P110, a peptide inhibitor of Drp1-Fis1 interaction, reduces pathology in numerous models of neurodegeneration, ischemia, and sepsis without blocking the physiological functions of Drp1. Since peptides have pharmacokinetic limitations, we set out to identify small molecules that mimic P110’s benefit. We map the P110-binding site to a switch I-adjacent grove (SWAG) on Drp1. Screening for SWAG-binding small molecules identifies SC9, which mimics P110’s benefits in cells and a mouse model of endotoxemia. We suggest that the SWAG-binding small molecules discovered in this study may reduce the burden of Drp1-mediated pathologies and potentially pathologies associated with other members of the GTPase family.The large cytosolic GTPase, dynamin-related protein 1 (Drp1), mediates both physiological and pathological mitochondrial fission. Cell stress triggers Drp1 binding to mitochondrial Fis1 and subsequently, mitochondrial fragmentation, ROS production, metabolic collapse, and cell death. Because Drp1 also mediates physiological fission by binding to mitochondrial Mff, therapeutics that inhibit pathological fission should spare physiological mitochondrial fission.  Dynamin-related protein 1 (Drp1) mediates physiological and pathological mitochondrial fission, and the latter can be selectively blocked by a peptide inhibitor. Here, the authors identify a small molecule that mimics the benefits of this peptide inhibitor in cells and a mouse model of endotoxemia. Damaged mitochondrial components are directly involved in pro-inflammatory signaling and healthy mitochondria are in turn damaged by inflammatory signals, creating a robust pro-inflammatory vicious cycle11. When cell stress is high or prolonged, mitochondria undergo excessive fission leading to mitochondrial fragmentation, reduced mitochondrial membrane potential, increased ROS, and reduced ATP production, which triggers apoptosis14. Therefore, pharmacologic inhibition of pathological mitochondrial fission is a promising strategy to quell overactive inflammation and prevent mitochondrial dysfunction in a broad range of cell types and diseases17.Mitochondrial dysfunction during inflammatory cell stress has been identified as an important driver of diseases with unmet clinical needs, such as sepsis18. Drp1 is recruited to the mitochondria by partner proteins Fis1, Mff, MiD49, and MiD5119. Although Drp1 is an attractive drug target, its activity may not be dispensable. Drp1-Mff-mediated fission is important for maintaining mitochondrial quality and inhibition of this interaction is cytotoxic20. In vivo, Drp1-Mff inhibition pharmacologically or genetically accelerates the pathology associated with neurodegenerative disease21. In contrast, Drp1-Fis1 interaction mediates pathological conditions; a rationally designed heptapeptide inhibitor of Drp1-Fis1 interaction, P110, selectively inhibits pathological and not physiological mitochondrial fission22. P110 treatment reduces the pathologies associated with a variety of diseases, including Huntington’s disease (HD)27, amyotrophic lateral sclerosis (ALS)28, Alzheimer’s disease (AD)29, Parkinson’s disease (PD)31, scrapie32, cardiac ischemia33, stroke34, inflammatory bowel disease35, and sepsis38, in mouse and patient-derived cell models. Importantly, P110 has no apparent toxicity in mice even after five months of sustained delivery at 5 mg/kg/day28. Fis1 may also have physiological roles through its interaction with other proteins; however, Drp1-Fis1 interaction is specifically induced during inflammatory cell death39. In addition to the lack of phenotype with pharmacologic inhibitors under basal conditions, Fis1 knock-down with siRNA does not have an impact on basal mitochondrial fission40, and Fis1-KO has only a minor phenotype41. In contrast, Drp1-Mff interaction is found in both basal and stress conditions and Mff-KO has a profound effect on basal mitochondrial fission resulting in severely elongated mitochondria41. Mff may also recruit Drp1 during cell death and some genetic models indicate that Mff knock-down can reduce cell death in culture and infarct size during ischemia-reperfusion injury42. However, inhibition of Mff is not a viable strategy to reduce inflammatory cell death since pharmacologic and genetic perturbation of Mff is toxic in several cell-types and in vivo21. This is because Mff-Drp1 interaction governs essential functions under basal conditions whereas Fis1-Drp1 interaction does not.The primary driver of mitochondrial fragmentation is Drp1, a large cytosolic GTPase that translocates from the cytosol to the outer mitochondrial membrane, where it oligomerizes and mediates mitochondrial fission through a constricting loop43. The P110 heptapeptide (DLLPRGS) was rationally designed from such a short homology sequence between hDrp1  and hFis1 22. Cross-linking of the P110 heptapeptide to TAT47–57 enabled peptide delivery into cells in culture and in vivo. The term P110 hereafter refers to the TAT conjugated P110 heptapeptide. Although P110 has shown great benefit in animal models of numerous human diseases, its translation to a clinical intervention has been limited because P110 is susceptible to serum and cellular proteases and is not orally bioavailable.We have previously shown that different proteins that interact in an inducible manner often share short sequences of homology that represent competitive sites of inter/intra-molecular interactionsP110’s therapeutic effect. However, the feasibility of a small molecule mimetic is uncertain, since P110’s binding site is unknown and protein-protein interactions are often large, shallow surfaces that are difficult to inhibit with small molecules44. Here, we elucidate P110’s mechanism of action and map P110’s binding site. We then identify a small molecule that mimics P110. Our study demonstrates that small molecule inhibitors of protein-protein interactions can be generated for Drp1 and that the P110-binding site is an unrecognized allosteric backdoor for Drp1 inhibition that is selective for pathological fission. Our work also suggests that this allosteric strategy may be generalized to other members of the GTPase protein family.We seek to identify small molecules that could mimic P110 causes partial inhibition of Drp1 GTPase activity in vitro22, indicating a direct binding of P110 to Drp1. However, P110’s inhibitory kinetics were unknown. A TAT-free cyclic DLLPRGS  decreased both Vmax and Km of Drp1 GTPase activity ; BDY-P110 had an increased affinity for Drp1 (Kd ± SEM) in the presence of the non-hydrolysable GTP analog, GMPPCP (apparent Kd = 18 nM ± 23 nM) and GTP (apparent Kd = 58 nM ± 46 nM), as well as with GDP (apparent Kd = 219 nM ± 219) and it did not bind apo-Drp1 at the concentrations tested  is derived from the first half of switch I (49DLLPRGTGIVT59), an unstructured loop that is involved in GTP hydrolysis in a variety of GTPases45 47 suggested that the endogenous P110 region swings by ~9 Å, making backbone contacts with amino acids R53 and G54 to the GTP α-phosphate , and, as expected, the nucleotide-binding pocket was ranked first with the largest volume and most favorable propensity of the ligand-binding score (PLB: 3.97). We next performed Essential Site Scanning Analysis49 on PDB: 3W6P structure to identify the regions on the protein that can function as druggable sites that would modulate Drp1 GTPase activity. As expected, some residues lining the nucleotide-binding site had high druggable z-scores , the alpha helical residues from F104-E111 (G-domain α3), and the G5 element, including residues R247-V258 5.9 Fig. . We nameP110,SC1,SC3, and SC9 act as uncompetitive inhibitors of WT Drp1; at 1 µM they reduced both Km and Vmax of Drp1 , suggesting that SC3 may have some activity on the double mutant Drp1.Like rp1 Fig. . The D49SC9 Fig. . Like cPant Fig. . SC3 inhP110-binding site, we conducted a competitive Drp1-binding assay, by assessing the amount of labeled cP110 that co-sediments with oligomerized Drp1. In the vehicle condition (CT), 1 nM BDY-cP110 binds Drp1-GMPPCP oligomers and is pulled from the supernatant during centrifugation, resulting in decreased fluorescence in the supernatant. Pre-treating with a large excess of unlabeled cP110 (10 µM) blocks all P110-specific BDY-cP110 binding to Drp1, resulting in an increased BDY signal in the supernatant  and acts as a positive control in our assay. All three compounds also reduced the co-sedimentation of BDY-cP110 with recombinant Drp1, whereas Mdivi-152, a weak Drp1 inhibitor, did not  to cause pathological mitochondrial fission and dysfunction36. As before, we found mitochondrial fragmentation after 16 h of LPS treatment , 36% (LPS + SC9), and 38% (no LPS). SC1 caused only a marginal recovery in fused state cells . SC9 was more effective in decreasing the percentage of cells with fragmented mitochondria. SC9 reduced the percent of fragmented cells from 22% (LPS + Veh) to 9% (LPS + SC9), relative to 4% in the absence of LPS; P110 and SC1 caused only a slight decrease in the fragmented phenotype from 22% to 18%. In contrast to the other compounds, SC3 greatly exacerbated the LPS effect, decreasing the proportion of cells with fused mitochondria from 20% to 3% and more than doubling the percent of cells with fragmented mitochondria from 22% to 55%  and 41% (LPS + SC9). In contrast, SC1 and SC3 had no beneficial effect on the LPS-induced drop in mitochondrial membrane potential  levels  to 28% (LPS + P110) and to 14%  when measuring inhibition of LPS-induced mitochondrial depolarization after 24-h  at doses up to 50 mg/kg. We next assessed SC9 levels following a single intraperitoneal injection (20 mg/kg). At 15-min post-injection, SC9 plasma levels reached 5.8 μg/ml (or 12.4 μM) which is ~17x the EC50 in culture, indicating that we achieved a pharmacologically relevant dose. SC9 did not significantly penetrate the brain when measured after 15 min; the brain to plasma ratio of SC9 was about 2%  at 0 h and 4 h post-injection, we found 1.6% of peak SC9 levels at 16 h after the first injection, suggesting a longer half-life in LPS-treated mice  at 4 h after this dose of LPS, but not at later timepoints  in mice challenged with 0.267 mg LPS  with co-administration of ice Fig. . Althougnts Fig. . The effLPS Fig. . Most imLPS Fig. . 0.3 mg SC9 and vehicle-treated mice, we collected plasma 4 h after 0.3 mg LPS injection and carried out the mouse immune monitoring 48-plex ProcartaPlex panel. Compared to vehicle-treated mice, levels of inflammatory factors were down ~19% in SC9-treated mice relative to vehicle-treated LPS  although this effect was non-significant  and RAW 264.7 cells (mouse macrophage-like cells) treated with SC9 or P110 produced less mitochondrial ROS in the presence of LPS . Peroxide induced a 2X increase in mitochondrial ROS and SC9 decreased ROS production to the level of control  rescue treatment 4-hours post-LPS treatment (0.3 mg) caused an increase in survival  and did not affect body weight . We identified a compound, 45, but the large SWAG site, exposed by GTP binding, has not yet been explored in any GTPase. The SWAG could represent an inducible protein-protein interaction site for protein partners to directly modulate GTP affinity and GTPase activity through switch I/GTP stabilization. For Drp1, this SWAG site may represent the Fis1-selective binding site or may allosterically expose the Fis1-binding site elsewhere on Drp1; these two possibilities are under investigation. Regardless, we find molecules that bind to SWAG inhibit Drp1-Fis1 interaction and are effective and selective inhibitors of pathological and not physiological mitochondrial fission.Much effort has focused on the GTP-binding site near switch IP110 sequence in hDrp1 (amino acids 49-55) is within switch I, and it forms a mobile loop alternating between occupying SWAG and stabilizing GTP. We provide several lines of evidence demonstrating that P110, which we identified as a selective inhibitor of Drp1-Fis1 interaction and mitochondrial dysfunction22, shares a common mechanism with SC9: (1) both P110 and SC9 exert similar uncompetitive inhibitory kinetics on Drp1 