Multiple signaling pathways regulate the kinase GSK3β by inhibitory phosphorylation at Ser9, which then occupies the GSK3β priming pocket and blocks substrate binding. Since this mechanism should affect GSK3β activity toward all primed substrates, it is unclear why Ser9 phosphorylation does not affect other GSK3β-dependent pathways, such as Wnt signaling. We used biochemical reconstitution and cell culture assays to evaluate how Wnt-associated GSK3β is insulated from cross-activation by other signals. We found that the Wnt-specific scaffold protein Axin allosterically protects GSK3β from phosphorylation at Ser9 by upstream kinases, which prevents accumulation of pS9-GSK3β in the Axin•GSK3β complex. Scaffold proteins that protect bound proteins from alternative pathway reactions could provide a general mechanism to insulate signaling pathways from improper crosstalk. Glycogen Synthase Kinase 3β (GSK3β) is a potential therapeutic target for a range of diseases , but tarBoth Wnt and growth factor/insulin signaling pathways regulate GSK3β, but these pathways do not cross-activate . In Wnt Previous work in the field suggests two potential biochemical mechanisms that could insulate Wnt signaling from insulin and growth factor signals. First, by tethering GSK3β and the Wnt substrate β-catenin together, the Axin scaffold could rescue enzyme activity from the inhibitory effects of Ser9 phosphorylation . A seconkcat, KM, and kcat/KM as described previously  binds GSK3β with a similar affinity as full-length Axin1-826 . Additiogth Axin . Althougds GSK3β  and cannds GSK3β . Axin381miniAxin . This reOne possible structural explanation for the functional data is that the Ser9 phosphorylation site adopts an inaccessible conformation when Axin binds GSK3β. However, Ser9 is located in a flexible N-terminal tail that is unresolved in x-ray crystal structures with or without Axin bound, providing no clear indication for structural changes upon Axin binding . An alteTo determine if Axin prevents pS9-GSK3β accumulation in vivo, we overexpressed Axin in HEK293 cells. In the absence of Axin overexpression, we observed significant basal levels of pS9-GSK3β, in agreement with previous results . When AxThe observation that Axin protects GSK3β from PKA phosphorylation in vitro is consistent with previous in vivo co-immunoprecipitation experiments suggesting that Axin-associated GSK3β is not phosphorylated at Ser9 . Beyond 127-135 peptide ILSRRPSYR and mouse PKA were cloned and expressed as previously described  and a C-terminal His6 tag. The catalytic subunit of mouse PKA was expressed from pET15b with an N-terminal His-tag (addgene #14921) , along with the human CREBescribed . All seq #14921) .pS45-β-catenin was produced by coexpression with CK1α, as previously described . Lambda E. coli cells by inducing with 0.5 mM IPTG overnight at 18 °C. Constructs with N-terminal MBP and C-terminal His6 tags  were affinity purified with HisPur Ni-NTA resin (Thermo Scientific) and amylose resin (NEB). The PKA catalytic subunit was purified on Ni-NTA resin. pS45-β-catenin was produced by coexpression with CK1α, as previously described  and then dialyzed overnight at 4 °C in the presence of 500 nM TEV protease. The solution containing the cleaved peptide was separated from the amylose resin, concentrated, and purified by size exclusion chromatography using a Superdex 75 10/300 GL column  to remove TEV protease. Chromatography fractions were monitored by Coomassie-stained SDS-PAGE as the peptide is not detectable by UV. The final purified Axin peptide is GS-Axin381-407-SGR-His6.For quantitative kinetic and binding assays, all Wnt pathway, CREB, and PKA proteins were expressed in Rosetta (DE3) pLysS escribed . The Axikcat/KM that is due to an ~fivefold increase in kcat (Unphosphorylated GSK3β and GSK3β_S9A were produced by coexpression with lambda phosphatase and affinity purified with HisPur Ni-NTA resin (Thermo Scientific). Treatment of GSK3β and GSK3β_S9A with lambda phosphatase produces an ~fivefold increase in  in kcat . To prod in kcat . The secg. Protein concentrations were determined using a Bradford assay (Thermo Scientific). pS45-β-catenin was further purified by size exclusion chromatography using a Superdex 200 Increase 10/300 GL column  to remove a copurifying fragment before being dialyzed into 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, and 2 mM DTT, aliquoted, and stored at –80 °C. A Coomassie gel showing the purity of the proteins in this work is shown in Purified proteins were dialyzed into 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, and 2 mM DTT at 4 °C, aliquoted and stored at –80 °C. If necessary, proteins were concentrated using 10000 or 30000 MWCO Amicon Ultra-15 Centrifugal Filter devices at 4 °C, 2000×2, and 0.05% IGEPAL) at 25 °C in 60 µL total volume. Reactions were initiated by adding ATP to a final concentration of 100 µM. This ATP concentration is saturating for all reactions  antibody  that recognizes pS133-CREB127-135 (GSK3β-phosphorylated β-catenin was detected using a primary anti-Phospho-β-Catenin (Ser33/37/Thr41) antibody  that recognizes triply phosphorylated pS33/pS37/pT41-β-catenin . PKA-phoB127-135 . For all127-135 reactions the endpoint is pS133-CREB127-135, phosphorylated to completion by PKA. The pS133-CREB127-135 standard was prepared in a reaction with 50 nM CREB127-135, 100 nM PKA, and 200 µM ATP in kinase assay buffer at 25 °C for 20 hr.Concentrations of phosphorylated product in each reaction were determined by comparing western blot signal intensities to an endpoint standard containing 50 nM product phosphorylated to completion . For pS4Vobs) vs. [substrate] to the Michaelis-Menten equation kcat and KM reported in kcat/KM were obtained by fitting an alternative form of the equation kcat/KM was obtained from the slope of linear fit to the plot of Vobs vs. [substrate].Initial rate measurements were obtained from three independent reactions . Phosphorylated product levels from quantitative western blots were analyzed using Image Studio Lite 5.2.5 (Li-Cor) and kinetic parameters were determined by fitting to the Michaelis-Menten equation or to a linear equation using Kaleidagraph 4.1.3. Initial rates for each reaction were determined by fitting a linear model to a graph of [product] vs time. Kinetic parameters were determined by fitting plots of initial rates . Cell cultures were tested monthly for mycoplasma contamination (Southern Biotech #13100–01).Cell culture experiments were performed with HEK293 cells . Twenty-four hr following transfection, cells were starved by replacing media with 1% FBS DMEM. Twenty-four hr after starvation, cells were treated with DMSO (2 μL in 10 mL 1% FBS DMEM). Serum starvation and DMSO treatment were included in our protocol for consistency with previously published experiments . Total GSK3β was detected using a primary anti-GSK3β antibody . The secondary antibodies were IRDye 800CW Goat Anti-Rabbit IgG antibody (Li-Cor #926–32211) for pS9-GSK3β and IRDye 680RD Donkey Anti-Mouse IgG antibody (Li-Cor #926–68072) for total GSK3β. pS9-GSK3β and total GSK3β levels were analyzed using Image Studio Lite 5.2.5 (Li-Cor).Protein expression plasmids  generate This study presents a valuable and elegant kinetic analysis of the GSKbeta activity as a function of phosphorylation and Axin binding - providing insights into critical steps of Wnt pathway signaling. The results will be of big use to the broader signaling community. The work will be of broad interest to cell biologists and biochemists. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting the paper "The Axin scaffold protects the kinase GSK3β from cross-pathway inhibition" for consideration by Comments to the Authors:eLife within its current scope. Both reviewers agree that the elegant kinetics analysis of GSKb activity as a function of phosphorylation and Axin binding is thorough and will be very useful for the broader scientific community. However, the main criticism shared by the reviewers, is that the study does not present a big conceptual advance in mechanistic understanding of how the Axin scaffold shields GSKb from phosphorylation and contributes to the insulation of this kinase from other activating stimuli. If you are confident that such evidence could be provided within the revisions time frame, eLife would welcome the resubmission and will do its best to send the revised manuscript for assessment to the same reviewers. However, if such evidence cannot be provided in a reasonable time frame, we encourage you to submit your study for consideration for publication in a more specialized journal.We are sorry to say that, after consultation with the reviewers, we have decided that this work will not be considered for publication by Reviewer #1 (Recommendations for the authors):– At the start of the Results section and in Figure 2, the definitions of kCAT and Km should be given in a bit more detail (at present the reader is only provided with a reference to the 2020 paper). This will make the story a bit easier to read for non-biochemists. For example, lines 106-109 are not intuitive to follow.– The added value of the kinetic schemes in each of the figure panels is unclear as the link to specific biological/signal transduction steps is not made clear.– The cartoons in Figure 1 could also be improved to clarify the questions/knowledge gaps and specific mechanistic options.– The authors convincingly show that AXIN1 can play a role in shielding GSK3 from auto- inhibition. As it stands, the impact of this work on the field of WNT/CTNNB1 signaling is likely to remain limited. This is mainly due to the reason that the mechanism by which AXIN1 shields the WNT/CTNNB1 signaling pool of GSK3 from pSer9 inhibition remains unresolved. Based on the fact that a mini AXIN1 (i.e. an AXIN1 fragment) behaves the same as WT AXIN1, the authors conclude that AXIN1 likely causes allosteric changes on GSK3 but is less likely to block PKA from binding. They cannot conclusively show this, however, as they do not have evidence in favour of one or the other explanation.– The impact of this work would be greater if the authors could indeed discriminate between the two possible mechanisms they present . [Editors’ note: The authors appealed the original decision. What follows is the authors’ response to the first round of review.]Reviewer #1 (Recommendations for the authors):– At the start of the Results section and in Figure 2, the definitions of kCAT and Km should be given in a bit more detail (at present the reader is only provided with a reference to the 2020 paper). This will make the story a bit easier to read for non-biochemists. For example, lines 106-109 are not intuitive to follow.kcat and KM values at the start of the Results section (lines 61-62). We have also added content and a reference to clarify lines 106-109 (now lines 83-86). Given the space restrictions for the Short Report format our explanations are brief, we request editorial guidance if further elaboration is necessary.We have added additional explanation of the significance of – The added value of the kinetic schemes in each of the figure panels is unclear as the link to specific biological/signal transduction steps is not made clear.Kinetic schemes are included with each figure panel to enable clear comparisons between different reactions. We appreciate the reviewer’s point that it would be helpful to connect each scheme to a biological step, but we suggest that significant space would be necessary to place each reaction in a biological context and would largely reproduce information that is available in Figure 1.– The cartoons in Figure 1 could also be improved to clarify the questions/knowledge gaps and specific mechanistic options.We have added a panel to Figure 1 to clarify the mechanistic models and we have corrected a mistake that was noted by Reviewer #2. We would be happy to further revise the figure if the editors or reviewers have additional suggestions.– The authors convincingly show that AXIN1 can play a role in shielding GSK3 from auto- inhibition. As it stands, the impact of this work on the field of WNT/CTNNB1 signaling is likely to remain limited. This is mainly due to the reason that the mechanism by which AXIN1 shields the WNT/CTNNB1 signaling pool of GSK3 from pSer9 inhibition remains unresolved. Based on the fact that a mini AXIN1 (i.e. an AXIN1 fragment) behaves the same as WT AXIN1, the authors conclude that AXIN1 likely causes allosteric changes on GSK3 but is less likely to block PKA from binding. They cannot conclusively show this, however, as they do not have evidence in favour of one or the other explanation.We thank the reviewer for this important comment which details the central concern raised in the review process. To address this point, we have collected additional biochemical data that conclusively shows that the Axin shielding effect is allosteric and not a steric block. We demonstrated that a minimal, 27 amino acid Axin peptide produces the same GSK3β shielding behavior as full length Axin and miniAxin. The minimal Axin peptide does not sterically occlude the GSK3β phosphorylation site. This data is included in a revised Fig 4A and described on lines 151-157 of the revised manuscript.– The impact of this work would be greater if the authors could indeed discriminate between the two possible mechanisms they present .We agree with the reviewer on this central point and have conducted additional experiments that conclusively resolve the issue. These experiments are described above and included in the revised manuscript .


