Escherichia coli. Since the effects of interactions depend on coexpression and stoichiometries, cellular levels of all GGDEF/EAL domain proteins were also quantified and found to vary dynamically along the growth cycle. Instead of detecting specific pairs of interacting DGCs and PDEs, we discovered a tightly interconnected protein network of a specific subset or “supermodule” of DGCs and PDEs with a coregulated core of five hyperconnected hub proteins. These include the DGC/PDE proteins representing the c-di-GMP switch that turns on biofilm matrix production in E. coli. Mutants lacking these core hub proteins show drastic biofilm-related phenotypes but no changes in cellular c-di-GMP levels. Overall, our results provide the basis for a novel model of local c-di-GMP signaling in which a single strongly expressed master PDE, PdeH, dynamically eradicates global effects of several DGCs by strongly draining the global c-di-GMP pool and thereby restricting these DGCs to serving as local c-di-GMP sources that activate specific colocalized effector/target systems.The bacterial second messenger bis-(3′-5′)-cyclic diguanosine monophosphate (c-di-GMP) ubiquitously promotes bacterial biofilm formation. Intracellular pools of c-di-GMP seem to be dynamically negotiated by diguanylate cyclases  and specific phosphodiesterases . Most bacterial species possess multiple DGCs and PDEs, often with surprisingly distinct and specific output functions. One explanation for such specificity is “local” c-di-GMP signaling, which is believed to involve direct interactions between specific DGC/PDE pairs and c-di-GMP-binding effector/target systems. Here we present a systematic analysis of direct protein interactions among all 29 GGDEF/EAL domain proteins of  E. coli, together with our measurements of cellular c-di-GMP levels, challenges both aspects of this current concept. Knocking out distinct DGCs and PDEs has drastic effects on E. coli biofilm formation without changing the cellular c-di-GMP level. In addition, rather than generally coming in interacting DGC/PDE pairs, a subset of DGCs and PDEs operates as central interaction hubs in a larger "supermodule," with other DGCs and PDEs behaving as “lonely players” without contacts to other c-di-GMP-related enzymes. On the basis of these data, we propose a novel concept of “local” c-di-GMP signaling in bacteria with multiple enzymes that make or break the second messenger c-di-GMP.c-di-GMP signaling in bacteria is believed to occur via changes in cellular c-di-GMP levels controlled by antagonistic and potentially interacting pairs of diguanylate cyclases (DGCs) and c-di-GMP phosphodiesterases (PDEs). Our systematic analysis of protein-protein interaction patterns of all 29 GGDEF/EAL domain proteins of  Escherichia coli K-12 has 29 GGDEF/EAL domain proteins, including 12 DGCs and 13 PDEs -cyclic diguanosine monophosphate (c-di-GMP) activates genes involved in biofilm formation or directly stimulates the biosynthesis and secretion of the exopolysaccharides found in the extracellular matrix of bacterial biofilms. Cellular c-di-GMP is controlled by antagonistic enzymes, i.e., diguanylate cyclases (DGCs [with GGDEF domains]) and specific phosphodiesterases (PDEs [which can feature either EAL or HD-GYP domains]). Most bacterial species contain multiples of these enzymes as well as enzymatically inactive degenerate members of these protein families –4. Thus,–′-cyclic  13 PDEs . Despite 13 PDEs –9. In a 13 PDEs ––13.E. coli  and a DGC (DgcM) directly interact with each other as well as with the transcription factor MlrA. Depending on the expression and activity of yet another antagonistic DGC/PDE pair (DgcE/PdeH), c-di-GMP can accumulate and can then be bound and cleaved by the “trigger PDE” PdeR. This releases DgcM and MlrA from direct inhibition by PdeR, which allows DgcM/MlrA-driven expression of the biofilm regulator CsgD   . In ordeE. coli—present in the 12 active DGCs as well as in degenerate versions that can occur alone (CdgI) or are linked to active or inactive EAL domains—we considered and systematically tested the possibility of heterodimerization between all these GGDEF domains. Since protein-protein interactions and their potential consequences  depend on the cellular concentrations of the interaction partners, we also determined the cellular levels of all GGDEF/EAL domain proteins throughout all phases of the growth cycle of E. coli.Another relevant regulatory interaction is the homodimerization of GGDEF domains, which is absolutely required to activate DGCs and usually seems to depend on signal input via an N-terminal sensory domain , 19. HowE. coli is far more complex than was anticipated. Rather than being organized in small modules of DGC/PDE pairs that act in parallel, c-di-GMP signaling seems to operate in a complex hierarchical protein interaction network with a few hubs or master controllers. These can engage in multiple contacts to more-peripheral components, with the major physiological output of this network being biofilm extracellular matrix production. In addition, there are “lonely players,” i.e., other DGCs and PDEs not engaging in mutual interactions. Overall, the protein interaction landscape of c-di-GMP signaling revealed by our study, together with our data on the cellular c-di-GMP levels in various dgc and pde mutants, led to a new model of local c-di-GMP signaling and provided the basis for precise hypotheses on the regulation and function of distinct GGDEF/EAL domain proteins in E. coli that will be crucial for the design of future studies.Taken together, our data show that c-di-GMP signaling in a bacterium with multiple DGCs and PDEs such as in vivo, we used a bacterial two-hybrid (2H) system, in which potentially interacting proteins or domains are fused to the N-terminal domain of the phage lambda cI repressor  and to the N-terminal domain of the alpha subunit of E. coli RNA polymerase (RNAP)  .Previous studies have provided evidence that certain interacting DGC/PDE pairs can specifically affect distinct output functions , 17, 20.om pTRG) . InteracE. coli stationary-phase sigma factor RpoS (σS) and RssB, a RpoS-binding proteolytic targeting factor  show very low if any expression at all under standard conditions  or the cytoplasmic regions of N-terminally membrane-attached DGCs and PDEs were expressed from the two 2H vectors (including vector swaps). Putative interactions of all DGCs and the degenerate GGDEF domain protein CdgI with all PDEs and proteins carrying degenerate EAL domains  were assayed and normalized using the well-studied interaction between g factor , 23, as g factor  are shownditions .10.1128/mBio.01639-17.1FIG S1 E. coli K-12. The Bacterio-Match 2H system interaction data correspond to those shown and described in the legend to FIG S1, TIF file, 3.9 MB.The 2H interaction landscape of all 29 GGDEF/EAL domain proteins of Copyright © 2017 Sarenko et al.2017Sarenko et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01639-17.2FIG S2 E. coli K-12. Exemplary Bacterio-Match 2H system interaction data for PdeR combined with all DGCs and the degenerate GGDEF domain protein CdgI are shown as raw data (in CFU counts) (A) and after normalization for the strong interaction between RpoS and RssB determined in parallel (B). Corresponding data sets were generated for each of the 29 GGDEF/EAL domain proteins (data not shown) and are summarized in numerical form  for the reciprocal vector configurations as described for panels C and D. Data for DgcI and CdgI are missing in panel C because overproduction of these proteins from pTRG, which has a higher copy number than pBT, was toxic. Download FIG S2, TIF file, 4.5 MB.Graphical and numerical data sets of 2H interaction experiments with all 29 GGDEF/EAL domain proteins of Copyright © 2017 Sarenko et al.2017Sarenko et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the A principal result from this 2H analysis was the finding that DGCs and PDEs do not come in specific interaction pairs. Rather, a few DGCs and PDEs show multiple interactions in a tightly interconnected network, whereas others do not seem to make significant contacts. The small set of promiscuously interacting proteins, or “interaction hubs” , consisted of DgcE, DgcM, and DgcO as well as PdeR and PdeG  and after  normalization for RpoS/RssB interaction. The fully membrane-integrated N-terminal domains of DgcN and DgcE were not included in the analysis. A similar data set corresponding to the interaction between isolated domains of PdeR and those of DgcM was previously reported  using thCopyright © 2017 Sarenko et al.2017Sarenko et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the PdeR can undergo interactions with other GGDEF domains   . While CE. coli) . We obseE. coli) . Finally10.1128/mBio.01639-17.4FIG S4 E. coli, DgcT and DgcX, with the latter not occurring in E. coli K-12 but occurring in enteroaggregative E. coli. The isolated CdgI GGDEF domain was purified, and binding of radiolabeled c-di-GMP or GTP was assayed by UV cross-linking (B), with nonradiolabeled c-di-GMP added at the indicated concentrations. No binding of radiolabeled GTP (at the same concentration as the radiolabeled c-di-GMP) was observed (last lane). Download FIG S4, TIF file, 9.8 MB.CdgI is a c-di-GMP-binding protein. CdgI consists of a fully membrane-inserted N-terminal MASE4 domain linked to a GGDEF domain with an intact I-site but a degenerate A-site (A). The same MASE4-GGDEF domain architecture is also found in two active DGCs of Copyright © 2017 Sarenko et al.2017Sarenko et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Interaction of certain proteins can potentially play a physiological role only if these proteins have a chance to “see” each other, i.e., if they are expressed under the same physiological conditions. In addition, a significant functional consequence of direct protein interactions—whether in the form of activation, inhibition, or sequestration—depends on adequate stoichiometries of the binding partners. In order to systematically measure cellular levels of all GGDEF/EAL domain proteins, we inserted a FLAG-tag-encoding sequence at the 3′ end of all corresponding genes in the chromosome. All proteins were quantified by immunoblot analysis along the entire growth cycle of these strains in complex medium .10.1128/mBio.01639-17.5FIG S5 578 levels of 0.3, 1, and 2.5 as well as after 12 h of incubation and overnight (oN) incubation, samples were taken for immunoblot analysis (using antibodies against the 3× FLAG tag). Each row shows a representative image of cellular 3× FLAG-tagged protein levels (left panel) and the respective samples of purified C-terminally 3× FLAG-tagged PdeL applied in different amounts  as the standard protein for quantification on the same gel (right panel). Although PdeL reference samples were always run on the same gel side by side with the cellular samples, images of these reference samples were cut and ordered with respect to the ranges of their concentrations . Each experiment was done in at least two biological replicates. The asterisk (*) indicates unspecific bands, which also appeared in the control samples from unflagged W3110 on the same immunoblot. Download FIG S5, TIF file, 5.4 MB.Complete immunoblot data set for the determination of cellular levels of detectable GGDEF/EAL domain proteins. Derivatives of strain W3110 carrying 3× FLAG-tagged fusions at the 3′ ends of the GGDEF/EAL genes as indicated in the respective rows were grown in LB at 28°C. At ODCopyright © 2017 Sarenko et al.2017Sarenko et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli K-12, 25 were found to be expressed. Only four proteins remained at levels below detection: DgcT and CdgI, which are known to be subject to CsrA repression , DgcC, PdeG, PdeR, PdeB, and PdeD—and corresponds to the previously reported RpoS regulation of their genes . In cont of RpoS .lacZ fusions, which essentially reflect transcriptional activities of the corresponding genes as reported earlier , which reflects reduced levels of curli and cellulose. The ΔdgcC mutation resulted in a concentric ring morphology indicative of impaired cellulose biosynthesis but normal curli production  of 1, which was transiently raised 2-fold during the transition into stationary phase (at an OD578 of 3). This corresponds to surprisingly low cellular c-di-GMP concentrations of approximately 40 and 80 nM, respectively. In contrast, the ΔpdeH mutant reached >10 pmol/mg total cellular protein (corresponding to a cellular c-di-GMP concentration of approximately 0.6 μM). This indicates that under these conditions, which coincide with its major expression, PdeH is the major PDE affecting the overall cellular c-di-GMP pool. In contrast, the ΔdgcE, ΔdgcM, ΔdgcC, and ΔpdeR mutations—despite their equally pronounced effects on macrocolony morphology and curli and/or cellulose production—did not significantly affect the cellular c-di-GMP level in otherwise wild-type backgrounds. This is a clear indication that these three DGCs as well as PdeR act in a local manner instead of controlling the cellular c-di-GMP level.Cellular c-di-GMP concentrations were determined for mutants that showed clear-cut effects on curli and cellulose production . In the ΔpdeH mutant, we knocked out all dgc genes in the ΔpdeH background. Eliminating several DGCs reduced c-di-GMP levels by 20% to 50%  analysis, the study presented here produced a large data set corresponding to possible protein-protein and domain-domain interactions among the 29 GGDEF/EAL domain proteins of E. coli K-12. 