GTPase activity; (2) their effect is lost when two amino acids associated with the SWAG, D49, and R53, are mutated to alanine; (3) SC9 competitively inhibits P110 binding to Drp1; (4) both SC9 and P110 inhibit LPS-induced Drp1-Fis1 association in cells, without affecting Drp1 association with its physiological mitochondrial adaptor, Mff; (5) they both inhibit mitochondrial fragmentation and dysfunction in cells treated with LPS; (6) they both inhibit LPS-induced pathology in an in vivo endotoxemia model. Whereas the SC9 safety study was conducted in both male and female mice, the mouse endotoxemia studies were conducted in female mice only. Future studies will determine sex-specific LPS-induced pathologies, the contribution of co-morbidity factors, and their contribution to the efficacy of SC9 in endotoxemia models.The 44. However, in this case, GTP binding induces a conformational change in Drp1 that exposes the druggable SWAG site, which has sufficient depth to accommodate a small molecule or peptide protein-protein interaction inhibitor.The rational design of a protein-protein interaction modulator is challenging as large flat surfaces without obvious clefts often mediate these interactionsP110 to Drp1 and the conformation of P110 bound within the pocket, we chose not to take the ligand-based approach for generating a pharmacophore. Instead, the protein structure-based pharmacophore approach was used. We generated an 8-point pharmacophore model with 3 donors, 3 acceptors, 1 donor or acceptor, and 1 aromatic center or pi ring center and an excluded volume to model the switch I-adjacent groove. Eight features in a pharmacophore model for virtual screening is unusually high; most studies use 4 or 5-point pharmacophores. However, we rationalized that the 8-feature pharmacophore with several donor and acceptor features could more faithfully mimic a heptapeptide, with several backbone and sidechain atoms that can function as donors and acceptors. We also determined that certain conformations of P110 can satisfy 6 out of these 8 pharmacophore features when excluded volume criteria were ignored (to take induced fit into account). Screening for molecules that satisfied all 8 features resulted in a low hit rate. Therefore, we next screened for molecules that satisfied any 7 of the 8 features. Unsurprisingly, several hits from the screen resembled peptides and sugars with many polar groups. However, there were some drug-like molecules that we explored further.In the absence of information on the mode of binding of 52. However, recent studies have shown that the therapeutic effect of mdivi-1 may be attributed to its inhibition of complex I54 or mitochondrial calcium uptake55, not specifically to Drp1 inhibition. Drpitor1a, a more recently discovered competitive GTPase inhibitor, inhibits mitochondrial fragmentation and exerts therapeutic potential in cells and a cardiac ischemia-reperfusion injury mouse model56. However, since Drp1 has essential functions outside of the stress response57, the long-term safety of agents that inhibit Drp1 function indiscriminately is uncertain. Indeed, Drp1 loss-of-function variants are not tolerated in vivo; homozygous variants are embryonically lethal, and loss of function heterozygous variants can lead to severe neurological dysfunction in mice58. Neuron-specific and cardiac Drp1 knockout and knock-down mouse models led to progressive cell degeneration and dysfunction indicating that Drp1 is not dispensable for brain or cardiac functions62. At least 75% of Drp1 protein levels were required for normal cardiac function in mice indicating limited spare capacity60. Drp1 is required for mitochondrial quality control in neurons and functions synergistically with Parkin, an important mitophagy protein63. In humans, de novo variants that inhibit function result in severe neuro-muscular diseases64. Furthermore, it seems Drp1 has functions outside of mitochondrial division and mitophagy including peroxisomal fission65 and synaptic endocytosis66. This has spurred interest in selective protein-protein interaction inhibitors, such as P110 and SC9, which blocks Drp1’s pathological functions without inhibiting its physiological functions. Although P110 is a promising potential therapeutic with a wide range of potential applications and excellent safety features28, its pharmacokinetic properties are limiting in applications that require long-term dosing or oral bioavailability.Although small molecule inhibitors of Drp1 have been described, their specificity and therapeutic utility are uncertain. Mdivi-1 was the first described Drp1 inhibitor67, the assays employed in these studies would not have discovered SC9. SC9 is a potent inhibitor of pathological mitochondrial fragmentation yet it is only a weak partial inhibitor of the GTPase activity in vitro (causing only 10–25% inhibition). The published screen that identified uncompetitive compounds used low GTP concentrations (0.1 mM) and set a 30% inhibition cut-off67. At this GTP concentration, SC9 does not have any inhibitory activity in vitro; supraphysiologic concentrations of GTP (1 mM) are required to detect the 10–25% inhibition with SC9. Yet, SC9 has a potent therapeutic effect in cells with a nM-range IC50. This finding likely dissociates the link between GTPase inhibition in vitro and therapeutic activity in vivo.While both competitive and uncompetitive small molecule inhibitors of Drp1 GTPase activity have been describedP110 and SC9. This mechanism would explain why both compounds inhibit stress-mediated mitochondrial fission but do not affect physiological mitochondrial fission, a feature that likely contributes to the good long-term safety observed following a five-month treatment with P11028. By contrast, Mff-mediated mitochondrial fission seems to act independently of LPS or our inhibitors. This finding further supports the hypothesis that Mff mediates physiological mitochondrial fission whereas Fis1 mediates mitochondrial fragmentation associated with pathological stimuli, such as LPS. We note that the partial GTPase inhibition measurement was conducted using a recombinant Drp1 with a His-tag, which has been shown to impact Drp1 self-assembly68. In theory, this tag could alter the inhibitory properties of P110 and SC9. However, in studies using a tag-less recombinant Drp1 and Drp1 immunoprecipitated from SHSY-5Y cells we observed a similar partial inhibition effect by P11022.We hypothesize that inhibition of Drp1-Fis1 protein-protein interaction, which we confirmed using proximity ligation assay, is the feature responsible for the therapeutic effect of both SC9 in inhibiting LPS-induced mortality in mice (Veh. 80% vs. SC9 33%) was comparable to that of P110 (Veh. 77% vs. P110 25%)36. However, SC9's pharmacological features are not yet optimal; it is relatively large , is too polar to get into the brain, and has a short half-life. Therefore, current efforts are focused on identifying analogs of SC9 with improved pharmacologic properties, including a longer half-life and improved blood brain barrier penetrance. It is not clear why SC1 and SC3, which mimic at least some of the features of P110 in vitro, do not mimic P110 effects in cells. SC9 has fewer rotatable bonds (7) compared to SC1 (14) and SC3 (10). SC1 is not selective to Drp1 since it inhibits dynamin-2 activity. SC3 also has low predicted solubility (0.03 mg/ml) and may aggregate. SC1 and SC3 could also have poor plasma membrane penetrance, off target toxicity, or lower stability in culture during 24-h incubations. Exploring these possibilities and conducting further medicinal chemistry to improve the pharmacological features of SC9 remain outside the immediate scope of this study. Increasing blood-brain barrier penetrance of SC9 may be required to mimic the anti-neurodegeneration effects previously achieved with P110.The benefit of 69, it is unclear which cell types in vivo are the key mediators of the therapeutic benefit. While SC9 mimics P110 in blocking mitochondrial dysfunction using the recently published cardiomyocyte model36, likely, SC9 will also mimic P110 in inhibiting innate immune cell activation26. Immune cells, such as macrophages, monocytes, NK cells, B cells, effector T cells, and astrocytes, respond robustly to danger signals and have been identified as key drivers of inflammatory disease70. When these cell types are activated, they fragment their mitochondria and shift to aerobic glycolysis70. Intriguingly, inhibition of Drp1, and other interventions that maintain oxidative phosphorylation, have been found to suppress the activation of pro-inflammatory immune cells and promote the anti-inflammatory state70. This agrees with the ProcartaPlex immune monitoring panel analysis that we conducted; of which the top three hits include IL-13, IL33R, and M-CSF, all of which have been implicated in innate immune cell activation and the proinflammatory response73. The concordant increase in IL-10 suggests the formation of OXPHOS-competent anti-inflammatory M2 macrophages74. This hypothesis is supported by experiments done here on RAW cell macrophages. Further experiments on innate immune cells and other cell types are needed to confirm this hypothesis. Nevertheless, our work here shows that Drp1-Fis1 inhibitors, such as P110 and SC9, which preserve mitochondrial function by inhibiting excessive fission, may be important tools in the emerging field of immunometabolism. As such, SC9 represents a class of anti-inflammatory small molecules with the potential to address mitochondrial dysfunction in a wide range of diseases.How inhibition of excessive mitochondrial fission attenuates LPS-induced endotoxemia in vivo is not yet known. Since many cell types and tissues undergo mitochondrial fragmentation in response to pathogen-associated molecular patterns (PAMPs), danger-associated molecular patterns (DAMPs), cytokines, and chemokinesP110 cargo  and BODIPY-TMR conjugated cyclic P110 , each at 99% purity, were synthesized by CPC Scientific . cP110 uses a glutaryl linker from the C-terminal lysine to the N-terminus of DLLPRGSK. For the cP110-BODIPY-TMR (BDY-cP110), the BODIPY was conjugated to a cysteine added at the C-terminus (DLLPRGSKC), and the peptide was cyclized with a glutaryl bridge between the C-terminal lysine and the N-terminus. Linear BODIPY-TMR-AGGDLLPRGS  was synthesized by AnaSpec . TAT47–57-conjugated P110 for in culture studies  was synthesized by Ontores . SC1,SC3, and SC9, with a minimum purity of 90%, were obtained through Mcule  from various chemical suppliers. Chemical characterization of the compounds was performed at Enamine Ltd  and is provided below. The spectral data is available in the supplementary information file  . Mdivi-1 was obtained from Sigma . For the endotoxemia model, lipopolysaccharide (LPS) was sourced from Sigma .Cyclic 1H NMR  δ 8.31 , 7.68 , 7.49 , 7.09 , 6.90 , 6.49 , 5.99 , 3.55 , 3.11 – 3.04 , 2.35 – 2.30 , 2.30 – 2.24 , 2.06 , 1.59 – 1.52 . 13C NMR  δ 155.79, 155.70, 139.07, 138.63, 130.32, 119.73, 112.07, 110.72, 66.65, 56.33, 56.31, 53.83, 37.86, 37.81, 27.14, 17.65. HRMS (m/z): calc. for C23 H38 N6 O4 (M + ) 462.2955, obs. 462.2951.1H NMR  δ 9.66 , 7.78 , 7.75 – 7.70 , 7.46 – 7.42 , 7.29 , 7.26 – 6.96 , 4.82 , 3.89 , 3.26 , 2.98 , 2.73 . 13C NMR  δ 173.03, 172.67, 169.21, 166.99, 164.41, 156.84, 139.22, 131.73, 130.66, 129.19, 128.65, 126.96, 126.14, 121.73, 65.10, 44.49, 31.80, 28.80. HRMS (m/z): calc. for C23 H22 Cl N7 O3 (M + ) 479.1473, obs. 479.1464.1H NMR  δ 10.97 , 7.33 , 7.24 – 7.18 , 7.16 , 7.09 , 5.15 – 5.02 , 4.12 – 4.00 , 2.90 , 2.62 , 2.54 . 