To address the issues of not accurately identifying ice types and thickness in current fiber-optic ice sensors, in this paper, we design a novel fiber-optic ice sensor based on the reflected light intensity modulation method and total reflection principle. The performance of the fiber-optic ice sensor was simulated by ray tracing. The low-temperature icing tests validated the performance of the fiber-optic ice sensor. It is shown that the ice sensor can detect different ice types and the thickness from 0.5 to 5 mm at temperatures of −5 °C, −20 °C, and −40 °C. The maximum measurement error is 0.283 mm. The proposed ice sensor provides promising applications in aircraft and wind turbine icing detection. Aircraft and wind turbine icing is a common natural phenomenon . AircrafCurrently, accurate ice detection is still a significant challenge. First of all, it is hard to increase sensor measurement accuracy and range since there are many impact factors. These impact factors include the metrics, such as mean volume diameter (MVD), liquid water content (LWC), airflow speed (V), ambient pressure (P), and icing ambient temperature (T), which lead to three ice types, e.g., glazed ice, rime ice, and mixed ice, and thus affect the measurement accuracy of ice sensors seriously due to different physical characteristics of the ices such as density, dielectric constant, optical characteristics, and adhesion.Nowadays, the most commonly used icing detection method includes the representative technologies of piezoelectric ice sensors and resonant frequency ice sensors. Piezoelectric ceramic sensors, piezoelectric lead zirconate titanate sensors, and piezoelectric film sensors are three kinds of piezoelectric ice sensors. By delivering a sinusoidal scanning voltage to the driver, the piezoelectric ceramic sensor and the piezoelectric lead zirconate titanate sensor capture the response signal and detect the icing by evaluating the wavelet packet energy of the signal ,13. The Fiber sensor provides a solution for the ice detection problem for wind turbines and aircraft. There are some advantages of fiber-optic ice sensors including their small size and ability to be installed on the curve surface. Thus, they can be installed on the leading edge of wind turbine blades or wings but also can identify the ice type and obtain accurate icing information. Using the scattered and reflected light in ice, Aris A. Ikiades et al. proposed a method to measure the ice thickness in real time in 2007 . FurtherTo address the problems mentioned above, we propose a smaller fiber-optic ice sensor that can identify the ice type and measure ice thickness in the present study. The total reflection was applied in the probe structure design of the fiber-optic ice sensor. The numerical simulation and test validation were carried out to determine the light intensity and ice thickness relationship.Based on the assumption of the quasi-Gaussian distribution, the ray tracing method was applied to simulate the optical field from the fiber-optic ice sensor, including the light reflection at the ice–air interface and the light scattering within the ice ,20,21.REC, REF, and SCA denote the receiving, reflecting, and scattering of light, respectively.The fiber-optic ice sensor primarily detects the backscattered light inside the ice and reflected light at the ice–air interface as the light intensity. Equation (1) presents the received light intensity, including Equations (2) and (3), which present the mathematical models of reflection light intensity and the scattering light intensity, respectively.The proposed fiber-optic ice sensor is shown in The photoelectric conversion and amplifier circuit are key components of the fiber-optic ice sensor, as shown in The detection process of the sensor is modeled by the ray tracing method. The luminous power of the light source is 0.24 W . The output voltage of the sensor is recorded by a photoelectric conversion circuit. The record interval is 30 s, until the ice thickness reaches 5 mm.GLA, RIM1, and RIM2 represent glazed ice, rime ice 1, and rime ice 2, respectively.Similar to the simulations, we define the light intensity modulation function (M) in which the measured output voltage for RF2 is divided by that for RF1, as shown in As mentioned above, the ice type can be achieved by using the output voltage from RF3. Then, we can detect the ice thickness by using the light intensity modulation function in each ice type case. To verify the mathematical model, we detect the ice type and thickness again, as shown in (1)The ice type can be achieved by using the output voltage from RF3. Then, we can detect the ice thickness by using the light intensity modulation function in each ice type case.(2)The ice sensor can measure the glazed ice thicknesses from 1 to 5 mm. For the rime ice, the measuring range of ice thickness is from 0.5 to 5 mm.(3)The maximum and minimum ice thickness measurement errors are 0.283 mm and 0.087 mm, respectively, for the measuring range of 0.5 to 5 mm at −5 °C, −20 °C, and −40 °C.A novel fiber-optic ice sensor was proposed based on the total reflection principle and scattering methodologies. Numerical simulation and experimental validation were performed. Some conclusions are shown as follows:


Vibrio parahaemolyticus. The use of probiotics to control V. parahaemolyticus is recognized as a good alternative to antibiotics for avoiding the development of antibiotic-resistant bacteria. In this study, two strains of B. HLJ1 and B. C1 with strong inhibitory activity on V. parahaemolyticus were isolated from aquaculture water and identified as Bacillus subtilis and Bacillus pumilus, respectively. Both B. HLJ1 and B. C1 lacked antibiotic resistance and virulence genes, suggesting that they are safe for use in aquaculture. In addition, these two strains can tolerate acid environments, produce spores, secrete extracellular enzymes, and co-aggregate as well as auto-aggregate with V. parahaemolyticus. B. HLJ1 and B. C1 produced the same anti-V. parahaemolyticus substance, which was identified as AI-77-F and belongs to amicoumacins. Both B. C1 and B. HLJ1 showed inhibitory activity against 11 different V. parahaemolyticus and could effectively control the growth of V. parahaemolyticus in simulated aquaculture wastewater when the concentration of B. C1 and B. HLJ1 reached 1 × 107 CFU/mL. This study shows that B. HLJ1 and B. C1 have great potential as aquaculture probiotics.The adoption of intensive farming has exacerbated disease outbreaks in aquaculture, particularly vibriosis caused by  Vibrio parahaemolyticus, one of the primary causative agents of vibriosis, can cause acute hepatopancreatic necrosis disease (AHPND)  was co-cultured with isolated strains in 96 microwell plates  as Goulden et al. described  was examined, and there was a multiplicative power relation between fluorescence and the 17802 [GFP] number at the first 12 h (y = 6.6895xV. parahaemolyticus was classified into three categories: low inhibitory activity (25% ≤ Inhibition rate of V. parahaemolyticus < 50%), moderate inhibitory activity (50% ≤ Inhibition rate of V. parahaemolyticus < 75%), and high inhibitory activity (Inhibition rate of V. parahaemolyticus ≥ 75%).The inhibitory effect of strains on Bacillus species have been observed to display fairly conserved 16S rRNA sequences compared to other genera, and the use of this taxonomic marker is sometimes insufficient to define the species  at 105, 106, and 107 CFU/mL, respectively. When the initial concentration of isolated strains was set at 1 × 105 CFU/mL, the strains had relatively low inhibitory activity against V. parahaemolyticus and the inhibition rates were less than 25%—except for strain JZ12 which showed low inhibitory activity against the growth of V. parahaemolyticus with the inhibition rate against V. parahaemolyticus at 40.88%. The strain JZ12 shows moderate inhibitory activity against the growth of V. parahaemolyticus and the inhibition rate of JZ12 against V. parahaemolyticus was 51.29% when the initial concentration of isolated strains increased to 1 × 106 CFU/mL. In this initial concentration (1 × 106 CFU/mL), the inhibition rates of two strains  against V. parahaemolyticus were 32.61% and 37.88%. At the highest initial concentration (1 × 107 CFU/mL), strains JZ12 and M. JZ5 showed moderate inhibitory activity against V. parahaemolyticus and the inhibition rates of V. parahaemolyticus were 61.41% and 74.67%, respectively. The strains B. C1, B. HLJ1, M. CY1, M. SY7, and M. HLJ3 showed high inhibitory activity against the growth of V. parahaemolyticus, the inhibition rates of the five strains against V. parahaemolyticus were 100%, 100%, 100%, 100%, and 95.04%, respectively.  