2H system-based approaches are known to generate certain fractions of false positives. On the other hand, they also miss many relevant interactions and may even systematically underestimate numbers of “real” interactions  proteins interconnected by multiple interactions in a c-di-GMP signaling supermodule network (As demonstrated here for  network  and (ii) network —serve as network .E. coli. The role of the two other multiconnected proteins of the supermodule core, i.e., DgcO and PdeG, is currently less clear, and their input into the c-di-GMP supermodule may be conditional.Despite our systematic and unbiased collection of protein interaction data, which did not take into account any previous knowledge about specific protein functions, the hyperconnected five-protein core of the supermodule network turned out to include precisely those components—i.e., DgcE, PdeR, and DgcM—that make up the c-di-GMP-driven switch which turns on CsgD expression and thereby the production of the biofilm matrix components curli and cellulose . This isdgcE mutation, just as the pdeR and dgcM mutations, has a major effect on matrix production  were observed for a highly nonrandom pattern of 12 pairs among the 171 possible heteromeric combinations , this may directly affect its DGC activity. For degenerate GGDEF domains such as GGDEF domains . Under s domains  and overis toxic  and C. Uis toxic , which rDgcT and the degenerate GGDEFCsrD domain   , 42. ThuE. coli differ by more than 3 orders of magnitude  they do so in a highly dynamic equilibrium in which PdeH counteracts the DGCs that are expressed; and (iii) these DGCs are in an active state—i.e., activation via their N-terminal domains does not need any rare or exotic signals.When y phases , c-di-GMy phases . In cont E. coli —had a st E. coli ; it was  E. coli . This obdgcE mutation does not alter the cellular c-di-GMP level in the presence of PdeH but reduces cellular c-di-GMP levels 4-fold in a pdeH mutant background (Kd) value for c-di-GMP binding by various effectors is usually in the high nanomolar to low micromolar range  DgcM  DgcO is a member of a specific degradosome complex that also includes the c-di-GMP-controlled PNPase  DgcM , 14 and d PNPase , 16; andd PNPase . Identifd PNPase , 51. Witoduction . Even inGMP pool  and yet GMP pool .In conclusion, our systematic analysis of all 29 GGDEF/EAL domain proteins has provided novel evidence for highly specific local c-di-GMP signaling by (i) revealing a tightly interconnected protein network of a specific subset or “supermodule” of DGCs and PDEs that also show functional and regulatory association and (ii) demonstrating a lack of correlation between global cellular c-di-GMP levels and the phenotypes generated by knocking out these DGCs and PDEs. Our results led to a novel concept of local c-di-GMP signaling and, as discussed above, also provide the basis for numerous detailed hypotheses on the molecular mechanisms involved. These can now guide future studies that will focus on particularly interesting members of this network, such as, e.g., DgcE, DgcO, and CdgI and also DgcC, with its highly specific control of cellulose biosynthesis.E. coli K-12 strains W3110 (bcsQ (which in the cellulose-negative W3110 is the stop codon TAG) was changed to the sense codon TTG , which is >90% P1 cotransducible with bcsQwt  and which can be flipped out using FLP recombinase  are full orf deletion/resistance cassette insertions generated in W3110 and were previously described (pdeL::cat is a full orf deletion/resistance cassette insertion constructed via one-step inactivation (csgB::lacZ reporter fusion was described before (att(λ) location of the chromosome of a W3110 derivative containing a Δlac(I-A)::scar deletion via phage λRS45 -flanked resistance cassettes initially introduced in various genetic constructs were eliminated using FLP recombinase , and theCells were grown in LB medium  under coE. coli macrocolony biofilms were visualized at ×10 magnification with a Stemi 2000-C stereomicroscope . Digital photographs were taken with an AxioCam ICC3 digital camera coupled to the stereomicroscope, which was operated using AxioVision 4.8 software (Zeiss).HIS3 gene. This suppresses histidin auxotrophy of the E. coli reporter strain (a derivative of XL1-Blue MRF′) in a manner that can be fine-tuned by adding the His3 inhibitor 3-amino-1,2,4-triazole (3-AT) (The Bacterio-Match II two-hybrid system (Agilent Technologies) uses hybrid proteins linked to the NTD of lambda cI (expressed from pBT) and to the bacterial RNA polymerase alpha-NTD (expressed from pTRG), with coexpression of interacting proteins leading to expression of the e (3-AT) . If not d-thiogalactopyranoside), 25 µg/ml chloramphenicol, 12.5 µg/ml tetracycline, and several supplements as described in the manual. In parallel, 200 µl was plated on selective plates also containing 3-AT. When cells grew insufficiently even on nonselective plates, they were plated at a higher titer. Cells on nonselective plates were grown for 24 h at 37°C. Cells on selective plates were grown for 24 h at 37°C followed by 48 h at 28°C. The colonies obtained were counted, and the ratio of the CFU counts on selective plates to the counts on nonselective plates was calculated. Interaction activity was normalized to the strong and well-studied interaction of the sigma factor RpoS and its proteolytic targeting factor RssB  potentially leading to growth on selective minimal media (lacking histidine), a 5 mM concentration of the competitive inhibitor 3-AT was added to the plates. For a qualitative readout, cotransformants first obtained on nonselective plates can be patched on selective plates, with growth of the patches being only visually assessed. Alternatively, for a more quantitative readout, cotransformant colonies can be obtained and counted directly on selective plates relative to nonselective plates . For thetor RssB , which wlacIqΔlacZYA::scar and ΔpdeL::scar mutations. Cells were grown at 37°C to an OD578 of 0.1, IPTG (0.1 mM) was added, and incubation proceeded for 3.5 h. Cells were harvested and resuspended in immunoprecipitation (IP) buffer  that included proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) (1 mM) and were sonicated in a cooling bath. Insoluble material was removed by centrifugation. IP buffer/PMSF was added to the supernatant to reach a final volume of 750 µl, and the reaction mixture was mixed with 250 µl of a magnetic microparticle suspension (Sigma) for preadsorption and incubated for 3 h at 4°C under conditions of rotation. The tubes were placed in a magnetic separator, and the supernatant was transferred into a new tube containing 50 µl (packed gel volume) of an anti-FLAG M2 magnetic bead suspension (Sigma) previously equilibrated once in TBS  and twice in TBS–5% milk powder (TBSM). The immunoprecipitation of FLAG fusion protein was carried out overnight at 4°C under conditions of rotation. Washing steps and elution of FLAG fusion protein from the magnetic beads by competition with the 3× FLAG peptide (Sigma) were performed according to the anti-FLAG M2 magnetic bead technical bulletin (Sigma).C-terminally 3× FLAG-tagged PdeL, for use as the standard for protein quantification on immunoblots, was purified from a pCAB18-derived plasmid  in a W31E. coli Fi8202  was added, and incubation continued for 4 h. Cells were harvested and lysed by passage through a French press. The soluble protein fraction was used for protein purification according to a standard protocol . Proteins were dialyzed in diguanylate cyclase reaction buffer .The N-terminally His6-tagged cytoplasmic GGDEF domain of CdgI was purified from a plasmid derived from pQE30xa (Qiagen) in i Fi8202 . Strains578 levels of 0.3, 1, and 2.5 and after 11 or 12 h of incubation and overnight [oN] incubation). Samples corresponding to up to 320 µg of total cellular protein were precipitated with 10% trichloroacetic acid (TCA) for at least 20 min on ice. After washing was performed once with ice-cold acetone, the protein pellets were resuspended in SDS-PAGE sample buffer and incubated for 10 min at 70°C and for 10 min at 100°C. Samples were adjusted such that all samples derived from the same culture had similar amounts of total protein. These were then loaded along with different concentrations of the purified standard protein  and run on 10% SDS–polyacrylamide gels. 3× FLAG fusion proteins were detected by immunoblotting as previously described  or [α-32P]GTP  to purified proteins in vitro was assayed by UV cross-linking according to the method described in reference Binding of [α-578 level, culture samples were harvested, concentrated into 1.5 ml LB, and pelleted again . Sample extraction and analysis of c-di-GMP by liquid chromatography/tandem mass spectrometry (LC-MS/MS) were performed as described previously  as a substrate and is reported as millimoles of o-nitrophenol per minute per milligram of cellular protein .Protein sequence alignments were performed with Clustal Omega and phylogenetic analyses with Simple phylogeny (both accessible at