13C NMR  δ 170.04, 165.89, 164.57, 159.86, 155.45, 153.39, 150.52, 146.68, 136.61, 128.98, 127.47, 126.33, 110.79, 94.37, 44.82, 35.37, 35.02, 24.85, 16.88. HRMS (m/z): calc. for C21H22N8O3S (M + ) 466.1536, obs. 466.1528.http://prody.csb.pitt.edu/tutorials/essa_tutorial/. Python 3 was used to execute python codes for ESSA. The output Protein Data Bank (3W6P_gnm_zs.pdb) file was opened in MOE and a protein surface model was generated. The surface was colored according to the Z-scores (reflecting the effect of interaction with a ligand) of residues using a custom Scientific Vector Language code in MOE. The minimum Z-score was colored yellow, the intermediate Z-score was set to 0.7 and was colored cyan, and the maximum Z-score of 2 or above was colored magenta.ESSA was performed on PDB: 3W6P, as described in 30 was used to screen for small molecules that satisfy 7 out of 8 pharmacophore features without causing clashes with the protein.Molecular modeling and virtual screening were carried out using MOE Software (version 2019.01). PDB: 3W6P was loaded into MOE and the system was prepared using QuickPrep functionality in MOE at the default setting. QuickPrep functionality optimizes the hydrogen bond network and performs energy minimizations in the system. After the system was prepared, Receptor 3W6P.A, Metal 3W6P.A and Ligand 3W6P.A was kept, and all other molecules were removed. Site Finder functionality in MOE was used to calculate all the putative ligand binding sites. Dummy atoms were created at the site adjacent to Switch I (SWAG) identified by the Site Finder. All the residues within 4.5 Å from the dummy atoms were defined as pocket residues. AutoPH4 tool was used to generate the 8-feature pharmacophore with excluded volume criteria at the dummy atom site in the apo generation mode. CoCoCo librarywww.swissadme.ch). The compounds with unfavorable chemical groups flagged by Brent and PAINS filtered were removed.The list of small molecules obtained from the above in silico screen was prepared using the Wash functionality at the dominant protonation state at pH 7.0 and was energy-minimized, using the Energy Minimize function in MOE. The structures, Receptor 3W6P.A, Metal 3W6P.A and Ligand 3W6P.A was used for the docking analyses. The small molecule hits were docked at the dummy atom site on Receptor 3W6P.A. Triangle Matcher method was used for placement and the Rigid Receptor method was used for refinement, using London dG and GBVI/WSA dG scoring functions, respectively. Binding Score (S) was used to rank-order the molecules based on predicted affinity. The online Swiss ADME tool was used to compute the predicted physiochemical properties of the hit compounds  by Gibson cloning . Proteins were expressed in BL21 E. coli. cultured at 37 °C to an OD600 of 1.2. Protein expression was then induced with 0.1 mM IPTG, following an overnight incubation at 37 °C. Bacteria were then lysed with BugBuster Master Mix supplemented with 20 mM imidazole, 0.4 mM PMSF, and protease inhibitor cocktail  and adjusted to pH 7.8. Insoluble debris was pelleted by centrifugation at 30,000 × g for 30 min and the supernatant was flowed twice through Nickle His GraviTrap Columns . The column was washed with 20 mL wash buffer  and eluted in elution buffer . The buffer was exchanged with a Zeba™ Spin Desalting Column, 7 K MWCO, 10 mL  loaded with HEPES 20 mM pH 7.8, 0.25 M NaCl, 1 mM DTT, and protease inhibitor cocktail and mixed and stored in 25% glycerol. Protein concentration was estimated with nanodrop, and purity was checked with gel electrophoresis and Coomassie stain. Drp1 mutants were generated with the Agilent Quick Change II site directed mutagenesis kit  and were expressed in parallel under identical conditions. Protein purity and yield were comparable between mutants.The cDNA for Drp1 isoform 3 (DNM1L) was gifted by Professor Janet Shaw (University of Utah)Recombinant Drp1 isoform 3 GTPase activity was measured with Sigma Malachite Green Phosphate Assay Kit . Drp1 was diluted to 200 nM in 100 mM KCl, 10 mM MgCl2, and 1 mM DTT and incubated with 2 µM test compounds for 30 min while rotating at room temperature. This solution was then added to 0.2 ml PCR Tubes,  preloaded with varying concentrations of GTP , resulting in a final concentration of 100 nM Drp1 and 1 µM compounds. Plates were then incubated for up to 1 h at 37 °C. To keep GTP concentration under 250 µM the reactions above 250 mM GTP were diluted with water and loaded into clear 96-well assay plates . Then, the malachite green reagent was added. Absorbance was measured at 620 nm after 30 min, using a SpectraMax fluorescence microplate reader . The reaction Kcat was estimated using a phosphate standard curve, which was generated by adding known concentrations of phosphate to the same GTP-containing reaction solution and assayed in parallel on each plate. The standard accounts for non-enzymatic GTP hydrolysis. The data are expressed as production of inorganic phosphate [Pi μM]/min/[Drp1 μM] (and assay carried out at pH = 7.5).Recombinant Drp1 was serially diluted from 1 µM to 5 nM in 50 mM KCl, 3 mM MgCl2, 1 mM DTT, 0.01% NP40 Substitute , and protease inhibitor cocktail (as above). 200 µM GMPPCP , 200 µM GTP , or 200 µM GDP  and then 10 nM (BODIPY-TMR)-AGGDLLPRGS  were mixed into each fraction and 10 µL immediately plated in a black 384-well, small volume, microplate  with 6 replicates per condition. The plate was briefly centrifuged and then the BODIPY-TMR fluorescence polarization was measured with a Tecan Infinite M1000 microplate reader , after a total of 30-min incubation at room temperature. The peptide only condition was not subtracted from the data and represents the 0 points on the linear scale x-axis.cP110 or compounds at room temperature with rotation. Then, 500 µM GMPPCP (as above) and 1 nM BODIPY-TMR -cP110 were added  for a final volume of 100 µL. Drp1 was allowed to oligomerize at room temperature for 1 h while protected from light. Samples were spun at 21,000 × g at 4 °C for 1 h. Directly after centrifugation, 3 × 10 µl of the supernatant were carefully removed and placed into a Greiner 384 well black microplate . BODIPY-TMR fluorescence (544 ex./570 em.) was measured with a Tecan Infinite M1000 microplate reader (as above).Recombinant Drp1 was diluted to a final concentration of 500 nM in 100 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.1 mg/ml 99% bovine γ-globulins , and protease inhibitor cocktail (as above), and was incubated for 30 minutes with 10 µM 2O2  for 3hrs. MitoSOX in HEK-293, RAW 264.7, and MEFs were measured with the SpectraMax fluorescence microplate reader and normalized to the cell number using Hoechst 33342 1:10,000.For the mitochondrial membrane potential assay, clear bottom black 96-well TC plates  were seeded with H9c2(2-1) cells  at 8000/well and allowed to adhere overnight in 100ul DMEM/10% FBS. The cells were washed with PBS and incubated for 24hrs in DMEM/10% FBS with LPS (2 µg/ml) and test compounds. This solution was removed, and the cells were incubated with a staining solution containing Hoechst 33342 1:20,000  and 100 nM Tetramethylrhodamine , in FluoroBrite DMEM/4 mM Glutamine. Plates were incubated for 25 min at 37 °C, washed 3× with 150 µL warm HBSS , and then placed in 33 uL warm FluoroBrite DMEM/4 mM Glutamine. Fluorescence readings (top) were taken with a SpectraMax fluorescence microplate reader . For mitochondrial ROS production assay, cells were prepared as above except cells were stained with Hoechst (as above) 100 nM MitoTracker Deep Red FM , and 5 µM MitoSOX Red Mitochondrial Superoxide Indicator . After LPS treatment, cells were washed (as above) and then plated in 100 µL FluoroBrite DMEM/4 mM Glutamine and imaged at 10× magnification in a Keyence BZ-X700 light microscope  outfitted with a live-cell incubator. Before the experiment, a program with points pre-set at the center of each well with autofocus on the MitoTracker channel. Images were analyzed in bulk with a simple ImageJ macro (version 1.53t). The MitoTracker signal was used as a mask to extract and quantify the total MitoSOX signal (integrated density) in single cells and this was normalized to the total MitoTracker footprint. Mitochondrial ROS signal in RAW 264.7 cells (ATCC: TIB-71) and HEK-293 cells (ATCC: CRL-1573) was measured after a 6 h incubation with LPS (2 µg/mL). Mouse embryonic fibroblasts : 789–802) were treated with 200 µM Hz-stacks were collected from random points on the plate in high resolution mode for exactly 20 min. Z-stacks were converted to full focus maximum intensity projections in Keyence BZ-analyzer and then filtered with an ImageJ macro to yield both background-subtracted and binarized images. The data were then blinded to the analyzer until all analyses were completed. To correlate mitochondrial morphology with TMRM values of single cells, cells were manually selected in ImageJ (version 1.53t) and measurements were computed in both the binarized MitoTracker channel and a TMRM channel masked by the binarized MitoTracker channel. Mitochondrial size is expressed as the 2D area per binary MitoTracker particle in the 2D maximum intensity projection. Data were expressed as the total TMRM signal (integrated density) per MitoTracker positive area per cell. All complete cells absent of mitochondrial overlap with an adjacent cell were collected from the image for analysis. Only MitoTracker objects greater than 4 pixels were used for analysis to eliminate noise.To quantify mitochondrial morphology and membrane potential at high magnification, 160,000 × H9c2 cells were plated on a poly-D-lysine coated MatTek 35 mm glass bottom dish  and allowed to adhere for 24-hours in DMEM/10% FBS. The conditions were staggered by 30 min to ensure the identical time of incubation. One by one, the dishes were washed with PBS and incubated in FluoroBrite DMEM/4 mM glutamine/10%FBS supplemented with LPS (2 µg/ml) and test compounds at 2 µM for 16 h. The media was then removed and replaced with FluoroBrite DMEM/4 mM glutamine/10%FBS supplemented with 100 nM MitoTracker Deep Red FM (as above), 100 nM Tetramethylrhodamine (as above), and Hoechst 33342 1:20,000 (as above), and incubated for 25 min at 37 °C. For the staining solution stock, 1000x dye stock mix diluted in DMSO mix was precisely aliquoted and kept at −20 °C or on dry ice under foil until just before each sequential staining media prep. Cells were washed three times in warm HBSS and then placed in warm FluoroBrite DMEM/4 mM glutamine/10%FBS supplemented with LPS 2 µg/mL and test compounds at 2 µM. The cells were imaged in a Keyence BZ-X700 light microscope outfitted with a live-cell incubator. Multicolor 60x z-stacked since the mitochondria are not all parallel to the bottom of the cell; a full cell z-stack captures polymerized Drp1 foci on all the mitochondria in the cell. For the PLA assay, the Duolink Red kit  was used as described in the manufacture’s protocol. Primary antibodies were used as above with the addition of 1:66 rabbit anti-MFF . The PLA foci images were background-subtracted and thresholded to create a binary image. The total PLA foci area per cell area was quantified for individual cells manually by an observer blind to the conditions using ImageJ (version 1.53t).160,000 × H9c2 cells were plated on a poly-D-lysine coated MatTek 35 mm glass bottom dish (as above) and allowed to adhere for 24-h in DMEM/10% FBS. Cells were washed with PBS and plated in DMEM/10% FBS supplemented with LPS (2 µg/ml) and test compounds at 2 µM. After 23 h, cells were re-dosed with an 2 µM compounds and LPS and incubated for 1 h. Cells were then washed 1× with PBS and fixed with 4% formaldehyde  in PBS for 10 min. Cells were then washed once with PBS supplemented with 22 mg/ml glycine and then permeabilized with 0.1% trition-X-100 in PBS supplemented with 22 mg/ml glycine for 10 minutes. Cells were then washed 3× with PBS. For Drp1 foci immunostaining, cells were blocked at room temperature in PBS Tween 0.1% (PBST) + 1% BSA (1 h) and then incubated overnight in a humidified chamber with 40 uL of 1:50 mouse anti-Drp1  and 1:66 rabbit anti-Fis1 . Cells were washed 3 × 5 min with PBST and incubated with 1:1000 secondary antibodies goat anti-Rabbit IgG (H + L) DyLight 488  and goat anti-Mouse IgG (H + L) DyLight 650  in PBST + 1% BSA. Cells were washed for 3 × 5 min with PBST and then mounted with 50 uL of mounting media with DAPI . The Fis1 staining (used to focus only on Drp1 polymers that are associated with the mitochondria) was thresholded to create a binary mask to extract Drp1 foci that localize to mitochondria. The Drp1 stain was background subtracted to isolate signal from foci over the background. The total integrated density of the Drp1 foci signal per total mitochondrial area per cell was quantified by segmenting each cell manually by an observer blind to the conditions using ImageJ. The images for Fig. To determine Drp1 protein levels, 100 × 20 mm Vented TC plates  were seeded with H9c2 at 400000/plate in 10 mL DMEM/10% FBS. The cells were then incubated for 24hrs in with LPS (2 µg/ml) and test compounds, at 2 µM, or vehicle. Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit . Proteins (15ug) were resuspended in Laemmli sample buffer containing 2‐mercaptoethanol, loaded on SDS-PAGE, and transferred onto 0.45 μm nitrocellulose membrane. as described before . Membranes were probed with the indicated antibody and then visualized by ECL. Images acquired with Bio-Rad ChemiDoc Imagers  were analyzed with Image Lab Software (version 6.0.1) to determine relative band intensity. The antibodies used in this study were: anti-Drp1 rabbit monoclonal antibody ; anti-α-Tubulin mouse monoclonal antibody . Secondary antibodies including anti-mouse IgG  and anti-rabbit IgG  were obtained from GE Healthcare . Drp1 KO MEFs were validated using rabbit anti-Drp1 monoclonal antibody  and mouse anti-β-Actin  using the same protocol as above. Detailed antibody validation profiles are available from the aforementioned source companies.SC9 was confirmed at the Stanford University Mass Spectrometry core facility (SUMS). The SC9 methanol solution was run on LC-ESI/MS on the Waters Acquity UPLC and Thermo Exploris 240 Orbitrap mass spectrometer . A 15-minute gradient was used, switching from water (0.1% formic acid) to acetonitrile (0.1% formic acid). The column, a Zorbax SB-C18 2.1 × 50 mm, 2.7 u from Agilent , was maintained at 50 °C at a flow rate of 0.3 ml/min. Effluent from the first 2 min of the run was diverted to waste. The injection volume was 2 µL. Spectra were collected in full scan MS mode with polarity switching, Orbitrap resolution 120000, a mass range of 100–1000 Da.The identity of Mouse serum was collected via cardiac puncture. 0.5 M EDTA pH 8.0 coated tubes and syringes were used to prevent clotting. Serum was allowed to clot for 30 min at room temperature and then centrifuged at 1000 g for 10 min at room temp. The supernatant was collected. The 48-plex mouse ProcartaPlex immune monitoring panel was run at the Stanford Immune Monitoring Center in duplicate according to the manufacturer’s instruction. Standards were used to estimate ng/μl and values were normalized to vehicle mean.SC9 was dissolved in DMSO and diluted to the working standards with ACN. The MRM transition under the ESI positive mode for SC9 was m/z 467 > 221, internal standard (Carfentanil-d5) has an MRM transition m/z 400- > 113. The mass spectrometer was a Sciex API5500 with a Shimadzu 20 AD HPLC system. The HPLC column was a Phenomenex Kinetex 2.6 µm, XB-C18, 50 × 2.1 mm. The A mobile phase was 0.1% formic acid in water, the mobile phase B was ACN. The flow rate was 0.5 mL/min. 50 mL of mouse plasma was mixed with 50 uL of internal standard (Carf.-d5 5 µg/mL) and 0.4 mL of ASCN, mixed, and centrifuged at 14000 RPM for 5 min at 4 °C. 25 mL of the upper phase was transferred to an HPLC injection vial with 250 mL of water. For brain tissue samples, 9 parts of 50% methanol/50% water were added to 1 part of the tissue and homogenized with beads at 6500 RPM for 40 s. 50 mL of the homogenate was used for sample analysis as described above.Pharmacokinetic plasma measurements were conducted by Keystone Bioanalytical . Mice were kept on under a 12 h/12 h light/dark cycle, room temperature (~22 °C), and ~50% humidity.SC9 stock solution (50 mg/mL in DMSO) or plain DMSO was added. The final concentration of DMSO in the injection solution was 2.3%. The experimental conditions were blinded (marked A or B) and a different scientist administered 0.2 mL of LPS solution via intraperitoneal injection, taking note of which cages got A or B and the information was then sealed in an envelope. After 4 h, the same protocol was used to re-dose with SC9 or DMSO (10 mg/kg) in 0.1 ml PBS (no LPS). Another scientist, blinded to which cages were A and which were B, conducted the subjective scoring. Mice were weighed and scored every 8 h for 72 h with the murine sepsis score as previously described in Shrum 2014. The respiratory distress score was taken as the sum of the respiratory rate and respiratory quality score from Shrum 2014. Mice were euthanized if they developed severe tremors.Female BALB/cAnNCrl mice (Strain Code 028), 7 weeks old, were purchased from Charles River . LPS doses from 0.2 mg to 0.325 mg were tested (corresponding to 10–16.67 mg/kg based on the average weight of the group). LPS at the indicated concentration was reconstituted in sterile PBS, mixed well, and then divided equally into two tubes. To each tube, a freshly prepared n = 6 per condition  were dosed with SC9 (50 mg/kg) IP and subsequently observed for 72-h. This duration was chosen since that is the typical length of our sepsis experiments. At the end of the observation period, mice were sacrificed, and blood was collected via cardiac puncture. Samples were sent to the Stanford University core animal pathology facilities for standard toxicity screening. Results included biomarkers for hematologic, hepatic, renal, and musculoskeletal damage.C57Bl6/J mice  with All animal experiments were carried out under the protocols (APLAC-33002) approved by the Institutional Animal Care and Use Committee of Stanford University.t-test was used to compare the two groups. The Grubbs’ test was used to determine if any outliers should be removed. For murine sepsis score data, a t-test was used to determine the significance between the two groups at the 24-h time point. For the mouse survival analysis, the 0.3 mg LPS dose was considered, and the Log-rank (Mantel-Cox) test was used to determine significance. The Gehan-Breslow-Wilcoxon test was also automatically generated by Prism and this test concurred with the first test. For the 48-Plex mouse ProcartaPlex panel, a volcano plot without multiple test correction was used to find immune markers of interest.All data were analyzed in Prism. For the GTPase assay, three Michaelis-Menten curves were used to estimate Km and Vmax. A one-way ANOVA with Dunnett’s multiple comparisons test was used to determine statistical significance between multiple groups. A Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


A. hydrophila infection is not well known. Using zebrafish (Danio rerio) kidney macrophages (ZKM), we report that A. hydrophila infection upregulates wnt2, wnt3a, fzd5, lrp6, and β-catenin (ctnnb1) expression, coinciding with the decreased expression of gsk3b and axin. Additionally, increased nuclear β-catenin protein accumulation was observed in infected ZKM, thereby suggesting the activation of canonical Wnt signaling in A. hydrophila infection. Our studies with the β-catenin specific inhibitor JW67 demonstrated β-catenin to be pro-apoptotic, which initiates the apoptosis of A. hydrophila-infected ZKM. β-catenin induces NADPH oxidase (NOX)-mediated ROS production, which orchestrates sustained mitochondrial ROS (mtROS) generation in the infected ZKM. Elevated mtROS favors the dissipation of the mitochondrial membrane potential (ΔΨm) and downstream Drp1-mediated mitochondrial fission, leading to cytochrome c release. We also report that β-catenin-induced mitochondrial fission is an upstream regulator of the caspase-1/IL-1β signalosome, which triggers the caspase-3 mediated apoptosis of the ZKM as well as A. hydrophila clearance. This is the first study suggesting a host-centric role of canonical Wnt signaling pathway in A. hydrophila pathogenesis wherein β-catenin plays a primal role in activating the mitochondrial fission machinery, which actively promotes ZKM apoptosis and helps in containing the bacteria.Canonical Wnt signaling plays a major role in regulating microbial pathogenesis. However, to date, its involvement in  A. hydrophila is a Gram-negative, rod-shaped bacterium that is ubiquitous in aquatic environments. The bacterium is predominantly pathogenic to poikilothermic invertebrates and vertebrates, including fish [A. hydrophila is also an important human pathogen causing gastroenteritis and various other systemic infections [A. hydrophila a pathogen of growing concern [ing fish . In fishfections . The abi concern ,3.A. hydrophila pathogenesis involves the concerted activation of numerous signaling cascades [A. hydrophila infection in several organisms [cascades ,5,6,7. Rrganisms ,9,10, burganisms . It relirganisms . Multiplrganisms .Canonical Wnt signaling shuffles between “off” and “on” states. In the “off” state, cytoplasmic levels of β-catenin are maintained at an exceptionally low level by the β-catenin destruction complex composed of adenomatous polyposis coli (APC), axin, casein kinase 1α (CK1α), and glycogen synthase kinase 3 (GSK3), which target β-catenin to proteolytic degradation. The ‘on’ state is initiated once the Wnt ligands bind Fzd and LRP5/6, which stabilizes β-catenin through inhibiting the β-catenin destruction complex . ThereafChlamydia pneumoniae and Toxoplasma gondii [Pseudomonas aeruginosa and Clostridium difficile [Being a key component of cell and tissue homeostasis, canonical Wnt signaling has also gained recognition as an innate immune attribute, orchestrating interactions between the host immune system and invading pathogens . Howevera gondii ,17, the ifficile ,19, suggAmongst the several downstream factors that β-catenin regulates, reactive oxygen species (ROS) generation is an important phenomenon , althougA. hydrophila pathogenesis.Several reports have reviewed the role of mitochondria as a central hub in innate immunity . Among tdnm1l expression and inhibiting mfn expression [A. hydrophila-induced mitochondrial fission.Mitochondrial dynamics are the balance between mitochondrial fusion and fission, which is paramount to maintaining cell fate. Mitochondrial dynamics are regulated by intricate signaling that involves the pro-fission molecule dynamin-related protein1 (Drp1) and the pro-fusion molecules, mitofusin 1 and 2  . It has pression ,32. Howepression , but theA. hydrophila infection leads to functional alterations in the mitochondria with pro-apoptotic implications [A. hydrophila-infected macrophages [A. hydrophila pathogenesis.We previously reported that ications . To furtrophages . Althougrophages ,37, the A. hydrophila. Our findings establish an intriguing axis of β-catenin-ROS-mtROS–Drp1 that culminates in ZKM apoptosis promoting A. hydrophila clearance.Zebrafish kidney contains both immune and renal cells, and ZKM are inherently phagocytic and serve as an attractive model to study bacterial infections . In the Zebrafish experiments were conducted following the guidelines set forth by the Animal Ethics Committee, University of Delhi (DU/ZOOL/IAEC-R/2013/32) and were performed according to the protocols approved by the Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA), Govt. of India.Selectively bred zebrafish born from one mother were kept in the Animal House Facility, University of Delhi at 28–30 °C under a 14 h light/10 h dark diurnal photoperiod. Prior to setting up the experiments, fish (6–7 months old) were acclimatized to laboratory conditions for 15 days. Throughout the study, fish health was monitored regularly, and they were maintained as described earlier .A. hydrophila (strain 500297) used in the study was gifted from Dr. T. Ramamurthy, the National Institute of Cholera and Enteric Diseases, India. The pathogenicity of the isolate was checked, and the bacteria were grown to log phase in brain–heart infusion broth  containing 100 µg/mL ampicillin overnight at 30 °C with proper aeration [aeration .g for 10 min at 4 °C, with the supernatant discarded, the pellet re-suspended and layered on a discontinuous percoll density gradient (34/51%), and centrifuged at 4000× g for 10 min at 4 °C. The phagocyte-rich fraction was collected, washed, and left for overnight adherence in 24-well culture plates at 30 °C in a CO2 incubator. The