clustered into the same branch, and M. SY7 clustered with L. garvieae (NR113268.1), so the M. C1 and M. SY7 were identified as L. garvieae. The M. HLJ3 and Enterococcus faecalis (NR115765.1) clustered into the same branch and M. HLJ3 could be identified as E. faecalis. Both L. garvieae and E. faecalis are conditional pathogenic bacteria that cannot be used as aquaculture probiotics. B. C1 and B. HLJ1 could be identified as Bacillus by 16S rRNA, and the phylogenetic tree constructed based on the gyrB gene could reveal that B. C1 clustered with B. pumilus GBSW2 (GU568234.1) (B. subtilis Y98b (JX513934.1)  (B. subtilis NRS6167 (OX419562.1)  A and B. 13934.1) B. The phP054310) C and B. 19562.1) D. TherefB. cereus to B. subtilis, the plasmid carrying resistance to tetracycline, pBC16, which was originally isolated from B. cereus, could be subsequently transformed in B. subtilis and stably maintained. The minimum inhibitory concentration (MIC) of antibiotics against B. C1 and B. HLJ1 were measured using the broth microdilution method, and the susceptibility of the strains to the corresponding antibiotics was assessed using the CLSI guidelines. B. C1 and B. HLJ1 were sensitive to most of antibiotics required by EFSA (FEEDAP 2012), but B. C1 showed intermediate sensitivity to erythromycin and B. HLJ1 showed intermediate sensitivity to chloramphenicol  and B. subtilis (spore production rate 80%) reported in the previous study [In aquaculture, probiotics are often added to feeds for use, and high temperatures are often used in feed production to improve palatability and kill pathogens. To prevent inactivation by high temperatures, probiotics must be heat resistant . The tolronments . With lous study ,38.B. subtilis SK07 and B. amyloliquefaciens SK27 can produce amylase and protease as well as cellulase, and B. licheniformis ABSK55 can produce protease and cellulase. When they were added to water or feed at a concentration of 109 CFU/mL, they could promote the growth of shrimp [Extracellular enzyme production is an important feature of good probiotics, which provide beneficial effects to the host in terms of food absorption and improve aquaculture water quality by degrading organic waste, including amylase, protease, and cellulase ,40. As sf shrimp .B. tequilensis, B. velezensis, and B. subtilis after the first 2 h , while also having the same efficacy after 24 h [V. parahaemolyticus were 43.4% and 36.7% after the first 2 h, while they were 65.6% and 74.3% at 24 h, respectively + ion at m/z 390.1542 Da and 390.1541 Da  at three initial concentrations in simulated aquaculture wastewater, respectively. When the initial concentration of B. HLJ1 was 1 × 105 CFU/mL, it showed low inhibitory activity against the growth of V. parahaemolyticus. When the initial concentration of B. HLJ1 was 1 × 106 CFU/mL, it could reduce V. parahaemolyticus by more than two orders of magnitude (2.35 lg) at 48 h. When the initial concentration of B. HLJ1 was 1 × 107 CFU/mL, B. HLJ1 reduced V. parahaemolyticus by about three orders of magnitude (2.67 lg) at 12 h, by more than four orders of magnitude (4.45 lg) at 24 h, and completely inhibited V. parahaemolyticus at 48 h  at 12 h, by more than four orders of magnitude (4.14 lg) at 24 h, and completely inhibited V. parahaemolyticus at 48 h . B. C1 and B. HLJ1 were identified as B. pumilus and B. subtilis by using the gyrB and pycA sequences. Many studies have reported the inhibitory effect of Bacillus on pathogenic bacteria [B. licheniformis exhibits strong inhibitory activity against A. hydrophila and feeding diets supplemented with B. licheniformis (108 CFU/g of feed) improved the immunity of Labeo rohita and its resistance to A. hydrophila [B. velezensis had inhibitory effects on V. parahaemolyticus, A. veronii, and Plesiomonas; the inhibition effect increases with the rising concentration of B. velezensis and when the concentration reaches 2.7 × 108 CFU/mL, the inhibition effect is significant [V. parahaemolyticus at a concentration of 107 CFU/mL. The inhibition activity against V. parahaemolyticus was influenced by the concentration, a finding similar to that previously reported [In this study, two probiotics that inhibit bacteria . The prodrophila . Anothernificant . In thisreported .nheA, nheB, nheC, hblA, hblC, hblD, and entFM). The safety shown by B. HLJ1 and B. C1 makes them prospective for use as probiotics in aquaculture.In aquaculture, probiotics should not carry antibiotic-resistant genes and be non-pathogenic to aquaculture animals . It has Bacillus is mainly due to its ability to produce endospores, so the heat resistance of Bacillus is generally assessed by evaluating its ability to produce spores [Heating is frequently used in aquaculture feed production to improve palatability and kill pathogens . Therefoe spores . In thisB. methylotrophicus can produce amylase, lipase, and protease, promoting the growth of fish [V. parahaemolyticus. This finding is consistent with a previous study showing that Bacillus has the ability to auto-aggregate and co-aggregate with A. hydrophila and E. faecalis [The production of extracellular enzymes is one of the criteria for assessing probiotics, which promote the growth and development of aquatic animals as well as improve the quality of aquaculture water ,40. It h of fish . In this of fish  found th of fish . In thisfaecalis .V. parahaemolyticus substances produced by B. C1 and B. HLJ1 were identical to AI-77-F. The AI-77 series compounds, first isolated and identified from B. subtilis AI-77 by Shimojima et al. [Alternaria tenuis Sg17-1 and investigated its inhibitory effect on human malignant tumor A375-S2 and Hela cells, but the effect was very weak. Shi et al. [B. subtilis subsp. Inaquosorum and found that it had potential inhibitory activity against quorum sensing (QS) and could be used in the development of a novel anti-microbial agent to treat pathogenic infections. In this study, it is the first report that AI-77-F has inhibitory activity against V. parahaemolyticus.One of the main mechanisms by which probiotics inhibit pathogens is through the secretion of antibacterial substances . Based oa et al. , belong a et al.  isolatedi et al.  isolatedV. parahaemolyticus. Both B. C1 and B. HLJ1 showed inhibitory activity against 11 different V. parahaemolyticus and could effectively control the growth of V. parahaemolyticus in simulated aquaculture wastewater when the concentration reached 1 × 107 CFU/mL. The results showed their great potential as probiotics for application in different aquaculture environments to control V. parahaemolyticus.Compared to laboratory conditions, aquaculture environments are more complex, nutrient-poor, and contain different V. parahaemolyticus, and the most effective dose of both probiotics B. C1 and B. HLJ1 to control V. parahaemolyticus was 107 CFU/mL. Based on the most effective doses of B. C1 and B. HLJ1 against V. parahaemolyticus and the commercial market price in China, the cost of using B. C1 and B. HLJ1 for effective control of V. parahaemolyticus in aquaculture can be estimated to be about USD 0.0241/m3 [V. parahaemolyticus. As a result, there is great potential for using B. HLJ1 and B. C1 as aquaculture probiotics.In conclusion, B. HLJ1 and B. C1 have inhibitory activity against .0241/m3 . The saf


Calcaneal Sanders type II or III fractures are highly disabling with significant burden. Surgical treatment modalities include open reduction and internal fixation (ORIF) techniques and a variety of minimally invasive surgical (MIS) approaches. ORIF techniques are associated with complications and traditional MIS techniques need extensive intraoperative fluoroscopic procedures. The present study aims to investigate the effects of three different minimally invasive internal fixation (MIIF) techniques used to treat Sanders type II intra-articular calcaneal fractures using finite element analyses.A 64-row spiral computed tomography scan was used to observe the calcaneus of a healthy adult. The scanning data were imported into Mimics in a DICOM format. Using a new model of a Sanders type II-B intra-articular calcaneal fracture, three minimally invasive techniques were simulated. Technique A involved fixation using an isolated minimally invasive locking plate; Technique B used a minimally invasive locking plate with one medial support screw; and Technique C simulated a screw fixation technique using four 4.0-mm screws. After simulating a 640-N load on the subtalar facet, the maximum displacement and von Mises stress of fragments and implants were recorded to evaluate the biomechanical stability of different fixation techniques using finite element analyses., with Technique C having the least stress.After stress loading, the maximum displacements of the fragments and implants were located at the sustentaculum tali and the tip of sustentaculum tali screw, respectively, in the three techniques; however, among the three techniques, Technique B had better results for displacement of both. The maximum von Mises stress on the fragments was < 56 Mpa, and stress on the implants using the three techniques was less than the yield strengthAll three techniques were successful in providing a stable fixation for Sanders type II intra-articular calcaneal fractures, while the minimally invasive calcaneal locking plate with medial support screw fixation approach exhibited greater stability, leading to improved enhancement for the facet fragment; however, screw fixation dispersed the stress more effectively than the other two techniques. Calcaneal fractures are one of the most challenging and divisive injuries to treat . They arAlthough a variety of surgical approaches have been used to treat displaced intra-articular calcaneal fractures, the extensile lateral approach has been the most common technique over the past 30 years. However, a reportedly ~ 25–30% of soft tissue complications remains a great challenge when using this technique , 7. WithA 28-year-old male patient weighing 64 kg and height of 1.71 m without any history of foot and ankle trauma, deformity, or other diseases was enrolled in the study. The 64-row computed tomography (CT) scanner  was used to scan and provide three-dimensional (3D) reconstruction of the calcaneus in a 1-mm-thick scanning layer. The computed tomography (CT) images in the matrix of 512 × 512 (DICOM format) were obtained after scanning.The models used were developed using the protocols listed below.Based on the selected CT images, the 3D model of calcaneus was established using threshold segmentation. The CT images were imported into MIMICS, and the bony structure was extracted to obtain a complete set of calcaneus data, which were then exported in STL format. In Geomagic Studio, the calcaneus data were further processed for smoothing, slacking and feature elimination. According to the typical pattern of a Sanders type II-B calcaneal fracture, the calcaneus was divided into four fragments as follows to generate a 3D geometric model: anterior fragment fragment 1), medial fragment including medial facet of subtalar joint and sustentaculum tali (fragment 2), lateral fragment with lateral facet of subtalar joint (fragment 3), and the calcaneal tuberosity fragment (fragment 4). The fragments were processed for curved surface to generate a nonuniform rational basis spline (NURBS) surface. Then, the model of the Sanders type II-B intra-articular calcaneal fracture  were scanned to build geometric models through inverse processing approach. The screw thread was simplified, and the forward geometric