There are three classes of interferons  that can contribute to the development and maintenance of various autoimmune diseases, including systemic lupus erythematosus (SLE). Each class of interferons promotes the generation of autoreactive B cells and SLE-associated autoantibodies by distinct signaling mechanisms. SLE patients treated with various type 1 interferon-blocking biologics have diverse outcomes, suggesting that additional environmental and genetic factors may dictate how these cytokines contribute to the development of autoreactive B cells and SLE. Understanding how each class of interferons controls B cell responses in SLE is necessary for developing optimized B cell- and interferon-targeted therapeutics. In this review, we will discuss how each class of interferons differentially promotes the loss of peripheral B cell tolerance and leads to the development of autoreactive B cells, autoantibodies, and SLE. An interplay of immune dysregulation and environmental triggers causes autoimmunity in individuals with genetic susceptibility to various autoimmune diseases. B cell-mediated autoimmune diseases are characterized by autoantibodies that target healthy tissues and cause pathology. In systemic lupus erythematosus (SLE), patients develop autoantibodies that are reactive to nuclear antigens , including DNA (deoxyribonucleic acid), nucleosomes, ribonucleoproteins, and RNA (ribonucleic acid). Due to the abundance of nuclear autoantigens throughout the body that can be targeted by ANAs, SLE patients often develop a complex disease affecting multiple organ systems. Nuclear autoantigens can be released from dying cells by environmental insults, which then lead to the induction of pro-inflammatory cytokines, including interferons. Normally, interferon-mediated inflammatory responses are required for immune protection against various pathogens, but an elevated interferon response is also a hallmark of SLE .Interferons are organized into three classes by target receptor binding, and these interferon receptors elicit distinct signaling cascades in specific immune cell populations . Environ2a, IFNαR2b, or IFNαR2c). However, each T1IFN family member can elicit distinct signaling cascades due to their co-receptor availability, duration of binding to the receptor complex, and binding orientation [Type 1 interferons (T1IFN) in mammals include interferon alpha (α), beta (β), omega (ω), delta (δ), nu (ν), kappa (k), tau (τ), and epsilon (ε) ,11. Humaentation ,14. Durientation . T1IFN sentation .The type 2 interferon (T2IFN) family in mice and humans consists of only one member, interferon gamma (IFNγ), that binds to a single receptor complex (IFNγR) that consists of interferon gamma receptor 1 (IFNγR1) and 2 (IFNγR2) . IFNγ siThe type 3 interferon (T3IFN) family consists of four cytokines in humans, including IFN-λ1 , IFN-λ2 , IFN-λ3 , and IFN-λ4. Mice only have two T3IFNs, called IFN-λ2 and IFN-λ3 . These cThe peripheral B cell pool consists of immature transitional B cells and mature B cells. Transitional B cells are subdivided into transitional type 1 (T1), type 2 (T2), and type 3 (T3). Mature B cells develop from transitional cells and are broadly categorized into two distinct subsets with diverse functions: innate-like (IL-B) B cells and conventional follicular B-2 cells. IL-B cells can be further divided into marginal zone B cells (MZBs) and B-1 B cells. IL-B cells are self-renewing polyreactive B cells that generate rapid but low-affinity antibody responses. In contrast, B-2 cells are antigen-specific naïve B cells in secondary lymphoid organs that require strong B cell receptor (BCR) activation to generate a slow, high-affinity antibody response to specific antigens . As descB-1 cells are abundant in the peritoneal cavity and barrier tissues, where they can be rapidly mobilized during infection. B-1 cells express high levels of PRRs and express poorly diversified V(D)J genes that allow for a low-affinity, T-independent, and polyreactive natural IgM antibody responses during the early stages of infection. B-1 cells can also generate IgM+ memory B cells, but the bulk of long-lived antibody responses are generated via B-2 founder cells. CD5+ B-1 cells  are potentially self-reactive because they bypass the pre-BCR checkpoint of B cell development. In addition, a subset of B-1a cells can bind to apoptotic cell debris, potentially allowing them to be activated in response to self-antigens . HoweverMZBs are localized at the splenic interface between circulating blood and the lymphatic system. Like B-1 cells, MZBs carry a polyreactive BCR, high levels of surface IgM, high levels of complement receptors (CD21 and CD35) and PRRs and are a key source of natural IgM. MZBs also transport antigens from the blood to follicular dendritic cells (FDCs) in the B cell follicle to activate antigen-specific B-2 cells  and helpB-2 cells reside in the follicles as inactive (naïve) cells, but they can be activated by cognate T cell help to become both low-affinity, short-lived antibody-producing cells and high-affinity, long-lived antibody-producing cells. During an immune response, T cells activated by antigen presenting cells (APCs) undergo co-stimulatory interactions with activated antigen-specific B-2 cells at the B cell and T cell border, allowing these B cells to initially differentiate into extrafollicular antibody forming cells (AFCs), which produce low affinity, short-lived IgM and class-switched antibodies. Later in the response, activated B-2 cells enter germinal center (GC) reactions, where they undergo rapid proliferation and somatic hypermutation of the immunoglobulin genes, promoting an affinity-based selection process . The GC In SLE, self-reactive B cells can persist as long-lived plasma cells or memory B cells, and some studies suggest that most self-reactive antibody-forming cells in SLE patients are derived from non-autoreactive precursors ,42. In aIn a typical immune response, a separation of labor occurs between the innate-like B cell subsets and the B-2 B cell subsets. Innate-like B cells are activated without T cell help and are responsive predominantly to TLR stimulation, whereas B-2 B cells require T cell help to generate highly specific antibody responses. In SLE, interferon signaling activates both groups of B cells, and promotes crosstalk between T-independent and T-dependent B cell responses.Although previous studies described a strong correlation between B-1a cell expansion and lupus pathogenesis in murine SLE models , the rol+ MZ macrophages and allow for the migration of MZB cells into the B cell follicle [Previous studies have highlighted the role of MZB cells and their regulation by T1IFNs in SLE mouse models. Elevated levels of circulating T1IFNs have also been shown to promote the activation and accumulation of plasmacytoid dendritic cells (pDCs) in several mouse models of SLE, including the Wiskott–Aldrich syndrome (WAS) chimera model of B cell-driven autoimmunity , B6.Nba2follicle . These ffollicle , suggestfollicle ,51,53. IIfnar1 gene in GC B cells using the Igh-γ1 Cre (Cγ1-Cre) system in B6.Sle1b mice, we also delineated a GC B cell-specific role of T1IFN signaling in promoting the spontaneous GC response and loss of tolerance [Whereas pathogen-induced AFCs and GCs generate antibodies against pathogens, SLE patients and murine models of SLE develop spontaneous GCs and AFCs that produce disease-causing autoantibodies ,58,59,60olerance . We furtolerance . Enhanceolerance . Furtherolerance , prolifeolerance . CollectIn addition to the role of T1IFNs in controlling spontaneous GC, AFC, and autoantibody responses at secondary lymphoid organs, T1IFN signaling was shown to induce CXCL13 production by fibroblasts to generate ectopic GCs during the immune response to influenza infection, and the combination of IFNβ and STING signaling recruited B cells to the lung in the absence of influenza virus . AlthougAlthough a role for T1IFNs in SLE patients and in murine models of SLE is well established, several groups recently identified B cell-intrinsic roles for T1IFN signaling in autoimmune B cell responses and disease development that varied depending on the mouse model studied . By dele-/- mice and identified a role for T1IFN signaling and T1IFN-produced by pDCs in the development of anti-dsDNA antibody responses and SLE-like disease observed in these mice [In addition, the Mountz group highlighted the role of endogenous IFNβ expression in transitional type 1 (T1) B cell survival and development in BXD2 lupus-prone mice and circulating B cells in SLE patients ,68. The ese mice . Interesese mice . Altogetese mice ,71,72 fuDnase1l3- and Trex1-deficient mice, and Ifih1 mutant mice, also have elevated titers of autoantibodies [Type 1 interferon signaling is controlled by several pro-inflammatory and regulatory pathways, and genetic mutations in these signaling intermediates are associated with a class of mendelian diseases called type 1 interferonopathies. Mutations in nucleic acid sensing receptors, DNA repair proteins, complement pathway intermediates, and proteosome regulators are all associated with this class of inflammatory diseases. Many type 1 interferonopathy patients develop a SLE-like disease with a single gene mutation, whereas SLE is classically defined as a polygenic disease that is largely activated by environmental triggers. SLE-like autoantibody profiles are also present in patients with type 1 interferonopathies, but our understanding of B cell-specific phenotypes in these patient cohorts is still limited. Several mouse models of interferonopathies, including tibodies . Therefosan/san (sanroque) mice [san/san mice  signaling ameliorated autoimmune-associated lymphadenopathy and autoantibody production, and provided robust protection against the development of end-stage organ disease in SLE-prone MRL/lpr mice . In humaue) mice . This grsan mice . bcl6 in B cells, promoting autoimmune GC, Tfh, and autoantibody responses and the development of immune-complex nephritis. Concurrent with the study by Jackson et al., we showed the requirement of B cell-intrinsic IFNγ signaling in spontaneous GC, AFC, Tfh, and antibody responses in both non-autoimmune B6 and autoimmune-prone B6.Sle1b mice [Consistent with findings from the sanroque model, Jackson et al. found increased IFNγ production in the WAS chimera model of B cell-mediated SLE . These ae1b mice . In thise1b mice .Optimal STAT1-mediated immune responses require phosphorylation of both tyrosine 701 (STAT1-pY701) and serine 727 (STAT1-pS727). By crossing autoimmune-prone B6.Sle1b and SLE disease-prone B6.Sle1b.Yaa mice to the B6.STAT1-S727A mutant strain , in whic+T-bethi age-associated B cells (ABCs) play an important role in autoimmunity development and are expanded in several autoimmune diseases including SLE [+T-bet+ ABCs in SLE patients, including the accumulation of TLR7-driven and T-bet-expressing IgDnegCD27negCD11c+CXCR5neg (named DN2) pre-antibody secreting cells (pre-ASCs) [hi human DN2 cells to promote TLR7/TLR8 and IL-21 driven differentiation [CD11cding SLE ,82,83. Tding SLE ,84. Althding SLE ,84. In tding SLE , and tarre-ASCs) . Consistre-ASCs) . They funtiation . At presAnother subset of cytokine-producing B cells, called B effector cells (Be1), was previously reported to be regulated by both T1IFN and T2IFN signaling. T1IFN and T2IFN signaling in B cells was shown to coordinate the generation of Be1 cells from naïve and memory B cell precursors . After a+CD5+CD1d+ regulatory B cells in mice [While IFNγ signaling induces a pro-inflammatory type 1 immune response, it concurrently negatively regulates immunoregulatory pathways, including inhibition of regulatory B cell responses. Regulatory B cells (Bregs) are a subset of B-2 cells that secrete IL-10, IL-35, and TGFβ to inhibit inflammatory responses ,92,93,94 in mice . We demo in mice . Interes in mice , consist in mice ,100,101. in mice . These dAn elevated expression of interferon lambda (IFNλ) has been associated with several autoimmune diseases, including SLE, rheumatoid arthritis, Sjogren’s syndrome, and systemic sclerosis ,106,107.+ memory B cells and the synergistic effects of IFNλ and TLR stimulation on B cell activation suggest that T3IFN signaling could play a crucial role in regulating ABC or DN2 cell activity in SLE, although a link between IFNλ signaling and ABCs or DN2s has not been established.IFNλR1 expression was shown to be upregulated on human naïve and memory B cells , and stiGoel et al. showed that IFNλ was elevated after epicutaneous treatment of a TLR7 agonist, and broadly promoted systemic autoimmune responses as indicated by splenomegaly, increased white blood cells, decreased hemoglobin and platelets, myeloid expansion, neutrophil NET production, and T cell activation . SurprisSLE is a complex B cell-mediated autoimmune disease caused by genetic and environmental factors. Here, we have discussed the roles of all three classes of interferons  in the regulation of autoimmune B cell responses, production of autoantibodies, and development of lupus nephritis. Existing literature from various autoimmune- and SLE-prone mouse models suggests a minor to moderate model-dependent B cell-intrinsic role of T1IFN signaling in autoimmune B cell responses and development of lupus nephritis ,120,121.


Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains  of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties. Botulinum neurotoxins (BoNTs) are extremely toxic to humans by causing flaccid paralysis of botulism. The catalytic light chain (LC) of BoNTs is the warhead of the toxin, which is mainly responsible for BoNT’s neurotoxic effects. As an endopeptidase, LC is delivered by the toxin to inside neurons where it specifically cleaves neuronal SNARE proteins and causes muscle paralysis. While the currently available equine and human antitoxin sera can prevent further intoxication, they do not promote recovery from paralysis that has already occurred. We strike to develop single-domain variable heavy-chain (VHH) antibodies targeting the LC of BoNT/A (LC/A) and BoNT/B (LC/B) as antidotes to inhibit or eliminate the intraneuronal LC protease. Here, we report the identification and characterization of large panels of new and unique VHHs that bind to LC/A or LC/B. Using a combination of X-ray crystallography and biochemical assays, we reveal that VHHs exploit diverse mechanisms to interact with LC/A and LC/B and inhibit their protease activity, and such knowledge can be harnessed to predict their specificity towards different toxin subtypes within each serotype. We anticipate that the new VHHs and their characterization reported here will contribute to the development of improved botulism therapeutics having high potencies and broad specificities. Botulinum neurotoxins (BoNTs) are extremely potent toxins to humans and cause flaccid paralysis. These toxins are widely distributed in nature and relatively simple to produce in quantity. For these reasons, BoNTs are listed by the Centers of Disease Control and Prevention (CDC) as Tier 1 Select Agents. BoNTs also are highly variable in nature with at least seven major serotypes reported (A-G), most of which forms a group with multiple variant subtypes. This extreme natural variability seriously complicates the development of botulism treatments. Currently the only available therapeutics for botulism are equine or human antitoxin serum products which are administered intravenously to patients exposed to toxin. Efforts are well underway to develop monoclonal antibody (mAb) antitoxins with the goal to replace current antiserum products . While aSeveral groups are working to develop the next generation of botulism antidotes with the goal to inhibit or eliminate the intraneuronal protease that causes paralysis. Our group seeks to develop single-domain antibodies (sdAbs) to be employed as components of biomolecular antidotes that target the various BoNT proteases. These sdAbs are derived from the heavy-chain-only antibodies produced by camelid animals. The heavy chain variable regions of these antibodies, called VHHs or nanobodies, are small sdAbs of ~14 kDa that are tightly folded, stable, and highly amenable to recombinant expression and engineering as multimers or as fusions to other vehicles. VHHs also have a preference for binding to conformational epitopes that often are functional sites on proteins such as enzyme active sites or receptor-binding sites. Multimers of toxin-neutralizing VHHs have been employed in the generation of botulism antitoxins and shown to have substantially improved potencies ,7, parti2+-endopeptidase that specifically cleaves neuronal SNARE proteins and is mainly responsible for BoNT’s neurotoxic effects, while the heavy chain (HC) mediates toxin attachment to neurons and delivers the LC into the cytosol. Two recent reports demonstrated that animals with symptomatic botulism can be rescued by treatments with biomolecular antidotes consisting of an atoxic BoNT delivery vehicle fused to VHHs that bind and inhibit the LC of the intoxicating BoNT .,25.15,25Comparing the structures of SNAP25-bound LC/A with the structures of LC/A in complex with protease-inhibiting VHHs reveals diverse mechanisms by which these VHHs inhibit LC/A cleavage by competing with SNAP25 binding at different sites. Specifically, strong LC/A protease inhibiting VHHs could be classified into four groups: (1) three VHHs bind to the α-exosite of LC/A ; (2) two bind to anchoring site I (JPU-A11 and JPU-D12); (3) two bind anchoring site II (JPU-C1 and JPU-G3); and (4) two bind the β-exosite of LC/A (JPU-A5 and JPU-C10). Consistent with our competition studies, VHHs in each of these four groups belong to the same competition group and bind to overlapping epitopes . Structu2 on LC/A  with a surface complementarity (SC) score of 0.731 [2 with an SC score of 0.686 . The weaker binding of JPU-G3 in comparison to JPU-C1 may explain its lower protease inhibition potency. Interestingly, the CDR3 of both VHHs engage overlapping epitopes near the 160-loop of LC/A in nearly opposite orientations. As a result, the three LC/A-binding residues are in reverse orientation on JPU-C1 (residues 101LED103) and JPU-G3 (residues 104DEL106) (VHHs JPU-C1 and JPU-G3 both bind to the anchoring site II of LC/A and inhibit LC/A protease . JPU-C1 of 0.686 . JPU-G3 4DEL106) .Further structural analysis indicates that the CDR3 of JPU-C1 and JPU-G3 use a similar mechanism to interact with the SNAP25-binding residues on LC/A and therefore to preclude SNAP25 binding even though their CDR3 reaches into the anchoring site II from opposite orientations . For exa2 per molecule with a relatively high SC score of 0.775, while JPU-A11 interaction leads to a BSA of 984 Å2 and an SC score of 0.671  including this region was previously considered as uncrystallizable because it is flexible and prone to aggregation . We idenBoNTs are found in nature as at least seven different serotypes, and within each serotype are also found a substantial number of subtype variants. For therapeutic botulism antitoxins or antidotes to be broadly effective, they must be capable of protecting individuals from this diverse array of potential threats. Studies have shown that mAbs can be identified having broad specificity for natural subtype variability ,40 and tAll procedures using alpacas were conducted in accordance with the guidelines approved by the Institute Animal Care and Use Committee at Tufts Cummings School of Veterinary Medicine under protocols G2013-85, G2017-18 and G2019-142.Table 1). LC/B-binding VHHs were selected in two processes. The initial VHHs were obtained by capturing LC/B to plastic with BLc-B10 VHH [Table 2). BLc-B10 proved to poorly capture LC/B so the discovery process was repeated using JND-E4 as the capture VHH, which led to selection of many additional LC/B-binding VHHs (JSG series in Table 2). Screening for LC-binding VHHs was performed by ELISAs employing VHH-captured LC/A or LC/B. All VHH panning and screening was performed using standard lab procedures [Two alpacas were immunized with purified, full-length LC/A protein and two alpacas immunized with full-length LC/B as previously reported ,12. VHH-ocedures .E. coli thioredoxin and purified by standard lab procedures [50 values, i.e. the VHH concentration that resulted in 50% peak binding. Competition assays were done as first described in [VHHs identified in the discovery process were subcloned into the pET-32 expression vector, expressed as fusions to ocedures . Bindingocedures ) and empocedures  and apparibed in  with somribed in .Tables 1 and 2.All VHHs were tested for their potencies to inhibit LC/A and LC/B protease activity. Assays employed multiple substrates. For SNAP25 cleavage, we employed either recombinant YFP/SNAP25/CFP  or Repcon5 . For VAMLC/B(M1–S425), LC/B(M1–K441), anti-BoNT/A VHHs , and anti-BoNT/B VHHs  were cloned into pGEX-6p-1 for expression following the N-terminal GST and a PreScission cleavage site. LC/A(M1–T420), LC/A P2–K438), ALc-H7, ALc-B8, ciA-D12, and ciA-F12 were expressed and purified as described previously [, ALc-H7,E. coli strain BL21-Star (DE3) (Invitrogen). GST-tagged JPU-A5 was expressed in the E. coli strain Origami B (DE3) (Novagen). Bacteria were cultured at 37°C in LB medium containing ampicillin. Temperature was reduced to 18°C when OD600 reached ~0.6. Expression was induced with 1 mM IPTG and continued at 18°C for 16 hr. Cells were harvested by centrifugation and stored at –20°C until use.GST-tagged LC/A, LC/B, and VHHs  were expressed in 4). Each protein complex was concentrated to ~5 mg/ml using Amicon Ultra centrifugal filters (Millipore) and stored at –80°C until further characterization or crystallization.For protein purification, bacteria were re-suspended in a buffer containing 50 mM Tris (pH 8.0), 400 mM NaCl, and 0.4 mM PMSF and lysed by sonication. All GST-tagged proteins were purified using Glutathione Sepharose 4B resins  in 50 mM Tris (pH 8.0), 400 mM NaCl, and eluted from the resins after on-column cleavage using PreScission protease. The proteins were further purified by Superdex-200 Increase or Superdex-75 SEC in 10 mM HEPES (pH 7.4) and 150 mM NaCl. The LC–VHH complexes were made by mixing the purified LC and VHHs at a molar ratio of 1:1.5 for 1 hr on ice, followed by purification using Superdex-200 Increase SEC  and high-throughput crystallization screen kits (Hampton Research and Qiagen). Extensive manual optimizations were performed at 18°C when proteins were mixed with reservoir solution at 1:1 ratio.The best single crystals of sLC/A–JPU-A5–ALc-H7–JPU-C1–JPU-D12–ciA-F12 were grown by the sitting-drop vapor diffusion method at a protein concentration of 8 mg/ml with a reservoir solution containing 20% 2-propanol, 0.1 M sodium citrate, 20% PEG4000, pH 5.6.Crystals of fLC/A–ALc-B8–JPU-C10–JPU-G3–JPU-D12–ciA-F12–ciA-D12 were obtained by the sitting-drop vapor diffusion method at a protein concentration of 6 mg/ml with a reservoir