrochemical impedance spectra were measured using an AUTO LAB (AUT302N).Crystal structure, absorption, microscopic structure, morphology, surface state, and energy level were characterized using a powder X‐ray diffractometer , UV–vis spectrophotometer (Jasco V‐780), high‐resolution transmission electron microscope , field‐emission scanning electron microscope , and Thermo‐Fisher machine (ESCLAB 250XI) with either a monochromatic Al KThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Lysine methylation is among the key posttranslational modifications to histones that contribute to epigenetic regulation. SMYD3 is a lysine methyltransferase that is essential for the proliferation of a range of tumorigenic cells. The findings that SMYD3 is significantly upregulated in most colorectal carcinomas, hepatocellular carcinomas, and breast cell carcinomas support a model in which its aberrant expression modifies established patterns of gene expression, ultimately driving unrestrained proliferation. Herein, we dissect the unique structural features of SMYD3 relative to other SET enzymes, with an emphasis on the implications for selective design of therapeutics for the clinical management of cancer. Further, we illustrate the ability of inhibitors targeting the SET domain of SMYD3 to reduce the viability of colorectal and lung carcinoma cells. Post-translational histone modifications, including methylation, acetylation, phosphorylation, and ubiquitination, modify chromatin structure to alter DNA accessibility and the resulting gene transcription. Histone lysine methylation is a predominant epigenetic mechanism in all eukaryotic organisms, and modulates a wide range of cellular functions, including cell differentiation and proliferation. Aberrant histone lysine methylation has been associated with a range of human diseases, including cancers.Su(var)3–9 Enhancer of Zeste and Trithorax (SET) protein domain, which exists in over 100 human proteins [Histone lysine methylation has been shown to be catalyzed exclusively by the proteins ,3,4,5. Tproteins ,7,8,9. TMyeloid-Nervy-DEAF-1 (MYND) domain. The MYND domain is a zinc-finger motif that further modulates protein–protein interactions, and separates the N-SET and I-SET subdomains. These SET and MYND Domain-containing proteins (SMYDs) have been shown to be essential for a wide range of developmental processes. Of particular interest is SMYD3, which is normally expressed predominantly during embryogenesis. SMYD3 mainly catalyzes H3K4 methylation, and its transcriptional regulation has been reported as a major component in RNA polymerase complex synthesis. SMYD3 can also trimethylate H3, H4, and H5, giving it a broad range of effects in the development of skeletal and cardiac muscle tissue, as well as in mediating estrogen-receptor gene expression [A specific family of SET-domain containing proteins also contain the pression ,11,12,13pression ,15,16,17pression ,19,20. Npression ,19,20.In this study, we present and compare the tertiary structures of the subdomains in SMYD3 to the SET domains in other proteins to inform the development of specific small molecule inhibitors that could be useful in the understanding and treatment of SMYD3-positive cancers. We recently developed a panel of SMYD3 inhibitors, one of which (Inhibitor-4) effectively restricted the proliferation of breast cancer cells . Here, w® software.SMYD3 protein was purified, and X-ray crystallography data was acquired in our previous publications ,22. Our Candidate SMYD3 inhibitors were designed in silico as previously described . In that4 cells were plated in 6-well plates with various concentrations of SMYD3 Inhibitor-4 for 48 h. Then, cells were washed with PBS, trypsinized, and stained with Trypan blue to exclude died cells. Cells were counted using a TC20 automated cell counter. Viability curves were drawn using GraphPad Prism 6 software.To assess overall cell viability, 3 × 102 and 37 °C for two weeks. Colonies were fixed with 75% ethanol, and stained with 0.2% Crystal Violet dye. Colonies were counted, and survival fraction was calculated as the number of colonies relative to the untreated group. Curves were drawn using GraphPad Prism 6 software.To assess the ability of the cells to proliferate and survive under long-term treatment with Inhibitor-4 , 1000 cells were plated in a 6-well plate, and treated with various concentrations of SMYD3 Inhibitor-4. Then, cells were incubated in a humidified incubator at 5% COFrom the N- to C-terminal, SMYD3 consists of six subdomains: the N-SET, MYND, I-SET, core SET (C-SET), post-SET, and C-terminus domains. These domains are depicted according to our 2.3 Å solution A. SMYD3 Based on comparisons in sequence conservation, and buttressed by our analysis of its crystal structure, we demonstrated that the SET domain of SMYD3 consists of two non-contiguous elements derived from the N- and C-terminal halves of the SET sequence. Termed N-SET and C-SET, respectively, these are highly conserved, and display remarkable structural overlap with other known SET domains. We overlayed our solved SMYD3 N-SET domain with those of other SET proteins, and found