models of the screws were established. The geometric parameters of the implants were imported with the screw of the plate system set as a φ3.5-mm rod, and other screws as φ4.0-mm rods. Then, the coordinate system was set up, and the geometrical parameters were imported.In the Geomagic DesignX tool, three different minimally invasive fixation techniques were simulated. The three techniques, named A, B, and C are described below, respectively. Technique A: The fracture was stabilized using only a minimally invasive calcaneal locking plate with two screws in the anterior fragment, three screws in the lateral and medial fragment with one screw fixation for the sustentaculum tali, and three screws fixation in the tuberosity. Technique B: The fracture was fixed with a minimally invasive calcaneal locking plate using the same method as Technique A, but with the addition of a medial support screw from the medial tuberosity to the medial fragment underneath the medial facet. Technique C: The fracture was simulated as a screw fixation with one from the lateral tuberosity to the anterior fragment; one from the medial tuberosity to the medial fragment underneath the medial facet and two from the lateral fragment to the medial fragment, one of which was fixed in the sustentaculum tali Fig. A–C.Fig. The fixation model was imported into ANSYS  finite element analysis software. The material properties are defined according to the parameters given in Table The friction coefficient between the fragments was set as 0.2, while the friction coefficient between the fragments and implant was set as 0.5. The calcaneocuboid facet and the contact area between the calcaneus and ground was sustained. It has been reported that stress on the Achilles tendon sustains about 50% of the force of the foot . Using tThe stress loading in the present study comprised two parts—the subtalar facet sustained a 640-N load, while an upward load of 160 N was sustained on the Achilles tendon insertion of calcaneal tuberosity. The mesh generation was chosen in the directory tree, the mesh of fracture model was set as 2 mm, while the mesh of implant was set as 1 mm, and then the tetrahedral mesh was established. The final finite element analysis was resolved, and the displacement and stress of fragments and implants were recorded.The criteria considered to determine the results were as follows : (1) scrThe maximum displacement of the fragments from the three fixation methods was as follows: (1) Technique A: fragment 1 was 0.20 mm, fragment 2 was 0.55 mm, fragment 3 was 0.40 mm, and fragment 4 was 0.15 mm; (2) Technique B: fragment 1 was 0.18 mm, fragment 2 was 0.40 mm, fragment 3 was 0.29 mm, and fragment 4 was 0.12 mm; (3) Technique 3: fragment 1 was 0.19 mm, fragment 2 was 0.47 mm, fragment 3 was 0.36 mm, and fragment 4 was 0.15 mm. The maximum displacement of all fragments was at the medial facet fragment  Technique A: 0.42 mm, (2) Technique B: 0.29 mm, and (3) Technique C: 0.41 mm. Displacement was located at the tip of sustentaculum tali screw Figs. . HoweverAfter stress loading, the maximum von Mises stress on the fragments from the three techniques was as follows: (1) Technique A: fragment 1 was 26.02 MPa, fragment 2 was 11.42 MPa, fragment 3 was 10.03 MPa, and fragment 4 was 18.07 MPa; (2) Technique B: fragment 1 was 19.73 MPa, fragment 2 was 10.84 MPa, fragment 3 was 10.11 MPa, and fragment 4 was 17.10 MPa, and (3) Technique C: fragment 1 was 21.39 MPa, fragment 2 was 10.75 MPa, fragment 3 was 13.84 MPa, and fragment 4 was 17.69 MPa. The maximum stress with all three techniques was on the anterior fragment  Technique A: 286.62 MPa, (2) Technique B: 133.67 MPa; and (3) Technique C: 102.86 MPa. Stress was better dispersed using Technique C  [Open reduction and internal fixation or arthrodesis for calcaneus via a large incision may lead to various complications, including infections, neurovascular damage and poor wound healing, especially in cases of the high-risk foot with severe soft tissue injuries . Therefon = 104)  and repoIn last decade finite element analyses have been extensively used in evaluation, optimization, and developing new methods for effective management of fractures, especially for comparison of different fixation techniques for calcaneal fractures . FindingWith refined modeling methods, improved validation efforts, and large-scale systematic analyses, finite element analysis is poised to advance the understanding of fracture fixation failure, enable optimization of implant designs, and improve surgical guidance.Previous studies have developed several intra-articular calcaneal fracture models –28. In tUnder physiological loading, we found that the maximum displacements of the fragments and implants were located at the sustentaculum tali and the tip of sustentaculum tali screw, respectively, which implied the relative weakness of the medial column and the need for a more stable fixation technique; however, Techniques B and C decreased the tendency for displacement of medial and lateral fragment, and the locking plate with a medial support screw fixation demonstrated a better result. We considered that with support for the medial column, the stability of the lateral fragment could increase subsequently, which would enhance the entire stability of the subtalar joint; therefore, we concluded that the medial support screw was necessary to augment the fixation of the facet. Similarly, the displacement of other fragments and implants was also less using Technique B than using the other two techniques and suggested that the fixation of a minimally invasive calcaneal locking plate with a medial support screw would provide better biomechanical strength for Sanders type II intra-articular calcaneal fractures.Moreover, we found that Technique C yielded better support of the facet than the Technique A. We considered that the greater stability was achieved from the distribution of the screws. That is, three screws were inserted at different points for fixation of the subtalar joint, which included one screw in the axial plane to support the medial column. As the medial facet fragment was enhanced, another screw from the lateral facet to the medial facet subsequently provided greater stability, as previously mentioned; therefore, we concluded that the medial support was also critical for stabilizing the facet and that the medial screw plays an important role in this fixation method. We also suggested that regardless of which fixation technique is chosen, at least two screws should be applied to fix the subtalar facet to guarantee its stability.From the maximum von Mises stress, we found that the stress of each fragment after using the three different fixation techniques was < 56 MPa, which indicated a low risk of screw loosening. In addition, the maximum von Mises stress of the implant after using the three techniques was less than its yield strength. We concluded that all of these techniques could achieve a stable fixation with less potential for implant breakage or failure. Although some difference in fragment stress existed among the three techniques, the difference in implants appears more obvious. In the present study, the maximum von Mises stress of the implants was the lowest in screw fixation. We suggested that the screw distribution within different dimensions and the intramedullary fixation style may play important roles in providing better implant stress dispersion. Furthermore, we found that with a medial support screw, the stress of the implants after using Technique B was less in an isolated locking plate model, which indicated that the medial support screw was also essential for implant stress dispersion.The present study suffered some limitations that should be considered when interpreting its findings, particularly when generalizing the findings to the clinical practice of general population. The main limitations of the study are as follows: (1) We simulated only the Sanders type II-B intra-articular calcaneal fracture using a simple model; therefore, it is not known whether our findings could be applied to other types of fractures, such as comminuted fractures. Therefore, to validate our findings, further controlled studies should be conducted; (2) evaluation of the biomechanical effects using the finite element analyses had some intrinsic limitations; therefore, we could not consider the findings as definite evidence for our conclusion, and the mechanical stability of the different fixation methods must be verified using classic biomechanical experiments; (3) any changes in model design, parameter settings, screw orientation, and implant position might affect the final result and cause bias; (4) in clinical work, screw distribution and orientation would be adjusted according to the fracture lines and the degree of comminution, which may also alter the strength of fixation and stress distribution; (5) we simplified the screw as a solid rod. However, the cannulated screws are more likely applied for classic screw fixation and medial support screw fixation in clinical practice. Whether the biomechanical stability could be compromised with cannulated screws still remains in doubt. Furthermore, the compression effect of cannulated screws could not be simulated in this model; and (6) although we set the same elastic modulus for plates and screws, in reality, differences exist, especially in different implant brands and designs. To resolve these limitations, conducting further well-designed and controlled studies in clinical setting as well as in modeling environments is necessary.All three of the minimally invasive fixation techniques could provide stable fixation for Sanders type II intra-articular calcaneal fractures; however, a medial support screw would provide greater stability, especially with a minimally invasive calcaneal locking plate fixation. Thus, the insertion of a medial support screw should be recommended in clinical practice to enhance the facet fragment. In addition, classic screw fixation could most effectively disperse stress, which is a reliable and economic method and should continue to be applied.