solution containing 2.4 M sodium malonate, 0.1 M Bis-Tris, pH 7.0.Crystals of fLC/A–JPU-B9–JPU-A11–JPU-G11 were grown by the sitting-drop vapor diffusion method at a protein concentration of 5 mg/ml with a reservoir solution containing 25% PEG 4000, 0.1 M sodium cacodylate, pH 6.5.Crystals of sLC/B–JLJ-G3–JNE-B10 were obtained by the sitting-drop vapor diffusion method at a protein concentration of 5 mg/ml with a reservoir solution containing 0.2 M KI, 25% PEG 4000, 0.1 M MES, pH 6.5.Crystals of fLC/B–JSG-C1 were obtained by the sitting-drop vapor diffusion method at a protein concentration of 5 mg/ml with a reservoir solution containing 20% PEG 4000, 0.15 M ammonium sulfate, 0.1 M Hepes, pH 7.0.https://github.com/RAPD/RAPD) [S2 Table. Structures of the sLC/A–JPU-A5–ALc-H7–JPU-C1–JPU-D12–ciA-F12, fLC/A–ALc-B8–JPU-C10–JPU-G3–JPU-D12–ciA-F12–ciA-D12, and fLC/A–JPU-B9–JPU-A11–JPU-G11 complexes were determined by molecular replacement using the Phaser software [S2 Table. All structure figures were prepared with PyMol (http://www.pymol.org).All crystals were cryoprotected in their original mother liquor supplemented with 20–25% (v/v) ethylene glycol. The X-ray diffraction data for the crystals were collected at 100 K at the NE-CAT beamline 24-ID-E, Advanced Photon Source (APS). The data were processed with iMOSFLM  or XDS aPD/RAPD) . Data cosoftware  with LC/software  and the software  as the ssoftware , PHENIX software , and CCPsoftware  in an itsoftware . The strsoftware  and showS1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 FigAmino acid sequences of all of the LC/A-binding VHHs studied in this report. Sequences are aligned to conserved framework regions and CDRs are indicated.(PDF)Click here for additional data file.S2 FigAmino acid sequences of all of the LC/B binding VHHs studied in this report. Sequences are aligned to conserved framework regions and CDRs are indicated.(PDF)Click here for additional data file.S3 FigLC/A protease was incubated with the BoTest A/E reporter in the presence or absence of VHHs at a molar ratio of 5:1 VHH:LC/A. Incubations were performed at 37°C for either 10 or 60 minutes as indicated, and the reaction was terminated by boiling in SDS sample buffer. An equal aliquot of each sample was analyzed by performing western blots and the substrate and products were detected by HRP/anti-GFP antibodies.(PDF)Click here for additional data file.S4 FigLC/B protease was incubated with a recombinant YFP/VAMP/GFP reporter in the presence or absence of VHHs at a molar ratio of 2:1 VHH:LC/B. Incubations were performed at 37°C for either 60 or 180 minutes as indicated, and the reaction was terminated by boiling in SDS sample buffer. An equal aliquot of each sample was analyzed by performing western blots and the substrate and products were detected by HRP/anti-GFP antibodies.(PDF)Click here for additional data file.S5 FigSequence conservation of VHH-binding epitopes across BoNT/A or BoNT/B subtypes. (A–H) LC/A or (I–J) LC/B is drawn in surface. Identical, conserved, semi-conserved, and variable residues at the LC–VHH interface are colored, green, blue, purple, and red, respectively. (Right) The amino acid sequence alignment among BoNT/A or BoNT/B subtypes performed using Clustal Omega. Only the VHH-binding epitopes are represented.(PDF)Click here for additional data file.S6 FigRecombinant LC/A1, /A2, /A3 and /A4 and LC/B1 and /B8, each strep-tag fusion proteins, were captured to streptactin plates and VHH binding dilution ELISAs were performed as described in the Materials and Methods. The name of the VHH tested in each ELISA plot is indicated above the data. VHH binding data to each subtype is indicated in different colors and plotted as a function of VHH concentration.(PDF)Click here for additional data file.


In behavioral learning, reward-related events are encoded into phasic dopamine (DA) signals in the brain. In particular, unexpected reward omission leads to a phasic decrease in DA (DA dip) in the striatum, which triggers long-term potentiation (LTP) in DA D2 receptor (D2R)-expressing spiny-projection neurons (D2 SPNs). While this LTP is required for reward discrimination, it is unclear how such a short DA-dip signal (0.5–2 s) is transferred through intracellular signaling to the coincidence detector, adenylate cyclase (AC). In the present study, we built a computational model of D2 signaling to determine conditions for the DA-dip detection. The DA dip can be detected only if the basal DA signal sufficiently inhibits AC, and the DA-dip signal sufficiently disinhibits AC. We found that those two requirements were simultaneously satisfied only if two key molecules, D2R and regulators of G protein signaling (RGS) were balanced within a certain range; this balance has indeed been observed in experimental studies. We also found that high level of RGS was required for the detection of a 0.5-s short DA dip, and the analytical solutions for these requirements confirmed their universality. The imbalance between D2R and RGS is associated with schizophrenia and DYT1 dystonia, both of which are accompanied by abnormal striatal LTP. Our simulations suggest that D2 SPNs in patients with schizophrenia and DYT1 dystonia cannot detect short DA dips. We finally discussed that such psychiatric and movement disorders can be understood in terms of the imbalance between D2R and RGS. In our brain, learning and memory are strongly modulated by dopamine (DA) signals. Even a short absence of DA (0.5–2 s), called “DA dip,” triggers long-term memory formation, the underlying processes of which are hitherto largely unknown. Here, we examined how the DA dips are processed through a biochemical signaling network to generate long-term memory. Computer simulation and theoretical analyses showed that the DA-dip signal is processed only if the levels of two key molecules, DA D2 receptor (D2R) and regulators of G protein signaling (RGS), are both delicately balanced. This balance seems to be achieved in the healthy brain, whereas imbalance between D2R and RGS levels appear in patients with schizophrenia and DYT1 dystonia, both of which may manifest abnormal long-term memory. The D2R–RGS imbalances hamper DA-dip detectability, and thus disturb long-term memory formation, which may result in the symptoms of schizophrenia and dystonia. The balance between D2R and RGS appear to be finely regulated in the healthy brain to underpin normal learning and memory. The 0.5-s DA dip is similar to the timescale of the G-protein signaling (0.1–1 s)  denotes time-varying molecular concentration, [X]tot denotes the total concentration, and [X]buff denotes the buffered concentration. [X]basal denotes molecular concentration at the basal state , [X]dip denotes molecular concentration at the DA-dip state (d[X]/dt = 0 under [DA] = [DA]dip). [DA]basal and [DA]dip are described in the subsection “Inputs.”All molecular interactions in the D2 model were represented by binding and enzymatic reactions under the mass assumption ,38,39. I complex . GTP andA binds to B to form A:B was expressed by the following equation:kon and koff are the rate constants for the forward and backward reactions, respectively. Here, koff/kon is known as the dissociation constant, Kd. Enzymatic reactions were modeled based on the Michaelis-Menten (MM) equation:S, E, and P denote substrate, enzyme, and product, respectively, and Km and kcat are the Michaelis constant and product turnover rate, respectively. We did not consider E–S complexes for simplicity, similarly to a previous study [A binding reaction in which us study .t1/2 ~ 0.1 s) [DA], as follows :Tdip is the duration of a DA pause, tDA,delay is the onset time of the DA pause, [DA]dip is the bottom concentration of produced DA dip, and [DA]opto is the amplitude of DA signal by a single light pulse. kcat,DAT[DAT] and [DA]opto were determined so as to give an average concentration of 0.5 μM and a half-valued period of 0.1 s  :We next characterized the DA-dip signal as a square wave dip of [DA]basal = 0.5 μM and [DA]dip = 0.05 μM unless otherwise stated . Those levels of DA have been estimated based on a reference experiment (S1 Table) buff .In Iino et al. (2020), A2AR was continuously activated to produce Gant rate . The Golm the AC . The dyn2+. The pre–post pairing was constituted of 10-consecutive elemental pairs at 10 Hz :We also simulated a pre–post pairing-induced dynamics of Ca2+ influx via voltage-gated Ca2+ channels (VGCC) and N-Methyl-D-aspartate receptors (NMDAR). The Ca2+ bound to CaM, while free Ca2+ was uptaken by a Ca2+ pump. The Ca2+-CaM further bound to a specific site of AC. Detailed scheme and parameters of the Ca2+ signal are described in Urakubo et al. (2020) , was defined by the following equation :i, Golf, and Ca2+-CaM, respectively, and i = 1,…,9 denote the states of Ca2+-CaM. Here, i-free AC, i.e., Golf]buff/{Kd,Golf+[Golf]buff} denotes the binding ratio of AC to Golf. If [Golf]buff is a constant,The concentration of active-state AC, [ACprimed is simply Golf]buff. All simulation and analyses were conducted on the standard non-competitive model unless otherwise stated.Thus, ACactive], under the situation of 100%-competitive binding between Gi and the other two molecules, Golf and Ca2+-CaM :Golf: AC: Ca2+-CaMi] denotes the concentration of AC that binds to both Golf and Ca2+-CaM, but not to Gi. The Golf binding and Gi unbinding are shared requirements for the activities of AC1 and AC5 . We thus picked up the shared part of AC1 and AC5, as a primed state of AC for the activity, ACprimed, as:We defined the concentration of active-state AC,  = [DA]basal, and DA] = [DA]dip.In both non-competitive and competitive models, ACprimed using the