non-adherent cell fraction was discarded the following day, and the macrophages were collected by washing with chilled RPMI-1640 . ZKM purity was determined by flow cytometric analysis and staining with Wright Giemsa (90%), and the percentage viability (>90%) was determined by the 0.4% trypan blue dye exclusion method.Zebrafish were sacrificed using an excess of MS222, and the kidney was aseptically removed and placed in sterile PBS. Single-cell suspensions were prepared by homogenizing the kidney using an electric homogenizer for 30 s. The cell suspension was centrifuged at 400× A. hydrophila grown to log phase at different multiplicities of infection (MOIs) (ZKM: bacteria). A short spin of 5 min was given to facilitate bacteria–ZKM interactions, which were then distributed in 24-well culture plates and incubated for 1 h at 30 °C in the presence of 30 µg/mL chloramphenicol [For infection, ZKM were first washed in antibiotic-free RPMI 1640 supplemented with 10% FBS (Gibco-Invitrogen) and were then infected with phenicol . The conZKM were pre-treated separately with β-catenin inhibitors , NADPH oxidase (NOX) inhibitors , Drp1 inhibitor , Mitochondrial permeability transition pore (MPTP) inhibitor , mtROS inhibitor , caspase-1 inhibitor , and caspase-3 inhibitor , respectively, for 1 h prior to addition of the bacteria to the ZKM culture. l1b-siRNA were infected with A. hydrophila, and at indicated time points the resulting apoptosis was studied with various methods.The ZKMs pre-treated with or without the indicated inhibitors, or transfected with sc-siRNA or (i) DNA ladder assay: ZKM (5 × 106) were lysed at 24 h post infection (p.i.) using lysis buffer containing 0.2% Triton X-100, 10 mM Tris (pH 7.2), 1 mM EDTA (pH 8.0), and proteinase K (5 mg/mL). The genomic DNA was extracted, electrophoretically separated on 1.8% agarose gel along with 100 bp DNA ladder (Roche) and visualized under the UV transilluminator after staining with 0.5% ethidium bromide [ Tris pH .2, 1 mM (ii) Hoechst 33342 staining: ZKM (1 × 106) were collected at 24 h p.i., washed, and fixed in 3.7% paraformaldehyde. The fixed ZKM were then stained with Hoechst 33342 , mounted with fluoroshield (Sigma), and visualized under a fluorescence microscope . Three different fields having at least 100 cells were observed for determining the percentage of Hoechst-positive ZKM [(iii) Annexin V-Propidium Iodide (AV-PI) staining: ZKM (1 × 106) were collected at 24 h p.i., stained with AV-PI following the manufacturer’s protocol (BD Pharmingen), washed, mounted with fluoroshield, and visualized under a fluorescence microscope . Three different fields having at least 100 cells were observed for determining the percentages of AV+PI+, AV−PI+, and AV+PI− cells, respectively [ectively .6) pre-treated with or without the indicated inhibitors were infected with A. hydrophila and at indicated time points, ZKM were collected in TRI reagent . The total RNA was isolated following the manufacturer’s protocol. After checking the purity and integrity of RNA, cDNA was synthesized using a first-strand cDNA synthesis kit  from 1 μg of DNase-treated (RNase-free) RNA. Diluted cDNA (1 µL) was used as a template for RT-qPCR using the SYBR Green PCR Master Mix . The conditions for amplification were incubation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and at 60 °C for 1 min, and one cycle at 95 °C for 15 s, at 60 °C for 1 min, and finally at 95 °C for 15 s . The comparative ΔΔCT method was used to determine the expression levels of the analyzed genes, and relative fold changes were normalized against the housekeeping gene, actb1 [ZKM  and incubated for 20 min at 30 °C to facilitate the siRNA transfection complex formation. Next, ZKM (2 × 106) were gently mixed with the transfection complex and incubated at 30 °C in the presence of 5 % CO2 for 16 h, washed, and then infected with A. hydrophila. The efficiency of knockdown was confirmed by RT-qPCR analysis  as per the manufacturer’s instructions. Briefly, 5 µL scrambled siRNA (sc-siRNA) or analysis . The lis6) pre-treated with or without the indicated inhibitors, or transfected with sc-siRNA or il1b-siRNA were infected with A. hydrophila. At indicated time points p.i., the ZKM were lysed (0.1% Triton X-100), following which 20 µL MTT -2,5-diphenyltetrazolium bromide, 5 mg/mL, Merck) was added, and the lysate was then incubated for 5 h at 30 °C. The formazan crystals were dissolved in 100 µL dimethyl sulfoxide  and the absorbance was read at A595. The number of intracellular bacteria was enumerated by interpolating the obtained absorbance values on the standard curve [ZKM (1 × 10rd curve .6) pre-treated with or without the indicated inhibitors were infected with A. hydrophila, and 100 µL of the cell-free culture supernatant collected at indicated time points p.i. were added to each well and incubated for 12 h at 4 °C. The wells were then washed, and 100 µL of biotinylated detection antibodies (1:100) were added to each well and incubated for 60 min at 37 °C. The plates were washed in 1× wash buffer and 100 µL of HRP-conjugated secondary antibodies (1:100) were added to each well and incubated at 37 °C for 30 min. Following washing with 1× wash buffer, 90 µL of substrate was added and further incubated for 15 min at 37 °C. Finally, 50 µL of stop solution was added to each well, and the absorbance was read at A450 using a microplate reader . The β-catenin protein concentration was enumerated by interpolating the values on the standard curve.β-catenin concentration was measured using the CTNNβ1 ELISA Kit (Elabscience) using the chemicals that were supplied with the kit. Briefly, ZKM (2 × 106) were re-suspended in 500 µL of fractionation buffer , passed through a 27-gauge needle, and centrifuged at 720× g for 5 min. The nuclear pellet was then washed with the fractionation buffer, passed through the needle, and centrifuged at 720× g for 10 min. The supernatant was discarded and the pellet was re-suspended in Tris-buffered saline containing 0.1% SDS and then sonicated for 10 s in ice. Thereafter, the β-catenin levels were measured as described above.To obtain the nuclear fraction, ZKM (2 × 106) pre-treated with or without the specific inhibitors were infected with A. hydrophila. The ZKM were collected at indicated time points and superoxide ion measured using nitroblue tetrazolium . Briefly, the cell pellet was resuspended in 100 µL NBT and incubated for 5 h at 30 °C. The pellet was then dissolved in 70 µL DMSO (Sigma) and 60 µL KOH (2 M). The absorbance was read at A620 using a microplate reader [ZKM (1 × 10e reader .6) pre-treated with or without the indicated inhibitors were infected with A. hydrophila. The ZKM were collected at indicated time points p.i., washed, and incubated with 5 µM MitoSOXTM Red mitochondrial superoxide indicator (Molecular Probes) for 20 min in the dark. The pellet was resuspended in 100 µL 1× PBS, and the changes in fluorescence intensity were quantified at an excitation/emission of A510/A580 using a multi-mode microplate reader  [ZKM (2 × 10Germany) .TM were washed, and the pellet was resuspended in 100 µL 1× PBS and incubated with DAPI  for 15 min. The excess dye was washed and the ZKM were then mounted on a slide with fluoroshield, and three different fields were visualized using a fluorescence microscope .In a parallel study, ZKM treated with MitoSOXm were studied using rhodamine 123 dye (Molecular Probes) [6) pre-treated with or without the indicated inhibitors were infected with A. hydrophila. ZKM were collected at indicated time points p.i., washed, and incubated with rhodamine 123 (10 µM) for 30 min at 37 °C in dark. Excess dye was removed with repeated washing, and the fluorescence was recorded at excitation/emission of A511/A534 (BMG Labtech).The changes in ΔΨ Probes) . Brieflyc release was measured according to the mentioned protocol [6) pre-treated with or without the indicated inhibitors were infected with A. hydrophila. The cultures were terminated at indicated times p.i., homogenized in buffer A , followed by the addition of 2% glucose, and centrifuged at 2000× g for 10 min at 4 °C. The supernatant was treated with ascorbic acid (500 mg/mL) for 5 min and absorbance was read at A550 to detect the release of cyt c in the cytoplasm.The cyt protocol . Briefly6) pre-treated with or without the indicated inhibitors were infected with A. hydrophila, and cultures were terminated at 12 h p.i. The ZKM were incubated with MitoTracker Green (50 nM) for 30 min at 30 °C. After washing, DAPI was added for 20 min at 30 °C, and the ZKM were then washed again and allowed to air dry. Next, the slides were mounted, and three different fields were visualized under a confocal microscope . The quantitative analyzes of the confocal images were performed for two parameters: % ZKM showing fragmented mitochondria, and aspect ratio (AR). The AR was calculated using the ImageJ software.The mitochondrial morphology was analyzed with confocal microscopy using MitoTracker Green dye (Molecular Probes). ZKM (1 × 106) pre-treated with or without inhibitors, or transfected with sc-siRNA or il1b-siRNA were infected with A. hydrophila. The cultures were terminated at indicated time points p.i., and 100 µL of cell-free culture supernatant was collected and loaded onto IL-1β antibody-precoated wells. The wells were then sealed and incubated at 37 °C for 30 min. Following washing with wash buffer for 3 times, 50 µL of HRP-Conjugate reagent was added to each well; samples were incubated at 37 °C for 30 min and washed as mentioned above. A total of 50 μL of chromogen Solution A and 50 μL of chromogen Solution B were added to each well, mixed, and incubated at 37 °C for 15 min. The absorbance was recorded at A450 using a microtiter plate reader immediately after adding 50 μL of stop solution. The concentrations of IL-1β were determined through interpolating the values from the standard curve.IL-1β was quantified using the IL-1β ELISA kit . ZKM (2 × 106) pre-treated with or without inhibitors or transfected with sc-siRNA or il1b-siRNAs were infected with A. hydrophila. ZKM were washed and lyzed , and 50 μL of cell lysate was mixed with a 2× reaction buffer containing 5 μL of YVAD-pNA (caspase-1 specific substrate) and DEVD-pNA substrate (caspase-3 specific substrate). The samples were incubated for 1 h at 37 °C and the absorbance was recorded at A405 using a microtiter plate reader . The relative fold changes in caspase-1 and caspase-3 activities were plotted as compared to the control.ZKM , followed by Dunnett’s post hoc analysis using GraphPad software. The value of A. hydrophila-induced cell death has been reported in several organisms including fish [A. hydrophila (ZKM: bacteria), and cytotoxicity was studied using the trypan blue dye exclusion method at 24 h p.i. We observed a positive correlation between ZKM cytotoxicity and MOI  and late apoptotic  ZKM, along with an exceptionally few necrotic ZKM   B, therebpoptosis , and we 4 h p.i. D.A. hydrophila infection led to a significant decline in % Hoechst-positive and AV+PI−/AV+PI+ ZKM at 24 h p.i.  mRNA expression and observed a significant up-regulation in ctnnb1 expression with the peak fold change recorded at 6 h p.i.  for 1 h led to increases in mtROS production in the ZKM at 12 h p.i.  in infected ZKM. Drp1 favors mitochondrial fission, and we observed a significant increase in dnm1l mRNA expression with the maximum fold change observed at 12 h p.i.  . To expl2 h p.i. . Additiocted ZKM . Our fincroscopy E. The uncted ZKM E.A. hydrophila, and the expression of dnm1l, mfn1, and mfn2 mRNA were monitored at 12 h p.i. Pre-treatment with JW67 and MSAB were found to downregulate dnm1l expression and upregulate the expression of mfn1 and mfn2 at 12 h p.i. . Pre-treatment with CsA for 1 h inhibited cyt c release at 12 h p.i. . Although mtROS generation in infected macrophages has been reported both in vitro and in vivo, the underlying molecular mechanisms remain poorly defined. Moreover, reports investigating ROS-induced-mtROS production are limited in fish. We observed significant mtROS levels in drophila  and othedrophila , establidrophila . We notedrophila , we obsesod2 mRNA expression in A. hydrophila-infected ZKM, which recovered on inhibiting β-catenin signaling. Based on these results, we propose that β-catenin inhibits the activation of cellular antioxidant machinery leading to pronounced oxidative stress (NOX-mediated and mtROS), thereby eliciting ZKM apoptosis and A. hydrophila clearance.Sod2 plays an important role in maintaining the redox balance under different conditions of stress . Its rolm collapse has also been well documented [A. hydrophila pathogenesis. We report that mtROS induces the decrease of ΔΨm in A. hydrophila-infected ZKM. Mitochondrial dynamics plays an intimate role in apoptosis, and it has been suggested that the loss in ΔΨm favors mitochondrial fission, triggering apoptosis [m in mitochondrial fission. The key players regulating mitochondrial fission include Drp1, Mfn1, and Mfn2, respectively [A. hydrophila infection results in the upregulation in pro-fission dnm1l expression, and downregulation in anti-fission mfn1 and mfn2 expression, which was reversed in the presence of CsA and Mdivi-1. Inhibition of MPTP and mitochondrial fission also restored the intracellular growth of A. hydrophila. Additionally, inhibiting β-catenin signaling restored ΔΨm, decreased dnm1l expression, and increased mfn1 and mfn2 expression, with concomitant reductions in mitochondrial fission and increased intracellular A. hydrophila replication observed. Collectively, our results implicate that: (1) β-catenin induces mitochondrial fission using mtROS and ΔΨm as intermediates, and (2) mitochondrial fission leads to apoptosis of the infected ZKM and clearance of intracellular bacteria.Synchronism in mtROS production and ΔΨcumented ,49, but poptosis . We set ectively . We obsec accumulation. Our results corroborate previous findings depicting inhibition in mitochondrial fission leads to the reduced release of cyt c into the cytosol [A. hydrophila infection has been reported to induce cyt c release subsequently triggering apoptosis [c levels in the infected ZKM. Hence, our study reflects on the involvement of β-catenin in inducing mitochondrial fission leading to the release of pro-apoptotic cyt c into the cytosol.Mitochondrial fission results in the leakage of mitochondrial proteins in the cytosol, activating the downstream apoptotic signaling cascade . We obse cytosol ,72. Howepoptosis , the rolA. hydrophila pathogenesis [A. hydrophila-infected fish macrophages. These results are in synchrony with earlier studies on canonical Wnt signaling triggering caspase-dependent apoptosis in bacterial infections [The caspase-1/IL-1β signalosome has been implicated in inducing caspase-3-mediated apoptosis in ogenesis . Extendifections . TogetheA. hydrophila pathogenesis. We propose that β-catenin creates a pro-oxidant environment which triggers mitochondrial fission, culminating in caspase 3-mediated apoptosis of the infected ZKM, and the clearance of intracellular A. hydrophila (In conclusion, our study has established the pro-apoptotic and bactericidal role of canonical Wnt signaling in drophila . Our fin


Candida tropicalis strains with reduced xylitol dehydrogenase (XDH) activity to eliminate co-substrate requirement for corn cob-based xylitol-ethanol biorefinery.Xylitol has a wide range of applications in the pharmaceuticals, cosmetic, food and beverage industry. Microbial xylitol production reduces the risk of contamination and is considered as environment friendly and sustainable compared to the chemical method. In this study, random mutagenesis and genetic engineering approaches were employed to develop w/v) was fermented in bioreactor, the Ethyl methane sulfonate (EMS) mutated strain  showed 9.2% and XYL2 heterozygous (XYL2/xyl2Δ::FRT) strain  showed 16% improvement in xylitol production compared to parental strain . Furthermore, 1.5-fold improvement (88.62 g/L to 132 g/L) in xylitol production was achieved by C. tropicalis K21D after Response Surface Methodology (RSM) and one factor at a time (OFAT) applied for media component optimization. Finally, corncob hydrolysate was tested for xylitol production in biorefinery mode, which leads to the production of 32.6 g/L xylitol from hemicellulosic fraction, 32.0 g/L ethanol from cellulosic fraction and 13.0 g/L animal feed.The results suggest that when pure xylose (10% C. tropicalis K21D as a microbial cell factory for efficient production of xylitol and ethanol via an integrated biorefinery framework by utilising lignocellulosic biomass with minimum waste generation.This work, for the first time, illustrates the potential of The online version contains supplementary material available at 10.1186/s12934-023-02190-3. It has various applications in the nutraceuticals, pharmaceuticals, beverage and food industries owing multiple pharmacological values like deterrence of ear infections and dental cavities ., 30.Candrategies , 7. Howeentation , 31. In C. tropicalis K2M which was explained by more than onefold reduction in XDH activity. The main mechanism regulating xylitol build-up is a redox imbalance between NAD-dependent XDH and NADPH-dependent XR. Similar reports with improved xylitol production have also been stated in literatures. For instance, Kim et al. [K. marxianus ATCC 36907 from 14 to 25 g/L. In another study by Rao et al. [C. tropicalis was mutated with methylnitronitrosoguanidine (MNNG), and 10% increment in xylitol yield was attained using pure xylose. Moreover, in a recent study, Prabhu et al. [P. fermentans as compared to the wild type strain.When EMS mutagenesis was applied in this study, improvement in xylitol yield and productivity was achieved by m et al. , used EMo et al. , C. tropu et al. , showed XDH gene resulted in more than twofold reduction in XDH activity of C. tropicalis K21D which led to significant increase in xylitol levels. Further, the inhibitor tolerant experiment proves that C. tropicalis K21D strain is highly resistant to the mixture of inhibitors tested, indicating that detoxification steps are not required for this strain during fermentation in LCB, being economically beneficial in biomass conversion.Moreover, the knockout of single allele of C. tropicalis K21D revealed that our results exhibited promising xylitol yield compared to others reported in the literature, where the recombinant strains were developed with disruption or deletion of XYL2 gene. Zhang et al. 2021, constructed Kluyveromyces marxianus YZB194 by overexpressing NcXYL1 from Neurospora crassa and ScGAL2N376F from S. cerevisiae, and deleting XYL2, PGI1, KU70 and GPD1, with a xylitol yield of 0.83 g/L.h by co-utilizing glucose and xylose. In another report, the non-conventional yeast Meyerozyma guilliermondii ATCC 6260 showed three times higher xylitol yield as a result of the knockout of the XDH gene coupled with the cloning and overexpression of the XR gene [XYL2 gene deletion and heterologous expression of NADPH in C. tropicalis XZX-B4ZG obtained 0.82 g/L.h xylitol productivity [C. tropicalis K21D achieved a 90% xylitol yield without co-substrate requirement such as glycerol, allowing more cost-effective purification of xylitol.Xylitol fermentation in the Erlenmeyer flask with the engineered strain XR gene  using glC. tropicalis K21D strain, contrary to the wild type. Interestingly, a 1.5-fold increase in xylitol production was determined experimentally in C. tropicalis K21D strain using parameters 1.27 g/L urea, 1.19 g/L inoculum and 15.57% w/v xylose as predicted by model. The mathematical model used did not exhibit a lack of fit, and the F-test shows high statistical significance (p < 0.001) at 99% confidence level. Existing literature also suggests that media optimization and RSM can enhance the xylitol production by the yeast. Ling et al. [C. tropicalis HDY-02. Likewise, Yewale et al. [C. tropicalis to enhance the xylitol yield. These experiments illustrate the necessity of statistical tools in finding the critical factors affecting the yeast cell biomass and proliferation along with the desired product output.Factors such as nitrogen, inoculum and sugar concentration are determinant factors to successfully scale-up a bioprocess. In the present work, RSM has been used to determine the optimization reactions boosting xylitol production in the g et al. , used a e et al. , optimizC. tropicalis K21D fermentation was significantly higher than in previous studies. In one study, fermentation with detoxified corncob hydrolysate and C. tropicalis strain achieved 0.64 g/g xylitol yield after 48 h [C. tropicalis JH030 [P. fermentans on SCB pre-hydrolysate without detoxification.The process variables such as aeration, pH-controlled, mechanical agitation, and bioreactor configuration perhaps contributed to enhancing xylitol production in bioreactor experiments compared to flask experiments. When bioreactor experiments were performed using non-detoxified SCBH, the xylitol yield achieved with ter 48 h . Anotheris JH030 . More reS. cerevisiae NGY10 estimated here is much higher than reported in other literature on biomass hydrolysates. Earlier, 0.44 g/g ethanol yield was achieved with enzymatic hydrolysate of cotton stalk [S. cerevisiae VS3 and C. tropicalis JFH5 with ethanol and xylitol yield of 0.43 g/g and 0.65 g/g, respectively [Kluyveromyces marxianus to produce xylitol (0.82 g/g yield) along with the ethanol (0.41 g/g yield).To develop a sustainable and integrated process, ethanol and xylitol production was proposed in this study via the volarization of the corncob. The ethanol yield (0.47 g/g) by ectively . In anotectively , performCandida species has been proposed for SCP production in previous studies [C. utilis has the potential to be a useful alternative protein source (38 to 40%) in diets for Atlantic salmon. Using yeast protein as an animal feed or SCP might endorse notable value in the bio-refinery industries. It is possible to increase the process’s profitability by recovering yeast biomass and utilizing it as a protein source while synthesising both xylitol and ethanol.Complete conversion of LCB into valuable compounds such as xylitol, ethanol and animal feed via integrated biorefinery framework is important to make the process cost-effective . The sin studies . Øvrum H studies  reportedC. tropicalis strain K21D can be performed by a combination of metabolic, genetic, and process engineering strategies in future. For instance; altering metabolic pathway by modifying the xylose transporters and enzymes involved in xylose metabolism to improve the efficiency of xylose uptake and conversion to xylitol. Overexpression of XYL1  will further boost the xylitol production in this strain. Also, increasing the cellular redox balance by overexpressing enzymes involved in NAD(P)H regeneration will increase the xylitol biosynthesis. Furthermore, strong and inducible promoters could be used to optimize the expression of key genes involved in the xylitol pathway. Development of an efficient separation and purification method to recover xylitol from the fermentation broth will also be an effective approach for optimization process. Moreover, performing transcriptomic and proteomic analyses to understand the cellular response to genetic modifications and identify potential targets for further optimization and increasing xylitol yield.To further enhance xylitol production, optimization of an engineered In order to generate value-added products from various LCB, mass balance assessment plays an essential role in determining the bottlenecks of bioprocess . MountraCandida tropicalis K21D and corncob with minimum waste generation. The integrated method aided us in producing higher xylitol yield (0.82 g/g) and animal feed (6.8 g) from corncob hemicellulosic fraction. Moreover, the ethanol produced (32 g/L) from cellulosic fraction may significantly affect the economy of the xylitol production process. Considering the futuristic approach toward circular economy and biorefinery, this strain can be utilized as a potential bio-tool for the cost-effective production of xylitol using non-detoxified lignocellulosic biomass, thus providing value-added products