The difference in the carbon content affected mainly the difference in the phase fractions before deformation, where the higher carbon content increased the phase fraction of retained austenite (γ). However, the changes in the relative fraction of martensitic transformation with respect to the applied strain were found to be similar in both steels since the carbon concentrations in γ were similar regardless of different carbon contents. The phase stress of martensite was found much larger than that of γ or bainitic ferrite since the martensite was generated at the beginning of plastic deformation. Stress contributions to the flow stress were evaluated by multiplying the phase stresses and their phase fractions. The stress contribution from martensite was observed increasing during plastic deformation while that from bainitic ferrite hardly changing and that from γ decreasing.Two TRIP-aided multiphase steels with different carbon contents (0.2 and 0.4 mass%) were analyzed  TRIP-aided multiphase steels (TRIP steels) contain several tens of percent of metastable retained austenite (γ) that transform to martensite (α′) during deformation, leading to the improvement of strength and ductility6. There have been many studies concerning the effects of phase fraction, stability of γ, and chemical composition to the TRIP behavior of TRIP steels11. The martensitic transformation behavior and the stability of γ have been reported to show strong relations with the strain rate and temperature16. The studies of the effect of carbon content have been also performed using TRIP steels containing carbon of 0.2 and 0.4 mass% by tensile tests, and have predicted that load partitioning to the γ was largely concerned in the difference in deformation induced martensitic transformation due to the different carbon content16. However, these studies remained unclear, because the qualitative prediction were interpreted only from the observation using the stress-strain curves as done in many studies16, and the quantitative measurements of phase stresses were not performed.Transformation Induced Plasticity (TRIP) is well known as one of important effects in steel strengthening mechanismin situ neutron diffraction (ND)19 and X-ray diffraction (XRD) measurements21 during deformations have been confirmed as powerful tools. In situ ND studies during tensile deformation of TRIP and TWIP steels have been conducted using time-of-fight method25 and also angular dispersion method27. In previous reports26, the strengthening in TRIP steels was discussed mainly as a load partitioning only between bainitic ferrite matrix and γ, though the occurrence of martensitic transformation during deformation was confirmed from the decrease of phase fraction of γ. Because γ was found to be harder than bainitic ferrite, it was concluded that γ acts as an effective reinforcement responsible for large macroscopic stresses characteristic22. Meanwhile, Jacques et al.27, performed peak separations between bainitic ferrite and α′ by careful peak analyses on angular dispersion ND patterns with good statistics, and determined successfully phase stresses for bainitic ferrite, γ and α′. The α′ phase stress was found to be the largest, and its contribution to the strength was confirmed to be large enough though the phase fraction was small27. The phase fraction evolution was, however, not evaluated in situ during the deformation test for ND experiment. The phase fraction values measured using microscopy analysis and XRD in several deformed states were used to extrapolate the phase fractions to the deformation conditions of ND experiments. Lattice strains of α′ formed by the transformation were also successfully observed in a TRIP ultra-fine grained (TRIP-UFG) steel, when a neutron time-of-flight (TOF) diffractometer with high resolution was used for the measurement24. Decreases in the integrated intensities were observed in all γ-hkl peaks during Lüders deformation in the beginning of tensile deformation, where the deformation induced martensitic transformation preferentially occurred. Discussion concerning the validities of lattice strains and the quantitative evolution of phase stresses and phase fraction was however not performed.To understand the strengthening mechanism of multiphase steels during deformation, in situ TOF ND under continuous tensile deformation. The chemical compositions are shown in Table In this study, tensile deformation behavior and deformation induced martensitic transformation in two TRIP steels with different carbon contents (Steel A and Steel B) and moderate grains (3~9 μm for ferrite and 1~4 μm for γ) see Fig.  were stuin situ ND experiments during tensile loading. The Lüders deformation that was reported in the TRIP-UFG steel24 was not observed, which might be due to the larger carbon concentration in γ (σt) or true strain (et) shown in Fig. σ) or nominal strain (e) according to Figure mentclass2pt{minimin situ ND during tensile deformation with a displacement-stepwise manner, was suppressed. The larger carbon content led to increases in the elastic limit and the flow stress, while a decrease in the uniform strain. The work hardening rates inserted in Fig. Because the tensile deformation in plastic regime was conducted continuously, the stress relaxation that is often observed in the fγ) in Steel A and Steel B that were obtained from the Rietveld refinements of the ND patterns obtained before deformations. The fγ values are in good agreement with those obtained from the OM and the XRD measurements. Distinctions between ferrite and bainite in the ND patterns were difficult, because they have the base-center-cubic (BCC) structure and the similar lattice constant. They were assumed to behave as one phase which is called bainitic ferrite (α) from here after. The refined lattice parameters before deformation can be used to estimate the 28, asTable aγ,0 and aα,0 are the undeformed lattice constants of γ and α, respectively. The evaluated 24 having a carbon content similar to Steel B, the fγ values in the specimens used in this study were much smaller, whereas the Here, entclass1pt{minima30. Weak rolling textures were already found before deformation in both specimens that might be introduced during the sample preparation. The IPF images in 20% deformed states  were evaluated by normalizing the hkl integrated intensities obtained during deformation to those obtained before deformation and to specimen cross section reductions during deformation. Figure Irel values of several γ-hkl peaks for Steel B. The Irel values were almost unchanged during deformation in elastic regime regardless of the <hkl> and specimen orientation   evaluated in Steel A and Steel B. The fγ values decreased almost linearly with respect to the applied true strain. The fγ values remain much larger than zero though the specimens were broken. When the fα′ values were normalized to the fγ value before deformation, relative fractions of martensitic transformation  were evaluated and are plotted in Fig. fmt,rel for both specimens lay on the same line or curve, i.e., the increase rates of fmt,rel to the applied true strain in Steel A and Steel B were the same, except values for Steel B in a large strain region above 0.19. The final fmt,rel value was larger in Steel A  generated by the transformation in the α′ grains.The intergranular strains were clearly observed in the Phase stresses can be evaluated from phase strains according to the Hooke’s law, using the elastic constants and the Poisson’s ratios. Due to the geometry of the tensile test, which involves Eγ, Eα or Eα′ is the Young’s modulus for γ, α or α′, respectively. In this study, the values of 38 and BCC40 polycrystalline materials, respectively. The values of Here, fγ that caused larger stresses to yield macroscopically. The CPSMγ), because the appearance of α′ was confirmed at this stress level. The CPSMγ values in both specimens were similar. These experimental results, with also considering that the increase rates of fmt,rel to the applied true strain in Steel A and Steel B were similar  were studied using Two TRIP steels, consisting of different carbon content, were used in this study. The steels were prepared by vacuum melting, heat-treatment at 1473 K for 1.8 ks, hot rolling to 8 mm thickness, and cold rolling to 2.2 mm thickness. The steels were then solution treated at 1073 K for 180 s, air cooled to 998 K, rapid cooled to 673 K and subsequently held for 180 s, and then air cooled to RT, to produce microstructures containing ferrite, bainite and γ. Specimens used in this study were made from the steels after removal of 0.2 mm thick parts from both surfaces.in situ ND experiments during tensile deformation at room temperature. The tensile loading direction was adjusted being parallel to the rolling direction. To monitor the applied strain and the localized deformation that might occur, two strain gauges with 2 mm gauge length were glued on both sides of 6 mm wide surfaces in such a way that they were separated each other for about 25 mm. The experiments were conducted at TAKUMI44, a high resolution and high intensity TOF neutron diffractometer for engineering materials science at Materials and Life Science Experimental Facility of Japan Proton Accelerator Research Complex. The specimen was mounted horizontally in a loading machine which was installed at TAKUMI, in such a way that the ND patterns in the axial and transverse directions were measured simultaneously using two detector banks with the scattering angles of ±90° (see Fig. 2 and radial collimators viewing 5 mm wide, i.e., an average data from the center area of parallel part was obtained. The tensile deformation was conducted in a step-load controlling manner with 300 s holding in elastic regime, and in a continuous manner with a constant crosshead speed with an initial strain rate of 1.8 × 10−5 s−1 in the plastic regime. Diffraction patterns for the plastic regime were then extracted periodically with 300 s accumulating time for different applied strain or stress values.Plate specimens with the parallel part of 55 mm long, 6 mm wide and 1.8 mm thick were prepared for see Fig. . The ND 45. Phase strains and macroscopic lattice strains during loading tests are often estimated using average lattice constants determined from the Pawley method or Rietveld refinement37, but sometimes the intergranular stresses are difficult to be absorbed38 and the positions of peaks with large intensities become dominant contributions to the lattice constants30. In this study, the evaluation of lattice strain was performed by a single peak fitting using the Z-Rietveld, and three peaks were selected for each constituent phase. A convolution function of the pseudo-Voigt function and the rise-decay function was used to fit the peak profile (see Fig. 46 and ND studies24. The parameters of rise-decay function were fixed to keep the instrumental peak shape to be similar for α and α′, while the parameters of pseudo-Voigt function were refined during fitting. These achievements were not only because of the high resolution of neutron diffractometer used in this study, but also because of the high beam intensity that realized continuous plastic deformation to suppress the stress relaxation in the plastic regime. However, the peak separations were possible only for the patterns in the axial direction. The peaks of α and α′ in the patterns in the transverse direction became close each other due to the Poisson’s effect, and the peak separations became difficult to perform.Data analyses were performed by a single peak fitting method and a multi peak fitting method using a Rietveld software called Z-Rietveldsee Fig. . Peak seSupplementary information


Soft white wheat is used in domestic and foreign markets for various end products requiring specific quality profiles. Phenotyping for end-use quality traits can be costly, time-consuming and destructive in nature, so it is advantageous to use molecular markers to select experimental lines with superior traits. An association mapping panel of 469 soft white winter wheat cultivars and advanced generation breeding lines was developed from regional breeding programs in the U.S. Pacific Northwest. This panel was genotyped on a wheat-specific 90 K iSelect single nucleotide polymorphism (SNP) chip. A total of 15,229 high quality SNPs were selected and combined with best linear unbiased predictions (BLUPs) from historical phenotypic data of the genotypes in the panel. Genome-wide association mapping was conducted using the Fixed and random model Circulating Probability Unification (FarmCPU). A total of 105 significant marker-trait associations were detected across 19 chromosomes. Potentially new loci for total flour yield, lactic acid solvent retention capacity, flour sodium dodecyl sulfate sedimentation and flour swelling volume were also detected. Better understanding of the genetic factors impacting end-use quality enable breeders to more effectively discard poor quality germplasm and increase frequencies of favorable end-use quality alleles in their breeding populations. Triticum aestivum L.). Generally, hard wheat flours have higher gluten strength, damaged starch, and non-starch polysaccharides that lead to increased water absorption capacity, whereas soft wheat flours have lower gluten strength, damaged starch, and non-starch polysaccharides that lead to decreased water absorption capacity , water absorption, protein (gluten) strength, and milling quality differentiate the end-use quality of wheat , and their corresponding x- and y-type subunits account for 47-60% of the variation in bread making quality of wheat  on chromosomes 4A, 7A, and 7D control starch amylose composition in wheat, a key factor in white salted noodle texture  and the low molecular weight (LMW) loci on the long and short arms of chromosome 1A, 1B and 1D  can be detected in a panel of soft white winter wheat adapted to the Pacific Northwest and