following equation:Here, we quantified the DA-dip duration-dependent response of ACprimed〉Tdip represents the average increase in ACprimed during the DA-dip period  in Eq  [D2R]tot and 0.5 × [RGS]tot was used for the schizophrenia model, because D2 blockers need to occupy 70~85% of the total D2R to give clinical effects while avoiding side effects [D2R]tot and 2.0 × [RGS]tot was used for the dystonia model, because Bonsi et al. have observed a 0.7-fold D2R decrease and 1.6-fold RGS increase in the protein expression levels of the striatal detergent-resistant membranes (DRM) in a mouse model of DYT1 dystonia [Four pairs of [D2R]l day 7) . Such a l day 7) ,51. A pa effects ,53, and  effects ,32. A padystonia , and anodystonia . Note thFig 1) tot, was set to be 10 times higher than the standard concentration (S1 Table), ACprimed showed ~80% of the maximal activity even with the basal DA signal . In this case, there was only small room for further activation. By contrast, if [RGS]tot was set to be 10 times lower than the standard concentration, ACprimed did not show any activity even during the DA dip . We thus raised a next question: what are requirements on the parameters, i.e., molecular concentration and kinetic constants, for the DA-dip detection?The response of Fig 3A, top). Here, the DA-dip detectability was quantified by two variables: ACbasal and ACdip . ACbasal denotes the steady-state level of ACprimed under [DA] = [DA]basal . We observed ACbasal and ACdip if the concentration of one of the five constituent molecules, [D2R]tot, [RGS]tot, [AC]tot, [Gi]tot, and [Golf]buff, was varied ranging from 0.1-fold to 10-fold, while the concentrations of other molecules were kept unchanged . First, we observed that neither ACbasal nor ACdip was sensitive to [Gi]tot and [Golf]buff if they were higher than 4% of the standard concentration , while ACbasal and ACdip were both highly sensitive to [D2R]tot and [RGS]tot . This is because the concentrations of D2R and RGS determined the available amount of Gi-free AC for the activity. Here, for convenience, the regions that satisfies ACbasal < 30% and ACdip > 70% were highlighted as the regions that enabled DA-dip detection . In the cases of [D2R]tot, [RGS]tot, and [AC]tot, the regions that satisfied ACbasal < 30% (blue) and those that fulfilled ACdip > 70% (light blue) occupied the opposite ends of the concentrations, and the intersection of the two regions satisfying both of them were limited within narrow concentration ranges. Note that the requirement of higher [D2R]tot for smaller ACbasal has been shown as the requirement of higher [DA]basal in a previous study  [D2R]tot for higher ACdip has also been shown as the requirement of lower [DA]dip in another study tot and [RGS]tot were particularly narrow, we further plotted their two-way relationship in a 2D space . The DA-dip detectable region in [D2R]tot had a clear positive relationship with that in [RGS]tot; the higher [D2R]tot requires the higher [RGS]tot for the detection of a DA dip . Very interestingly, Bonsi et al. (2019) have shown that the expression level of striatal RGS9–2 has a similar positive relationship with that of D2R  in postnatal development of mice, during which both their expressions are increased basal = 0.5 μM and [DA]dip = 0.05 μM. The concentrations of DA were determined based on experimental measurements (S1 Table) [DA]basal is known to depend on the specific situation [DA]basal and [DA]dip, and found that they affected the regions of ACbasal < 30% and ACdip > 70%, and the region that satisfied both of them disappeared depending on the pair of [DA]basal and [DA]dip . Nevertheless, the DA-dip detectable region in [D2R]tot always had a positive relationship with that in [RGS]tot , and the D2R–RGS balance was always required regardless of the pair of [DA]basal and [DA]dip, if we considered analytical solutions that explain these boundaries .The requirement of the D2R–RGS balance appeared under buff led to a simultaneous increase in ACbasal and ACdip, which decreased the dynamic range of ACprimed . Conversely, if [Golf]buff was set to be low, the dynamic range was restored ; however, the maximal activity of AC became small. As a result, there appeared to be an optimal [Golf]buff for ΔACactive where S4C Fig). It was consistent with the simulation results in a previous study tot exceeded a certain level . Then, we plotted the DA-dip detectable area in the 2D space of [D2R]tot and [RGS]tot and found a slight dependence on [D2R]tot . We finally overlaid this plot with the requirements on ACbasal and ACdip . [D2R]tot and [RGS]tot were needed to fall in the overlapping region  for the DA-dip detection in LTP and/or the change of neuronal excitability . As expected, this region depended on [DA]basal and [DA]dip . The higher [DA]basal and lower [DA]dip were required for the dynamics range in the response of ACprimed, while the lower [DA]basal was better for the rapid response . The requirements of the D2R–RGS balance and high [RGS]tot were preserved regardless of the concentrations of DA.The DA-dip detection depends not only on the steady-state levels of ACprimed . We thusT1/2 were also preserved in the competitive model , and T1/2 in the competitive model further depended on the binding/unbinding reaction rate of Golf . This is because ACprimed represented the state of Gi-free and Golf-bound AC, and the Golf binding rate became a time liming process if the Golf binding process was slower than the Gi unbinding process. The reaction rate of Golf did not affect the requirement of the D2R–RGS balance , because it did not affect the steady-state level of Golf, but the dissociation constant, Kd,Golf, should affect it. Similar reaction-rate dependency has been examined by Bruce et al. (2019) [All the characteristics of e et al. 19 [20].RGS]tot and [D2R]tot , while it should also depend on the other type of parameters, i.e., kinetic constants (S2 Table). Similarly, we defined the DA-dip detectable region, i.e., ACbasal < 30%, ACdip > 70%, and T1/2 < 0.5 s, mainly for convenience, and they do not necessarily take exactly these values. We thus derived their analytical solutions to examine the overall parameter dependence of ACbasal and ACdip, and T1/2.The D2 model revealed that DA dip could be detected only in a restricted range in the space of [[D2R]tot . Howeverd[P]/dt ~ [E]kcat if Km << [S], to the GTP/GDP exchange of Gi-GDP, i.e.,To enable it, we first made simplification on the enzymatic reactions based on the MM formulation. We introduced the catalyst-saturated approximation, Km,exch,Gi << [Gi:Gβγ],(a) Km,exch,Gi is the Michaelis constant, and [Gi:Gβγ] is the substrate concentration. The constraint (a) was based on the facts that Km,exch,Gi ~ 10 nM (S2 Table) and [Gi]tot ~ 10 μM (S1 Table). Thus, in almost all the situations, Km,exch,Gi was much lower than [Gi·Gβγ]. Similarly, we introduced the first-order rate approximation, d[P]/dt ~ [E][S]kcat/Km if Km >> [S], to the GTP hydrolysis of Gi, i.e.,where Km,hyd,Gi >> [Gi-GTP],(b) Km,hyd,Gi is the Michaelis constant, and [Gi-GTP] is the substrate concentration. The constraint (b) was based on the parameters that Km,hyd,Gi ~ 12 μM and [Gi]tot ~ 10 μM . Only a subpopulation of [Gi]tot forms [Gi-GTP]; thus, Km,hyd,Gi > [Gi]tot > [Gi-GTP], and Km,hyd,Gi > [Gi-GTP]. We further set the following constraints:where D2R]·[DA] / [D2R:DA] = Kd,DA, where Kd,DA = koff,DA/kon,DA,(c) [Gi-GDP] (see section A in S1 Appendix).(d) t1/2 ~ 30 ms, S2 Table) tot, kDA,basal = kcat,exch [DA]basal / , and Golf = [Golf]buff/Kd,Golf (see S1 Appendix). ACdip was also obtained by replacing kDA,basal with kDA,dip. The analytical ACbasal and ACdip were both well fitted with the simulated ACbasal and ACdip, respectively , and the analytical ACbasal and ACdip were the functions of [RGS]tot, [D2R]tot, and [AC]tot, but not the function of [Gi]tot. This is because the constraint (a) simplified the Gi-dependent V1 (Eqs (section A in S1 Appendix) into a Gi-independent form (Eqs (S43) and (S44), section A in S1 Appendix). The constraint (a), Km,exch,Gi << [Gi:Gβγ], was invalid in the small range of [Gi]tot; thus, the analytical and simulated ACbasal were mismatched under [Gi]tot < ~0.04 μM .Based on the constraints (a-d), we successfully obtained the steady-state ratio of Gws , Eq (24) was still too complicated to provide an intuitive understanding. We thus further simplified Eq  well described the simulated ified Eq  by consikRGS << koff,GiGDP, koff,GiGTP denotes the situation that Gi-GTP hydrolysis is much slower than the Gi-dissociation rate from AC, and kRGS >> koff,GiGDP, koff,GiGTP denotes the reversed situation. Eqs , and they are both linear functions of [RGS]tot (∝ kRGS); thus, ACbasal can be characterized by a transition between the two linear functions . The gradients and D2R-intercept depend on ACbasal, [DA]basal, [AC]tot, and kinetic constants. One of the asymptotic functions, Eq , but it is due to the D2R-intercept, and indeed linear in the linear space. The other asymptotic function  appeared to be linear, because it has no D2R- or RGS-intercepts. ACdip was also derived where kDA,basal was replaced with kDA,dip, and showed the same characteristics .Here, ion. Eqs  and 15)kRGS << ktion ACprimed(t), T1/2, where ACprimed(t = T1/2) =  / 2 after a sudden decrease of [DA] at t = 0 s . Unfortunately, the dynamics of ACprimed(t) was governed by exponentials of exponential functions, which has a complicated form (gamma functions) in the analytical solution. To avoid it, we further introduced three additional constraints to the D2 model:We next considered the half maximal time of  0 s ACisite]·[Gi-GTP]/[ACisite:Gi-GTP] = Kd,GiGTP,(e) [ACisite]·[Gi-GDP]/[ ACisite:Gi-GDP] = Kd,GiGDP,(f) [AColfsite]·[Golf-GTP]/[ACisite:Golf] = Kd,Golf (only for the competitive model),(g) tot . Similarly, the constraint (g) is not valid if kRGS is much larger than the rate of AC-Golf unbinding, as it also appeared .The derivations of Eqs , 17), a, a17), aWe finally simplified Eq (27) by considering if ACbasal = 0% and ACdip = 100%, or ACbasal + ACdip = 100%. The asymptotic Fig 5C, red dotted line) if ACbasal < 30% and ACdip > 70% . We thus obtained an analytically closed form of the isoline of T1/2 in the DA-dip detectable region. In Eq (RGS]tot (∝ kRGS). Thus, [RGS]tot must be higher than the isoline for the shorter DA-dip detection.Here, n. In Eq , T^1/2 iACbasal,ACdip, and T1/2. ACbasal and ACdip were both characterized by the transition between two asymptotic linear functions , and T1/2 also had an approximate closed form . The boundaries of ACbasal and ACdip were linear regardless of the model parameters as far as they satisfied the constraints (a–d), and such a linear relationship was also seen in developing mice  tot . We thus examined two-way relationships between [AC]tot and [D2R]tot  as well as between [AC]tot and [RGS]tot . The DA-dip detectable region in [AC]tot showed a positive relationship with that in [D2R]tot , and a negative relationship with that in [RGS]tot . These [AC]tot dependences originated from the sequestration of Gi-GTP for the inhibition of AC. If Gi-GTP was set not to be sequestrated by AC, i.e., V4, …, V7 were removed only from Eq (S3) , the [AC]tot dependences were completely eliminated , while the relationship between [RGS]tot and [D2R]tot was still preserved . Furthermore, the lower [AC]tot was, the lower [AC]tot dependences appeared  because the smaller amount of Gi-GTP was sequestered by AC.The DA-dip detectability also depended on tot and 0.5 × [RGS]tot , and found the similar response of ACprimed to the DA dips , although it showed lower sensitivity to the shorter dips. On the contrary, a schizophrenia model that had 4.0 × [D2R]tot and 0.5 × [RGS]tot  did not detect the DA dips because of the excessive amount of Gi-GTP. It caused the excessively low ACprimed at the basal state and its lower and slower increase . Similarly, a dystonia model that had 0.5 × [D2R]tot and 2.0 × [RGS]tot  showed high ACbasal and weak responses to the DA dips . In summary, the D2 models for healthy adult and healthy infant responded to DA dips with sufficiently large dynamic ranges , although the healthy-infant model was less sensitive to shorter DA dips. The schizophrenia model showed the slower and smaller responses of AC against DA dips , and the dystonia model showed the rapid but small response .We first confirmed that the D2 model with the standard set of parameters , and they can be understood using a schematic picture . At the basal state, DA-dependent D2R activity works like a faucet (tap) that provides Gi-GTP with a constant rate . The production rate, V1, is almost independent of [Gi:Gβγ] because [Gi]tot (~9 μM) is much higher than Km,exch,Gi ). The produced Gi-GTP is drained through RGS, where the draining rate, V2 + V8, is proportional to [Gi-GTP] + [AC:Gi-GTP] (Eqs (S40) and (S41), section A in S1 Appendix) because the GTP hydrolysis is unsaturated (constraint (b)). The difference in these rates makes a pool of Gi-GTP . In the healthy-adult model, the pooled Gi-GTP almost completely inhibits AC, which is represented by a ball . Here, the Gi-soaked part of AC is inhibited, and the Gi-free part is disinhibited . During the DA-dip, the production of Gi-GTP almost stops , and the level of Gi-GTP is decreased for the activation of AC for LTP and/or the change of neuronal excitability. In the schizophrenia model, the larger [D2R]tot makes the production rate of Gi-GTP, V1, much higher than its draining rate, V2 + V8 . These imbalances caused the decrease the levels of DA-dip detection .Apparently, their DA-dip detectability was disrupted by the excessive or insufficient levels of G V2 + V8 , leadingFig 7). Compared to the healthy-adult model , the healthy-infant model showed the smaller level of DA-dip detection in ACacitve because the DA dips had a short period of 0.5 s . The schizophrenia model did not show any increase in ACactive , while the dystonia model detected the DA-dip, but the timing-independent component of ACacitve was high . Because of the high timing-independent ACactive, the peak-to-basal ratio of ACactive in the DYT1 dystonia became the lowest , indicating the small signal-to-noise ratio even if the activity of the downstream signaling can adapt to the high timing-independent levels of ACactive.Finally, we examined the timing detection for AC1 in D2 SPNs in the healthy adult, healthy infant, schizophrenia, and DYT1 dystonia . The time windows for the delay of DA dip/burst were similar to those in AC1 , and the dystonia model rather showed DA-burst detection, because the longer DA burst completely inhibited the increase in ACactive due to the adenosine burst .Following a previous study , we alsoHere, we showed that a D2R–RGS balance was required for the detection of DA dips in D2 SPNs, where the DA-dip detection is important for the LTP and/or change of neuronal excitability. High-level RGS was further required for the detection of short (~0.5 s) DA dips. These requirements were satisfied in healthy development but disrupted in our models of schizophrenia and DYT1 dystonia.The D2 model highlights the importance of RGS. In particular, a type of RGS, RGS9–2, is known to be specifically and abundantly expressed in the striatum ,34,63. TFig 6)  (= [Gi-GTP] + [AC:Gi-GTP]), but only dependent on [RGS]. Then, [GTP] would become an increasing or decreasing function depending only on [RGS], and Gi took only one of two stable states, i.e., fully hydrolyzed or non-hydrolyzed states. ACprimed could not show a rapid increase from the fully non-hydrolyzed state of Gi , nor detect a short DA-dip. The kinetics constants and molecular concentrations seem to be arranged for effective DA-dip detection, which may be a design principle in the biological system, as often quoted in the field of systems biology tot and [RGS]tot for the healthy and pathologic conditions were determined only for exemplifying purpose , could also be related to the D2R–RGS imbalance. The depletion of DA is associated with Parkinson’s disease, and the DA depletion is restored by administration of a DA precursor, L-DOPA . The L-Dolf and Gi  . (TIF)Click here for additional data file.S5 FigDA]basal is set to be 0.125, 0.25, …, 4 μM (left), and the areas of ACbasal < 30% (top) and T1/2 < 0.5 s (bottom) are plotted in the space of [RGS]tot and [DA]tot. Dotted lines denote the analytical solutions, and colored areas denote the simulation results. (B) [DA]dip is set to be 0.0125, 0.025, …, 0.4 μM (left), and the areas of ACdip > 70% (top) and T1/2 < 0.5 s (bottom) are plotted.(A) buff are kept constant.  ACbasal, ACdip, and T1/2 in the standard non-competitive model (A), and competitive model (B). Green dotted line in panel B denotes τ = 0.5/log 2 (T1/2 = 0.5 s).Here, the binding/unbinding reaction rate of G(TIF)Click here for additional data file.S7 FigACbasal < 30% (blue), ACdip > 70% (light blue), and T1/2 < 0.5 s (pink) in the standard non-competitive model (A). These areas are plotted in the spaces of [D2R]tot versus [RGS]tot , [D2R]tot versus [AC]tot , and [RGS]tot versus [AC]tot . Dotted lines denote their analytical solutions. (H–N) Same as panels A–G, but the standard D2 model is modified so that AC sequesters Gi, i.e., V4, V5, V6, and V7 are removed only from Eq (S3) but not from Eq (S12).(A–G) Areas of (TIF)Click here for additional data file.S8 Figi-GTP , while the Gi-GTP is drained through RGS with a speed (V2 + V8) that is proportional to the amount of Gi-GTP. Difference in the fluxes generates a pool of Gi-GTP that inhibits AC, which is represented by a sunk ball (AC). During the period of a DA dip (right), the supply of Gi-GTP nearly completely stops, and the pooled Gi-GTP is drained rapidly. AC is then disinhibited to be activated by Golf and Ca2+-CaM. (B-E) The Gi inhibition of AC in the healthy adult, healthy infant, schizophrenia, and dystonia, all of which are described in Fig 6A−6E. (C) In the healthy-infant model, the influx and efflux of Gi-GTP are both small, and the smaller efflux causes delayed disinhibition of AC during a DA dip. (D) In the schizophrenia model, hyperactive D2R provides a larger amount of Gi-GTP, thus AC is fully inhibited and its disinhibition is delayed. (E) The larger efflux of Gi-GTP in the dystonia model results in the chronic activity of AC.(A) At the steady state (left), DA-bound D2R provides a constant flux of G(TIF)Click here for additional data file.S9 Figolf) coincides with a DA dip/burst with a variety of delay times. (A) Schematic of AC5 signaling. (B) Adenosine burst as a square wave of Golf . (C) Square-wave dip and burst of DA . (D-G) Example traces of the AC5 activities , and DA-dip/burst delay dependence of the maximal ACactive (bottom) in the healthy-adult, healthy-infant, schizophrenia, and dystonia models. In the example traces, adenosine and DA signals were given during the periods indicated by blue and red bars, respectively. Similar simulation (adenosine versus transient DA dips) has been conducted by Nair et al. (2015)  where NA = 6.02 ×1023 (Avogadro constant).Densities of membrane molecules should have the unit of membrane area (/μm(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 AppendixACbasal and ACdip. (C) Derivation of analytical T1/2.(A) Definition of the D2 model. (B) Derivation of analytical (PDF)Click here for additional data file.