to industries and reducing environmental waste generated during production.This study reveals a novel integrated biorefinery approach using C. tropicalis K2, was isolated and characterized in our previous study [2PO4: 2.0 g/L, (NH4)2SO4: 1.0 g/L, MgSO4.7H2O: 1.0 g/L, xylose 5%).The strain, us study  , 0.25 M, 0.50 M, 0.75 M and 1.0 M in 2 mL tube and incubated at 30 °C . The EMS mutagenesis reaction was ended by addition of 0.5 mL sterile sodium thiosulfate (5% w/v) to the mutated cultures, followed by incubation at room temperature (15 min). Cells were pellet down at 4000 rpm (5 min), washed with sodium thiosulfate (5% w/v) and resuspended in sterile milliQ water (100 µL). Finally, the cells were plated on the YEPX plate containing 5% w/v xylose, incubated for 48 h (30 °C), and selected colonies were screened for xylitol production. For this, 1.0 OD cells of each selected mutant were inoculated in 1.5 mL of fermentation media in 24 well plates and xylitol production was analyzed after 96 h of incubation (30 °C) by spectrophotometric method  as described [EMS mutagenesis of  earlier . Brieflyescribed .XYL2 gene was deleted by SAT1-flipperstrategy as described previously [XYL2 coding sequence was PCR amplified from C. tropicalis K2 genomic DNA using XYL_2 and XYL_3, XYL_4 and XYL_5 primers, respectively  and KpnI and XhoI (3′ homologous region) restriction enzymes and cloned in to pSFS2A vector to generate pSFS2A-XYL2-I plasmid. The pSFS2A-XYL2-I was digested with SacI and XhoI, the resulting linear DNA, 5′XYL2-FRT-SAT1-FLP-FRT-3′XYL2, was utilised as the disruption cassette and transformed into the C. tropicalis K2 using lithium acetate (LiAc) method [SAT1 cassette was confirmed by using XYL_11 and XYL_6 (5′ integration primers) and XYL_7 and XYL_10 (3′ integration) primers  amplification. The presence of internal amplicon as well as 5′ and 3′ integration of SAT1 cassette confirmed the deletion of XYL2 single allele. Further, SAT1 cassette was removed fromthe C. tropicalisK21D strain using the maltose induction medium . Overnight grown cells (50 X) were plated on YPD for a single colony, and marker-less C. tropicalis K2 single allele deleted cells were confirmed using negative selection on nourseothricin plate [The eviously . Briefly) method . Transfoin plate .®-24), at maximum speed  with intermittent cooling followed by centrifugation . The cell lysate was then used for measuring XDH and XR enzyme activities [Cell extracts were prepared by resuspending the harvested cells (10 mL) in 500 µL of homogenization buffer  and glastivities  as well tivities . XR and tivities . One unid-glucose: 20 g/L) containing acetic acid (3.0 g/L), HMF (1.0 g/L) and furfural (1.0 g/L) individually as well as in a cocktail . Spotting assays were performed on SD agar plates, as reported earlier [Inhibitor tolerant assays were executed in 5.0 mL SD medium , nitrogen sources {ammonium sulphate, ammonium chloride, peptone and urea } and seed inoculum  were optimized by using one factor at a time (OFAT) strategy at 30 °C for 144 h (180 rpm) in Erlenmeyer flask. The medium components  selected were further optimized by Response Surface Methodology (RSM) (Design Expert 13.0 (DE 13) software, Stat-Ease, Inc., Minneapolis, MN, USA) and Box Behnken Design (BBD) as described earlier [Initial xylose concentration  protocol was used for hemicellulose, cellulose, and lignin content estimation in corncob  .v/v sulphuric acid was added to the powdered biomass (100 g) and autoclaved at 121 °C (60 min). The resulting liquid fraction 1 after centrifugation and filtration (pH 5.5) is then utilized as fermentation media for xylitol production by C. tropicalis K21D. Furthermore, for ethanol production, the remaining cellulignin  from the acid pre-treatment was subjected to alkaline pre-treatment (121 °C for 60 min), filtered, and the cellulosic residue (solid fraction 2) obtained was enzymatically hydrolysed with CTec2 commercial cellulase  (5 FPU/g of 10% w/v dry biomass) for 72 h (50 °C) [Saccharomyces cerevisiaeNGY10. The samples were taken periodically at every 6 h of interval for analysis by HPLC (High performance liquid chromatography). The fermentation medium containing pure glucose (70 g/L), yeast extract 5 g/L, magnesium sulphate 0.374 g/L, ammonium sulphate 3.74 g/L, dihydrogen potassium phosphate 2.10 g/L, calcium chloride 0.5 g/L was used as control.Acid pre-treatment of corncob was performedas described earlier . Briefly (50 °C) . The resw/v) was performed with C. tropicalis K2, C. tropicalis K2M and C. tropicalis K21D strains. Further, the bioreactor fermentation was performed with high xylitol-producing C. tropicalis K21D strain to validate RSM experiment with fermentation media containing pure xylose (15.57% w/v) in 1% YNB and urea. Fermentation was also conducted with wild-type strain C. tropicalis K2 at similar conditions using xylose (15.57% w/v) for comparison. In addition, fermentations on non-detoxified acid pre-treated corncob hydrolysate (200 mL) were performed with C. tropicalis K21D and wild type strain C. tropicalis K2 after supplementation with urea. For this, overnight grown cells (0.7 g/L) in YEPX were added to the respective fermentation media and experiments were performed at 0.5 vvm aeration having continuous impeller speed of 300 rpm with controlled pH 5.5 and temperature 30 °C. The samples were analysed by HPLC at every 6 h. The dry cell weight (DCW) was determined after drying the cells for 24 h at 80 °C and expressed in g/L. Xylitol yield (g/g) was measured by the ratio of xylitol (g/L) produced vs xylose (g/L) utilized. The maximum xylitol productivity (g/Lh) was calculated as reported earlier[Xylitol fermentation was executed in a bench-top bioreactor , integrated with pH control, aeration, dissolved oxygen (DO) and temperature sensors with a working volume of 200 mL. Initially, bioreactor fermentation using pure xylose  detector  by HPLC . The proANOVA  was executed, and t-test was done to detect significant differences (p < 0.05).Additional file 1:Figure S1. Sugar released after enzymatic hydrolysis of various acid and alkali pretreatment of corncob for ethanol production.Additional file 2:Table S1. List of primers used in this study.Additional file 3:Figure S2. Gene deletion strategy in C. tropicalis K2: deletion of XYL2 gene in C. tropicalis K2 includes three steps. In the first step, PCR amplification of 5′UTR and 3′UTR overhang and construction of deletion cassette with SAT1 as a marker and their integration into cassette. Second step includes the transformation of this deletion cassette in the WT cells and integration of the cassettevia homologous recombination. The final step is to validate the gene deletion via genomic DNA PCR. Primer 1: XYL_26, Primer 2: XYL_11, Primer 3: XYL_10, Primer 4: XYL_7, Primer 5: XYL_8, Primer 6: XYL_9.


N,N-dimethylethylenediamine (DMED), in positive-ion ESI-MS/MS. The findings indicate that when these compounds contain an allylic hydroxyl group that is located distal to the terminal DMED moiety in the case of resolvin D1, D4, and lipoxin A4, an aldehyde (-CH=O) is predominately formed, which arises from the breakdown in between vicinal diols, whereas, in the case of an allylic hydroxyl group that is located proximal to the DMED moiety, as in resolvin D2, E3, lipoxin B4, and maresin 2, an allylic carbene (-CH=CH-CH:) is formed. These specific fragmentations could be used as diagnostic ions for characterizing the above seven PUFAs. As a result, it was possible to detect resolvin D1, D2, E3, lipoxin A4, and B4 in sera (20 μl) obtained from healthy volunteers by multiple-reaction monitoring using LC/ESI-MS/MS.A characteristic fragmentation was observed for PUFAs that contain allylic vicinal diol groups , which were derivatized with  Resolvins D and E, maresins, and lipoxins constitute important classes of specialized proresolving mediators, and the former three are biosynthesized from n-3 fatty acids while the origin of the last one is n-6 fatty acids , 2. ThesN,N-dimethylethylenediamine (DMED) that contain allylic vicinal diols, namely, Resolvin D1 (RvD1), resolvin D2 (RvD2), and resolvin D4 (RvD4); 5S, 6R-Lipoxin A4 (LxA4) and 5S, 14R-Lipoxin B4 (LxB4); maresin 2 (Mar2), and resolvin E3. They specifically gave either aldehyde (-CH=O) or allylic carbene (-CH=CH-CH:)-terminal fragment ions as the result of breakdown in between the vicinal diol groups. These characteristic fragment ions were used to screen these PUFAs in human serum.We report herein on a characteristic fragmentation of PUFAs derivatized by S, 6R-Lipoxin A4-19,19,20,20,20-d5; and Resolvin D1-21,21,22,22,22-d5 were supplied by Cayman Chemical Co. . 18S-Resolvin E3 and 18R-Resolvin E3 (RvE3R) were prepared as described previously , dimethylformamide , 2,6-di-tert-butyl-4-methylphenol, triethylamine, DMED, and 2-chloro-1-methylpyridinium iodide were purchased from Sigma-Aldrich Co. LLC . RvD1, RvD2, and RvD4; LxA4 and LxB4; maresin 1 and Mar2; 9,10-DiHOME, 12,13-DiHOME, 5eviously , 16. HPLSamples of human sera , 585-male (20 y.o.), 587-male (36 y.o.), 588-male (32 y.o.), 589-male (36 y.o.), 590-male (34 y.o.), 591-male (32 y.o.), 593-female (28 y.o.), 594-female (25 y.o.), 597-female (34 y.o.), and 600-female (30 y.o.)) without personally identifiable information were purchased from BioIVT . This study was approved by the Ethics Committee of the Institute for Protein Research, Osaka University (No. 2021-1-1). Human studies abided by the Declaration of Helsinki principles.The PUFAs were derivatized by reaction with DMED according to a previously reported method  with minThe calibration curves, correlation coefficients, accuracies, and linear ranges for the seven types of PUFAs based on the characteristic fragment ions were obtained , which sS, 6R-Lipoxin A4-19,19,20,20,20-d5(17.48 pg) and Resolvin D1-21,21,22,22,22-d5 (8.19 pg) as the internal standards. The prespiked sample was mixed with 100 μl of a 90% ACN/10% MeOH solution containing 0.01 mM 2,6-di-tert-butyl-4-methylphenol, vortexed, allowed to stand at 4°C for 30 min, and then centrifuged for 15 min at 12,000 rpm. The supernatant was diluted eight times with ultrapure water and then loaded on a Bond Elut C18 column (50 mg) that had been washed with 1 ml of aqueous 70% ACN/0.3% FA and equilibrated with 2 ml of aqueous 5% ACN/0.3% FA. After washing the column three times with 1 ml of 5% ACN/0.3% FA, the PUFAs were eluted with 800 μl of aqueous 45% ACN/0.3% FA. The eluates were evaporated to dryness, derivatized with DMED as above, and subjected to LC-MS/MS for the MRM analysis (see below).The PUFAs were isolated from serum according to a previously reported method with minor modifications , 18. Twem/z values for the precursor and fragment ions, that are listed in The LC-MS/MS analysis was performed using an Agilent 1290 Infinity II and 6470 triple quadrupole MS, consisting of Q1 (MS1), Q2 (collision-induced dissociation [CID] area), and Q3 (MS2), equipped with an ESI ion source . Chromatographic separation was achieved with an Agilent Eclipse Plus C18 RRHD 2.1×100 mm, 1.8 μm column, Lot No. B22496 , the inlet of which was connected to a heat exchanger  , which was maintained at 60°C and connected to an InfinityLab Quick Change inline filter . The mobile phase consisted of solvent A (0.3% FA in ultrapure water) and solvent B (0.3% FA in 20% MeOH and 80% ACN). The elution gradient was as follows: 25.0–28.0% B (0–5.0 min); 28.0–45.0% B (5.0–10.0 min); 45.0–80.0% B (10.0–11.0 min); 80.0% B (11.0–12.0 min); 80.0–25% B (12.0–12.01 min); 25.0% B (12.01–14.0 min). The injection volume was 20 μl for all samples. The MRM mode was used for detecting the derivatized PUFAs by using the transitions, pairs of 2) gas pressure of 30 psi, drying gas (N2) temperature and flow rate of 240°C and 13 L min-1, respectively, and a sheath gas temperature of 250°C was used. Product-ion spectra were obtained by injecting 2.5 ng of each of the DMED-derivatized PUFA into the LC-MS/MS, which was operated under the same conditions as above. Data were acquired using an Agilent MassHunter Acquisition system and processed using Agilent MassHunter Quantitive Analysis.The collision energy voltages were set at 10, 15, or 20 eV and were optimized for each analyte see . The fraThrough measurements of various types of PUFAs, we found that PUFAs that contain allylic vicinal diols  gave theIn order to assess the usefulness of the above diagnostic ions for characterizing PUFAs with allylic vicinal diols in a bio-sample, we applied the above fragment ions  as the dS-Resolvin E3, RvE3R, LxA4, and LxB4 among the eight types of the PUFAs to be detected in human serum pyridinium (AMPP)  and in their configurations. Thus, in order to clearly identify and quantify these molecules by MRM analysis, especially those in a complex mixture, it is necessary to use characteristic fragment ions that are specific for each of them  as the dm (AMPP) , 14 signal diols . This, hal diols . Howeveral diols .The characteristic fragmentation occurring at the allylic vicinal diols, which is derived from a dehydration process promoted by a mobile proton , is usefAll data are presented within the article and Supporting information.