that SNPs with pleiotropic effects on numerous end-use quality traits will also be identified. The objective of this study was to assess a regionally adapted germplasm panel for significant MTAs to several end-use quality traits. These results should further elucidate the genetic factors controlling soft wheat end-use quality.This association mapping panel was developed using 480 advanced soft white winter wheat breeding lines and released cultivars from Pacific Northwest breeding programs  selected from 1992 to 2014. Experimental lines were included, if they had at least six environmental observations in the Wheat Analysis System database from the USDA-ARS Western Wheat Quality Laboratory in Pullman, WA. The population contained lines with either a club or a lax head type. Club, Soft White (lax head), and Western White (a mixture of the two) are the three market sub-classes traded in commerce. Out of the 480 original lines in the panel, only 469 lines were eventually used in the association analysis. Three were phenotypically identified as hard white winter lines and an additional eight lines were excluded from the analysis due to poor DNA quality or missing marker covariate information. The degree of relatedness between these 469 lines is presented in Supplementary File Historic phenotypic data on all end-use quality traits were obtained from the Wheat Analysis System database in the USDA-ARS Western Wheat Quality Laboratory in Pullman, WA, USA. Environments were considered to include both crop year and field locations, for a total of 462 individual environments. These field locations included sites in Montana, Idaho, Oregon, and Washington from 1992 to 2014. Each genotype was represented by a single sample per environment. The dataset was unbalanced; not all experimental lines were grown in all environments, except for one location in 2014, where the entire association mapping panel was grown in one complete field replication in Pullman, WA.Testing procedures from the American Association of Cereal Chemists International  sedimentation test assessed gluten strength of flour samples (Approved Method 56-60.01), and the flour swelling volume test assessed the starch composition of flour samples (Approved Method 56-21.01). The four solvent retention capacity (SRC) tests assess different aspects of end-use quality using the following solvents: water, lactic acid, sucrose, and sodium carbonate. These different solvents provide insight into certain end-use quality traits: the water SRC measures overall flour absorption, the lactic acid SRC measures glutenin quality, the sucrose SRC measures non-starch polysaccharides and gliadins, and the sodium carbonate SRC measures damaged starch content Gaines, .Smaller-scale laboratory tests for dough rheology and overall end-use quality are especially useful to differentiate between the quality traits of later generation breeding lines. In soft wheat, sugar snap cookie diameter (Approved Methods 10-50.05) and Japanese sponge cake volume  and these were removed prior to any statistical analyses. The summary statistics  were calculated for each phenotypic trait with Proc UNIVARIATE in SAS 9.3  Table . Due to h2) was calculated as the ratio of the additive variance to the total phenotypic variance using the R package rrBLUP  between alleles at two loci according to Weir . The intersection of the LOESS curve fit and critical value of r2 was considered as the estimate of the extent of LD.Linkage disequilibrium was calculated using 15,229 annotated markers and MAF greater than 5% in JMP Genomics 6.0. LD for markers on the same chromosome was measured using the squared allele-frequency correlation  calculated in GAPIT2 .Prior to association analysis, the SNPs were filtered to exclude those with greater than 20% missing data and those with a minor allele frequency (MAF) < 0.05 leaving a total of 15,229 high quality SNP markers. The remaining missing data were imputed using BEAGLE 3.3.2  glutenin data were added into the GWAS model as covariate when the trait was significantly influenced. The proportion of explained phenotypic variance by the SNP was calculated as follows:Due to the diverse backgrounds of the lines included in the association mapping panel and the large influence that the high molecular weight (HMW) glutenins can have upon soft wheat end-use quality, the panel was screened for alleles at yi is the observed phenotype value, and where h2) estimates ranged from 16 to 89%. High h2 traits (above 70%) included kernel hardness, break flour yield, lactic acid SRC, water SRC and cookie diameter. Low h2 traits (below 35%) included grain protein, flour protein and most of the baking parameter traits except for cookie diameter.Significant phenotypic variation was present in this association mapping panel for the measured end-use quality traits Table . Each ofr = 0.99) was observed between mixograph peak time and mixograph width. All milling traits were positively correlated to each other and ranged from r = 0.58–0.79. Cookie diameter was significantly correlated with all traits except test weight. Cookie diameter was positively correlated with all milling traits, and additionally flour swelling volume, and had a negative correlation with all the other traits.Significant phenotypic correlations, either positive or negative, were observed within and across trait categories (Supplementary Table r = 0.88) (Supplementary Table r = 0.61 with lactic acid SRC and r = 0.49 with flour SDS). On the other hand, sucrose, carbonate and water SRC had negative PC2 values. Carbonate and sucrose SRC relate to the level of starch damage and arabinoxylan content, respectively. Water SRC estimates global water absorption, with higher values indicating among other things, more starch damage. These traits had positive correlations, but carbonate and water SRC were the most highly correlated (r = 0.78).The complexity of the correlation matrix for all end-use quality traits was visualized using PCA Figure . The firGlu-D1 2+12) compared to 16% with the Dx5 + Dy10 subunit , 17% of which had r2 > 0.2. Genome-wide LD decay was observed using a scatterplot of LD (r2) plotted against inter-marker distance (cM)  showed a substantial genetic differentiation between subpopulations, which was also consistent with their genetic distances , 3B (IWB18115) and 5A (IWB4446) explained up to 9% of the phenotypic variation. More than 80% of the genotypes carried the alleles for higher test weight in IWB18115 and IWB4446, whereas, only 21% had the favorable allele of IWB77391  were observed in chromosomes 2B (IWB30179) and 4A (IWB51337), together accounting for 16% of the trait variation. An MTA that explained 6% of the variation in kernel hardness was detected in chromosome 5A (IWB80508). The majority of both the club and lax head-type sub-groups  carried the allele for softer kernel.Thirteen MTAs for grain characteristics were detected across nine chromosomes Table . Six MTAB IWB1811 and 5A (IWB66086 at 110.1 cM on 4A accounted for most of the variation (11%) in break flour yield as well as 8% of the variation in carbonate SRC and in cookie diameter. Other markers associated with break flour yield included IWB45033 (R2 = 0.09) at 40.6 cM on 5B and IWB54370 (R2 = 0.05) at 55.6 cM on 7B. IWB78525, an MTA for milling score on 5B, was 2 cM from the break flour yield MTA. Four out of five MTA for higher flour yield had R2 values ≥ 20%, and two of these, IWB9175 and IWB27057, were positioned in the short and long arms of chromosome 1B, respectively. Aside from flour yield, IWB27057 was also strongly associated with water SRC (R2 = 0.22). The strongest MTA (IWB60199) for flour yield was positioned at 68 cM on 4B and the allele for higher flour yield was highly represented (84%) in the club wheats , flour yield (5) and milling score (1) (Table IWB3277 (1D) and IWB57207 (7B) were both associated with flour SDS sedimentation and lactic acid SRC. IWB3277, on the short arm of chromosome 1D, accounted for at least 44% of the phenotypic variation in these traits and the alleles for lower values were present in 74% of the genotypes (Supplementary Table IWB14950 (79.8 cM) and IWB56695 (78.5 cM) on chromosome 1B were also associated with flour and lactic acid SRC, respectively. The MTA for water SRC with the greatest effect (R2 = 0.27) was positioned at 26.5 cM on chromosome 5A (IWB10273). Water SRC and cookie diameter shared a locus on chromosome 1B at 8.4 cM (IWB7122) explaining 9-12% of the phenotypic variation. The favorable allele for flour ash (lower ash content is preferred) on chromosome 1A (IWB35825) was present in only 6% of the genotypes, mostly coming from the Oregon State University wheat breeding program (Supplementary Table IWB31459) and the allele associated with lower protein was present in 79% of the genotypes. A large effect flour protein MTA was positioned at 138 cM on chromosome 5A and explained 20% of the phenotypic variation. The minor alleles of the four MTAs forThe number of marker-trait associations for flour quality traits were greatest for flour SDS sedimentation, lactic acid SRC and water SRC, with 5 SNP markers each Table . IWB3277IWB35089 (1B), had the largest effect R2 = 0.22  to capture a wider range of performance in end-use quality traits that represented past and current soft winter wheat germplasm in the Pacific Northwest. The use of BLUPs accommodated departures from normality, while at the same time accounting for year and environmental effects that are common in unbalanced multi-location trials  for single kernel hardness on chromosome 5A. A similar locus in the same region on chromosome 5A, identified by wPt-1165, was also detected by Bordes et al. (IWB80740) was < 1 cM on the consensus map to a kernel hardness QTL (QHa.wak) identified in a bi-parental population derived from a cross between the club head cultivar “Coda” and the lax head cultivar “Brundage”  for flour yield near (~4 cM) IWB80508 on chromosome 5A which explained about 20% of the phenotypic variation. In addition, co-localized MTAs for single kernel hardness and water absorption SRC were also detected at 57 cM on chromosome 6BS.The main goal of this study was to dissect the underlying genetics controlling end-use quality traits of soft wheat, a class of wheat that is different from the hard wheat preferred for baking bread  was also reported in Sun et al. (IWB77420) for kernel weight and size is only 28%  locus for club head type. IWB77420 was 2 cM proximal to the flanking marker of the C locus, wmc144  on 5AL was associated with both traits and is close (less than 3 cM) to a previously reported flour QTL (QFpro.wak)  and milling score (IWB78525) were identified less than 2 cM apart in chromosome 5B. A QTL for kernel hardness (QHa.wak) was also reported in this region and contributed to variation in break flour yield , which explained 27% of the variation in flour yield, was represented in 17 and 84% of the genotypes with lax and club head types, respectively (Table Rht-B1) on 4B, we do not think there is a pleiotropic effect of dwarfing genes on flour yield. The majority (85%) of lines with lax head type carry the semi-dwarf allele Rht-B1b, mainly because this allele has historically been used in combination with selection for better emergence potential in lower rainfall regions of the Pacific Northwest. The club wheat lines, which have been bred and selected for higher flour yield, have a higher predominance of the semi-dwarf allele Rht-D1b, mainly because they have not undergone the intense selection for better emergence as the lines with lax heads have. Thus, although there is a correlation between dwarfing genes and flour yield, we do not believe it is causal.Milling performance directly translates into greater profit margins for flour millers. Hence, breeding programs aim for soft wheats with high break flour yield, flour yield, and milling score. The moderate to high heritability estimates and positive correlations among milling traits suggest simultaneous progress from selection can be achieved. Selecting alleles that increase these traits could lead to higher milling scores  in chromosome 1B. Soft wheats with lower water absorption are important