Manipulation of protein synthesis is commonly applied to uncover protein functions and cellular activities. Multiple inhibitors with distinct mechanisms have been widely investigated and employed in bio-related research, but it is extraordinarily challenging to measure and evaluate the synthesis inhibition efficiencies of individual proteins by different inhibitors at the proteome level. Newly synthesized proteins are the immediate and direct products of protein synthesis, and thus their comprehensive quantification provides a unique opportunity to study protein inhibition. Here, we systematically investigate protein inhibition and evaluate different popular inhibitors, i.e. cycloheximide, puromycin, and anisomycin, through global quantification of newly synthesized proteins in several types of human cells . The inhibition efficiencies of protein synthesis are comprehensively measured by integrating azidohomoalanine-based protein labeling, selective enrichment, a boosting approach, and multiplexed proteomics. The same inhibitor results in dramatic variation of the synthesis inhibition efficiencies for different proteins in the same cells, and each inhibitor exhibits unique preferences. Besides cell type- and inhibitor-specific effects, some universal rules are unraveled. For instance, nucleolar and ribosomal proteins have relatively higher inhibition efficiencies in every type of cells treated with each inhibitor. Moreover, proteins intrinsically resistant or sensitive to the inhibition are identified and found to have distinct functions. Systematic investigation of protein synthesis inhibition in several types of human cells by different inhibitors provides valuable information about the inhibition of protein synthesis, advancing our understanding of inhibiting protein synthesis. Protein synthesis inhibition is extensively utilized in bio-related research, and different inhibitors have been investigated and applied to various studies. However, the synthesis inhibition efficiencies of different proteins even by one inhibitor and the differences among different inhibitors remain to be explored. We systematically study protein synthesis inhibition and several popular inhibitors through comprehensive quantification of newly synthesized proteins. Besides the overall protein inhibition efficiencies by these inhibitors, the efficiencies of individual proteins by each inhibitor are obtained. The same inhibitor causes dramatic differences in synthesis inhibition of different proteins in the same cells. Furthermore, protein synthesis inhibition shows cell type- and inhibitor-specific effects. This work provides valuable information to the scientific community, helping scientists to choose a right inhibitor.Protein synthesis is essential to nearly every biological process, including cell growth and proliferation. Dysregulation of protein synthesis is related to various diseases, such as cancer and cognitive disorders . Hence, These inhibitors are commonly used to globally inhibit protein synthesis, and they can result in different inhibition efficiencies for the synthesis of individual proteins. For example, it was reported that ANM had the inhibition efficiency of only 65% for CYP2B1 when >95% of total protein synthesis was terminated in rat hepatocytes, but Puro inhibited ∼95% CYP2B1 expression while achieving a relatively low overall protein synthesis inhibition . SystemaRecently, the development of ribosome profiling (Ribo-Seq) allows for the transcriptome-scale translation efficiency evaluation through sequencing and quantifying the mRNA fragments protected by the ribosomes during the translation process. This method was utilized to investigate the effects of inhibitors on the protein translation process . HoweverIn this work, we systematically and quantitatively study the synthesis inhibition efficiencies of proteins by different popular inhibitors  through global analysis of newly synthesized proteins in four types of widely used human cells . Comprehensive and quantitative analysis of these proteins was achieved by integrating metabolic labeling, bioorthogonal chemistry-based enrichment, and multiplexed proteomics. Moreover, we incorporated a boosting approach to enhance the identification of newly synthesized proteins with low abundance. In total, >5,000 newly synthesized proteins were accurately quantified, and their inhibition efficiencies were obtained. The results demonstrate that protein synthesis inhibition efficiencies are markedly different among the cell types even for the same inhibitor, and the efficiencies for individual proteins in the same type of cells by the same inhibitor also vary dramatically. Among these inhibitors, Puro is the most nondiscriminatory, while CHX is relatively less effective in inhibiting the synthesis of ribosomal proteins despite its overall high potency and popularity. The correlations between the inhibition efficiencies of individual proteins and their various physicochemical and biological properties are further investigated. This work provides unprecedented and valuable information about the inhibition of protein synthesis, facilitating a better understanding of protein synthesis regulation. Both the inhibition efficiencies of individual proteins by each inhibitor and the different inhibition efficiencies of the same proteins in different cell types are beneficial for future biomedical studies.Quantification of newly synthesized proteins in cells with the treatment of each inhibitor can provide straightforward evidence for protein synthesis inhibition. Previously, methods such as bioorthogonal noncanonical amino acid tagging (BONCAT) and multiplex isobaric tagging/noncanonical amino acid tagging (MITNCAT) were developed to investigate newly synthesized proteins . Most ofTo enhance the identification of newly synthesized proteins with a short treatment time, a boosting approach was integrated into the current workflow. Based on a previous report, if the amount of the booster sample is too high (e.g. >20×), the quantification accuracy may be affected . Given tTo systematically evaluate the commonly used protein synthesis inhibitors , we globally identified and quantified newly synthesized proteins in various types of human cells  with each inhibitor Fig. , 28, andTo better evaluate the protein synthesis inhibition and different inhibitors, gel experiments were performed to choose the concentration of each inhibitor for the proteomics experiments Figs. . The conAs an example, CHX and ANM achieved ∼30% of the synthesis inhibition of NADH–ubiquinone oxidoreductase chain 2 (MT-ND2) in two types of cells (Jurkat and A549 cells), while Puro reached >85% inhibition of this protein in the same types of cells Fig. B. This oAlthough the mechanisms of these three inhibitors have been well studied, their inhibition efficiencies for individual proteins have never been systematically quantified and evaluated. Based on the total intensities of the reporter ions for each treatment, the overall inhibition efficiencies in MCF7 cells are 85.9%, 85.9%, and 86.7% for CHX, Puro, and ANM, respectively Fig. . OverallTo systematically evaluate the inhibition efficiencies of individual proteins in different cellular components and protein complexes by each inhibitor, we performed Gene Ontology (GO) analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID)  and clusGenerally, the performances of the three inhibitors are similar for proteins in the same cellular components and protein complexes Fig. D. ConsidWe further analyzed the inhibition efficiencies of proteins in four types of cells by CHX. For different types of cells treated with the same inhibitor, much more variations of the inhibition efficiencies were observed Fig. . For exaProteins in the Golgi were significantly less inhibited in all four cell types by CHX and all other experiments except for the Puro treatment in MCF7 cells Fig. E. The GoTotally, 5,617 newly synthesized proteins were quantified Figs. A and S3AThe distribution of the synthesis inhibition efficiencies under each treatment showed that different inhibitors had various inhibitions for different proteins in the same type of cells, and when compared between the cell types, each inhibitor also resulted in different efficiency distributions Fig. E. For inTo better understand proteins with various inhibition efficiencies across all experiments, we defined proteins with top 25% efficiencies as sensitive to a specific inhibitor in one cell type (i.e. one experiment). Similarly, those with the bottom 25% efficiencies were annotated as resistant proteins . In totaThe regulation of protein synthesis is critical for proteostasis. Dysregulation of protein synthesis is related to multiple diseases, and protein synthesis inhibitors are powerful for disease treatment . HoweverWe next performed more analysis to further understand proteins sensitive or resistant to the inhibition. First, proteins annotated as sensitive/resistant in two out of three treatments in one cell type were selected to exclude inhibitor-specific effects. Then proteins annotated as sensitive/resistant in three out of four cell types were further selected as generally sensitive/resistant to synthesis inhibition to minimize cell type-specific effects Fig. . OverallWe analyzed the abundances of proteins sensitive or resistant to the inhibition ) and fouN-glycosylation  exhibited great resistance to the inhibition in two types of suspension cells, but it was sensitive in A549 and MCF7 cells Figs. . Immune sylation , also deWe found all proteins with great differences between any two types of cells and performed GO analysis Fig. . Each paThe performance variation of the three inhibitors has been documented for single proteins , but sysThe ribosome is the target of these three inhibitors, even though their inhibition mechanisms are different. The ribosome biogenesis under the inhibitor treatment may be related to the resistance of the inhibition, which further affects the overall inhibition efficiency. GO enrichment analysis revealed that the cytosolic large ribosomal subunits were significantly less sensitive to CHX than both ANM and Puro Fig. . For PurTogether, these results indicated that CHX was relatively less effective in inhibiting cytosolic ribosomal proteins than Puro and ANM, typically the cytosolic large ribosome subunits. One possible reason is that the CHX treatment can rapidly accumulate mRNAs related to ribosome biogenesis , 47, whiThe inhibition of protein synthesis is frequently utilized to unravel protein functions and cellular activities , 32. AltBy integrating metabolic labeling and bioorthogonal chemistry, newly synthesized proteins were selectively enriched, allowing for their comprehensive analysis by MS while avoiding the masking effect from many highly abundant existing proteins . MoreoveThe inhibition efficiencies of proteins in different cellular compartments and protein complexes were systematically investigated in cells with the treatment of each inhibitor. For instance, nucleolar and ribosomal proteins are significantly more inhibited in every treatment, but proteins in the Golgi and endosome are less inhibited. The rRNA in the ribosome is generated in the nucleolus , and theIn conclusion, we systematically quantified the inhibition efficiencies of individual proteins in four types of human cells by three inhibitors, which contain a wealth of valuable information about protein synthesis inhibition. The correlations between the inhibition efficiencies of individual proteins and their various physicochemical and biological properties were further studied. The functions and multiple properties of proteins intrinsically sensitive or resistant to the inhibition were compared. Among resistant proteins, they are more hydrophobic and have less disordered regions. Further analysis revealed that the protein abundances and the translation efficiencies determined by Ribo-Seq are positively correlated with the inhibition efficiencies of corresponding proteins , 38. IntA summary of key methods is given below , respectively. Labeled cells were then harvested and lysed. Two biological replicated experiments were performed for each treatment.Cells were cultured in normal media until they reached ∼70% confluency. Then cells were grown in the methionine- and lysine-depleted media for 30 min to deplete free methionine and lysine before being cultured in the AHA, heavy lysine containing media for 1.0 h. The concentrations of AHA and heavy lysine are the same as those of methionine and lysine in normal media. During the depletion of free lysine and methionine and cell labeling, cells were treated with CHX, Puro, ANM, or DMSO with a final concentration of 6.9 µg at 4°C for 10 min, and the supernatant was kept. The clarified lysates were incubated with DBCO-conjugated magnetic beads at 4°C overnight. Proteins were then reduced and alkylated. Nonspecifically bound proteins were removed by stringent wash, and enriched newly synthesized proteins were digested by Lys-C overnight at 37°C with shaking. Digested peptides were desalted and lyophilized before TMT labeling and pooling. Pooled samples were fractionated using high-pH reversed-phase high-performance LC (HPLC) into 25 fractions in 10 mm ammonium formate (pH = 10). The collected fractions were further purified by the StageTip method. Quantitative LC–MS/MS analysis was performed with reversed-phase HPLC coupled with an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific).The cell lysates were clarified by centrifugation at 24,000 pgad168_Supplementary_DataClick here for additional data file.