By vaccinating SARS-CoV-2 naïve individuals who have already received two doses of COVID-19 vaccines, we aimed to investigate whether a heterologous prime-boost strategy, using vaccines of different platforms as the booster dose, can enhance the immune response against SARS-CoV-2 virus variants. Participants were assigned into four groups, each receiving different combination of vaccinations: two doses of BNT162b2 followed by one dose of BNT162b2 booster (B-B-B); Combination of BNT162b2 (first dose) and CoronaVac (second dose) followed by one dose of BNT162b2 booster (B-C-B); two doses of CoronaVac followed by one dose of CoronaVac booster (C-C-C); two doses of CoronaVac followed by one dose of BNT162b2 booster (C-C-B). The neutralizing antibody in sera against the virus was determined with live virus microneutralization assay (vMN). The B-B-B group and C-C-B group demonstrated significantly higher immunogenicity against SARS-CoV-2 Wild type (WT), Beta variant (BV) and Delta variant (DV). In addition, the B-B-B group and C-C-B group showed reduced but existing protection against Omicron variant (OV). Moreover, A persistent rise in vMN titre against OV was observed 3 days after booster dose. Regarding safety, a heterologous prime-boost vaccine strategy is well tolerated. In this study, it was demonstrated that using vaccines of different platforms as booster dose can enhance protection against SARS-CoV-2 variants, offering potent neutralizing activity against wild-type virus (WT), Beta variant (BV), Delta variant (DV) and some protection against the Omicron variant (OV). In addition, a booster mRNA vaccine results in a more potent immune response than inactivated vaccine regardless of which platform was used for prime doses. Coronavirus disease 2019 (COVID-19) is an upper respiratory tract infection caused by SARS-CoV-2 and has disrupted our daily lives since the end of 2019, leading to unprecedented global vaccination plans aiming to end the pandemic. Different vaccine platforms were approved for large-scale vaccination, such as inactivated virus vaccines (CoronaVac), viral vector vaccines (ChAdOx1nCoV-19 vaccine), mRNA vaccines (BNT162b2 vaccine), subunit vaccines (S-Trimer vaccine), etc. ,4. With As the current available vaccines were developed based on SARS-CoV-2 wild-type virus (WT), a heterologous prime-boost approach, using different combinations of COVID-19 vaccine candidates, was proposed. Such an approach was demonstrated to be effective in animal models and humans, and different combinations of vaccine platforms and sequences of the combinations demonstrated different effectiveness ,8,9. It This is a prospective cohort study performed in the Hong Kong West Cluster Hospitals under the Hospital Authority in Hong Kong. Twenty-three SARS-CoV-2 naïve healthy individuals who completed two doses of COVID-19 vaccine for at least 6 months were recruited and were given a booster vaccine dose (third dose) according to their preference. Their blood samples were collected before booster vaccination (baseline) and at least 3 days after the booster dose. In addition, 14 participants who had already received the booster dose for more than 3 days were also recruited and had their blood collected . All 37 The nurse administered the vaccines as an intramuscular injection according to the participant’s choice. Recruited participants were then assigned to 4 groups based on the vaccine platforms of their prime dose and booster dose: participants primed with 2 doses of BNT162b2 and received 1 booster dose of IM BNT162b2 (0.3 mL) (B-B-B); participants primed with BNT162b2 (first dose) and CoronaVac (second dose) and received 1 booster dose of IM BNT162b2 (0.3 mL) (B-C-B); participants primed with 2 doses of CoronaVac and received 1 booster dose of IM CoronaVac (0.5 mL) (C-C-C); participants primed with 2 doses of CoronaVac and received 1 booster dose of IM BNT162b2 (0.3 mL) (C-C-B).CoronaVac  is a purified inactivated SARS-CoV-2 vaccine candidate developed with CN2 strain of SARS-CoV-2 . BNT162b2, the cytopathic effect (CPE) was examined and the antibody titre was determined by the highest dilution with 50% inhibition of CPE (Blood was taken from the participants before the booster dose (baseline) and at least 3 days post-vaccination for the antibody assay. As described by our previous study, live virus microneutralization assay (vMN) was performed in the Biosafety level 3 facility of HKU to determine the level of neutralizing antibody in sera . Serial n of CPE .To assess the safety and adverse events of the booster dose, participants were asked to record any adverse events for 7 days after vaccination.The primary endpoint of this study was the vMN geometric mean titre (GMT) against WT, BV, DV and OV. The secondary endpoints were GMT fold increase and safety. For safety, severe adverse events (SAEs) were defined as death, disabling or life-threatening conditions related to vaccine; adverse events include fever (>38 °C), chills, headache, tiredness, nausea, vomit, diarrhea, muscle pain, joint pain, facial dropping, skin rash or injection site reactions .p < 0.05, the result was statistically significant. SPSS statistics  and GraphPad PRISM 9 were used for statistical computation.A statistical inference of normally distributed continuous variables was performed using t-tests and one way ANOVA, including demographic parameters (age), GMT and GMT fold increase. Categorical variables were analyzed using Pearson’s chi-squared test and Fisher’s exact test. When n = 15, median age = 53 years); participants primed with BNT162b2 (first dose) and CoronaVac (second dose) who received one booster dose of BNT162b2 ; participants primed with two doses of CoronaVac who received one booster dose of CoronaVac ; participants primed with two doses of CoronaVac and who received one booster dose of BNT162b2 . There was no statistically significant difference in age (p = 0.54), sex ratio (p = 0.888), comorbidities (p = 0.395) and number of days after the booster dose when the blood was sampled (p = 0.244) between all the groups , after administration of the booster dose, B-B-B group  and C-C-B group  had a significant higher Geometric mean titre (GMT) than C-C-C group   in sera was determined by vMN. For SARS-CoV-2 wild type (WT) virus, sera from participants in the B-B-B group had a significantly higher level of antibody than the other groups (.3–72.1) a. The GM.3–72.1) .For vMN titre against Beta variant (BV), there was no significant difference in the baseline GMT level across the groups. After administration of booster dose, B-B-B group  had a significant higher GMT level than C-C-C group . The GMT level of B-C-B group , C-C-B group  was also elevated but was not significantly higher than the GMT level of the C-C-C group b. The GMRegarding immunity against the Delta variant (DV), there was some pre-existing nAb against the virus in sera of the B-B-B and C-C-C groups. After booster dose, the B-B-B group  and C-C-B group  showed a significantly higher level of GMT than the C-C-C group . There was also an elevated GMT level in the B-C-B group  but it was not significantly higher than that of the C-C-C group c. The GMp = 0.077) but not the B-C-B group and C-C-C group (The immunogenicity of the booster dose against the Omicron variant (OV) is markedly reduced. For immunity against Omicron variant (OV), there was no pre-existing immunity in any of the group. After the booster dose, the GMT level was relatively higher in the B-B-B group  and the C-C-B group  than the C-C-C group  and the B-C-B group  d, but th-C group .In terms of changes in the vMN titre against the Omicron variant after booster dose, the GMT titre is 5  at baseline, which raised to 9.52 (95% CI 4.76–19) at day 3–7, 26.4  at day 8–14 and 23.5  after day 14. Surprisingly, the GMT titre after day 14 was similar to that at day 8–14 . This map = 0.733). Other injection site reactions such as redness (p = 0.523), swelling (p = 0.560) were also reported. Interestingly, injection site itchiness was reported and was significantly higher in the B-C-B group (p = 0.037). Other common adverse events reported include headache (p = 0.063), tiredness (p = 0.763), muscle pain (p = 0.899) and joint pain (p = 0.483). Fever (p = 0.174), chills (p = 0.196) and diarrhea (p = 0.103) were reported in a few cases. There was no report of severe adverse events , a booster dose of mRNA vaccine BNT162b2) offers more potent neutralizing activity against the Delta variant compared to using inactivated vaccine as a booster dose . Similar62b2 offeAs shown in this study, individuals who received two doses of inactivated vaccine and an inactivated booster dose had a less potent immune response against virus variants; therefore, these individuals may consider receiving a mRNA booster dose for better protection against virus variants. In terms of safety, our study shows that there were no statistically significant differences in terms of side effects between different combinations of vaccines except for itchiness, suggesting that a heterologous prime-boost vaccines strategy has a similar safety profile compared to a homologous prime-boost vaccine strategy, and is well tolerated.The limitation of this study is the small sample size, which lead to wide 95% confidence intervals of GMT. In addition, some of the patients were recruited after they received the booster dose and their baseline neutralizing antibody level before vaccination was lacking. For future work, cellular immunity induced by a heterologous prime-boost strategy should be evaluated, and the recruited patients should also be followed up to monitor the changes in neutralizing antibody level in long term.Our study found that a heterologous prime-boost approach using one booster dose of mRNA vaccine (booster dose) can enhance protection against SARS-CoV-2 variants, including wild type, Beta variant, Delta variant and Omicron variant. Although the response against Omicron variant (OV) is less potent compared to other variants, recipients of the third booster dose vaccine can still benefit from it. In conclusion, our study demonstrated that combination of vaccine platforms can be a potential vaccine strategy against emergence of virus variants.