in baking cookies. Upon further validation, these MTAs can be used to select soft wheats with improved total milling performance.To our knowledge, the two large effect MTAs for total flour yield on the short and long arms of chromosome 1B have not been reported, especially r = 0.87), which are both predictors of gluten strength. MTAs for flour SDS sedimentation and lactic acid SRC were detected on chromosomes 1BS and 1DS, respectively, near known glutenin genes Glu-B1 and Glu-D3, respectively  for total flour yield which was also detected in this region was consistent with the significant correlations among these traits. No additional MTAs were detected in the region of Glu-D1 because we included variation at Glu-D1 as a covariate in the association analysis. The MTA (IWB57207) on chromosome 7BS is potentially a novel locus controlling flour SDS sedimentation and lactic acid SRC. The high frequency of this MTA in this panel makes it a valuable resource for introducing new alleles with diverse genetic backgrounds.The strength of correlation among pairs of SRC traits were consistent with the clustering of MTA in certain genomic regions such as flour SDS sedimentation and lactic acid SRC (IWB35842), associated with flour ash on chromosome 1A, was likely the same as the QTL for flour ash identified from a double haploid mapping population  associated with flour swelling volume on 5A is potentially an important locus due to its large effect and availability especially in genotypes with lax head type.Water SRC co-localized with other quality traits on 1BS and 1BL, and with cookie diameter and total flour yield. Higher water SRC is partly a consequence of starch damage from milling and non-starch polysaccharides, including arabinoxylans  of the MTAs for cookie diameter, however, illustrated the complexity of this trait. A combination of different MTA from the traits mentioned above would be an effective strategy to achieve substantial gains. A regression analysis was used to model cookie diameter using only phenotype data, markers, or a combination of both. There was a 7% improvement in the R2 of the model when combining marker and phenotypic data in predicting cookie diameter , making it easier to test and determine the value of these MTAs in different breeding programs. Typically, end-use quality assessment is conducted later in the breeding cycle because these tests are time consuming and require a large amount of grain. MTAs with larger effects can potentially be used in marker-assisted selection to increase selection efficiency by reducing the time and cost in screening a larger number of plants. But for markers with minor effects, MAS would be inferior to phenotypic selection.One advantage of performing association mapping in elite and adapted lines rather than on a diversity panel is the opportunity to directly utilize the identified alleles in regional breeding programs. Genotypes that carry the favorable alleles for different end-use quality MTAs can be cycled back into breeding programs as parental material to exploit transgressive segregation, pyramid desirable alleles, and ensure potential genetic gains in succeeding generations. KASP markers of the reported MTAs are commercially available (Genomic selection is a breeding method used to predict the breeding value of individual genotypes using genome-wide markers (Bernardo, KJ: Analyzed the phenotypic data; JG: Analyzed the phenotypic and genotypic data and drafted the manuscript; MH: Performed GWAS; YZ and ZZ: Provided expert guidance in performing GWAS; CM, KG-C, ZZ, and AC: Edited the manuscript; CM and AC: Obtained funding for the project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The government of India has, over the past decade, implemented the “integration” of traditional, complementary and alternative medical (TCAM) practitioners, specifically practitioners of Ayurveda, Yoga and Naturopathy, Unani, Siddha, Sowa-rigpa, and Homoeopathy (collectively known by the acronym AYUSH), in government health services. A range of operational and ethical challenges has manifested during this process of large health system reform. We explored the practices and perceptions of health system actors, in relation to AYUSH providers' roles in government health services in three Indian states – Kerala, Meghalaya, and Delhi. Research methods included 196 in-depth interviews with a range of health policy and system actors and beneficiaries, between February and October 2012, and review of national, state, and district-level policy documents relating to AYUSH integration. The thematic ‘framework’ approach was applied to analyze data from the interviews, and systematic content analysis performed on policy documents.We found that the roles of AYUSH providers are frequently ambiguously stated and variably interpreted, in relation to various aspects of their practice, such as outpatient care, prescribing rights, emergency duties, obstetric services, night duties, and referrals across systems of medicine. Work sharing is variously interpreted by different health system actors as complementing allopathic practice with AYUSH practice, or allopathic practice, by AYUSH providers to supplement the work of allopathic practitioners. Interactions among AYUSH practitioners and their health system colleagues frequently take place in a context of partial information, preconceived notions, power imbalances, and mistrust. In some notable instances, collegial relationships and apt divisions of responsibilities are observed. Widespread normative ambivalence around the roles of AYUSH providers, complicated by the logistical constraints prevalent in poorly resourced systems, has the potential to undermine the therapeutic practices and motivation of AYUSH providers, as well as the overall efficiency and performance of integrated health services. •The roles of AYUSH (TCAM) providers in public facilities in India are perceived discrepantly.•Work sharing is dissimilarly interpreted by different health system actors.•Interactions among practitioners of different systems of medicine are fraught.•Logistical, infrastructural, technical, and administrative provision for TCAM is low.•Policy and budgetary support for TCAM are inadequate for field development. This unit, originally established as the Department of Indian Systems of Medicine and Homoeopathy in 1995, was renamed the Department of AYUSH in 2003, and governed the provision and practice of Ayurveda, Yoga and Naturopathy, Unani, Siddha and Sowa-Rigpa, and Homoeopathy (AYUSH) in India. It was elevated to a Ministry in November 2014. A Draft National Policy on AYUSH is in development in 2016 .The Over the years, the integration of AYUSH providers into the public health system of India has proceeded in different ways, and to varying extents in the different states of India, partly due to different state interpretations of the policies. Integration as policy and health systems reforms requires attention to health goals and stakeholder roles, multi-level reform, and a reorientation of systems values . ReportsWe conducted a study in three states of India to examine the operational and ethical challenges of AYUSH mainstreaming. The integration of the different systems of medicine in the public health system has at its centre the practitioners of the different systems of medicine. This paper presents findings on health policy and system actors' practices and perceptions related to AYUSH providers’ roles in government health services.22.1The protocol for this study received ethics approval from the Institutional Ethics Committee of the Public Health Foundation of India. The study was conducted in Kerala, Meghalaya, and Delhi. These states were chosen based on their: history of TCAM practice; the entrenchment and cultural consonance of certain systems of TCAM in their communities; differing administrative set-ups for the governance of AYUSH practice; and proximity to the centre of national policymaking in New Delhi. Kerala administers Ayurveda and Homoeopathy through distinct directorates, and does not co-locate AYUSH and allopathic practitioners. In contrast, in Delhi and Meghalaya, co-location of AYUSH and allopathic practitioners is common, although separate facilities for the different systems of medicine also exist. Certain AYUSH systems have an enduring presence in Kerala and Delhi, whereas several local healing traditions, such as Khasi and Garo medicine, rather than AYUSH systems, are inherent in Meghalaya .2.2We applied an action-centred approach of policy implementation analysis  in whichWe employed two principal techniques of data-collection: in-depth interviews; and review of policy documents. In addition, researchers’ observations of the infrastructural arrangements and interpersonal interactions in the healthcare facilities were documented, and explored further in the interviews.Reviewed policy documents included: stated national, state, and district policies for the mainstreaming of AYUSH; inter-office and intra-office memoranda on transfers, posting, in-service training, facilities, and grievances related to AYUSH personnel and supplies; and publicly available material on the internet. We mapped policy content using a framework developed for the assessment of governance architecture, functions, and policy and implementation gaps in an examination of regulation of healthcare in India .Interviewees were drawn from a range of health policy and system actors involved in the mainstreaming of AYUSH, selected purposively based on principles of maximum variability , in termInterviews were audio-recorded with the respondents’ permission, and only notes taken when permission for audio-recording was not granted. The majority of the interviews were conducted in English, and some in a mixture of English and local languages.2.3A priori codes were generated, based on the interview guide, before data collection began. Cross-coding by at least two investigators was performed on a random selection of interviews, to ensure standardization in the application of codes. To the a priori codes were added emergent codes, dealing with meanings, values and rationale, developed jointly by the research team from studying the data. Analytical codes, corresponding to operational and ethical enablers and challenges in integration, were then jointly developed by the researchers from studying the patterning of emergent themes. Following coding, data were extracted, charted, and interpreted.Systematic content analysis of the policy documents was performed. Interviews were transcribed by research assistants, and checked for accuracy by the investigators. They were then analysed using the ATLAS.ti Version 7 software (Scientific Software Development GmbH). Data were processed using the thematic ‘framework’ technique that combines both inductive and deductive approaches . A prior3This section describes the perceptions and expectations of the various health system actors of the role of an AYUSH provider, the dissimilar interpretations of work sharing of AYUSH practitioners and other health system actors, and the shortfalls in awareness of, and support for, AYUSH practice. Findings from this study relating to the facilitators and barriers of integration of TCAM providers in the public health system of India are reported elsewhere .3.1Role expectations varied by stakeholder category, as well as by region. Discrepancies among the perceptions of health system actors were greatest in regions where AYUSH providers were co-located with, and supervised by, allopathic providers, i.e., in Delhi and Meghalaya. In Kerala, where allopathic, Ayurvedic and Homoeopathic establishments were housed in separate physical facilities, and administered by distinct directorates, role expectations of AYUSH practitioners were not as much a matter of conflict. 