To evaluate the clinical and radiological results after fixation of the first metatarsal head (MTH) with one or two screws as part of the third-generation minimally invasive Chevron–Akin osteotomy (MICA) for hallux valgus deformities.Between August 2020 and November 2021, 55 MICA procedures , 22 with two (MICA2), 33 with one screw (MICA1) were performed for mild to severe hallux valgus deformities. Exclusion criteria were a concomitant pes adductus (Sgarlato angle > 20°) or hindfoot/midfoot deformities requiring treatment. In 27 cases, additional procedures on the forefoot  were necessary. Pre- and post-operatively, hallux valgus angle (HVA) and intermetatarsal I/II angle (IMA) were measured. Clinically, subjective satisfaction, range of motion (ROM) of the first metatarsophalangeal joint (MTPJ), and pain level (NRS score) were evaluated. The minimum follow-up was 12 months.Displacement of MTH was 70–90% on average, all osteotomies showed full consolidation at latest follow-up. In one case of either group, a slight subsidence of MTH was documented. The radiological and clinical parameters showed no differences between the groups. The pain level improved by an average of three points. The mobility of the MTPJ showed a slight reduction in nine cases after three months  which persisted in three cases. Fifty-two of 55 patients (95%) would opt again for the operation.Fixation of the first MTH with a single bicortical screw in MICA with moderate lateralization of MTH shows stable anchoring and good clinical results. The routine use of a second metatarsal screw can be omitted. Hallux valgus surgery can achieve good to very good results in over 90% of cases with modern, open procedures . ClassicAlthough excellent results can be achieved with MICA, there are also typical complications such as prolonged postoperative swelling of the foot, painful protracted secretions of the portals due to residual bone debris and sometimes disruptive foreign material . The disClinical experience suggests, there is usually sufficient stability in the area of the metatarsal osteotomy if one bicortical screw is inserted and translation of the MTH is less than 100% of the shaft width . Hence, The present study was designed as a retrospective analysis with prospective follow-up of our institutional foot and ankle registry data. The study was conducted as a proof-of-concept in accordance with the declaration of Helsinki and was approved by the local ethical review board . All patients with symptomatic mild to severe hallux valgus deformity  and correction with MICA in the period between August 2020 and November 2021 were included . Patients with a follow-up of < 12 months after surgery, a concomitant pes adductus (Sgarlato angle > 20°) and additional interventions on the midfoot and/or hindfoot were excluded. Pre- and post-operatively, at the different follow-up appointments , various clinical and radiographic data were collected. Clinically, the mobility of the first metatarsophalangeal joint (MTPJ) was documented and its difference (pre- to postoperatively) was calculated . Additionally, the pain level –10 (worst possible pain)) was recorded and the patient was asked: “Are you satisfied with the result of the operation?” (Answer options: yes/unsure/no). Satisfied patients were specifically asked: “Are there any restrictions or complaints in the area of the operated hallux at all?” If this question was answered in the affirmative , then this was classified as “forgotten bunion” (in accordance to a perfect result after joint arthroplasty ). RadiogThe operation was performed according to previously described techniques  under reThe same care was recommended for both groups. To avoid excessive swelling, the foot should be elevated frequently for the first 2 weeks. A flat postoperative shoe with a rigid sole  was used for 6 weeks, crutches were recommended for two weeks. Redression of the big toe with bandages was performed for 2 weeks, thereafter elastic taping was recommended for further 4 weeks. Full weight bearing of the heel was allowed immediately after the operation, the big toe could be beared after 3 weeks. Sports with high forefoot stress (e.g. jogging) were not allowed for the first 10 weeks.t-test and a Mann–Whitney U-test were performed for normal and non-normal distributed data, respectively, to describe significant differences between the groups. Statistical significance was assumed for all p values < 0.05. Correlation between different variables was determined using Pearson’s correlation coefficient (PCC). Correlation was classified as minimal (PCC ≤ 0.25), low (0.26 < PCC < 0.5), moderate (0.5 ≤ PCC < 0.7), high (0.7 ≤ PCC < 0.9), and excellent (PCC ≥ 0.9) [https://www.python.org/) and the scipy-library (https://scipy.org/).Normal distribution was verified using D’Agostino–Pearson testing. An independent C ≥ 0.9) . All anaModerate stiffness of more than > 10° in comparison to the preoperative range of motion occurred in 8 MICA1, 24%) and 4  cases after 3 months and persisted in two cases in the MICA1 group, and one case (5%) of the MICA2 group (6%) after 12 months. There were no cases of severe stiffness in any of the groups (defined as reduction in ROM > 30 degrees). MICA1 and MICA2 surgeries achieved similar improvements in pain score . The number of patients that would agree to undergo the operation again and the degree of satisfaction with the surgery  were similar in both groups. The rate of forgotten bunion was slightly higher in the MICA1 group . Correlation analysis revealed no high or excellent correlations of any parameters in either group , and 19 cases of MICA2 . All osteotomies healed within the study period Fig. . The HVADelayed wound healing occurred in three cases of either group , healing occurred in all cases with conservative measures. Deep infection was found in one case of MICA2 (5%) at the level of the distal metatarsal screw. Here, removal of the screw, local debridement and systemic antibiotics for 2 weeks were successful. Slight subsidence of the metatarsal osteotomy occurred in 1 case of each group , the final result was regular and both patients were satisfied after 12 months. Recurrence of the deformity with HVA > 20° and/or IMA > 10° occurred in one case of each group . No cases of hallux varus were observed. Significantly more patients were dissatisfied with the implants in MICA2  than in MICA1 , in all cases removal of the screw and resection of the bump was successful.The present study showed no significant differences regarding the clinical–radiological results and consolidation rates in third-generation MICA fixed with either one or two metatarsal screws. Nevertheless, we found a clear tendency to less disruptive material conflict in the single-screw MICA. Taken together, it was clearly shown that MICA is an effective and successful method in correcting hallux valgus deformities in the short-term follow-up, regardless of whether one or two metatarsal screws are used.Two meta-analyses including numerous publications have been able to demonstrate the effectiveness of MICA in the context of hallux valgus correction , 10. ComThe primary goal of hallux valgus corrective surgery is to align the first toe and thus regain physiologic loading within the MTPJ. HVA was corrected from 30.9 to 10.2 degrees in MICA1 and from 30.8 to 11.0 degrees in MICA2. IMA decreased from 15 to 5.5 degrees in MICA1 and from 14.6 to 5.8 degrees in MICA2. These corrections are reported to the same amount from other authors performing third-generation MICA . Hence iAs already mentioned, one important advantage of MICA in comparison to open techniques are lower rates of soft tissue complications and less stiffness of the first MTPJ  Stiffnesp value < 0.05 in scores may not necessarily translate into a considerable change for patients clinically [Our study has some limitations. First, it is a retrospective case series with a relatively small sample size, although the number of patients is well comparable to other studies dealing with MIS hallux valgus correction . Second,inically . To get This study proves that third-generation MICA is a very safe procedure, which offers a stable osteosynthesis and, due to the extra-articular osteotomy level, a very good postoperative mobility of the MTPJ. Placement of a second metatarsal screw may not be necessary if intraoperative stability of the metatarsal osteotomy is present. Nevertheless, if any doubt regarding stability of the metatarsal osteotomy is encountered, a second screw should always be inserted, in accordance to orthopedic philosophy: “in dubio, pro bolzo”.


NLRP3 is a prototypical sensor protein connecting cellular stress to pro-inflammatory signaling. A complex array of regulatory steps is required to switch NLRP3 from an inactive state into a primed entity that is poised to assemble an inflammasome. Accumulating evidence suggests that post-translational mechanisms are critical. In particular, phosphorylation/dephosphorylation and ubiquitylation/deubiquitylation reactions have been reported to regulate NLRP3. Taken individually, several post-translational modifications appear to be essential. However, it remains difficult to understand how they may be coordinated, whether there is a unique sequence of regulatory steps accounting for the functional maturation of NLRP3, or whether the sequence is subject to variations depending on cell type, the stimulus, and other parameters such as the cellular context. This review will focus on the regulation of the NLRP3 inflammasome by phosphorylation and dephosphorylation, and on kinases and phosphatases that have been reported to modulate NLRP3 activity. The aim is to try to integrate the current understanding and highlight potential gaps for further studies. RecogFor a long time, activation of the NLRP3 inflammasome was described as a two-step process: A first step, called “priming”, leading to transcriptional up-regulation of inflammasome genes including those encoding pro-IL-1β and NLRP3 itself, and a second step called “activation”, featuring assembly of the inflammasome components. In 2012, two studies , 10 prove.g., stimulus, cell-type, cellular context, or genetic polymorphisms?Many kinases are reported to regulate NLRP3. In several instances, acceptor phospho-sites in NLRP3 were identified, and phosphatases acting at some of the phosphorylated sites have been proposed. Different strategies were used for producing phospho-NLRP3, which may diversely and only partially reflect the actual physiological process. In fact, there is still very little understanding of the overall phospho-modulatory process and several questions have remained unanswered. Are all reported post-translational modifications genuine sites of regulation? Which are the critical ones? Are some sites redundant? What is the sequence of events? Could PTMs be subject to variations, according to, i.e., how the score may be played, is presented, informed by recent structural insights.This review will summarize the phospho-sites identified to date as if they would be notes in the NLRP3 “score”; the kinases and phosphatases that are reported to regulate NLRP3 activation are represented as orchestra players that are able to read and play the score. A potential scenario of how the phospho-regulatory process of NLRP3 may be integrated, 2A first read-through of the NLRP3 score will provide a two-dimensional sequence scan of the protein from N- to C-terminus, pointing out the part to play (NLRP3 subdomain) and the bars with an identified phospho-site. It will also state which leading instrument in the kinase and phosphatase sections may play the phospho-notes in the score 2.Part to play: N-terminal part of PYD domainBar: Serine 5 mouse: Serine 3Kinase section lead: Protein kinase B (AKT)Phosphatase