The renal outer medullary potassium (ROMK) channel accounts for the major K+ secretory route in collecting ducts during basal conditions. Activation of the angiotensin II (Ang II) type 1 receptor (AT1R) by Ang II is known to inhibit ROMK activity under the setting of K+ dietary restriction, underscoring the role of the AT1R in K+ conservation. The present study aimed to investigate whether an AT1R binding partner, the AT1R-associated protein (ATRAP), impacts Ang II-mediated ROMK regulation in collecting duct cells and, if so, to gain insight into the potential underlying mechanisms. To this end, we overexpressed either ATRAP or β-galactosidase , in M-1 cells, a model line of cortical collecting duct cells. We then assessed ROMK channel activity by employing a novel fluorescence-based microplate assay. Experiments were performed in the presence of 10−10 M Ang II or vehicle for 40 min. We observed that Ang II-induced a significant inhibition of ROMK in LacZ, but not in ATRAP-overexpressed M-1 cells. Inhibition of ROMK-mediated K+ secretion by Ang II was accompanied by lower ROMK cell surface expression. Conversely, Ang II did not affect the ROMK-cell surface abundance in M-1 cells transfected with ATRAP. Additionally, diminished response to Ang II in M-1 cells overexpressing ATRAP was accompanied by decreased c-Src phosphorylation at the tyrosine 416. Unexpectedly, reduced phospho-c-Src levels were also found in M-1 cells, overexpressing ATRAP treated with vehicle, suggesting that ATRAP can also downregulate this kinase independently of Ang II-AT1R activation. Collectively, our data support that ATRAP attenuates inhibition of ROMK by Ang II in collecting duct cells, presumably by reducing c-Src activation and blocking ROMK internalization. The potential role of ATRAP in K+ homeostasis and/or disorders awaits further investigation.Adjustments in renal K Ang II/or AT1R; . Accordiiets LK; , and AT1ervation , underliin vivo with AT1R in all nephron segments, from the Bowman’s capsule to the inner medullary collecting duct cells . ATRAP colocalizes ct cells . It has ct cells . Howeverct cells  and thatct cells . The impct cells .+ homeostasis remains to be established. Therefore, the present study aimed to test the hypothesis that ATRAP impacts Ang II-mediated ROMK regulation and if so, to gain insight into the potential underlying mechanisms.However, the potential role of ATRAP on Ang II-regulation of KReagents were obtained from ThermoFisher Scientific  unless otherwise noted. VU591, a specific ROMK inhibitor , and dex2 tissue culture flasks in a 1:1 mixture of Dulbecco’s modified Eagle’s medium/Ham’s F12 medium (DMEM/F12) supplemented by 100 U/ml penicillin, 100 μg/ml streptomycin, 0.5 mM sodium pyruvate, 5 μM dexamethasone, and 5% (v/v) heat-inactivated fetal bovine serum (FBS) as recommended by the supplier. Cultures were incubated in a humidified 95% air-5% CO2 atmosphere at 37°C and subcultured by 0.25% trypsin-EDTA. Cells were seeded onto 24-well microplates for immunoblotting experiments or 96-well microplates for FluxOR assay experiments. Non-transfected cells were grown to confluence and then serum-starved for at least 72 h. Lysates from M-1 cells were immunoblotted with antibodies against the α subunit of ENaC and ROMK for cell line characterization (M-1 collecting duct cells (ATCC CRL-2038) were kindly provided by Dr. Per Svenningsen  and maintained in 25-cmrization ,B. Band rization .2 of cell culture, 2 μg of plasmid DNA and 1-2 μl lipofectamine 2000 were added to the serum-free medium for 24 h. Cells were then maintained in a serum-free medium for at least more 48 h before conducting functional experiments.M-1 ATRAP coding sequence was amplified using 5' primer and 3' primer containing Hind III and Not I restriction sequences, respectively (5'-ACACAAGCTTGGGATGGAGCTGCCTGCCGTGAAC-3' and 5'-ACACGCGGCC-GCGGTACCCCCGGGGGGCAGCTTG-3') and cloned into pcDNA6/V5-His vector (ThermoFisher). pAcGFP1-C1 (Clontech) was used to assess lipotransfection efficiency. Cultures were serum-deprived at ~90% confluence and lipotransfected with pcDNA6/V5-His/galactosidase (LacZ) control vector, encoding a β-galactosidase protein, or with pcDNA6/V5-His/ATRAP vector, both of them tagged to V5-His at their C-termini. For every 2 cm+ channel and transporter activity and takes advantage of recently developed thallium-sensitive fluorophores . The assay is based on the fact that thallium influx serves as a surrogate for Krophores . The FluM-1 cells were solubilized by radioimmunoprecipitation assay (RIPA) buffer , and 10 mM Tris-HCl pH 8.0), supplemented with 5 mM EDTA, 1 mM EGTA, 0.2 mM PMSF, and protease cocktails . Phosphatases inhibitor cocktails [P5726 and P0044 (Sigma-Aldrich)], at 1:150 dilution, were also added to RIPA buffer when assessing phospho-c-Src expression. Protein supernatants were obtained by centrifuging cell lysates at 14,000 rpm for 20 min. Protein quantification was performed using the BCA or Lowry assay. The appropriate amount of lysate was denatured by Laemmli buffer . About 7.5–12%, SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described previously . Protein−10 M Ang II for 40 min before biotinylation protocol as previously described in detail  before exposing cells to vehicle or Ang II (10−10 M). As observed in + conductance. Finally, M-1 cells were also treated with Ang II or vehicle for 40 min in the presence or absence of 10 μM VU591. As illustrated in + conductance by ~45 ± 3% in comparison with vehicle-treated cells, underlining the critical role of the ROMK channel in apical K+ conductance in M-1 cells. Similar to the findings of + conductance by ~30 ± 6% compared to vehicle-treated M-1 cells. Importantly, VU591 and Ang II cotreatment did not induce an additive inhibitory effect on K+ conductance, supporting the notion that Ang II-mediated modulation of K+ transport in M-1 cells is due to the regulation of ROMK activity.In order to assess whether the Ang II-mediated effect on Kp < 0.05), but not in ATRAP-overexpressing cells (~15 ± 2%), when compared to vehicle-treated LacZ-overexpressing M-1 cells.The efficiency of M-1 transfection was evaluated by the emission of fluorescence in mock‐ or GFP-transfected M-1 cells and the efficacy of overexpression of ATRAP and recombinant β-LacZ was confirmed by immunoblotting using an anti-V5 antibody ,B. We th+ conductance observed in ATRAP-overexpressing M-1 cells is present at baseline conditions (8 ± 3% inhibition vs. vehicle-treated LacZ-overexpressing cells). In fact, when this difference at basal levels is taken into account, the magnitude of ROMK inhibition mediated by Ang II in ATRAP-overexpressing cells is only 7 ± 3% when compared to vehicle-treated ATRAP-overexpressing cells, an effect significantly smaller than that observed in LacZ-overexpressing cells . It was observed that Ang II treatment caused a reduction of ROMK cell surface expression in only one out of four samples from ATRAP-overexpressing cells. As seen in p < 0.05 vs. LacZ-overexpressing cells). Finally, we also assessed total ROMK expression normalized to GAPDH expression, and no difference was observed among experimental groups .Albeit ATRAP overexpression ignaling . The autIt is known that ROMK inhibition by c-Src is not directly caused by channel phosphorylation. Previous studies demonstrated that ROMK inhibition by exogenous c-Src was only seen in cell-attached patches but not in isolated patches. This inhibitory effect was associated with phosphorylation of tyrosine 337 of the channel and was abolished by agents that inhibit endocytosis . Therefo+ conservation through ROMK inhibition is an essential mechanism for K+ homeostasis without evoking other electrolyte imbalances. When such a regulatory mechanism is not enough to maintain K+ balance during LK diets, the consequent decreases of extracellular K+ levels induce hyperactivation of NCC transporter, thus linking K+ restriction to hypertension (+ levels and could impact not only K+ homeostasis but also pressure outcomes in K+-restricted diets. In this regard, + intake in healthy Malaysian adults. Subjects with the genotype AA, associated with lower expression of AT1R (+ intakes were low. Also, + restriction may also be associated with genetic variations in other RAS components, including ATRAP.Whereas the ATRAP’s impact on sodium balance and volemic homeostasis has been shown in different studies, particularly during Ang II-induced hypertension , its imprtension . Therefo of AT1R , showed + homeostasis and blood pressure control could resemble some effects of losartan treatment observed during poor-K+ diets (+-rich DASH diet amplifies losartan treatment response, particularly in African-American subjects, so that the combined treatment is more than additive (+ diets could be blunt during diets with a modest or more severe K+ restriction.The impact of differential regulation of ATRAP on ROMK regulation and, consequently, on KK+ diets . As aforK+ diets . This coadditive . It is kadditive . Notablyadditive . Taking + secretion capacity of collecting ducts. Interestingly, in vitro observations give us direct evidence of ATRAP’s ability to attenuate AT1R-mediated ROMK inhibition, further in vivo works will be important for assessing how ATRAP is modulated during changes in potassium intake and how CCD-specific ATRAP transgenic animals respond to potassium dietary changes, for instance. These analyses will address ATRAP’s physiological impact on potassium balance and might help us to explain previous physiological observations in this field.On the other hand, during HK diets, ATRAP might be an important component determining the K+ handling. As depicted in + conductance is inhibited in LacZ-overexpressing M-1 cells treated with Ang II. In contrast, this response is blunted in M1 cells overexpressing ATRAP. Accordingly, this differential response is accompanied by a decrease in the expression of ROMK channels at the plasma membrane of LacZ-overexpressing M-1 cells, but not in ATRAP-overexpressing cells. Finally, blunted response to Ang II in ATRAP-overexpressing M1 cells is associated with diminished c-Src phosphorylation signaling, a pathway that seems to be critically involved in ROMK endocytosis. Future studies should be performed in animal models to evaluate the impact of differential ATRAP expression in K+ homeostasis during different changes of K+ dietary content. Moreover, it would be interesting to assess how polymorphisms in ATRAP could be associated with changes in plasma K+ levels and/or in blood pressure during K+-poor diets in different human populations.In conclusion, our study demonstrates that ATRAP can counteract Ang II-mediated inhibition of ROMK activity and might then be a relevant protein regulating KThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.AG and NR designed the study. JP carried out the experiments, analyzed the data, wrote the manuscript, and prepared the figures. All authors contributed to the article and approved the submitted version.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.