3.1.1For AYUSH practitioners appointed to government positions on either contractual or permanent bases, the national government mandated outpatient consulting in the respective AYUSH modality. Additionally, AYUSH providers were expected to participate in the national health programmes and administer the allopathic modules contained in the programmes, following training in these specific modules . State g3.1.2“If they give me training in my field, I am open. I am comfortable in my field, right. I am not comfortable giving allopathic medicine. They want us to perform duties, like night duties, emergency, which I cannot handle … I won't do any justice to the patient, right. So I tried telling her [allopathic supervisor] this, but still it's very hard to sit down and have a talk.” {AYUSH practitioner (contract)}AYUSH practitioners entered the public health workforce expecting to practise only their own system of medicine, mostly as outpatient consulting. Many expressed a desire to admit inpatients for AYUSH treatment, and some co-located facilities reported planning such an inclusion in their wards. Besides this, AYUSH providers expected to conduct the AYUSH component of the health camps organized by their facility, and, following training, to participate in national health programmes. Most expressed disinclination to take up components of allopathic practice, conscious of their lack of expertise in allopathic medicine, and their ineligibility to practice a system of medicine that they were not qualified in (an activity known as cross-practice). They reflected on how cross-practice would compromise the quality of care they could offer patients.… paani bharne wale se batti jalwana.” [getting a water-carrier to light the lamps] {AYUSH practitioner (permanent)}“The AYUSH practitioner is analogized to a water-carrier, and the task of lamp-lighting is likened to the delivery of allopathic healthcare. This reflects expectations of service that are dissonant with the roles that AYUSH practitioners see themselves as having.The following metaphor used to describe the role of AYUSH practitioners emerged in an interview, and encapsulates this perception:“For one year after I was posted here, I did not have any medicines. And I was sitting in the same room as the allopathic doctors. So, I used to help them … take cases and prescribe [allopathic] medicines.” {AYUSH practitioner (contract)}“I believe that for us, OPD [outpatient consulting] is not as important. In the health sector, family planning is important … We have stayed up till 1 a.m., in villages, counselling people on no-scalpel vasectomy.” {AYUSH practitioner (contract)}Some AYUSH providers were willing to learn and practise allopathic prescription in outpatient consulting, and perform night duties, and obstetric and emergency services. This was particularly true of those who did not have adequate provision for AYUSH practice, and had to share the premises of their allopathic colleagues. Moreover, some AYUSH providers expressed deep commitment to promoting community health, beyond prescribing medications, either allopathic or AYUSH, and engaged proactively in public health endeavours, such as family planning counselling.AYUSH providers reflected on the different loyalties that they felt: to the welfare of patients; to the system of medicine that they were trained in; to the personnel team at the facility that they were appointed to; and to the country's public health. Many communicated frequent internal tussles among these loyalties, when inadequate provision for AYUSH practice, problematic communication, and interpersonal tension pulled them in different directions, unable to perform their roles as intended, and unwilling to compromise the care of the patient or the reputation of the professional team.3.1.3“There are local healing traditions that are part of the culture. To introduce, and more than that, to impose an alien system [AYUSH systems in addition to allopathy] is not right, when the traditional system is not being given any support.” {Key informant}Non-AYUSH TCAM practitioners, mostly local traditional healers, were largely outside the ambit of the government, national and regional, and not formally included as TCAM practitioners in the public health system, on either contractual or permanent bases.In some regions, local healers were organized into associations that advocated their system of medicine, and pooled resources to provide care to the population in need. These practitioners distinguished AYUSH systems from the allopathic, as well as from their own healing practices. However, they did not necessarily distinguish among the different systems of AYUSH. They did not evince any expectations of the AYUSH provider's role in the public health system beyond AYUSH consulting.3.1.4“See, some people love to work. Like the [AYUSH] doctor in <name of a facility>. Everyone is so happy with him. We want someone like him here.” {Allopathic supervisor}“When they have a D-R in front of their names [when they are called doctors], why can't they prescibe simple medicines like paracetamol and antibiotics? Even the staff nurse can do it, why can't they? We [allopathic doctors] are always available on phone to advise them.” {Allopathic supervisor}The activities expected by allopathic medical officers in charge of public health facilities, as the responsibilities of their AYUSH colleagues, varied across facilities, based largely on the volume of the inflow of patients and the capacity of the allopathic workforce at the facility to attend comfortably to it. In facilities with a low doctor-patient ratio, activities included, besides AYUSH consulting and participation in national health programmes and health camps: allopathic outpatient consulting; conducting childbirth; and performing night duties and emergency allopathic services, under remote supervision, i.e., in accordance with telephonically delivered advice from the allopathic supervisor. These expectations revealed the allopathic supervisors’ need for assistance rather than complementary practice. This need and its fulfillment were also expressed as approval of AYUSH practitioners known to take up and discharge health facility tasks other than AYUSH services voluntarily, as also in resentment of AYUSH practitioners reluctant to cross-practice.3.1.5“He is the most faithful doctor. When the allopath is not there, he manages the whole PHC [Primary Health Centre].” {Health system administrator}“AYUSH doctors help with sterilization. Together [with allopathic doctors], they motivate people.” {Health system administrator}In line with the state's and district's policy, health system administrators expected AYUSH personnel to engage in outpatient consulting in their respective AYUSH modalities, and additional work as specified, for which the district or state generally provided training. The reputation of individual AYUSH providers seemed to rely heavily on their enthusiasm for work other than AYUSH services at the health facility. AYUSH personnel who took up night duties, family planning counselling, obstetric services, and emergency services under allopathic supervision, and organized health camps, were appreciated widely.“AYUSH doctors are not allowed to conduct deliveries.” {Health system administrator}“AYUSH doctors can treat malaria patients only with pills, not injections. Complicated cases should be referred.” {Health system administrator}On the other hand, some administrators decried the expectation of non-AYUSH services from AYUSH practitioners.3.1.6To a large extent, community members did not distinguish between allopathic and AYUSH doctors. This non-distinction was predominant in facilities where AYUSH supplies were low or absent, and the AYUSH provider helping with or engaging fully in allopathic practice. In some co-located facilities, the AYUSH providers had developed a reputation for effective treatment, enthusiasm, and cooperation with the facility staff, and had a high flow of patients in the AYUSH outpatient department. In other facilities, some community members were reported to request non-AYUSH procedures, such as injections of allopathic medications, from AYUSH providers.3.2“See, in our state, I think doctor population will be quite low. So inclusion of this thing [AYUSH] has reduced the load, the patient load, in the allopathic doctors.” {Allopathic practitioner}“Eight AYUSH doctors are to be appointed for School Health. The government knows that they cannot get MBBS [allopathic] doctors easily.” {Health system administrator}“I don't think he [AYUSH doctor] benefits from us, because we don't understand his drugs. We benefit because he helps us.” {Allopathic practitioner}All the interviewees evinced the understanding that sharing the work involved in ensuring population health was the rationale behind the purposive inclusion of AYUSH providers in the public health system of India. However, work sharing was interpreted variously by different stakeholders, the greatest contrast demonstrated between the views of the AYUSH practitioners on contracts, and their allopathic supervisors, administrative superiors, who were permanent employees. AYUSH practitioners expected to complement the work of their allopathic counterparts through AYUSH practice. However, some allopathic supervisors expected allopathic services (under supervision) from the AYUSH doctors to supplement their allopathic practice, and the latter felt the pressure to oblige, or the stress of resisting such expectations. The concept of integration emerged as one not understood uniformly by all the actors in the system: some considered the inclusion of personnel with AYUSH credentials sufficient , whereas others deemed that AYUSH had been included only when AYUSH was being practised by a qualified provider in the health facility .3.3Numerous circumstances of poor communication, preconceived notions, shortfalls in provision, and power imbalances have been reported in the medically pluralistic public health system of India. These form the context, and possibly the antecedents and influencers, of the discrepancies observed in the expectations of the roles of AYUSH providers.3.3.1The roles of the AYUSH providers were ambiguously articulated, and very often not facilitated by the supplies, infrastructure, and personnel support required for optimal AYUSH practice. Some AYUSH providers reported that there was little or no communication of their appointment to the facility staff, including their assigned supervisors. There were practically no official channels for regular communication among practitioners and administrators of different systems of medicine. A few primary and community health centres reported a practice of plenary staff meetings, which gave the AYUSH practitioners and their facility colleagues the opportunity to interact with one another, and organize the allocation or sharing of infrastructure, personnel or other resources. There were some associations open only to AYUSH practitioners, with voluntary and discretionary membership, which facilitated deliberations, particularly on issues of recompense, supplies, and administrative hardships faced by the members, and helped convey these issues to the governing departments for resolution.3.3.2“Informally patients are referred from allopathic treatment to Ayurvedic treatment, like for joint pains. We don't get much chance to interact with allopathy doctors. Patients themselves come here for treatment after allopathic treatment.” {AYUSH practitioner (contract)}In co-located health facilities, there were no official procedures set up for cross-referral of patients and feedback on referred patients. AYUSH doctors reported referring cases that they deemed they could not handle, e.g., ‘emergencies’, to their allopathic counterparts. Referral of patients between practitioners of different systems of medicine was infrequent, and based entirely on personal initiative, often the patients', and the collegiality of the relationships among the staff within individual facilities. AYUSH practitioners who did not enjoy collegial relationships with their allopathic colleagues did not receive any referrals from them.In co-located facilities relatively free from logistical pressures, and with better formal and informal communication among the practitioners of different systems of medicine, patients were frequently cross-referred; practitioners discussed patients’ progress, and also sought treatment from one another, for themselves and their families.3.3.3“One of our staff nurses took some AYUSH treatment … AYUSH or Homoeo, I am not sure.” {Health system administrator}Across all categories of interviewees, the majority expressed or demonstrated a lack of awareness of different systems of TCAM. This was true even of AYUSH practitioners, who generally exhibited unfamiliarity with systems of AYUSH other than their own. None of the interviewees displayed a nuanced understanding of the different systems of AYUSH. Several interviewees conflated the descriptions of different TCAM systems, including AYUSH, local health traditions, and home remedies. The acronym AYUSH, which stands for six distinct systems of medicine, was frequently confused with Ayurveda, one of the component systems, even by senior health system administrators.