section lead: Protein Phosphatase 2A (PP2A)Stutz et al.  used an in vitro phosphorylation assay using immuno-precipitated AKT and HEK293T cell lysates expressing NLRP3. Next, cellular extracts and confocal imaging showed that AKT kinase inhibition leads to enhanced ASC oligomerization after LPS ± nigericin stimulation of BMDMs. The levels of NLRP3 were reduced after stimulation in THP1 cells when AKT was knocked down. Thus, AKT could have a dual and complementary role during NLRP3 activation by preventing NLRP3 degradation while inhibiting its oligomerization/inflammasome assembly process. Degradation of NLRP3 requires ubiquitin-dependent mechanisms involving E3 ligases such as TRIM31  both for recruitment to mitochondria via the antiviral signaling protein MAVS, and for activation . The subPart to play: PYD domainBar: Tyrosine 32 mouse: Tyrosine 30Kinase section lead: unknownPhosphatase section lead: Phosphatase and Tensin homolog (PTEN)Nlrp3-knockout BMDMs showing that the presence of Pten is indispensable for WT NLRP3 to restore inflammasome activation. The PTEN-dependent dephosphorylation of Y32 was demonstrated using an anti-phosphospecific antibody. Priming BMDMs with LPS induced phosphorylation at Y32, which was completely dephosphorylated upon subsequent activation with nigericin. Dephosphorylation did not occur in Pten-knockout BMDMs. The critical role of dephosphorylation at Y32 during inflammasome activation was further evidenced by reconstitution of Nlrp3-knockout BMDMs with NLRP3Y32E, and by the isolation of BMDMs from knock-in mice harboring the Nlrp3Y30E allele, which both showed deficient inflammasome activation upon activation with nigericin, MSU, or ATP.By using recombinant FLAG-tagged NLRP3 immunoprecipitated after expression in HEK 293T cells, Huang et al.  identifiThe kinase responsible for the PTEN-sensitive phosphorylation of Y32 in NLRP3 has yet to be reported.Part to play: PYD/NACHT interdomainBar: Tyrosine 136 mouse: Tyrosine 132Kinase section lead: Ephrin type-A receptor 2 (EphA2)Phosphatase section lead: unknownEphA2-knockout mice confirmed the role of EphA2 in the regulation of inflammasome activation and the resulting lung inflammation.The Ephrin type-A receptor 2 (EphA2) transmembrane tyrosine kinase is selectively expressed in airway epithelial cells in human and mouse. Knock-down of EphA2 in epithelial cells was shown to up-regulate IL-1β and IL-18 production after stimulation with the RNA virus reovirus . In keepin vitro kinase assays with pulled-down proteins, and endogenously using airway epithelial cells from control or EphA2-knockout mice primed with LPS or infected with reovirus. Y132 was identified as the most critical phosphorylation site among five Tyrosine residues predicted as phospho-sites with high confidence scores by the NetPhos prediction server . Deficiency in EphA2, or Y132F mutation of NLRP3, resulted in increased inflammasome activation , which led the authors propose Y132 (Y136 in human) as the EphA2 phosphorylation site in NLRP3. This, however, remains to be more firmly evidenced.Pull-down experiments in airway epithelial cells indicated that EphA2 interacts with the LRR domain of NLRP3, but only after the cells have been infected with reovirus. Immunofluorescence imaging revealed endogenous NLRP3 colocalized with EphA2 in the cytosol, and this colocalization was enhanced with reovirus infection. Direct phosphorylation of NLRP3 by EphA2 was demonstrated by cellular co-expression studies, Part to play: PYD/NACHT interdomainBar: Tyrosines 136, 140, 143, 168 mouse: Tyrosines 132, 136, 164Kinase section lead: Bruton tyrosine kinase (BTK)Phosphatase section lead: unknownin vitro or in reconstituted HEK293 cellular system, which may not recapitulate inflammasome biology in physiological cellular systems. In addition, the strict requirement for BTK kinase activity remains questionable as experiments made use of 60 μM of ibrutinib . In addition, whether the four tyrosine residues identified in this work are direct substrates of BTK under physiological conditions awaits more definitive evidence.Collectively, BTK is emerging as a potential kinase for regulating the NLRP3 inflammasome during activation, by modification of a protein motif involved in subcellular localization and inflammasome assembly. The partial effects observed upon nigericin treatment suggest that this pathway of activation operates independently of BTK. Why stimulation by other means, Part to play: PYD/NACHT interdomainBar: Serine 198 mouse: Serine 194Kinase section lead: cJun N-terminal kinase 1 (JNK1)Phosphatase section lead: unknownvs. WT in their reconstitution assay. In immortalized mouse bone marrow-derived macrophages (iBMDMs) with endogenous Nlrp3 knock-down, stable transfection of the S194A variant showed abrogated NLRP3 activation following stimulation with nigericin, ATP or MSU, by comparison to WT NLRP3 controls. Similar findings were obtained with ex vivo BMDMs from S194A NLRP3 knock-in mice. Subjecting these mice to an MSU-induced peritonitis model and to an LPS-induced sepsis model showed that S194 phospho-mutation reduced IL-1β levels, reduced peritoneal extrudate cell numbers, and conferred higher resistance to sepsis, thus providing compelling evidence for the importance of this serine residue for mouse NLRP3 inflammasome function. Phosphorylation at S198 occurred early during the priming step . Combining the CAPS (Cryopyrin-associated periodic syndromes) variant T346M, which needs only priming for activation, together with a S198A mutation abolished LPS-induced inflammasome activation. However, the S198D NLRP3 mutant protein still required priming for activation, suggesting this key phosphorylation does not recapitulate the whole priming process.S198 lies in a loop at the interface between the NACHT and LRR domains, recently coined the “activation loop” . In theiin vitro kinase assays to identify JNK1 as the kinase responsible for NLRP3 phosphorylation at S198. Finally, they showed that phosphorylation of S198 by JNK1 allows for de-ubiquitylation of NLRP3 by BRCC3, an essential step for NLRP3 inflammasome activation.The authors then used a combination of prediction tool analyses, inhibitory approaches  as well as Unpublished data from our laboratory have confirmed that the S198D mutant protein drives stronger inflammasome activation compared to WT NLRP3  based on a reconstitution assay related to that published by Song et al. . In our Part to play: NACHT domainBar: Serine 295 mouse: Serine 291Kinase section lead #1: Protein kinase A (PKA)Kinase section lead #2: Protein kinase D (PKD)Phosphatase section lead: unknownIn 2016, a study by Mortimer et al. addressed the long reported inhibitory effect of prostaglandins such as PGE2 on inflammasome activation , showingUsing Prosite, they identified two potential PKA phosphorylation sites in NLRP3: S295 and S597 . The S295A variant was the only one blocking forskolin-mediated inflammasome inhibition, as measured by ASC oligomerization, IL-1β release, and ATP hydrolysis. In fact, this mutation enhanced inflammasome activity triggered by cAMP. Of note, the D305G mutation, proximal to S295 and known to cause NOMID, the most severe manifestation of CAPS, had a similar phenotype. It is hypothesized that these CAPS-susceptible NLRP3 mutations might potentially resist control by PKA-induced phosphorylation , 44.Further studies have confirmed a PKA-dependent inhibitory phosphorylation of NLRP3. Guo et al.  suggestee.g., nigericin, DAG levels increase at the Golgi and MAMs become adjacent to Golgi membranes, thus allowing for phosphorylation of NLRP3 by PKD. By using PKD inhibitors as well as mouse models of PKD deficiency, Zhang et al. uncovered the contribution of PKD enzyme activity for the release of active NLRP3 into the cytosol, capable of assembling with ASC. By ectopic expression of NLRP3 and PKD proteins (both NLRP3 WT and variant forms including point mutations at anticipated putative phosphorylation sites) they identified S293 within the nucleotide binding domain of NLRP3 (S295 in human) as the site sensitive to regulation by PKD. Confirmation of direct phosphorylation of endogenous NLRP3 by PKD has remained challenging because sensitive antibody tools are not available, and likely because the S291 (S293 in their report) can also be phosphorylated by PKA (see above). However, the importance of PKD in the regulation of NLRP3 was further highlighted in a recent study by Heiser et al. Phosphatase section lead: unknowne.g., Rac2) by bacterial toxins and virulence factors in human and mouse macrophages Phosphatase section lead: unknownMink1 knockout mice and showed that the lack of Mink1 reduces the level of NLRP3 activation following various stimuli. Next, they used LPS-induced sepsis and alum-induced peritonitis models with these mice, adding evidence for reduced NLRP3-dependent inflammatory responses in vivo. MINK1 deficiency did not influence the transcript levels of the NLRP3 inflammasome components, including that of pro-IL1β, suggesting that MINK1 may regulate the NLRP3 activation step. Reconstitution experiments in BMDMs from Mink1 knockout mice showed that the kinase domain of MINK1, not the regulatory domain is required to support inflammasome activation. Kinase activity of MINK1, however, only had a limited impact based on the study of the K54R MINK1 mutant. Pull-down experiments showed that the LRR domain of NLRP3 binds to MINK1, which is where S725 is located. Mink1-deficient BMDMs primed with LPS and stimulated with nigericin showed reduced phosphorylation of NLRP3. However, clear cut evidence for direct phosphorylation of S725 by MINK1 was not provided in this study. Phosphomimic variants of the S725 site  in NLRP3, expressed in Nlrp3 deficient BMDMs could reconstitute inflammasome activation whereas the constitutive unphosphorylated S725A variant failed to signal in this assay, thereby showing the importance of phosphorylation of the S725 site. Reconstitution experiments in HEK293 cells suggested that phosphorylation at S725 is important for NLRP3 oligomerization. ROS are known to activate MINK1 and accordingly, scavenging them reduces MINK1 activity. This coincided with the inhibitory effect of ROS scavengers on IL-1β and caspase-1 activity in BMDMs, which was not observed in the absence of MINK1. Similar data were obtained in THP1 cells supporting the hypothesis that MINK1 may connect ROS production to the modulation of NLRP3 activation.The PhosphoSitePlus database rated S728 as the most likely phosphorylated site in NLRP3 and there is evidence this site or possibly the nearby S735 can be phosphorylated , 39, 49.Part to play: canonical-LRR domainBar: Serine 806 mouse: Serine 803Kinase section lead: Casein kinase 1-α (CSNK1A1)Phosphatase section lead: unknownNiu et al.  used masNiu et al. then used a comprehensive panel of experiments leading to identification of Casein Kinase 1 alpha1  as the responsible kinase for phosphorylation of S806/S803. Phosphorylation of S806/S803 occurs during priming. Consistently, it was not phosphorylated in NLRP3 samples obtained from unchallenged expressing cells . SubsequThis work provided information about the contribution of NEK7 to the process of NLRP3 activation. The fact that the NLRP3 S803A mutant can recruit NEK7 (and BRCC3) upon stimulation while displaying only low activity suggests NEK7 recruitment is not sufficient for NLRP3 activation. Contrary to S806A, the S806D mutant was unable to recruit NEK7 and was completely inactive. Thus, dephosphorylation of S806 is an obligatory step. Identification of the relevant phosphatase is a subject of continuing studies by this group.Part to play: canonical-LRR domainBar: Tyrosine 861 mouse: Tyrosine 858Kinase section lead: unknownPhosphatase section lead: PTPN22Nlrp3knockout