Pseudomonas spp. including the soil bacterium Pseudomonas protegens Pf-5. In this study, we show that PltR, a known pathway-specific transcriptional activator of pyoluteorin biosynthesis genes, is necessary but not sufficient for pyoluteorin autoinduction in Pf-5. We found that pyoluteorin is perceived as an inducer by PltZ, a second pathway-specific transcriptional regulator that directly represses the expression of genes encoding a transporter in the pyoluteorin gene cluster. Mutation of pltZ abolished the autoinducing effect of pyoluteorin on the transcription of pyoluteorin biosynthesis genes. Overall, our results support an alternative mechanism of antibiotic autoinduction by which the two pathway-specific transcriptional regulators PltR and PltZ coordinate the autoinduction of pyoluteorin in Pf-5. Possible mechanisms by which PltR and PltZ mediate the autoinduction of pyoluteorin are discussed.Antibiotic biosynthesis by microorganisms is commonly regulated through autoinduction, which allows producers to quickly amplify the production of antibiotics in response to environmental cues. Antibiotic autoinduction generally involves one pathway-specific transcriptional regulator that perceives an antibiotic as a signal and then directly stimulates transcription of the antibiotic biosynthesis genes. Pyoluteorin is an autoregulated antibiotic produced by some  N-acyl-homoserine lactones, also known as quorum sensing signal molecules, which regulate cell-to-cell communication in many bacteria. In a classic model, N-acyl-homoserine lactone binds to its cognate response regulator and directly activates expression of N-acyl-homoserine lactone biosynthesis gene(s), which leads to an enhanced level of the signal molecule [Autoinduction, also called positive feedback regulation, is commonly used by bacteria to induce phenotypes required to respond promptly to a particular environmental condition ,2,3. Themolecule .In addition to the quorum sensing signal molecules, production of many antibiotics is also controlled by autoinduction ,13,14,15Pseudomonas species including P. aeruginosa [P. protegens (previously known as P. fluorescens) [P. protegens Pf-5. Adding nanomolar concentrations of pyoluteorin to Pf-5′s cultures enhanced pyoluteorin production and transcription of pyoluteorin biosynthesis genes [P. protegens CHA0 [P. aeruginosa M18 [2) with a chemical structure distinct from pyoluteorin  . Autoindis genes . Later, ens CHA0  and P. anosa M18 . Productnosa M18 , which bnosa M18 . PltR benosa M18 . Pyolutenosa M18 . Howeverluteorin A, is now cluster A, binds Pseudomonas protegens Pf-5 and its mutants were cultured at 27 °C on King’s Medium B agar [The bacterial strains used in this study are listed in m B agar , NutrienpltZ, a 235-bp DNA fragment containing pltZ promoter was amplified from the Pf-5 genome using oligonucleotide pair pltI-F4/pltI-R4  carried by the vector pME6010 [The expression construct pME6010-/pltZ-R2 . The exp pME6010 .XhoI-NdeI PCR fragment containing pltZ gene into pET28a digested by the same restriction enzymes. This DNA fragment was amplified from the genomic DNA of Pf-5 using oligonucleotide pair pltZ 5′/pltZ 3′  of 1.0, and then used to inoculate 200 μL NBGly plus kanamycin to obtain a start OD600 of 0.01. Each strain was grown in three wells of a 96-well plate, which was incubated in a microplate shaker at 27 °C with shaking at approximately 400 r.p.m. Bacterial growth was monitored by measuring the OD600. The green fluorescence of bacteria was monitored by measuring emission at 535nm with an excitation at 485nm and corrected for background by subtracting fluorescence emitted by control strain which has an empty vector of the reporter construct.The GFP-based transcriptional reporter assays were modified from a previous report . BrieflyE. coli BL21 (DE3). Cells were cultured in LB broth with 50 μg/mL kanamycin at 37 °C to an OD600 of 0.5, then induced by 0.4 mM IPTG, and incubated overnight at 20 °C. The cells were lysed in Tris-NaCl buffer . The PltZ proteins with a N-terminal 6xHis tag were purified using a Ni-NTA Purification System (Invitrogen) and dialyzed in Tris-NaCl buffer. The protein concentrations were determined using a DC Protein Assay .Protein overexpression and purification of PltZ was modified from previous method . BrieflypltIZ (TATTTTCTAATTTAAATTCAAATTGAATTTTAATTAGGCCTTGG) and a 66-bp DNA probe named pltRL (TCGGGGCTGTTTTGCCTTTGCGGATATGCAAAGGCCTTTTGCAAAAACGGCTATTCACAAGTGCAT), that contain the putative promoter region of pltI and pltL, respectively, were synthesized and labeled with biotins using a Biotin 3′ End DNA Labeling Kit (ThermoFisher). Labeled DNA probes (1 nM) were mixed with different concentrations of PltZ protein and incubated at 25 °C for 20 min in a 20 μL reaction. If needed, unlabeled DNA probes (1 μM) were used to compete with the labeled DNA in binding PltZ. Various concentrations of pyoluteorin were added in the reaction to test if this compound can release free DNA probes from the protein-DNA complex. The reaction samples were loaded to a 5% native PAGE gel  and electrophorized in 0.5× Tris Borate-EDTA (TBE) buffer at 80 V for one hour in a 4 °C cold room. The DNA samples were transferred onto a nylon membrane  which then was exposed to a UV light treatment at 254nm for 10min to fix the nucleotides and the images were developed using X-ray films .Electrophoretic Mobility Shift Assay (EMSA) experiments were performed at 25 °C using a LightShift™ Chemiluminescent EMSA Kit (ThermoFisher). A 44-bp DNA probe named Interaction of PltZ with pyoluteorin was tested by two approaches. In the first approach, different concentrations (0–57.5 μM) of pyoluteorin were added into a PltZ protein solution (39 μM) which was prepared in a Tris-HCl buffer . Absorbance of PltZ protein, pyoluteorin compound and their complex was measured in a 1-cm-path cuvette using a UV–vis spectrophotometer (Beckman DU730). The protein-compound solution which contains PltZ protein over saturated with pyoluteorin compound (57.5 μM) was loaded onto a Sephadex G-25 column (1 × 20 cm) preequilibrated and washed with 80 mL of the Tris-HCl buffer. The PltZ-pyoluteorin complex was eluted with 7 mL of the Tris-HCl buffer. The absorbance of purified PltZ-pyoluteorin complex was measured using the same method described above.In the second approach, Isothermal titration calorimetry (ITC) experiments were performed at 25 °C using a MicroCal VP-ITC microcalorimeter . Pyoluteorin was dissolved in methanol first and then diluted in the Tris-HCl buffer to a concentration of 100 μM. The titration assay included an initial 2 μL injection, followed by 44 (10 μL) injections of pyoluteorin into PltZ protein solution prepared above, accompanied by stirring at a constant rate. Protein samples and compound solutions were degassed by centrifugation at 14,000 r.p.m. for 20 min prior to the titration experiment. Data were processed using Origin 7.0 and fit to an independent binding model to calculate the thermodynamic parameters of interactions between PltZ and pyoluteorin.pltA mutant was used to exclude the influence of internal pyoluteorin on the expression of pyoluteorin biosynthesis genes. The promoter activity of pltL, the first gene of the pyoluteorin biosynthesis gene cluster, was measured using the reporter construct pL-gfp which contains the pltL promoter fused with a promterless gfp  is(are) needed for autoinduction of pyoluteorin.pltR, the plt gene cluster encodes another transcriptional regulator PltZ (pltZ genes repress the expression of linked transporter genes in P. aeruginosa strains M18 [P. protegens Pf-5) has not been demonstrated, and the function of PltZ in P. protegens has not been characterized. In this work, pltZ was deleted in the ΔpltA mutant of Pf-5, which generated a ΔpltAΔpltZ double mutant. Two reporter constructs, pZ-gfp and pI-gfp, containing the promoter of pltZ and pltI respectively fused with a promoterless gfp, were made to test if PltZ regulates the expression of itself or the linked transporter genes pltIJKNOP  pltZ repressed pltI promoter activity heterogeneously in strain SBW25  pyoluteorin released a pltIpromoter-containing DNA probe bound by PltZ  pyin SBW25 A; (3) Pl by PltZ .pltIJKNOP transporter gene expression by PltZ, together with the result that mutation of pltZ abolished autoinduction of pyoluteorin  identified in P. aeruginosa ATCC 27853 [pltI promoter region of Pf-5 (pltL promoter region (pltR and pltL of Pf-5 (P. aeruginosa ATCC 27853 [pltL directly.Another possible mechanism by which PltZ controls pyoluteorin autoinduction is that PltZ may directly bind to CC 27853  is also  of Pf-5 A. However region C, and no of Pf-5 D or P. aCC 27853 , suggestpltIJKNOP transcription. Results of this study also show that pltZ, together with the other pathway-specific transcriptional regulator pltR, is essential for autoinduction of pyoluteorin in Pf-5.In summary, we report that pyoluteorin serves as an inducer that binds to PltZ and relieves PltZ-mediated repression of