“AYUSH and Homoeo department, third floor”.This confusion was also evident, and transmitted to the public, in the boards put up at certain busy facilities, directing people to AYUSH consulting rooms, e.g., “Everybody thinks that AYUSH doctors cannot do this, cannot do that. But that is not the case. This doesn't feel good. We are seen with different eyes …. When we studied, there was a difference only in one subject. They studied pharmacology, and we studied materia medica. Every other subject was the same.” {AYUSH practitioner (contract)}“We preach scientific medicine. They don't have proof. I don't know the basis of their medicines. We neither discourage nor interfere. Their medicines are effective – I am saying this from personal experience. I don't know much about its science. We never had much to do with them.” {Allopathic practitioner}“Allopathic specialists can be given some orientation whereby they are made aware that these are the medicines in ISM [Indian Systems of Medicine] which you can prescribe. Not vice versa. An allopath is an ocean in herself. You cannot ask a pond to use the water of the ocean. You can ask the ocean.” {Health system administrator}Furthermore, most health system administrators and allopathic supervisors had no knowledge of the medical curriculum and training of the AYUSH providers appointed in their facilities, and therefore, often underestimated or overestimated their scope. The unfamiliarity with different systems of AYUSH often went hand-in-hand with a low value assigned to AYUSH practices and practitioners.3.3.4In several facilities, the geographical and infrastructural arrangements made for AYUSH practice granted less visibility to the consulting rooms, and less convenience in physical access for patients. For instance, AYUSH consulting rooms were moved from the ground floor of a public hospital to the third floor, accessible only through a stairway from a screened corridor, with the sole direction to this section being a hand-written note pasted on a wall next to the stairway. In a rural location, the AYUSH section was placed in a new building, with no directions posted at the main building. The mismatch between infrastructural provision and role requirements of AYUSH practitioners was cited by several study participants.AYUSH providers were seldom at the helm of organizations, or of individual public health facilities, except in regions where they were placed in exclusively AYUSH facilities or departments. AYUSH providers appointed to positions in hospitals and those appointed to various other public health clinics as contractual staff had supervisors who were allopathic doctors. Supervision took the form of documentation of attendance and channelizing of indents, requests, and official communications through the supervisor to administrative superiors. Allopathic supervisors and AYUSH providers working within hierarchical reporting structures, in the milieu of partial information and, frequently, disciplinary biases, often found themselves in strained, unfulfilling, and stressful working conditions.“These policies are made by health people [the Department of Health and Family Welfare], and AYUSH people are asked later.” {AYUSH practitioner (permanent)}“To function well, we need to have someone to dispense medicines, and at least one male and one female assistant to do panchakarma and massage, etc. We don't have anyone now. The post of assistant has been removed.” {AYUSH practitioner (permanent)}“Our colleges are not as strong. There's only enough money for salaries in the Research Councils, we need to fix this.” {Health system administrator – AYUSH }There were glaring budgetary and policy disparities between support for AYUSH systems on the one hand and the allopathic system on the other. Policymaking for AYUSH in India was not perceived as adequately participatory, and sufficiently responsive to the nature of the AYUSH systems, the needs of the personnel, and the preferences of the population. In fact, AYUSH, although officially a department in the Ministry of Health and Family Welfare, was often treated as an outside entity at key moments of planning and decision-making, even decision-making for AYUSH personnel. AYUSH providers described numerous lacunae in the provision of supplies and human resources support, such as the egregious delays and shortfalls in the supply of medicines, and the inconvenient elimination of the role of a technical assistant to help with clinical consultations and dispense prescriptions. Health system administrators and key informants also remarked on the low support for research in AYUSH, a situation presenting bleak prospects for the development of a strong evidence base for the efficacy of the various AYUSH systems.4This study, undertaken to explore operational and ethical challenges in the integration of AYUSH providers in the public health system of India, had the methodological strengths of a review of policy; in-depth interviews with a wide range of health system actors in three geographically distant, and administratively and culturally different states of India, with varying levels of indigeneity and entrenchment of different TCAM systems in the community; and observations and analysis by a team of researchers. The elicitation of participants' experiences and opinions, as well as the interpretation of the findings could have been limited by the researchers’ expertise and viewpoints.A major finding of this study was discrepant perceptions of the role of the AYUSH provider in government health facilities. National policy articulations closely matched AYUSH providers’ expectations of their role descriptions. In contrast, the perceptions held by health system administrators, and allopathic counterparts and supervisors differed, often greatly, from the perceptions of the AYUSH providers. Discrepant role expectations emerged as the nub of the interpersonal tension among health system actors in the integration of AYUSH into the public health system. These discrepancies had the most adverse impact in Meghalaya and Delhi, where co-location of practitioners of different systems of medicine was in operation, and, within these states, in the facilities shared by practitioners of different systems of medicine, placed in hierarchical administrative structures. In Kerala, where practitioners of different systems of medicine were not co-located, tensions related to discrepant role expectations did not play out as much in interactions and day-to-day functioning, although numerous other divergent perceptions of the different systems of medicine were expressed. Another context in which the discrepancies in perception were expressed was the variable entrenchment and indigeneity of the various AYUSH systems in the states studied. Thus, AYUSH was often confused with Ayurveda in Meghalaya, but not in Kerala, where people were much more familiar with Ayurveda. Local health traditions played a more prominent role in the discourse in Meghalaya than in the other states.Gaps in role descriptions for personnel, as well as in the articulation of referral and collaboration protocols at healthcare facilities  resultedThe most contentious of the expectations held about AYUSH providers’ functions revolved around cross-practice, specifically the practice of allopathic medicine by AYUSH providers. Participants in our study displayed a range of stances towards cross-practice: Certain allopathic supervisors of AYUSH practitioners in co-located facilities expected and encouraged allopathic practice by the AYUSH practitioners. AYUSH practitioners were mostly reluctant, or, in some cases, frankly opposed to prescribing allopathic medications, while a few were quite willing to do so. Administrators and academic experts, as well as several practitioners in this study, did not advocate cross-practice.Some proponents of cross-practice recommend the prescription of allopathic medications by AYUSH practitioners for medical emergencies, and for situations in which AYUSH medications are unavailable . Singh aSeveral researchers have commented on the hegemony of allopathic medicine across the world , and theGaps in infrastructural provision and supplies, including inadequacy of space, storage facilities, signage, medications, and office supplies, which reports over the years show have dogged AYUSH practices in mainstreaming endeavours  were repStudies conducted in integrated practices of biomedicine and TCAM in other countries  have revOther examinations of the functioning of co-located AYUSH and allopathic facilities have found little or no interaction, professional or social, among providers practising different systems of medicine at the same facility, no published protocol for cross-referral , little Communication between AYUSH providers and allopathic peers and supervisors in our study was often reported to be minimal, fraught with tension, or even avoided if possible , althoug4.1The role ambivalence of TCAM (AYUSH and non-AYUSH) practitioners, and the contestations among health system actors over the functions and contributions expected of TCAM practitioners, have implications for TCAM practice, TCAM provider morale, and overall health system efficiency and performance. The low policy and budgetary support for TCAM, as well as the logistical pressures upon TCAM practice, in addition, pose the threat of the loss of the integrity of individual TCAM systems in a notionally integrated health system, and the possible loss of bodies of TCAM knowledge and praxis. Interpreting ‘integration’ as a reclassification of TCAM techniques and products into biomedical categories, without the concepts underlying their use, has also raised concerns among researchers and advocates of medical pluralism .4.2Potential directions suggested by the findings of this study include unambiguous articulation of the roles and responsibilities of the TCAM providers appointed to public health facilities in medically pluralistic systems. In addition, clear delineation of the procedures to be followed in situations of delayed or inadequate infrastructural provision, supplies, and personnel support would be helpful. The provision of basic information to the clinical and support staff, on the system of TCAM to be practised at each facility, would aid in establishing peaceful coexistence of different systems of medicine at the facility, and help more patients access TCAM practitioners with ease. A regular and transparent routine of plenary staff meetings, and the promotion of professional associations to discuss strategies and solutions, would help keep communication channels open.Broader health system related recommendations to improve role clarity are of improved communication, referral protocols, and awareness-raising. Financial and political support, such as the revision of the budgetary allocation to the TCAM sector at national, state and district levels, to provide adequate support for education, research, community outreach, infrastructure and supplies, and staffing and training, are crucial to steer the public health system towards vibrant and effective pluralistic healthcare.Further work is called for on ascertaining the awareness and attitudes among health system actors regarding the different systems of medicine in practice in their communities. Future studies could also examine policymaking, and provision, for the practice of various TCAM systems in a medically pluralistic health system.