BMDMs reconstituted with WT or Y861 mutant forms of NLRP3. Increased inflammasome activation was observed with a non-phosphorylated mutant form of NLRP3 compared with abrogated activity when a phospho-mimetic mutant was employed.In THP-1 cells, PTPN22 was shown to be involved in NLRP3 activation , 53. IntNLRP3 stimuli increased the phospho-tyrosine content of immunoprecipitated NLRP3 from autophagosomes whereas this signal decreased in whole cell lysates . In factPart to play: canonical-LRR domainBar: Serines 894, 898 mouse: Serines 891, 895Kinase section lead: unknownPhosphatase section lead: unknownA recent study by Wang et al.  providedPart to play: canonical-LRR domainBar: Tyrosine 918 mouse: Tyrosine 915Kinase section lead: LynPhosphatase section lead: unknownLyn-/- BMDMs and recovered upon expression of LynWT but not upon expression of its inactive LynY397F variant. Furthermore, −/−Lyn BMDMs treated with LPS and then stimulated with ATP displayed reduced NLRP3 ubiquitylation and degradation and produced significantly higher IL-1β than WT BMDMs. Site directed mutagenesis of NLRP3 potential Lyn-targeted phosphorylated residues (predicted with NetPhos 3.1) in NLRP3 constructs used to reconstitute -/-Nlrp3 BMDMs led to identification of Y918 as the phosphorylation site. -/-Lyn mice were highly susceptible to LPS-induced septic shock and died within the first 24h after LPS whereas blocking NLRP3 activity with MCC950 rescued the mice from lethality.Tang et al.  noticed e.g., ATP, led to enhanced tyrosine phosphorylation whereas a previous report by Spalinger et al. Phosphatase section lead: unknownTranscriptional regulation of NLRP3 inflammasome components relies on canonical NF-κB. Consequently, inhibitors of IκKβ, the key kinase in this pathway, are effective blockers of inflammasome function. Besides, compelling evidence suggests IKKβ also plays a role during the activation phase of the NLRP3 inflammasome. Furthermore, the ubiquitin-ligase activity of TRAF6, a well-known regulator of IKKβ, contributes to non-transcriptional priming of NLRP3, by impacting on the inflammasome assembly process .+ vesicles , Inhibitor of Kappa B kinase epsilon (IKKε)Phosphatase section lead: Protein phosphatase 2A (PP2A)The work by Fischer et al.  describeAKT is a well described substrate of TBK1. However, neither inhibition of AKT activity nor the mutagenesis of S5 in NLRP3 into Alanine  could prevent the binding of TBK1 to NLRP3. The binding of endogenous active-, phospho-TBK1 and endogenous NLRP3 could be measured by proximity ligation assay in iBMDMs, peaking at 30-60 min following LPS stimulation. TBK1/IKKe inhibition did not impact on the interaction between NLRP3 and NEK7, but it strongly increased ASC speck formation. While an S5A NLRP3 variant expressed in BMDMs was insensitive to AKT inhibition, it remained sensitive to TBK1/IKKe inhibition and to PP2A inhibition, suggesting that the regulation afforded by TBK1/IKKe and by PP2A goes beyond modulation of the S5 site.3Several recent cryo-electro-microscopy studies revealed that full length NLRP3 (mouse and human) can present as double-ring barrels or cages representing multimeric forms of NLRP3 , 51, 67.Here, we will summarize the localization of the reported phosphorylation sites that can be retrieved from the available NLRP3 structures with the aim to evaluate their exposure and address possible evidence of critical interfaces during the regulation of the NLRP3 maturation process Figure 33.1All reported phosphorylation sites, with exception of those featured by the basic cluster, are within the buried interior of the inactive NLRP3 oligomer Figure 43.2PYD filament structure  on monomeric NLRP3 might add to its stability by introducing charge and potentially mediating PYD-NACHT interaction; phosphorylation of the PYD domain within the caged structure may or may not have an effect; phosphorylation of the PYD domain in the context of the fully assembled NLRP3 will interfere with the PYD-PYD interaction of the PYD-ASC filament and, by destabilizing the filament could potentially add to the disassembly of the inflammasome .3.3The tyrosine phosphorylation sites Y136, Y140, and Y143 in the first helix of the FISNA subdomain are embedded in a basic cluster required for binding to membranes, which is exposed on the surface of the inactive NLRP3 assembly Figure 4The potential phospho-sites S161 and S163, though never studied in detail, are noted by several studies , 39, 40.Serine residue S295 lies in the center of the NBD domain, at the NACHT/NACHT interface. It is solvent exposed within the interior of decameric NLRP3 Figure 43.4Tyrosine 168 is visible in both the EM and Xray structures. Its sidechain faces the flexible 190-210 loop region (activation loop) .Serine 198 lies in the activation loop of NLRP3, upstream of the NBD domain. This loop region  interacts with the LRR domain, which is hidden in the inner side of the NLRP3 barrel, further acting as a barrier to prevent premature activation. In fact, S198 is visible in the EM structure, very close to the first helix of the LRR domain 710–720) Figure 40–720 FigThreonine 659 belongs to the transition LRR, in the vicinity of an important binding interface with NEK7 (interface II) . Reconst3.5e.g., R1009). Therefore, its phosphorylation may stabilize the NLRP3 barrel and support TGN dispersion. It is located at the NEK7-binding interface on the concave surface of LRR, which explains why its dephosphorylation during NLRP3 activation is required to allow for NEK7 binding . Therefore, akin to the PYD domain, preventing or limiting protein-protein associations may be key for tight control of inflammasome activation.Remarkably, all phosphorylation reactions reported at the LRR interfaces are inhibitory (phosphorylation at S806 inhibits NEK7 binding but is required during priming). The LRR domain has the propensity to self-oligomerize (resulting in cage formation) and to be involved in protein-protein interactions  and ATPase activity (not detectable in decamers), which suggests a role of this phosphorylation in ATP exchange or in contributing to a change in the relative orientation of the NBD and LRR domains  in the FISNA activation loop is hidden in the inner side of the decamer, in close proximity to the outer helix α domains . Activatt manner , 13. Phot manner . Furthert manner . These ot manner Figure 6t manner  and the t manner  likely pPhosphorylation at S806 occurs during priming as shown upon treatment with various TLR regimens . It has Phosphorylation of several tyrosine residues, Y136, Y140, Y143 (BTK-mediated) upon activation is believed to reduce the affinity of NLRP3 for membranes due to charge. The three phosphosites likely affect the packing of helix ax (127–137) against the NBD which may provide the necessary flexibility to allow the PYD to exit from the decamer cavity during the transition from closed to semi-open decamer, a transition state that has not been observed as such but was suggested to happen when ATP transforms the NACHT domain of NLRP3 while still being in the decameric or dodecameric cage , 67. In Phosphorylation of NLRP3 monomers at S295 by PKA may prevent cage formation and binding to membranes such as MAMs. Following dephosphorylation, re-phosphorylation of NLRP3 S295 by PKD has been proposed to result from approximation of PKD and oligomerized NLRP3 following stimulation-induced and DAG-mediated enrichment of PKD at the Trans-Golgi membrane network. Because S295 phosphorylation was reported to inhibit the ATPase activity of NLRP3 , it mighFurther potential sites have been suggested but characterization remains too limited to understand when phosphorylation/dephosphorylation may occur and how it might fit in the sequence of events. Beside phosphorylation of S5 and S295, phosphorylation by TBK1 and IKKe downstream of TLR activation, at sites that remain to be identified might be another way to introduce a brake preventing premature activation of NLRP3 without a bona fide activation signal.i.e., late in the NLRP3 maturation process. The domains of interaction between NLRP3 and JNK1 and between NLRP3 and CK1α are yet undescribed  and was previously found to be phosphorylated . This siSeveral stress pathways can lead to NLRP3 inflammasome activation. A distinction must be made between extracellular agonists, such as nigericin and ATP, which act fast, and particulate agonists, such as MSU or silica, which have a slower onset. Some regulatory mechanisms have emerged that may apply differentially according to which type of agonist is used. For instance, nigericin-induced NLRP3 activation can be suppressed by PGE2, in a PKA-dependent manner, whereas PGE2-induced suppression of MSU and silica responses is not PKA mediated and thus, may not involve phosphorylation of NLRP3 at S295 . SimilarDifferential phosphorylation might also be a way to drive the inflammasome activation process towards various outcomes. For instance, phosphorylation of T659 by Pak1 induced by the bacterial virulence factor CNF1 led to secretion of IL-1β independently of Gasdermin D, which might rely on an alternative secretion path using plasma membrane ruffles that are formed when the Rac GTPase is activated by CNF1 .A given phosphorylation site might be phosphorylated by more than one kinase during the NLRP3 maturation process. For instance, independent studies have shown that both PKA and PKD can phosphorylate S295 in human NLRP3 –45, 47, As mentioned earlier, phosphorylation and ubiquitylation mechanisms of NLRP3 regulation are interconnected. Ubiquitylation-driven down-regulation of NLRP3 by Parkin was described and may represent a specific pathway for regulation of NLRP3 in dopaminergic neurons . Whethere.g., S728 lies in exon 5, S806 in exon 6, Y861 in exon 7), it can be expected that the maturation of NLRP3 variants will be differentially impacted.Finally, several splice variants of human NLRP3 have been identified, which have so far received little attention , 95, 96.+-dependent  and K+-independent (imiquimod) stimuli, whereas the non-caged pathway occurs only with the K+-dependent stimuli. Considering this, BTK, which is reportedly more relevant for NLRP3 activation by imiquimod compared to activation with nigericin (see above), might not be involved in the non-cage forming/monomeric NLRP3 pathway. Similarly, PKD impacts mostly the early phase of NLRP3 activation by nigericin  and kinases  may differentially control these processes remains to be evaluated.Further understanding of the organelle-specific interactome of NLRP3 including kinases and phosphatases should also clarify where the inflammasome can assemble to activate caspase-1. ASC specking activity is a widely used readout of inflammasome activation in cellular systems, which is believed to recapitulate inflammasome assembly and activation at the MOTC. However, recent work has shown that specks may also assemble independently of MTOC . AnotherIn conclusion, significant progress has been made to unravel the processes that contribute to NLRP3 functional maturation. Key studies have identified either NLRP3 residues that are post-translationally modified, quaternary structures that NLRP3 can adopt, or subcellular locations to which NLRP3 localizes during activation. The challenge ahead is to integrate these individual aspects of NLRP3 biology into a comprehensive model for stepwise NLRP3 licensing and activation. Structural information combined with subcellular information as well as interactome elucidation will undoubtedly help refine our understanding of the beautiful cellular machinery blossoming into an active inflammasome.FB: Conceptualization, Data curation, Methodology, Writing – original draft, Writing – review & editing. CD: Data curation, Software, Visualization, Writing – review & editing.