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Currently, no PMMA cement is commercially available to cover VRE. Daptomycin shows promising results in treating infection, offering a good safety profile and a reduced risk of developing resistance. The purpose of this in vitro study was to investigate the mechanical stability, handling properties, elution behavior, and antimicrobial effectiveness of PMMA cement loaded with three different daptomycin concentrations in comparison to commercially available antibiotic-loaded bone cement (ALBC). Methods: Mechanical properties and handling characteristics , HPLC elution, antimicrobial effectiveness with proliferation assay (DIN 17025), and inhibition zone testing were investigated. Results: All tested daptomycin concentrations met the ISO and DIN standards for mechanical strength. Loading of 40 g of PMMA cement with 0.5 g of daptomycin did not show any antimicrobial effectiveness, in contrast to 1.0 g and 1.5 g. PMMA cement with 1.5 g of daptomycin was the best in terms of elution and effectiveness, and it showed good ISO mechanical strength; ISO doughing was sticky for a little longer and setting was faster compared to the vancomycin-containing reference cement. Conclusion: PMMA cement containing 0.5 g of gentamicin and 1.5 g of daptomycin could be a good alternative to the already established COPAL Staphylococcus aureus (VRSA), vancomycin-resistant Enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and methicillin-resistant Staphylococcus epidermidis (MRSE), is rising [S. aureus (VISA), VRSA, and VRE is daptomycin; it also shows antimicrobial activity against anaerobic bacteria, but none against Gram-negative bacteria [Periprosthetic joint infections (PJIs) are a challenging complication in joint replacement surgery that often results in worse outcomes for the patients, especially when the causative pathogen is a multidrug-resistant germ  and/or ts rising ,11, and s rising . One antbacteria ,13. Daptbacteria .There is no daptomycin ALBC commercially available, and the manual admixture of daptomycin is highly expensive . In clinical practice, surgeons increase the vancomycin concentration up to 4 g per 40 g of polymer powder to treat vancomycin-resistant germs. But a massively increased antibiotic concentration in PMMA spacers negatively influences their mechanical stability , and theWith this investigation, we wanted to figure out what concentration of daptomycin can be added to PMMA cement to efficiently inhibit bacterial growth and, at the same time, deliver mechanical stability according to ISO standards.S. epidermidis (MRSE), the blank sample and PMMA cement loaded with 0.5 g of gentamicin and daptomycin (GD0.5) did not show any antimicrobial activity (S. aureus (VRSA), the blank sample and GD0.5 did not show antimicrobial activity (E. faecium (VRE), where only GD1.0 and GD1.5 showed antimicrobial activity (S. aureus (MRSA), the blank sample and GD0.5 did not inhibit bacterial growth, in contrast to GD1.0 and GD1.5. Overall, the antimicrobial efficacy of GD1.5 and GD1.0 did not differ widely, with GD1.5 showing the greatest effect on bacterial growth.For methicillin-resistant activity a. GD1.0 activity b, whereaactivity c. For me2, 1.0 g led to an inhibition zone of 2219 ± 346 mm2, and 0.5 g led to an inhibition zone of 2134 ± 198 mm2. GD1.5, GD1.0, and GD0.5 showed antimicrobial effectiveness against B. subtilis  compared to gentamicin (734.1 ± 48 µg) for GD1.5 (® G+V) was assessed for vancomycin elution  R+G), Ref2 (COPAL® G+V) showed the highest total amount of antibiotics eluted. Compared to the vancomycin release (® G+V), the total amount of daptomycin released was lower, indicating an overall better elution from Ref2 (COPAL® G+V) compared to the GD samples.The highest volume of gentamicin released from daptomycin + gentamicin-loaded PMMA cement samples was observed on day 1, followed by a continuous decrease in antibiotic release a. The geor GD1.5 b,c. Addi elution d, showin release d from Re® R+G (Ref1) showed a bending strength of 71 ± 1 MPa, surpassing the threshold of 50 MPa. COPAL® G+V (Ref2) was close to the threshold, with a bending strength of 58 ± 3 MPa. The ISO bending strength was highest for GD1.0 (72 ± 1 MPa), followed by GD0.5 (70 ± 2 MPa) and GD1.5 (67 ± 2 MPa), which therefore come with a higher bending strength than COPAL® G+V (Ref2). The bending modulus of Ref1 (2922 ± 66 MPa) was comparable to that of Ref2 (2900 ± 30 MPa), fulfilling the minimum threshold of 1800 MPa (All tested daptomycin-containing PMMA cement samples fulfilled the ISO and DIN requirements a. PALACO1800 MPa b. GD1.0 1800 MPa c. The co2) of DIN bending strength, with 69 ± 3 N/mm2, as did Ref1 with 81 ± 1 N/mm2 (2 (GD0.5) to 70 ± 3 N/mm2 (GD1.0) and 64 ± 4 N/mm2 (GD1.5). GD1.0 showed a comparable DIN bending strength to Ref2, whereas GD1.5’s was slightly below that of Ref2. The DIN impact resistance was measured for COPAL® G+V (Ref2) as 3.0 ± 0.3 kJ/m2, which was set as a reference. The DIN impact resistance is shown as the difference compared to COPAL® G+V (Ref2) (2), GD1.0 (3.2 ± 0.4 kJ/m2), and GD0.5 (3.1 ± 0.5 kJ/m2) exceeded the impact resistance of Ref2, while GD1.5 (2.6 ± 0.6 kJ/m2) showed the highest difference in DIN impact resistance. The higher the daptomycin concentration, the lower the measurements for DIN bending strength and impact resistance, indicating that a high daptomycin concentration in PMMA bone cement reduces its mechanical properties. The ISO bending strength for GD1.5 was lower compared to all other concentrations and the references.All GD cement samples fulfilled the requirements for mechanical stability according to DIN 53435. Ref2 exceeded the minimum threshold (50 N/mm 1 N/mm2 a. The DIV (Ref2) b. Ref1 (® G+V (Ref2), and even more so compared to PALACOS® R+G (Ref1) (GD1.5 was slightly faster-setting compared to COPALG (Ref1) , resultiAs PJIs still pose a threat to patients and the healthcare system, especially when caused by resistant germs, all kinds of support are needed to prevent PJIs or treat them in a two-stage revision protocol . Daptomy® G+V is already established for the prevention of PJIs, especially against the most frequent PJI germs S. aureus and S. epidermidis [Galleria melonella larvae biofilm model also showed a synergistic effect of combining gentamicin with daptomycin for the treatment of vancomycin resistant E. faecium [® G+V as a reference. Despite finding the highest vancomycin elution for day 1, the elution had already decreased by ~85% on day 2. We doubt that this vancomycin concentration, in a clinical setting, would be sufficient to meet the MIC of VRSA (MIC ≥ 16 µg/mL) and VRE (MIC ≥ 32 µg/mL) [In clinical practice, spacers for two-stage revision procedures are created by using industrially manufactured cement already containing vancomycin, or by manually admixing vancomycin with PMMA cement ,18,19. Tdermidis . The spedermidis . Additiodermidis . Antibiodermidis . Daptomydermidis , coming dermidis . Accordidermidis , knowleddermidis  figured dermidis . A firstdermidis . In our dermidis , who condermidis . The ovedermidis , showingdermidis , who con faecium . We obse faecium , indicat2 µg/mL) , but we To ensure patient safety, daptomycin must be sterilized with either ethylene oxide or gamma radiation. Our findings suggested that daptomycin-loaded bone cement should be sterilized using gamma radiation to maintain its antimicrobial effectiveness, because sterilization with ethylene oxide reduced the efficacy of daptomycin significantly.PIF Pocket Guide [® G+V (40 g) by another 2 g. But an increase in the addition of antibiotic powder beyond a total concentration of 10% results in a spacer cement that no longer fulfills the mechanical ISO requirements for bone cement [® R+G instead of using the commercially available COPAL® G+V. These spacers do not comply with the ISO requirements, as the bending strength for PALACOS® R+G + 4 g of vancomycin is below the minimum threshold of 50 MPa and comes with the potential risk of bone cement or spacer fracture [® R+G and COPAL® R+V, indicating a slightly faster setting time for GD1.5, which means a shorter application time window.To best treat MRSE, MRSA, or enterococci, the et Guide  recommene cement ,19,28. De cement  pointingfracture ,19,21,29fracture . The mecfracture ,22,30. Cfracture , determiOur investigations indicated that the performance of a PMMA bone cement containing 0.5 g of gentamicin and 1.5 g of daptomycin is the optimal choice considering its antibiotic effectiveness, antibiotic release, and mechanical stability . AccordiWe want to highlight that a surgeon ordering daptomycin from the pharmacy will receive a different product than the industrially used daptomycin. The clinical available “Cubicin” , in addi® R, PALACOS® R+G, and COPAL® G+V  were used. PALACOS® R is a plain PMMA cement without antibiotics, PALACOS® R+G contains 0.5 g of gentamicin, and COPAL® G+V contains 0.5 g of gentamicin combined with 2 g of vancomycin. PALACOS® R+G was loaded with 1.5 g, 1.0 g, and 0.5 g of daptomycin powder . PALACOS® R, PALACOS® R+G, and COPAL® G+V were used as references  and Tris-buffered minimum medium  were prepared and incubated with TCC 6633 . Two bonB. subtilis, inhibition zone tests were performed by an external certified lab (INNOVENT e.V. Jena). Data collection and statistical analysis were performed using IMPAGE pro V2 scanning software.To determine the influence of sterilization on the antimicrobial effectiveness of non-sterilized, ethylene-oxide-sterilized, and gamma-sterilized daptomycin against To determine the release profile of gentamicin and daptomycin dissolved from bone cement samples over 5 days (60 dissolution samples each), HPLC with MS/MS detection was performed ,39. As tTo ensure that the added daptomycin did not negatively influence the mechanical stability of the PMMA cement, mechanical tests for bending strength, bending modulus, and compressive strength were performed according to ISO 5833:2002 . To dete2. For the DIN bending strength, a bending force of 400 Ncm was applied to the bone cement samples until they broke. The bending strength was measured and calculated, and statistical analysis was performed according to DIN 53435 [The DIN impact strength and bending strength were also determined according to DIN 53435 . The recThe ISO setting time and ISO doughing time were assessed, and statistical analysis was performed .Our results suggest adding 1.5 g of daptomycin to PMMA cement combined with 0.5 g of gentamicin for PJI cases caused by vancomycin-resistant germs. All mechanical and handling properties, along with the elution profile and effectivity of 1.5 g of daptomycin added to 40 g of PMMA, fulfilled all clinical requirements. Due to its antimicrobial spectrum against vancomycin-resistant germs , a PMMA cement containing 0.5 g of gentamicin and 1.5 g of daptomycin instead of vancomycin could be a good option for the treatment of PJIs. Further investigations of its performance in biofilm models and against clinical isolates are recommended.