The RING-domain E3 ligases (RING E3s), a group of E3 ligases containing one or two RING finger domains, are involved in various cellular processes such as cell proliferation, immune regulation, apoptosis, among others. In the host, a substantial number of the RING E3s have been implicated to inhibit viral replication through regulating immune responses, including activation and inhibition of retinoic acid-inducible gene I-like receptors, toll-like receptors, and DNA receptor signaling pathways, modulation of cell-surface expression of major histocompatibility complex, and co-stimulatory molecules. During the course of evolution and adaptation, viruses encode RING E3s to antagonize host immune defense, such as the infected cell protein 0 of herpes simplex virus type 1, the non-structural protein 1 of rotavirus, and the K3 and K5 of Kaposi’s sarcoma-associated herpesvirus. In addition, recent studies suggest that viruses can hijack the host RING E3s to facilitate viral replication. Based on emerging and interesting discoveries, the RING E3s present novel links among the host and viruses. Herein, we focus on the latest research progresses in the RING E3s-mediated host–virus interactions and discuss the outlooks of the RING E3s for future research. In vertebrates, the immune system is composed of the innate and adaptive components. The innate immune system is a highly effective first-line of defense to detect and fight against invading pathogens, and it is initiated when pattern recognition receptors (PRRs) specifically recognize pathogen-associated molecular patterns (PAMPs) , 2. SubsTo accomplish such enormous and elaborative defense tasks, the immune system is under strict and precise regulation. One of the most important and extensive mechanisms is the ubiquitination, which plays a crucial role in the immune system . Three evia ubiquitination of several cellular proteins. In addition, recent studies suggest that viruses hijack the host RING E3s to promote viral replication through antagonizing IFN-mediated antiviral responses and ubiquitination of viral proteins.Since the host and viruses co-evolved over centuries, viruses have acquired tactics to antagonize the functions of the RING E3s at various levels to minimize host antiviral responses. Several studies have shown that viruses encode the RING E3s to facilitate the virus replication. Well-characterized examples include infected cell protein 0 (ICP0), an immediate-early proteins encoded by herpes simplex virus type 1 (HSV-1), which plays important roles during lytic and latent infections ; NSP1, aUntil recently, the immunity-related RING E3s have been reported increasingly in response to viral infections. However, potential roles of the RING E3s in host–virus interactions are still in infancy stage. Here, we review the mechanisms of the host RING E3s in regulating immune responses and the functions of the RING E3s encoded or hijacked by viruses in viral replication.Xenopus oocytes transcription protein TFIIIA, which contains a conserved RING finger motif, Cys–X2–Cys–X9–39–Cys–Xl–3–His–X2–3–Cys–X2–Cys–X4–48–Cys–X2–Cys (X could be any amino acid) (3HC4 (RING-HC), C3H2C3 (RING-H2), and C4HC3 (RING-CH) finger motifs  . In 1994no acid) . Accordis Figure . In 1999s Figure . Accordis Figure . Many RIThe RING E3s have evolved extensively, and based on phylogenetic analysis the RING E3s can be grouped into five different families, including tripartite motif-containing (TRIM), PA-TM-RING, RING between RING (RBR), membrane-associated RING-CH (MARCH), and RING-Ub interacting motif (UIM) families Figure . All of The human TRIM family consists of approximately 60 members and shares the RBCC motif, including one RING domain, one or two B-box domains, and one coiled-coiled domain. The C-terminus of TRIM proteins (TRIMs) is intrinsically divergent, allowing them to recruit different sets of corresponding proteins. Therefore, TRIMs are involved in a broad range of biological processes, such as autophagy, immunity, and cancers . RecentlThe MARCH family is structurally characterized by harboring one RING-CH domain and multiple TM domains. This family contains 11 members, i.e., MARCH-1 to -11, of which 9 members are TM proteins, and 2 members, MARCH-7 and -10, have no TM domain . FunctioThe PA-TM-RING family is defined by three conserved domains, the protease-associated (PA) domain, the TM domain, and the RING-H2 domain . These pThe RBR family (14 members in humans) is characterized by its conservative structure, where two RING domains are connected through an in-between-RING domain . These t2HC-type zinc finger, two C2H2-type zinc fingers, and one UIM-type domain -like receptors (RLRs), toll-like receptors (TLRs), and DNA receptors , 40. As via its RING domain and enhances the ubiquitination of TRAF3 and TRAF6 to positively regulate RNA virus-triggered IFN-β production , and laboratory of genetics and physiology 2, all of which contain RNA helicase domains sensing viral double-stranded RNA in the cytosol . The N-toduction . A notaboduction . As mentoduction .via K33-linked polyubiquitination at Lys379 of STAT1 in the nucleus to promote STAT1/STAT2 disassociation from DNA, inhibiting type I IFN signaling and antiviral responses  domain to transduce signals to downstream adaptors, including myeloid differentiation primary response protein (MyD88) and TIR-domain-containing adaptor protein inducing interferon-β-mediated transcription factor (TRIF) . The MyDMany sensors of cytosolic viral DNA have been identified, including cyclic GMP-AMP synthase (cGAS) , the helWhen pathogens conquer the innate immunity, adaptive immunity works under the control of humoral and cellular immunity. In the cellular immunity, T cell activation requires two elements: T cell’s receptors (TCRs) specially recognize MHC-bounded pathogen peptides and T cell ligands bind with co-stimulation factor provided by activated antigen presenting cells . Interes2H2 domain is involved in this process  motifs in their TM helices  VP35, a cofactor of the viral polymerase, plays a critical role in viral replication . TRIM6 iThe RING E3s-mediated orchestration of the immune system has contributed significantly to our understanding of host immune regulation. The RING E3s are divided into five subfamilies, each of which has its own special functions. The RING-UIM family is related to T cell activation, the MARCH family has been identified to inhibit antigen presentation through degrading cellular receptors and costimulatory molecules, and the TRIM family restricts virus replication through direct interactions with viral proteins. At present, there are few reports on RBR and PA-TM-RING family. Therefore, the identification of additional interacting proteins or substrates for RBR and PA-TM-RING families will uncover the novel functions of the RING E3s.2H2 and C2HC domains of the RING-UIM family. Third, as discussed in this review, it is obvious that multiple substrates can be targeted by the same RING E3, while multiple RING E3s can target the same substrate. The significance of RING E3s “redundancy” in substrate ubiquitination is poorly understood, which leaves a possibility that RING E3s may act as a cross talk to form an immunity-regulating network. In addition, the RING E3s exert various functions in a broad range of pathways such as carcinogenesis and autophagy. It would be important to consider the RING E3s’ functions as hubs connecting different signaling pathways or different systems. A deep understanding of Ub signaling would be extremely valuable for the future development of treatments for immune and inflammatory diseases.Although the functions and mechanisms of RING E3s-mediated immunity are continuously being reported, there are several fundamental questions that require future research. First, one of the most complex and challenging aspect is to elucidate how the RING E3s are activated upon virus infection and how their expression and localization are regulated during the stimuli in different cell types. Second, it is known that the RING domain credits RING E3s’ powerful E3 ligase function, while the function of other domains is poorly understood, such as CCurrently, most studies have primarily been focused on host RING E3s roles as antiviral factors, while RING E3s encoded or hijacked by viruses for evading the immune responses are largely undiscovered, the identification and characterization of these RING E3s has significantly advanced our understanding of persistent infection. It is generally known that ubiquitination can be reversed by deubiquitination enzymes (DUBs). DUBs can rescue proteins from Ub-mediated degradation and reverse the related biological process by removing Ub chains from the substrate. Novel DUBs and inhibitors of these viruses-encoded RING E3s identified will prevent viruses from escaping host defense.Taken together, the RING E3s-regulated immunity provides a novel perspective to better understand the host–virus interplay. Further detailed investigations are needed using the RING E3s as targets for the prevention and control of recurrent viral diseases.YZ and L-FL are the major contributors of the review. H-JQ and MM conceived and revised the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.