that N-SET domains in humans, plants, yeast, and viruses are composed of four β-sheets linked by a hairpin, between β-1 and β-2, and a loop, between β-2 and β-3 .The catalytic function of SET proteins is dependent upon their ability to transiently interact with an AdoMet cofactor. The crystal structure of other SET proteins has been solved in the presence of this cofactor, and a cluster of residues in the N-SET has been shown to cooperate with another conserved cluster in the C-SET to facilitate this AdoMet interaction ,30. ResiIn addition to the catalytic residue, F183, transposed from the N-SET, the C-SET region consists of two clusters that are absolutely essential for the catalytic activity of SET-dependent methyltransferases ,31. ThesFinally, the post-SET region of SET-dependent methyltransferases functions as a critical component of the active site by supplying an aromatic residue (Y257 in SMYD3) that anchors against the conserved SET core to form a hydrophobic channel. This channel is a common feature among SET-containing proteins, and serves as the interface along which the substrate associates [The structural similarity of the N-SET and C-SET regions is contrasted by a highly variable insert region (I-SET) in SET proteins. Positioned between the N-SET and the C-SET, the I-SET is a common feature among SET-domain proteins . Althouglarge subunit methyltransferase (Rubisco-LSMT) (2 fixation step of photosynthesis [The I-SET structure of SMYD3 is significantly longer than most I-SET domains A. In othco-LSMT) B. As itsynthesis ,32. The Though the I-SET region of SMYD3 does not appear to interact with the substrate near its methylation site, we predict that it may contact the substrate further upstream, as the I-SET comprises part of a groove along which the substrate extends C. AdditiSimilar to the N-SET and C-SET regions, the zinc finger-containing MYND domain in SMYD3 closely resembles MYND domains in other human proteins. The motif contains two zZinc-binding sites at either end of an alpha-helix that are stabilized by the presence of seven invariant cysteine residues, and an invariant histidine residue . A pointIn total, the protein ligand binding pocket is composed of parts of the C-SET, post-SET, and I-SET subdomains. Specific analysis of the hydrophobicity and hydrophilicity of the binding pocket was conducted to inform inhibitor design, and to aid in predicting the inhibitor binding location within the pocket. If the enzyme binding pocket is thought of as a conical funnel , it was found that the bottom-most ring of the funnel consists of almost entirely hydrophobic residues F183, I237, L204, I214, Y239, and Y257. The next-highest concentric ring of the funnel consists of almost entirely hydrophilic residues Q256, Q252, S182, T184, Q192, H366, P241, and the amino/carboxyl backbone groups of I179 and Y239. This arrangement yields a polar region around the middle of the binding pocket, and an oily region at the bottom of the binding pocket that could be exploited for inhibitor design B,D. As pIn our previous testing of Inhibitor-4, we demonstrated its ability to reduce the proliferation and viability of SMYD3-positive breast cancer cells without impacting wild-type breast epithelial cells . Using sEpigenetic control is tightly regulated in humans, and aberrant epigenetic marks are associated with several disease pathologies. Histone lysine methylation is a key epigenetic regulator, and is modulated chiefly by SET-domain-containing enzymes. Specifically, SMYD3 is a SET- and MYND-domain-containing enzyme that methylates both histone and non-histone targets. The overexpression of SMYD3 has been discovered in several cancer types, including breast, colorectal, and lung carcinomas. The inhibition of SMYD3 is promising for the therapeutic treatment of these cancer types.Structural overlays of similar proteins and protein domains, such as the overlays presented here, allow for in-depth analysis of functional residues, secondary structures, and enzyme binding pockets. This analysis sheds light on the similarities and differences in catalytic mechanisms across species, and can also inform the development of inhibitors for therapeutic intervention, or the study of protein function. Notably, our analysis revealed that the I-SET domain of SMYD3 is unique among other SET proteins, closely resembling the long I-SET domain of the ancient plant methyltransferase, Rubisco-LMNT.We previously developed a small molecule SMYD3 inhibitor using a random screen in silico, and demonstrated its efficacy in limiting the growth and survival of breast cancer cells . In thiThe DLD-1 colorectal cancer cell line has been shown to express lower levels of SMYD3 than some other colorectal lines , which mWe conclude that Inhibitor-4 is a promising inhibitor of SMYD3 and SMYD3-mediated breast, lung, and colorectal cancers. The structure–activity relationships explored here will inform compound modifications, and optimization of the inhibitor. Future work will also include the development of inhibitors for other enzymes implicated in disease, with an emphasis on enzymes that modulate the epigenetic landscapes of cells.